UvA-DARE (Digital Academic Repository) Thrombin ... · Thrombin-activatable fibrinolysis inhibitor and bacterial infections General rights It is not permitted to download or to forward/distribute

UvA-DARE is a service provided by the library of the University of Amsterdam (http://dare.uva.nl)

UvA-DARE (Digital Academic Repository)

Thrombin-activatable fibrinolysis inhibitor and bacterial infectionsValls Serón, M.

Link to publication

Citation for published version (APA):Valls Serón, M. (2011). Thrombin-activatable fibrinolysis inhibitor and bacterial infections.

General rightsIt is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s),other than for strictly personal, individual use, unless the work is under an open content license (like Creative Commons).

Disclaimer/Complaints regulationsIf you believe that digital publication of certain material infringes any of your rights or (privacy) interests, please let the Library know, statingyour reasons. In case of a legitimate complaint, the Library will make the material inaccessible and/or remove it from the website. Please Askthe Library: http://uba.uva.nl/en/contact, or a letter to: Library of the University of Amsterdam, Secretariat, Singel 425, 1012 WP Amsterdam,The Netherlands. You will be contacted as soon as possible.

Download date: 21 Mar 2019

https://dare.uva.nl/personal/pure/en/publications/thrombinactivatable-fibrinolysis-inhibitor-and-bacterial-infections(5e333238-bdc1-4193-adb7-a1abb388b0e7).html

Thro

mb

in-A

ctivatable

Fibrin

olysis In

hib

itor an

d B

acterial Infectio

ns

Me

rcede

s Valls Se

rón

20

11

Mercedes Valls Serón

Thrombin-Activatable Fibrinolysis Inhibitor and

Bacterial Infections

Thrombin-Activatable Fibrinolysis Inhibitor and Bacterial Infections


Thrombin-Activatable Fibrinolysis Inhibitor and Bacterial Infections Dissertation, University of Amsterdam, Amsterdam, The Netherlands Copyright © 2011, Mercedes Valls Serón All rights reserved. No part of this thesis may be reproduced or transmitted in nay form by any means, without permission of the author. Author Mercedes Valls Serón Cover Streptococci by www.fotolia.com Printed by Wöhrmann Print Service ISBN 9789085705758 Financial support for the printing of this thesis was kindly provided by The University of Amsterdam and by AMC Medical Research.

Thrombin-Activatable Fibrinolysis Inhibitor and Bacterial Infections

ACADEMISCH PROEFSCHRIFT

ter verkrijging van de graad van doctor

aan de Universiteit van Amsterdam

op gezag van de Rector Magnificus

prof.dr. D.C. van den Boom

ten overstaan van een door het college voor

promoties ingestelde commissie, in het openbaar

te verdedigen in de Agnietenkapel

op woensdag 16 november 2011, te 12.00 uur

door


geboren te Zaragoza, Spanje

Promotiecommissie

Promotores: Prof.dr. J. C. M. Meijers

Prof.dr. Ph. G. de Groot

Overige leden: Prof.dr. C. E. Hack

Prof.dr. F. Leebeek

Prof.dr. C. J. F van Noorden

Prof.dr. T. van der Poll

Prof.dr. A. J. Verhoeven

Faculteit der Geneeskunde

Financial support by The Netherlands Heart Fundation for the publication of this thesis is gratefully acknowledged.

A mis padres

Table of contents

CHAPTER 1: General introduction and outline of the thesis 9

CHAPTER 2: Recent developments in thrombin-activatable fibrinolysis 23

inhibitor research

CHAPTER 3: Thrombin-activatable fibrinolysis inhibitor is degraded by 45

Salmonella enterica and Yersinia pestis

CHAPTER 4: Binding characteristics of thrombin-activatable fibrinolysis 65

inhibitor to streptococcal surface collagen-like proteins A

and B

CHAPTER 5: Susceptibility of human TAFI-transgenic mice to 81

Streptococcus pyogenes

CHAPTER 6: Murine TAFI improves survival against Streptococcus pyogenes 93

CHAPTER 7: Summary and general discussion 109

CHAPTER 8: Nederlandse Samenvatting 116

Acknowledgements

List of publications

Introduction

Chapter 1

10

Introduction

Bacterial infections are initiated when bacteria and host come into contact. During the

infection process, the host responds to invading microbes with a number of different defense

mechanisms. In addition to physical barriers, the immune and hemostatic systems are

involved in destruction or contention of the infectious agent. Some bacteria or bacterial

products, however, can employ the hemostatic host response to their own benefit and cause

serious complications. Although different pathogenic bacteria seem to target different stages

of the coagulation and fibrinolytic systems, both systems often have a common consequence

which consists of disease propagation that lead to similar clinical pictures.

The hemostatic system

Hemostasis is a process which involves first blood clotting (coagulation) and subsequent brake

down of existing clots (fibrinolysis). Upon vessel injury, platelets adhere to the sub-

endothelial tissues and aggregate to form a haemostatic plug. At the same time, clotting is

initiated via the extrinsic or intrinsic pathway of coagulation, both of which lead to fibrin

formation through a common pathway, which forms the clot. The primary function of the

coagulation system is to stop bleeding of an injury until repair occurs.

The coagulation system consists of a number coagulation factors that circulate in plasma in

their inactive precursor forms. The extrinsic pathway (Figure 1) is triggered after an event of

injury of the blood vessel wall, when tissue factor (TF) is exposed to plasma and forms a

catalytic complex with coagulation factor VII, leading to the activation of factor X. Activated

factor X (FXa) will initiate the common pathway by assembly in the prothrombinase complex

with activated factor V. The prothrombinase complex (FVa-FXa) then cleaves prothrombin to

thrombin, which then cleaves fibrinogen to fibrin.

The intrinsic pathway (Figure 1) is initiated after activation of the contact system, a process

involving factor XI, factor XII, plasma kallikrein and high molecular weight kininogen. The

Figure 1. The coagulation cascade. The model is explained in detail in the text. The intrinsic and extrinsic pathways result in fibrin formation. Active (a) and inactive factors are represented by their roman numerals. TF: tissue factor.

Introduction

11

Ch

apte

r 1

contact system assembles on negatively charged surfaces, leading to the reciprocal activation

of FXII and prekallikrein. Activated FXII triggers the sequential activation of FXI, IX, and X, and

subsequent induction of the common pathway.

Overactive coagulation can result wide-spread thrombosis. Therefore, it has to be controlled

by anticoagulation systems. This is achieved through the anticoagulation and fibrinolytic

systems. Together, coagulation, anticoagulation, and fibrinolysis maintain a delicate

physiological balance.

The major anticoagulants include antithrombin (AT), tissue pathway inhibitor (TFPI), and

activated protein C (APC). AT is produced by the liver and inhibits several coagulation factors

such as thrombin, FVIIa, FIXa, and FXa [1]. TFPI is a serine protease that inhibits FXa. In the

presence of FXa, TFPI also inhibits the TF/VIIa complex [2]. Protein C is an inactive plasma

serine protease. When thrombin is produced, it can bind to thrombomodulin present on the

vascular endothelial surfaces. The thrombin/thrombomodulin complex can then cleave

protein C into APC. APC generation is enhanced by the endothelial cell protein C receptor

(EPCR) on the endothelial surface. APC, with cofactor protein S, can cleave and inactivate FVa

and FVIIIa to negatively regulate coagulation [3,4].

At the site of tissue injury, fibrinolysis is initiated when plasminogen is converted to plasmin

by tissue-type plasminogen activator (t-PA) (Figure 2).

Plasmin then degrades the fibrin clot into soluble fibrin degradation products. The C-terminal

lysine residues of fibrin, generated after limited plasmin cleavage, act as a template onto

which both t-PA and plasminogen bind. As a result of t-PA and plasminogen interaction with

fibrin, the catalytic efficiency is 100-1000 fold enhanced. Plasmin formation is regulated by a

thrombin-dependent activation of the plasma protein thrombin-activatable fibrinolysis

Figure 2. The fibrinolytic system. Fibrinolysis is initiated when plasminogen is converted to plasmin by

tissue-type plasminogen activator (t-PA). Fibrin then degrades the fibrin clot into soluble fibrin

degradation products. Activated thrombin-activatable fibrinolysis inhibitor (TAFIa) by thrombin (IIa)

together with thrombomodulin (TM) inhibits the formation of plasmin and the degradation of the

fibrin clot.

Chapter 1

12

inhibitor (TAFI). Activated TAFI (TAFIa) cleaves off the C-terminal lysine residues of the

partially degraded fibrin and thereby abrogates the fibrin cofactor function in the t-PA-

mediated plasmin formation. More detailed information about TAFI is described in Chapter 2.

Gram-positive: Streptococcus pyogenes

Like other members of the family Streptococcacae, streptococci are Gram-positive facultative

anaerobic organisms which occur in chains or in pairs. S. pyogenes display a β–hemolytic

pattern of growth on blood agar meaning that bacteria produce a complete hemolysis around

the colonies. S. pyogenes also contain the Lancefield serogroup A carbohydrate on their cell

surface, and are often referred to as group A streptococci (GAS).

Strain characterization of (GAS) has traditionally been based on serological identification of M

protein [5], T-protein and production of streptococcal serum opacity factor (SOF) [6].

Advances in DNA-sequencing technology in the last decade resulted in the development of a

method for determining the M type of GAS from the sequence of the gene encoding M

protein, the emm gene. More than 200 emm types are currently listed in the online C.D.C.

database (http://www.cdc.gov/ncidod/biotech/strep/strepindex.htm).

S. pyogenes is one of the most common and important human bacterial pathogens. Although

it causes relatively mild infections such as pharyngitis (strep throat) and impetigo they may

evolve to life-threatening invasive infections of deeper tissues, the blood stream, and multiple

organs like septicemia and toxic-shock syndrome [7].

S. pyogenes are responsible for an estimated 616 million cases of throat infection

(pharyngitis, tonsillitis) worldwide per year, and 111 million cases of skin infection (primarily

non-bullous impetigo) in children of less developed countries [8]. Based on these numbers,

the bacterium is among the 10 most mortality-causing human pathogens.

S. pyogenes produces several surface-bound and secreted virulence factors that give rise to

these complications. Virulence factors from S. pyogenes include: surface attached virulence

factors such as M proteins [9-11] , streptococcal collagen-like surface protein A and B [12,13]

and fibronectin-binding protein (Protein F1/Sfb1) [14,15]; capsule and cell wall (lipoteichoic

acid and hyaluronic acid [16,17]) and secreted virulence factors such as superantigens [18],

streptokinase [19,20], DNases [21], and streptococcal inhibitor of complement (protein

SIC)[22].

The probably best characterized surface attached virulence factors are the M proteins. M

proteins are composed of two polypeptide chains that form an alpha-helical coiled-coil

configuration. The emm gene encodes for M proteins and is regulated by the Mga regulon,

multiple gene regulator of GAS, which is maximally expressed during the logarithmic growth

phase and in vivo during the acute phase of infection [23,24].

A number of studies have shown that M proteins allow adherence to various host tissues and

extracellular matrix components, trigger internalization into host cells, can provide anti-

phagocytic properties and induce autoimmune reactions in rheumatic fever.

http://www.cdc.gov/ncidod/biotech/strep/

Introduction

13

Ch

apte

r 1

In addition to the M protein, streptococcal collagen-like surface protein A and B (SclA and

SclB), also contribute to cell adhesion and internalization. It has also been reported that SclA

from M type 41 activates the collagen receptor α2β1 integrin on fibroblasts and interacts with

the low density lipoprotein [25,26], high density lipoprotein [27], fibronectin and laminin [28].

In addition, SclA has been implicated in the inhibition of the alternative pathway of

complement [29,30]. More detailed information about SclA and SclB is described in Chapter 4.

In order to activate the clotting cascade, GAS have developed two mechanisms involving the

intrinsic and the extrinsic pathway of coagulation. M protein expressing bacteria can

assemble factors of the intrinsic pathway on their surface that will lead to fibrin formation. In

addition, soluble M1 and M3, and bacteria from the M1 and M3 serotype can activate the

extrinsic pathway by triggering tissue factor synthesis on isolated human monocytes [31,32]

and induce procoagulant activity on these cells.

Fibrin(ogen) plays multiple roles in the GAS/host interaction. The ability of S. pyogenes

surface to bind fibrinogen via fibrinogen-binding proteins (FgBPs) is believed to be important

in promoting bacterial adherence to host tissues during an infection and seem to have anti-

phagocytic function owing to their ability to impair deposition of complement. During

infection, the host generates fibrin at the local site of infection that can be used to wall off

the site of infection and limit pathogen invasion and spread. However, bacteria within a fibrin

network could be protected from the host defense machinery. To be able to circumvent the

thrombotic host defense, S. pyogenes expresses a number of molecules which confer the

bacterium ability to dissolve formed fibrin clots to facilitate bacterial spread. Streptococci are

proposed to gain fibrinolytic activity through direct binding of plasmin to specific surface

proteins or indirectly by sequential binding of fibrinogen and plasminogen [33].

Specific GAS surface proteins involved in plasminogen-binding are glyceraldehyde-3-

phosphate dehydrogenase (GAPDH) [19,34], streptococcal surface enolase (SEN) [35],

plasminogen-binding group A streptococcal M protein (PAM) [36] and PAM related protein

(Prp) [37]. Moreover, the GAS secreted streptokinase enables GAS to cleave plasminogen to

plasmin without proteolysis [20,38], which degrades connective tissue, extracellular matrix

(ECM), and fibrin clots [39,40].

Of importance for this thesis, SclA and SclB bind to TAFI and is subsequently activated at the

bacterial surface by plasmin and thrombin-thrombomodulin [41]. Furthermore, activation of

TAFI on the surface of S. pyogenes evoked inflammatory reactions by modulating the

kallikrein/kinin systems [42].

Thus, at different stages of the infectious process, S. pyogenes may recruit either thrombotic

or thrombolytic factors to meet the demand for bacterial survival and proliferation.

Gram-negative: Yersinia pestis and Salmonella enterica

Yersinia pestis and Salmonella enterica belong to the Gram-negative family of

Enterobacteriaceae. Both are invasive pathogens.

Chapter 1

14

Yersinia pestis

Yersinia species are anaerobic, non-spore-forming bacilli or coccobacilli. There are multiple

Yersinia species, including the three human pathogens Y. pestis, Yersinia pseudotuberculosis,

and Yersinia enterocolitica.

Y. pestis is the causative agent of plague, an illness that may manifests in bubonic,

pneumonic, or septicemic form. Plague is a zoonotic disease that affects rodents and is

transmitted to humans mainly through the bite of infected fleas. The reservoir of Y. pestis in

nature is wild rodents. Humans are accidental hosts and have no role in its long-term survival

in endemic regions [43].

Upon feeding on blood of infected animals, fleas acquire Y. pestis, which multiply and block

the flea’s foregut. The blocked fleas starve and frenetically bite other rodents and,

incidentally, also humans. [44]. The bacteria are injected into the subcutaneous tissue, where

they promote local proteolysis at the infection site and migrate through the subcutaneous

tissue to the lymph nodes [45,46] where it proliferates, causing bubonic plague.

Bacterial proliferation causes swollen lymph nodes, called buboes. Another route of infection

is via respiratory droplets from an infected mammal to another. The bacteria spread to lungs

within the droplets and multiply causing primary pneumonic plague. The third form of plague

is primary septicaemic plague, where a flea injects the bacteria directly into a blood vessel

[45]. Secondary pneumonic or septicaemic plague occurs if the bacteria spread from buboes

to lungs or to the blood stream, respectively.

Y. pestis has killed millions of humans in three pandemics. According to World Health

Organization (WHO), there are about 2.000 cases and 200 deaths per year, mostly in Africa

and Asia. Because the occurrence of human cases and local epidemics has increased during

the last decades, plague has been classified as a re-emerging disease (WHO).

The genome of Y. pestis consists of a chromosome and three virulence plasmids, a 70-kb pCD

(or pYV), a 96-kb pMT1, and a 9.5-kb pPCP1 [47]. Y. pestis has gathered only a few virulence

factors and they are mostly encoded in plasmids [48].

Y. pestis pathogenesis is mainly caused by the plasminogen activator (Pla). Pla is a

multifunctional virulence factor that belongs to the omptin family of outer membrane

proteases of the Gram-negative bacteria. The gene coding for Pla is located in the pPCP1

plasmid. It has been shown that bacteria that express Pla are highly virulent yielding an LD50

increase of 106 fold compared to the absence of Pla [45,49]. In addition, expression of Pla is

needed for establishment of pneumonic plague [50] and is necessary to initiate bubonic

plague [45].

Lipopolysaccharides (LPS) are found in the cell envelope of Gram-negative bacteria and are

the major components of the outer leaflet of their outer membrane. Typically, an LPS

molecule consists of three structural domains: the lipid A, which holds the endotoxic

biological activity; a non-repeating oligosaccharide core; and a polysaccharide, the O-specific

chain, also known as O-antigen [51]. LPS molecules containing the O-specific chain are termed

smooth LPS while the ones lacking O-antigen are known as rough LPS. Pla and other omptins

Introduction

15

Ch

apte

r 1

require rough LPS to be active [52,53]. Thus, the LPS composition plays an important role in

the proteolysis efficiency of omptins. Y. pestis is naturally rough, which enables the activity of

Pla [52,54,55]. Besides activating plasminogen, Pla also interferes with the regulation of the

fibrinolytic system by inactivating α2-antiplasmin. These two features of Pla, in addition with

its adhesive characteristics, promote uncontrolled proteolysis as well as damage of tissue

barriers at the site of infection [56]. Another proteolytic target of Pla is the serum

complement protein C3 [49]. In addition, Pla proteolytically degrades the main inhibitor of the

initiation phase of blood clotting TFPI, suggesting that inactivation of TFPI may accelerate

blood clotting [57]. Together these interactions facilitate bacterial dissemination.

Salmonella enterica

S. enterica is related to human disease. S. enterica ssp. enterica causes 99% of human

infections: serovars Typhimurium, Enteritidis, Typhi, and Paratyphi are the most common and

most studied serovars. S. enterica serovars Typhimurium and Enteritidis cause gastroenteritis,

and serovars Typhi and Paratyphi cause the life-threatening disease typhoid and paratyphoid

fever, which are severe systemic infections. Gastroenteritis is usually mild and self-limiting,

but the bacteria can spread to distant organs and cause systemic infection [58]. In more than

95% of the cases, the infection initiates as the host ingests food contaminated with

Salmonella cells, which pass the gastrointestinal tract and reach the small intestine [59].

Following the adhesion and colonization of the intestinal tract, bacteria invade the intestinal

mucosa. Upon crossing the intestinal barrier, S. enterica invades macrophages and multiplies

in specific vacuoles known as Salmonella containing vacuoles (SCV)[60] where the bacterium

survive and multiply. Thereafter, S. enterica spreads inside circulating macrophages via blood

to cause systemic disease [61,62]. The ability to survive in immune cells is a major

determinant of pathogenesis and a variety of virulence factors involved in this process have

been identified in S. enterica [63].

According to WHO, Salmonella-gastroenteritis affects millions of people annually, especially in

developing countries, and causes thousands of deaths, and Typhi affects 16-33 million people

with 216.000 deaths per year.

In addition to Pla, PgtE also require rough LPS to be active and is sterically inhibited by the O-

antigen [52,64]. Clinical isolates and cells grown in laboratory medium of S. enterica have LPS

O-antigen oligosaccharide chains and therefore PgtE is apparently inactive. In contrast,

Salmonella isolates from murine macrophages have LPS with altered structure where the LPS

O-antigen is strongly reduced [64,65]. Indicating that expression of PgtE is upregulated during

growth of Salmonella inside macrophages [64,65] since bacteria released from macrophages

exhibit a strong PgtE-mediated proteolytic activity [65].

PgtE from S. enterica Typhimurium cells isolated from SCV of mouse macrophages

proteolytically activates plasminogen to plasmin [49], inactivates the main physiological

inhibitor of plasmin, α2-antiplasmin [65], and mediates bacterial adhesion to extracellular

matrices of human cells [52]. In this way, PgtE mediates degradation of extracellular matrix

Chapter 1

16

components and generates localized proteolytic activity, which may promote migration of

Salmonella through extracellular matrices. PgtE also degrades alpha-helical antimicrobial

peptides [66], which may be important during intracellular growth of the bacterium.

Introduction

17

Ch

apte

r 1

Outline of the thesis

The major objective of the studies described in this thesis is to study the interactions between

TAFI and pathogenic bacteria.

The membrane proteases Pla Y. pestis and PgtE of S. enterica interact with the human

fibrinolytic system by activating plasminogen and inactivating α2-antiplasmin, and PgtE in

addition by activating proMMP-9. TAFI is a regulatory, anti-fibrinolytic protein linking the

coagulation and fibrinolytic systems. In chapter 2, an introduction is given to TAFI and its role

in fibrinolysis and inflammation is explained. In chapter 3, we investigated the effects of the

Gram-negative proteases Pla and PgtE on TAFI.

In chapters 4 to 6, several studies are summarized that investigated the interaction of the

Gram-positive bacteria, S. pyogenes with TAFI and the role of TAFI in the course of

experimental streptococcal infection in vivo.

The binding of TAFI to S. pyogenes is mediated by the surface proteins, SclA and SclB. In

chapter 4, we characterized the TAFI binding region that is involved in this interaction.

In chapters 5 and 6 we investigated whether TAFI is involved in the course and the outcome

of experimental murine S. pyogenes infection in vivo. To this end, humanized-TAFI and TAFI-

deficient mice have been used.

Finally, in chapter 7 the data of this thesis are summarized and discussed.

Chapter 1

18

Reference List

1 Roemisch J, Gray E, Hoffmann JN, Wiedermann CJ. Antithrombin: a new look at the actions of a

serine protease inhibitor. Blood Coagul Fibrinolysis 2002; 13: 657-70.

2 Broze GJ, Jr. Tissue factor pathway inhibitor and the revised theory of coagulation. Annu Rev

Med 1995; 46: 103-12.

3 Aird WC. Natural anticoagulant inhibitors: activated Protein C. Best Pract Res Clin Haematol

2004; 17: 161-82.

4 Esmon CT, Gu JM, Xu J, Qu D, Stearns-Kurosawa DJ, Kurosawa S. Regulation and functions of the

protein C anticoagulant pathway. Haematologica 1999; 84: 363-8.

5 Lancefield RC. Current knowledge of type-specific M antigens of group A streptococci. J Immunol

1962; 89: 307-13.

6 Johnson DR, Stevens DL, Kaplan EL. Epidemiologic analysis of group A streptococcal serotypes

associated with severe systemic infections, rheumatic fever, or uncomplicated pharyngitis. J

Infect Dis 1992; 166: 374-82.

7 Cunningham MW. Pathogenesis of group A streptococcal infections and their sequelae. Adv Exp

Med Biol 2008; 609: 29-42.

8 Carapetis JR, Steer AC, Mulholland EK, Weber M. The global burden of group A streptococcal

diseases. Lancet Infect Dis 2005; 5: 685-94.

9 Lancefield RC. Current problems in studies of streptococci. J Gen Microbiol 1969; 55: 161-3.

10 Oehmcke S, Shannon O, Morgelin M, Herwald H. Streptococcal M proteins and their role as

virulence determinants. Clin Chim Acta 2010; 411: 1172-80.

11 Smeesters PR, McMillan DJ, Sriprakash KS. The streptococcal M protein: a highly versatile

molecule. Trends Microbiol 2010; 18: 275-82.

12 Lukomski S, Nakashima K, Abdi I, Cipriano VJ, Ireland RM, Reid SD, et al. Identification and

characterization of the scl gene encoding a group A Streptococcus extracellular protein virulence

factor with similarity to human collagen. Infect Immun 2000; 68: 6542-53.

13 Lukomski S, Nakashima K, Abdi I, Cipriano VJ, Shelvin BJ, Graviss EA, et al. Identification and

characterization of a second extracellular collagen-like protein made by group A Streptococcus:

control of production at the level of translation. Infect Immun 2001; 69: 1729-38.

14 Talay SR, Valentin-Weigand P, Jerlstrom PG, Timmis KN, Chhatwal GS. Fibronectin-binding

protein of Streptococcus pyogenes: sequence of the binding domain involved in adherence of

streptococci to epithelial cells. Infect Immun 1992; 60: 3837-44.

Introduction

19

Ch

apte

r 1

15 Katerov V, Andreev A, Schalen C, Totolian AA. Protein F, a fibronectin-binding protein of

Streptococcus pyogenes, also binds human fibrinogen: isolation of the protein and mapping of

the binding region. Microbiology 1998; 144 ( Pt 1): 119-26.

16 Fischer W, Markwitz S, Labischinski H. Small-angle X-ray scattering analysis of pneumococcal

lipoteichoic acid phase structure. Eur J Biochem 1997; 244: 913-7.

17 Schwandner R, Dziarski R, Wesche H, Rothe M, Kirschning CJ. Peptidoglycan- and lipoteichoic

acid-induced cell activation is mediated by toll-like receptor 2. J Biol Chem 1999; 274: 17406-9.

18 Sriskandan S, Faulkner L, Hopkins P. Streptococcus pyogenes: Insight into the function of the

streptococcal superantigens. Int J Biochem Cell Biol 2007; 39: 12-9.

19 Broder CC, Lottenberg R, von Mering GO, Johnston KH, Boyle MD. Isolation of a prokaryotic

plasmin receptor. Relationship to a plasminogen activator produced by the same micro-

organism. J Biol Chem 1991; 266: 4922-8.

20 Lahteenmaki K, Kuusela P, Korhonen TK. Bacterial plasminogen activators and receptors. FEMS

Microbiol Rev 2001; 25: 531-52.

21 Buchanan JT, Simpson AJ, Aziz RK, Liu GY, Kristian SA, Kotb M, et al. DNase expression allows the

pathogen group A Streptococcus to escape killing in neutrophil extracellular traps. Curr Biol

2006; 16: 396-400.

22 Fernie-King BA, Seilly DJ, Willers C, Wurzner R, Davies A, Lachmann PJ. Streptococcal inhibitor of

complement (SIC) inhibits the membrane attack complex by preventing uptake of C567 onto cell

membranes. Immunology 2001; 103: 390-8.

23 Hondorp ER, McIver KS. The Mga virulence regulon: infection where the grass is greener. Mol

Microbiol 2007; 66: 1056-65.

24 Kreikemeyer B, McIver KS, Podbielski A. Virulence factor regulation and regulatory networks in

Streptococcus pyogenes and their impact on pathogen-host interactions. Trends Microbiol 2003;

11: 224-32.

25 Han R, Caswell CC, Lukomska E, Keene DR, Pawlowski M, Bujnicki JM, et al. Binding of the low-

density lipoprotein by streptococcal collagen-like protein Scl1 of Streptococcus pyogenes. Mol

Microbiol 2006; 61: 351-67.

26 Humtsoe JO, Kim JK, Xu Y, Keene DR, Hook M, Lukomski S, et al. A streptococcal collagen-like

protein interacts with the alpha2beta1 integrin and induces intracellular signaling. J Biol Chem

2005; 280: 13848-57.

27 Gao Y, Liang C, Zhao R, Lukomski S, Han R. The Scl1 of M41-type group A Streptococcus binds the

high-density lipoprotein. FEMS Microbiol Lett 2010; 309: 55-61.

Chapter 1

20

28 Caswell CC, Oliver-Kozup H, Han R, Lukomska E, Lukomski S. Scl1, the multifunctional adhesin of

group A Streptococcus, selectively binds cellular fibronectin and laminin, and mediates pathogen

internalization by human cells. FEMS Microbiol Lett 2010; 303: 61-8.

29 Caswell CC, Han R, Hovis KM, Ciborowski P, Keene DR, Marconi RT, et al. The Scl1 protein of M6-

type group A Streptococcus binds the human complement regulatory protein, factor H, and

inhibits the alternative pathway of complement. Mol Microbiol 2008; 67: 584-96.

30 Reuter M, Caswell CC, Lukomski S, Zipfel PF. Binding of the human complement regulators CFHR1

and factor H by streptococcal-collagen-like protein 1, Scl1, via their conserved C-termini allows

control of the complement cascade at multiple levels. J Biol Chem 2010.

31 Bryant AE, Hayes-Schroer SM, Stevens DL. M type 1 and 3 group A streptococci stimulate tissue

factor-mediated procoagulant activity in human monocytes and endothelial cells. Infect Immun

2003; 71: 1903-10.

32 Pahlman LI, Malmstrom E, Morgelin M, Herwald H. M protein from Streptococcus pyogenes

induces tissue factor expression and pro-coagulant activity in human monocytes. Microbiology

2007; 153: 2458-64.

33 Lottenberg R, Broder CC, Boyle MD, Kain SJ, Schroeder BL, Curtiss R, III. Cloning, sequence

analysis, and expression in Escherichia coli of a streptococcal plasmin receptor. J Bacteriol 1992;

174: 5204-10.

34 Pancholi V, Fischetti VA. A major surface protein on group A streptococci is a glyceraldehyde-3-

phosphate-dehydrogenase with multiple binding activity. J Exp Med 1992; 176: 415-26.

35 Pancholi V, Fischetti VA. alpha-enolase, a novel strong plasmin(ogen) binding protein on the

surface of pathogenic streptococci. J Biol Chem 1998; 273: 14503-15.

36 Berge A, Sjobring U. PAM, a novel plasminogen-binding protein from Streptococcus pyogenes. J

Biol Chem 1993; 268: 25417-24.

37 Sanderson-Smith ML, Dowton M, Ranson M, Walker MJ. The plasminogen-binding group A

streptococcal M protein-related protein Prp binds plasminogen via arginine and histidine

residues. J Bacteriol 2007; 189: 1435-40.

38 Braunwald E. Acute myocardial infarction--the value of being prepared. N Engl J Med 1996; 334:

51-2.

39 Ponting CP, Marshall JM, Cederholm-Williams SA. Plasminogen: a structural review. Blood Coagul

Fibrinolysis 1992; 3: 605-14.

40 Dano K, Andreasen PA, Grondahl-Hansen J, Kristensen P, Nielsen LS, Skriver L. Plasminogen

activators, tissue degradation, and cancer. Adv Cancer Res 1985; 44: 139-266.

Introduction

21

Ch

apte

r 1

41 Pahlman LI, Marx PF, Morgelin M, Lukomski S, Meijers JC, Herwald H. Thrombin-activatable

fibrinolysis inhibitor binds to Streptococcus pyogenes by interacting with collagen-like proteins A

and B. J Biol Chem 2007; 282: 24873-81.

42 Bengtson SH, Sanden C, Morgelin M, Marx PF, Olin AI, Leeb-Lundberg LM, et al. Activation of

TAFI on the surface of Streptococcus pyogenes evokes inflammatory reactions by modulating the

kallikrein/kinin system. J Innate Immun 2008; 1: 18-28.

43 Perry RD, Fetherston JD. Yersinia pestis--etiologic agent of plague. Clin Microbiol Rev 1997; 10:

35-66.

44 Duplantier JM, Duchemin JB, Chanteau S, Carniel E. From the recent lessons of the Malagasy foci

towards a global understanding of the factors involved in plague reemergence. Vet Res 2005; 36:

437-53.

45 Sebbane F, Jarrett CO, Gardner D, Long D, Hinnebusch BJ. Role of the Yersinia pestis plasminogen

activator in the incidence of distinct septicemic and bubonic forms of flea-borne plague. Proc

Natl Acad Sci U S A 2006; 103: 5526-30.

46 Sebbane F, Lemaitre N, Sturdevant DE, Rebeil R, Virtaneva K, Porcella SF, et al. Adaptive response

of Yersinia pestis to extracellular effectors of innate immunity during bubonic plague. Proc Natl

Acad Sci U S A 2006; 103: 11766-71.

47 Ferber DM, Brubaker RR. Plasmids in Yersinia pestis. Infect Immun 1981; 31: 839-41.

48 Wren BW. The yersiniae--a model genus to study the rapid evolution of bacterial pathogens. Nat

Rev Microbiol 2003; 1: 55-64.

49 Sodeinde OA, Subrahmanyam YV, Stark K, Quan T, Bao Y, Goguen JD. A surface protease and the

invasive character of plague. Science 1992; 258: 1004-7.

50 Lathem WW, Price PA, Miller VL, Goldman WE. A plasminogen-activating protease specifically

controls the development of primary pneumonic plague. Science 2007; 315: 509-13.

51 Raetz CR, Whitfield C. Lipopolysaccharide endotoxins. Annu Rev Biochem 2002; 71: 635-700.

52 Kukkonen M, Suomalainen M, Kyllonen P, Lahteenmaki K, Lang H, Virkola R, et al. Lack of O-

antigen is essential for plasminogen activation by Yersinia pestis and Salmonella enterica. Mol

Microbiol 2004; 51: 215-25.

53 Kramer RA, Brandenburg K, Vandeputte-Rutten L, Werkhoven M, Gros P, Dekker N, et al.

Lipopolysaccharide regions involved in the activation of Escherichia coli outer membrane

protease OmpT. Eur J Biochem 2002; 269: 1746-52.

Chapter 1

22

54 Prior JL, Parkhill J, Hitchen PG, Mungall KL, Stevens K, Morris HR, et al. The failure of different

strains of Yersinia pestis to produce lipopolysaccharide O-antigen under different growth

conditions is due to mutations in the O-antigen gene cluster. FEMS Microbiol Lett 2001; 197: 229-

33.

55 Skurnik M, Peippo A, Ervela E. Characterization of the O-antigen gene clusters of Yersinia

pseudotuberculosis and the cryptic O-antigen gene cluster of Yersinia pestis shows that the

plague bacillus is most closely related to and has evolved from Y. pseudotuberculosis serotype

O:1b. Mol Microbiol 2000; 37: 316-30.

56 Lahteenmaki K, Virkola R, Saren A, Emody L, Korhonen TK. Expression of plasminogen activator

pla of Yersinia pestis enhances bacterial attachment to the mammalian extracellular matrix.

Infect Immun 1998; 66: 5755-62.

57 Yun TH, Cott JE, Tapping RI, Slauch JM, Morrissey JH. Proteolytic inactivation of tissue factor

pathway inhibitor by bacterial omptins. Blood 2009; 113: 1139-48.

58 Coburn B, Grassl GA, Finlay BB. Salmonella, the host and disease: a brief review. Immunol Cell

Biol 2007; 85: 112-8.

59 Hohmann EL. Nontyphoidal salmonellosis. Clin Infect Dis 2001; 32: 263-9.

60 Gorvel JP, Meresse S. Maturation steps of the Salmonella-containing vacuole. Microbes Infect

2001; 3: 1299-303.

61 Mastroeni P, Grant A, Restif O, Maskell D. A dynamic view of the spread and intracellular

distribution of Salmonella enterica. Nat Rev Microbiol 2009; 7: 73-80.

62 Brown SP, Cornell SJ, Sheppard M, Grant AJ, Maskell DJ, Grenfell BT, et al. Intracellular

demography and the dynamics of Salmonella enterica infections. PLoS Biol 2006; 4: e349.

63 Groisman EA, Ochman H. How Salmonella became a pathogen. Trends Microbiol 1997; 5: 343-9.

64 Eriksson S, Lucchini S, Thompson A, Rhen M, Hinton JC. Unravelling the biology of macrophage

infection by gene expression profiling of intracellular Salmonella enterica. Mol Microbiol 2003;

47: 103-18.

65 Lahteenmaki K, Kyllonen P, Partanen L, Korhonen TK. Antiprotease inactivation by Salmonella

enterica released from infected macrophages. Cell Microbiol 2005; 7: 529-38.

66 Guina T, Yi EC, Wang H, Hackett M, Miller SI. A PhoP-regulated outer membrane protease of

Salmonella enterica serovar typhimurium promotes resistance to alpha-helical antimicrobial

peptides. J Bacteriol 2000; 182: 4077-86.

Recent developments in thrombin-activatable fibrinolysis inhibitor research Pauline F. Marx, Chantal J.N. Verkleij, Mercedes Valls Serón and Joost C.M. Meijers Mini Reviews in Medicinal Chemistry, 2009; 9 (10): 1165-73

Chapter 2

24

Abstract

Thrombin-activatable fibrinolysis inhibitor (TAFI) provides an important molecular link

between the coagulation and fibrinolytic systems. In this review, recent major advances in

TAFI research, including the elucidation of crystal structures, the development of small

inhibitors and the role of TAFI in systems other than hemostasis, are described and discussed.

Recent developments in TAFI research

25

Ch

apte

r 2

The basics about TAFI

The coagulation system is a strictly regulated series of enzymatic reactions that prevents

blood loss after vascular injury. The reactions ultimately lead to the formation of thrombin.

Thrombin converts soluble fibrinogen into a fibrin network, which is subsequently removed

by the fibrinolytic system during the healing process.

Thrombin-activatable fibrinolysis inhibitor (TAFI, recent reviews include: [1-10]) is a

glycoprotein with a molecular mass of 55 kDa that is synthesized in the liver and secreted into

the bloodstream in a zymogen form. TAFI is best known for its function in bridging the

coagulation and fibrinolytic cascades. TAFI is activated by the key component of the

coagulation system thrombin, either free or – more likely [11] – in complex with

thrombomodulin [12]. Alternative activators are plasmin [13-15] and neutrophil-derived

elastase [16]. The active form, the enzyme TAFIa, attenuates premature breakdown of the

fibrin clot. Hence its name with the accompanying acronym TAFI was chosen.

TAFIa functions by removing C-terminal lysine residues from partially degraded fibrin, which

act as binding sites for plasminogen and tissue-type plasminogen activator. This binding

facilitates the conversion of plasminogen into plasmin, the enzyme that degrades the fibrin

network of the blood clot.

Besides a function in fibrinolysis, TAFI also plays a role in inflammatory processes by

hydrolysis of bradykinin, osteopontin and the anaphylotoxins C3a and C5a (reviewed

elsewhere [10]). An overview of TAFI activation and TAFIa’s substrates, functions and

inactivation process is provided in Figure 1.

Due to more or less simultaneous discovery in various laboratories, the enzyme TAFIa was

also given different names, based on the biochemical features of the protein. TAFIa is a

member of the metallocarboxypeptidase subfamily, which is characterized by the presence of

a zinc atom in the active site that is required for the catalytic mechanism of the enzyme.

Metallocarboxypeptidases are further divided according to their substrate specificity into the

carboxypeptidases A (CPA), which preferentially hydrolyze aliphatic residues, and

carboxypeptidases B (CPB), which preferentially hydrolyze basic residues. TAFIa belongs to

the latter subfamily and is therefore sometimes referred to as plasma carboxypeptidase B.

The finding that TAFIa prefers to hydrolyse arginine residues, prompted other researchers to

call it carboxypeptidase R, where R stands for Arginine. Finally, TAFIa is a very labile enzyme,

hence it is also known as carboxypeptidase U, where the U stands for unstable.

The auto-regulation mechanism of TAFIa

Notwithstanding the high degree of homology between TAFIa and other members of the

carboxypeptidase B family (approximately 45%), TAFIa distinguishes itself clearly via an auto-

regulation mechanism which accounts for the enzyme’s short half-life. One of the first

Chapter 2

26

observations regarding TAFIa inactivation was that TAFIa is not inactivated by proteolysis

[17,18], and second that the catalytic zinc ion is not released in the inactivation process [19].

A third possibility was that the bond between the activation peptide and the catalytic domain,

amino acids 92 and 93, is cleaved during activation, but that the activation peptide remains

attached to the remainder of the protein. The actual release of the activation peptide could

then account for loss of activity. However, recently we were able to shown that the activation

peptide is not required for TAFIa activity and is not involved in stabilization of TAFIa, excluding

a role for the activation peptide in the inactivation mechanism [20].

Figure 1. Diagram of TAFI activation, TAFIa substrates, TAFIa functions and TAFIa inactivation. TAFI is

activated (closed arrows) by thrombin generated by the coagulation cascade, plasmin generated by the

fibrinolytic system, and elastase, that is released from neutrophils during inflammation, into TAFIa.

TAFIa, the active enzyme, converts several substrates (partially degraded fibrin, C3a, C5a, bradykinin and

osteopontin) to attenuate (open arrows) fibrinolysis or inflammatory processes. TAFIa inactivates

rapidly into TAFIai due to its structural instability after which it is proteolytically broken down and prone

to aggregation.

In the past decade, numerous studies were conducted to reveal the mechanism of TAFIa

inactivation by engineering more stable variants [17,18,21-26]. Extensive mutagenesis studies

revealed that all mutations that influence TAFIa stability are located in one segment of the

protein covering β-sheet 9 and α-helix 11 (residues 297-335). The most stable variant

generated today contains five point mutations (T325I, T329I, S305C, H333Y and H335Q) and it


27

Ch

apte

r 2

has a half-life of 180 times that of wild type TAFIa [25]. Remarkably, several of the more

stable mutants have an anti-fibrinolytic capacity that is less than expected from their increase

in half-life [21,25,26]. Although the reason for this observation is unknown, suboptimal

fitting of larger substrates due to structural changes distinct from the active site, may explain

this phenomenon.

Crystal structures explain the mechanism of TAFIa auto-regulation

Recently a breakthrough in the understanding of the auto-regulatory mechanism was made

when the crystal structures of various TAFI forms were solved [27-29]. Obtaining crystals

suitable for structure determination was a time consuming process due to the glycosylation

extent of the protein. TAFI has five putative N-glycosylation sites which account for the

heterogeneous appearance of the protein. Expression of TAFI in a particular cell line –

HEK293ES, which lacks N-acetylglucosaminoyltransferase-I – yielded a recombinant TAFI form

with homogeneous N-linked glycans. This engineering trick made it possible to grow properly

diffracting crystals and to solve the TAFI structure [27].

Similar to other members of the procarboxypeptidase A and B families [30-34], TAFI consists

of two structural domains, the activation peptide (first 92 amino acid residues) and the

catalytic domain [27]. In the zymogen, the activation peptide covers the active site preventing

substrates to enter the catalytic cavity and stabilizing a dynamic segment of the enzyme

moiety (residues 296-350). Proteolytic activation by for example thrombin, results in release

of the activation peptide and concomitant increase in dynamic segment mobility. The

increased dynamics lead to conformational changes that disrupt the catalytic site and

exposure of a cryptic thrombin-cleavage site at Arg302. An overall structure of TAFI is given in

Figure 2.

In agreement with this model, introduction of the stabilizing mutations T325I, T329I, H333Y

and H335Q, which results in a 70-fold more stable TAFIa form, or binding of the reversible

inhibitor GEMSA, which also stabilizes TAFIa, reduced the mobility of the dynamic segment

(Figure 3). Earlier research had already shown that Arg302 is the main site for proteolytic

breakdown of the enzyme moiety after it had lost the active conformation [17,18]. Our

structural data [27] were confirmed by two other studies published shortly after on the

crystal structures of bovine TAFI and TAFIa [28,29]. Recently, we observed that the

inactivated species, TAFIai, is prone to aggregation, forming large, insoluble protein

aggregates that are easily removed by centrifugation [20].

Chapter 2

28

Figure 2. Ribbon drawing of TAFI. TAFI (401 amino acid residues) has two structural domains, the activation peptide (blue) and the catalytic domain (green), including the catalytic zinc ion (magenta sphere) and the highly dynamic ‘flap’ (residues 296-350, orange).The dynamic region provides an explanation for the instability of the enzyme TAFIa. As a result of proteolytic activation of TAFI at Arg92 and the ensuing release of the activation peptide, the activation peptide is no longer capable to restrict the dynamics of the flap. Increased dynamics lead to loss of structural integrity and consequently to TAFIa inactivation. Inactivated TAFIa (TAFIai) is prone to proteolytic breakdown at Arg302 and aggregation

Figure 3. Inhibitor binding stabilizes TAFIa. Crystallographic data provided an explanation for the

stabilizing effect of reversible inhibitors, like 2-guanidino-ethyl-mercaptosuccinic acid (GEMSA), on

TAFIa. GEMSA binds in the catalytic cleft S1’ pocket where the carboxy-terminal arginine or lysine

residue of the substrate would bind. One carboxylate group of GEMSA coordinates the catalytic zinc ion

and the second carboxylate is coordinated by catalytic site residues Arg217 and Arg235. Additional

hydrogen bonds are formed with Asp348 and Asp349, while hydrophobic interactions are formed with

residues 299 and 340-349 that are all part of the dynamic flap of TAFI. The stabilizing effect of GEMSA

on the flap region supports the idea that flap dynamics and the instability of TAFIa are directly linked.

Dynamic flap, orange; catalytic domain, green; catalytic zinc ion, magenta sphere; GEMSA, cyan; oxygen,

red; nitrogen blue.


29

Ch

apte

r 2

Sugars: important post-translational modifications

The crystal structures also provided more information on the glycosylation status of TAFI.

Four N-linked glycans were observed in the structure, all located in the activation peptide, e.g.

Asn22, Asn51, Asn63, and Asn288. The fifth reported N-linked glycosylation site at Asn219

[35] is entirely buried, excluding glycosylation. Usage of different sources of TAFI,

recombinant or plasma-derived, may explain differences in glycosylation pattern, although

the fact that Asn219 was completely buried within the structure indicates that the

physiological significance of glycosylation of this residue is most likely limited. The role of the

four glycans is probably to increase the solubility of the protein, as non-glycosylated TAFI, as

well as TAFIa, which contains no sugars, have a poor solubility [35], and to ensure proper

folding and secretion of the protein. In addition, contacts between the glycans could play a

role in stabilizing the dynamic flap. In particular a complex N-glycan attached to Asn22 seems

sufficiently close to the dynamic flap to establish direct interactions [27]. A recent study

showed that the replacement of this Asn22 resulted in an increased intrinsic activity of the

zymogen [36], indicating that this particular glycan indeed forms interactions within the TAFI

molecule. It is however unclear if it interacts with the dynamic flap directly. Slight changes in

catalytic efficiency of the active form and anti-fibrinolytic potential of this glycosylation

mutant as well as one other, Asn63Gln, were also reported [36].

Intrinsic activity of the zymogen: no role in fibrinolysis

As mentioned earlier, the glycosylated activation peptide is cleaved off during the activation

process, but a recent paper suggests that not only the enzyme TAFIa, but also the zymogen

TAFI, exerts catalytic activity [37]. However, although it was shown that the zymogen displays

enzymatic activity towards small molecular substrates [37,38], it does not add significantly to

the attenuation of fibrinolysis [39,40].

TAFI as therapeutic target: inhibition versus stabilization

The crystal structure provides not only information on the TAFIa inactivation process, it also

paves the way for the development of rationally designed inhibitors and stabilizers for TAFIa

that can be used in a clinical setting in the future. Inhibition of TAFIa is an attractive new

concept of antithrombotic therapy as it is based on enhancing fibrinolysis rather than direct

inhibition of the coagulation cascade, thus limiting the adverse hemorrhagic side effects seen

with anticoagulant drugs. It may also find application as an adjunct to thrombolytics. Ideally, a

useful inhibitor is not only a potent inhibitor of TAFIa, but is also strongly selective for this

particular enzyme. The major blood component of interest in this respect is carboxypeptidase

N (CPN). CPN is a constitutively active enzyme circulating in the bloodstream that shares

TAFIa’s specificity for C-terminal basic residues. Although commonly regarded as an inhibitor

Chapter 2

30

of inflammatory processes – among CPN substrates are the anaphylotoxins C5a and C3a – an

anti-fibrinolytic function was recently ascribed to CPN [41]. Simultaneous inhibition of both

TAFIa and CPN may have adverse effects.

Some encouraging efforts were made in developing TAFIa inhibitors as well as in in vitro and

in vivo testing of the efficacy of TAFIa inhibitor therapy [42-48]. An alternative to the

structure-based design of small inhibitory molecular component is the production of

inhibitory antibodies and fragments thereof [49].

Among the inhibitors commonly used in in vitro experiments, and some also in animal studies,

are the carboxypeptidase inhibitor derived from potatoes (CPI), ε-amino caproic acid (ε-ACA),

guanidinoethyl-mercaptosuccinic acid (GEMSA), dithiothreitol (DTT), DL-2-mercaptomethyl-

3guanidino-ethylthiopropanoic acid (MERGEPTA) and zinc-chelators. One of the major

findings on TAFIa inhibitors is that reversible TAFIa inhibitors can both stimulate and inhibit

fibrinolysis [50,51]. A potential mechanism explaining this observation is that substrate-bound

TAFIa inactivates at a lower rate than free TAFIa [50,51] as the flexibility of the dynamic loop

is limited by interactions of the inhibitor with the dynamic flap, the catalytic residues and the

zinc ion [27] (Figure 3).

In contrast to thrombotic episodes, in the case of excessive bleeding, stabilizers of TAFIa

would increase the stability of a blood clot and prevent premature lysis. With the discovery of

TAFIa’s threshold mechanism of action [52,53] and the TAFI-325 polymorphism – either an Ile

residue or a Thr residue at position 325 – the impact of TAFIa stability became apparent

[54,55]. The TAFIa-325Ile variant is more stable than the more common TAFIa-325Thr variant

and this is also reflected in its antifibrinolytic potential [55]. Stabilization of TAFIa would

therefore potentially be a good therapeutic strategy for the

treatment of bleeding disorder.

The TAFI gene, polymorphisms and TAFI expression

Besides the TAFI-325 polymorphism (1057C/G), two other polymorphic sites in the coding

sequence of TAFI have been identified, TAFI-147 (505A/G, Ala or a Thr residue), which has no

known functional consequences, and a silent variation at position 678. In contrast, in the

promoter region of the TAFI gene and the 3’UTR, numerous additional single nucleotide

polymorphisms (SNPs) have been identified [56], many of which are in strong linkage

disequilibrium and some are in or in the proximity of potential transcription factor binding

sites [56,57]. Some polymorphisms are associated with clinical outcome, such as blood

pressure [58], angina pectoris [59], meningococcal disease [60], splanchnic vein thrombosis

[61], recanalization resistance [62] and recurrent pregnancy loss [63].

The gene encoding the 423 amino acids of pre-TAFI, CPB2, is located on chromosome

13q14.11 [64,65]. The 11 encoding exons stretch over approximately 48 kb of DNA [66] TAFI is

produced in the liver and seems to be under control of liver-specific transcription factors.

Research using the liver cell line HepG2 showed the importance of binding of nuclear factor Y


31

Ch

apte

r 2

and hepatocyte nuclear factor α for TAFI expression [67]. In mice, mRNA was detectable in

the liver, in a hepatocyte-specific, pericentral lobular distribution pattern [68]. TAFI was also

detected in human platelets and may have been produced in megakaryocytes rather than

taken up from the plasma [69].

A large number of studies over the years have been dedicated to the determination of the

plasma TAFI concentration in health and disease. In normal individuals, mean TAFI levels were

reported between approximately 75-275 nM [70-73], with a considerable variation of 45%-

150% of the mean value. The variation can in part be explained by the presence of different

genotypes, both because the variants are expressed at different levels and because some of

the polymorphisms affect the assays to detect TAFI. Because of the latter phenomenon –

discussed in more detail below – also the data on the influence of age, gender, ethnicity,

disease etc. and TAFI levels, is confusing and warrants further analysis in the near future. For

now it seems that approximately 25% of the variation can be explained by SNPs in the TAFI

gene [74], leaving a large percentage for non-genetic factors. Glucocorticoids were shown to

upregulate TAFI expression in vitro, whereas the interleukins IL-1β and IL-6 could down

regulate expression [75]. Since there are essential differences in the promoter sequences of

the human and mouse TAFI gene the mouse is not an optimal model system to study TAFI

gene regulation [2]. This may hamper progress in revealing the role of inflammation in

regulation of the CPB2 gene.

TAFI assays: not all assays measure the same

Although it is not quite clear yet what the impact of the polymorphisms is on development

and progression of various disorders, it is fact that many assays to measure TAFI and/or TAFIa

are compromised by the presence of various TAFI forms, especially the 325 polymorphism, in

the general and patient population [76,77]. Antibody-dependent assays suffer from affinity

differences between the TAFI-325-Thr and Ile form, and activity-based assays from a

difference in half-life. Nevertheless progress in this area over the past few years resulted in a

polymorphism-independent activity-based assay [78,79], an assay for the direct measurement

of functional TAFIa in plasma [80], the development of various new ELISAs specific for the

various TAFI fragments (zymogen, activation peptide, TAFIa/TAFIai) [81] and TAFI from

different species (human, mouse, rat) [82-84], and (global) fibrinolytic assays [85-87]. Also,

testing of novel substrates resulted in more selective TAFIa substrates that distinguish

between TAFIa and CPN [88]. Although not all these techniques are widely available, they are

valuable tools in TAFI research.

Chapter 2

32

TAFI from different species

The above mentioned assays for measuring TAFI of animal origin are important since the use

of experimental animals can yield valuable information. Also, TAFI from different species has

been cloned and characterized. The deduced amino acid sequence of rat TAFI is 83% [82]

identical to human TAFI. For mice this is 85% [68] compared to human, whereas the protein

sequences of rat and mice share 95% identity [82]. Although rat, mouse and human TAFI

share similar biochemical properties, the half-life of the rodents’ TAFI is shorter than the half-

life of their human counter part [68,82,89]. Also the plasma concentration of TAFI is lower in

these animals [68,82,90].

Some of the crystal structures of TAFI were solved using bovine TAFI, which has a sequence

identity of 77% with human TAFI, although little characterization of the functionality of this

protein in cows has been reported so far. Furthermore, the presence of TAFI was established

in pig, guinea pig, rabbit, dog, and baboon [90].

Functions: the interface between coagulation, fibrinolysis, inflammation and more

Besides in experimental animals, relations between TAFI levels and diseases were

investigated in human subjects. Recent advances on the role of TAFI in bleeding and

thrombotic disorders included the discovery that increased plasma TAFI concentrations are

associated with an increased risk for venous thrombosis and coronary artery disease [91,92]

and associations were found between TAFI levels and a number of disorders such as type-2

diabetes mellitus [93-96], hypertension [97,98], obesity [99], stroke [100-106], sepsis

[103,107-109], liver cirrhosis [110] and glomerulonephritis [111].

Patients with type 2 diabetes mellitus showed significantly higher TAFI levels compared to

non-diabetics [93] and TAFI levels were correlated with the urinary albumin excretion rate in

normotensive diabetes mellitus patients [94,96]. However, fasting TAFI levels were decreased

in normotriglyceridemic patients with type 2 diabetes compared to non-diabetes patients and

TAFI levels decreased postprandially in both groups [112].

The risk for ischemic stroke was also associated with elevated TAFI levels [101,102,105].

These patients showed elevated TAFI levels during the acute phase [100,106] and significantly

higher levels of TAFI were observed in stroke patients after recanalization by tissue-type

plasminogen activator infusion [103]. The baseline levels of TAFI, together with plasminogen

activator inhibitor 1, can predict the risk of symptomatic intracranial hemorrhage after tissue-

type plasminogen activator infusion [113].

In contrast, septic (both severe sepsis and septic shock) patients had significantly decreased

TAFI levels compared to controls [108]. Moreover, associations were found with TAFI and

mortality of meningococcal sepsis [107].


33

Ch

apte

r 2

With the engineering of TAFI knockout animals [84,114-116], the in vivo role of TAFI advanced

rapidly in the past few years. Compared to wild type animals, these mice were normal in

many respects, including survival, development, and fertility. Mao et al. reported that mice

lacking TAFI indeed have an enhanced endogenous fibrinolysis [117] and Wang et al. [116]

demonstrated the protective effect of TAFI deficiency in a ferric choride-induced occlusion

model of the vena cava. Similar results were obtained when TAFIa was inhibited by treatment

with carboxypeptidase inhibitor (CPI) [48,116]. Previously, enhanced in vivo thrombolysis was

already observed for TAFI deficiency in a background of plasminogen deficiency [118].

Besides the anti-fibrinolytic function of TAFIa, TAFI is also involved in inflammation and

wound healing [84]. The role of TAFI in inflammation is for example illustrated by the

observation that TAFI knock out mice, in contrast to control mice, were protected from liver

necrosis after intra peritoneal injection with Escherichia coli [119]. In another inflammation

model, this time with TAFI/plasminogen double knock out mice, the migration of leukocytes

towards the peritoneum was increased in the deficient animals compared to the wild types

showing the importance for TAFI in (plasminogen-dependent) cell migration in vivo [118].

Lately, we reported the binding of TAFI to the surface of a group A streptococci (M41

serotype) and subsequent activation at the bacterial surface via plasmin and thrombin-

thrombomodulin [120]. Furthermore, activation of TAFI on the surface of Streptococcus

pyogenes evoked inflammatory reactions by modulating the kallikrein/kinin system [121].

Additional in vivo experiments showed that the TAFI-deficient mice have a wound healing

problem [84], which may be related to the cell migration process mentioned above. In a skin

wound model [84], the keratinocyte migration pattern was disturbed, again pointing to a role

for TAFI in cell migration. Subsequent in vitro studies showed that TAFI inhibits endothelial

cell movement and tube formation [122].

However, it will take another while before the exact (patho)physiological roles of TAFI are

revealed, partly because interpretation of the data is difficult due to the genotype sensitivity

of many assays used in the past and still in use at the moment, and partially because some

studies were contradictory.

Concluding remarks

As outlined above, the TAFI research field has developed swiftly in the past few years and

now expands beyond hemostasis. The interest for the protein has increased since it is

recognized as a potential therapeutic target for novel intervention strategies. Inhibition of

TAFIa is expected to increase the efficacy of fibrinolytic therapy in thrombotic disorders.

Conversely, agents that improve or stabilize TAFIa, thereby down-regulating fibrinolysis, may

be useful for the treatment of bleeding disorders. In addition it may prove to be a target to

treat inflammatory or wound healing disorders.

Chapter 2

34

Reference List

1 Gils, A. Which carboxypeptidase determines the antifibrinolytic potential? J. Thromb. Haemost.,

2008, 6, 846-7.

2 Boffa, M. B.; Koschinsky, M. L. Curiouser and curiouser: recent advances in measurement of

thrombin-activatable fibrinolysis inhibitor (TAFI) and in understanding its molecular genetics,

gene regulation, and biological roles. Clin. Biochem., 2007, 40, 431-42.

3 Bouma, B. N.; Mosnier, L. O. Thrombin activatable fibrinolysis inhibitor (TAFI)--how does

thrombin regulate fibrinolysis? Ann. Med., 2006, 38, 378-88.

4 Willemse, J. L.; Hendriks, D. F. A role for procarboxypepidase U (TAFI) in thrombosis. Front

Biosci., 2007, 12, 1973-87.

5 Mosnier, L. O.; Bouma, B. N. Regulation of fibrinolysis by thrombin activatable fibrinolysis

inhibitor, an unstable carboxypeptidase B that unites the pathways of coagulation and

fibrinolysis. Arterioscler. Thromb. Vasc. Biol., 2006, 26, 2445-53.

6 Leurs, J.; Hendriks, D. Carboxypeptidase U (TAFIa): a metallocarboxypeptidase with a distinct role

in haemostasis and a possible risk factor for thrombotic disease. Thromb. Haemost., 2005, 94,

471-87.

7 Bouma, B. N.; Mosnier, L. O. Thrombin activatable fibrinolysis inhibitor (TAFI) at the interface

between coagulation and fibrinolysis. Pathophysiol Haemost Thromb, 2003, 33, 375-81.

8 Marx, P. F. Thrombin-activatable fibrinolysis inhibitor. Curr Med Chem, 2004, 11, 2311-24.

9 Rijken, D. C.; Lijnen, H. R. New insights into the molecular mechanisms of the fibrinolytic system.

J. Thromb. Haemost., 2008,

10 Leung, L. L.; Nishimura, T.; Myles, T. Regulation of tissue inflammation by thrombin-activatable

carboxypeptidase B (or TAFI). Adv. Exp. Med. Biol., 2008, 632, 61-9.

11 Binette, T. M.; Taylor, F. B., Jr.; Peer, G.; Bajzar, L. Thrombin-thrombomodulin connects

coagulation and fibrinolysis: more than an in vitro phenomenon. Blood, 2007, 110, 3168-75.

12 Bajzar, L.; Morser, J.; Nesheim, M. TAFI, or plasma procarboxypeptidase B, couples the

coagulation and fibrinolytic cascades through the thrombin-thrombomodulin complex. J. Biol.

Chem., 1996, 271, 16603-8.

13 Eaton, D. L.; Malloy, B. E.; Tsai, S. P.; Henzel, W.; Drayna, D. Isolation, molecular cloning, and

partial characterization of a novel carboxypeptidase B from human plasma. J. Biol. Chem., 1991,

266, 21833-8.


35

Ch

apte

r 2

14 Mao, S. S.; Cooper, C. M.; Wood, T.; Shafer, J. A.; Gardell, S. J. Characterization of plasmin-

mediated activation of plasma procarboxypeptidase B - modulation by glycosaminoglycans. J.

Biol. Chem., 1999, 274, 35046-52.

15 Marx, P. F.; Dawson, P. E.; Bouma, B. N.; Meijers, J. C. Plasmin-mediated activation and

inactivation of thrombin-activatable fibrinolysis inhibitor. Biochemistry, 2002, 41, 6688-96.

16 Kawamura, T.; Okada, N.; Okada, H. Elastase from activated human neutronpils activates

procarboxypeptidase R. Microbiol. Immunol., 2002, 46, 225-30.

17 Marx, P. F.; Hackeng, T. M.; Dawson, P. E.; Griffin, J. H.; Meijers, J. C. M.; Bouma, B. N.

Inactivation of activated thrombin-activable fibrinolysis inhibitor takes place by a process that

involves conformational instability rather than proteolytic cleavage. J. Biol. Chem., 2000, 275,

12410-5.

18 Boffa, M. B.; Bell, R.; Stevens, W. K.; Nesheim, M. E. Roles of thermal instability and proteolytic

cleavage in regulation of activated thrombin-activable fibrinolysis inhibitor. J. Biol. Chem., 2000,

275, 12868-78.

19 Marx, P. F.; Bouma, B. N.; Meijers, J. C. Role of zinc ions in activation and inactivation of

thrombin-activatable fibrinolysis inhibitor. Biochemistry, 2002, 41, 1211-6.

20 Marx, P. F.; Plug, T.; Havik, S. R.; Morgelin, M.; Meijers, J. C. The activation peptide of thrombin-

activatable fibrinolysis inhibitor: a role in activity and stability of the enzyme? J. Thromb.

Haemost., 2008,

21 Marx, P. F.; Havik, S. R.; Marquart, J. A.; Bouma, B. N.; Meijers, J. C. M. Generation and

characterization of a highly stable form of activated thrombin-activable fibrinolysis inhibitor. J.

Biol. Chem., 2004, 279, 6620-8.

22 Marx, P. F.; Havik, S. R.; Bouma, B. N.; Meijers, J. C. M. Role of isoleucine residues 182 and 183 in

thrombin-activatable fibrinolysis inhibitor. J. Throm. Haem., 2005, 3, 1293-300.

23 Knecht, W.; Willemse, J.; Stenhamre, H.; Andersson, M.; Berntsson, P.; Furebring, C.; Harrysson,

A.; Hager, A. C.; Wissing, B. M.; Hendriks, D.; Cronet, P. Limited mutagenesis increases the

stability of human carboxypeptidase U (TAFIa) and demonstrates the importance of CPU stability

over proCPU concentration in down-regulating fibrinolysis. FEBS J., 2006, 273, 778-92.

24 Ceresa, E.; De Maeyer, M.; Jonckheer, A.; Peeters, M.; Engelborghs, Y.; Declerck, P. J.; Gils, A.

Comparative evaluation of stable TAFIa variants: importance of alpha-helix 9 and beta-sheet 11

for TAFIa (in)stability. J. Thromb. Haemost., 2007, 5, 2105-12.

Chapter 2

36

25 Ceresa, E.; Peeters, M.; Declerck, P. J.; Gils, A. Announcing a TAFIa mutant with a 180-fold

increased half-life and concomitantly a strongly increased antifibrinolytic potential. J. Thromb.

Haemost., 2007, 5, 418-20.

26 Ceresa, E.; Van de Borne, K.; Peeters, M.; Lijnen, H. R.; Declerck, P. J.; Gils, A. Generation of a

stable activated thrombin activable fibrinolysis inhibitor variant. J. Biol. Chem., 2006, 281, 15878-

83.

27 Marx, P. F.; Brondijk, T. H.; Plug, T.; Romijn, R. A.; Hemrika, W.; Meijers, J. C.; Huizinga, E. G.

Crystal structures of TAFI elucidate the inactivation mechanism of activated TAFI; A novel

mechanism for enzyme auto-regulation. Blood, 2008, 112, 2803-9.

28 Sanglas, L.; Valnickova, Z.; Arolas, J. L.; Pallares, I.; Guevara, T.; Sola, M.; Kristensen, T.; Enghild, J.

J.; Aviles, F. X.; Gomis-Ruth, F. X. Structure of activated thrombin-activatable fibrinolysis

inhibitor, a molecular link between coagulation and fibrinolysis. Mol. Cell, 2008, 31, 598-606.

29 Anand, K.; Pallares, I.; Valnickova, Z.; Christensen, T.; Vendrell, J.; Wendt, K. U.; Schreuder, H. A.;

Enghild, J. J.; Aviles, F. X. The crystal structure of TAFI provides the structural basis for its intrinsic

activity and the short half-life of TAFIa. J. Biol. Chem., 2008, 283, 29416-23.

30 Barbosa Pereira, P. J.; Segura-Martin, S.; Oliva, B.; Ferrer-Orta, C.; Aviles, F. X.; Coll, M.; Gomis-

Ruth, F. X.; Vendrell, J. Human procarboxypeptidase B: three-dimensional structure and

implications for thrombin-activatable fibrinolysis inhibitor (TAFI). J. Mol. Biol., 2002, 321, 537-47.

31 Coll, M.; Guasch, A.; Avilés, F. X.; Huber, R. Three-dimensional structure of pocine

procarboxypeptidase B: a structural basis of its inactivity. EMBO, 1991, 10, 1-9.

32 Guasch, A.; Coll, M.; Aviles, F. X.; Huber, R. Three-dimensional structure of procine pancreatic

procarboxypeptidase A. A comparison of the A and B zymogens and their determinants for

inhibition and activation. J. Mol. Biol, 1992, 224, 141-57.

33 Garcia-Saez, I.; Reverter, D.; Vendrell, J.; Aviles, F. X.; Coll, M. The three-dimensional structure of

human procarboxypeptidase A2. Deciphering the basis of the inhibition, activation and intrinsic

activity of the zymogen. EMBO J., 1997, 16, 6906-13.

34 Gomis-Ruth, F. X.; Gomez, M.; Bode, W.; Huber, R.; Aviles, F. X. The three-dimensional structure

of the native ternary complex of bovine pancreatic procarboxypeptidase A with proproteinase E

and chymotrypsinogen C. EMBO J., 1995, 14, 4387-94.

35 Valnickova, Z.; Christensen, T.; Skottrup, P.; Thogersen, I. B.; Hojrup, P.; Enghild, J. J. Post-

translational modifications of human thrombin-activatable fibrinolysis inhibitor (TAFI): evidence

for a large shift in the isoelectric point and reduced solubility upon activation. Biochemistry,

2006, 45, 1525-35.


37

Ch

apte

r 2

36 Buelens, K.; Hillmayer, K.; Compernolle, G.; Declerck, P. J.; Gils, A. Biochemical importance of

glycosylation in thrombin activatable fibrinolysis inhibitor. Circ. Res., 2008, 102, 295-301.

37 Valnickova, Z.; Thogersen, I. B.; Potempa, J.; Enghild, J. J. Thrombin-activable fibrinolysis inhibitor

(TAFI) zymogen is an active carboxypeptidase. J. Biol. Chem., 2007, 282, 3066-76.

38 Willemse, J. L.; Polla, M.; Hendriks, D. F. The intrinsic enzymatic activity of plasma

procarboxypeptidase U (TAFI) can interfere with plasma carboxypeptidase N assays. Anal.

Biochem., 2006, 356, 157-9.

39 Foley, J. H.; Kim, P.; Nesheim, M. E. Thrombin-activable fibrinolysis inhibitor zymogen does not

play a significant role in the attenuation of fibrinolysis. J. Biol. Chem., 2008, 283, 8863-7.

40 Willemse, J. L.; Heylen, E.; Hendriks, D. F. The intrinsic enzymatic activity of procarboxypeptidase

U (TAFI) does not significantly influence the fibrinolysis rate: a rebuttal. J. Thromb. Haemost.,

2007, 5, 1334-6.

41 Walker, J. B.; Binette, T. M.; Mackova, M.; Lambkin, G. R.; Mitchell, L.; Bajzar, L. Proteolytic

cleavage of carboxypeptidase N markedly increases its antifibrinolytic activity. J. Thromb.

Haemost., 2008, 6, 848-55.

42 Adler, M.; Buckman, B.; Bryant, J.; Chang, Z.; Chu, K.; Emayan, K.; Hrvatin, P.; Islam, I.; Morser, J.;

Sukovich, D.; West, C.; Yuan, S.; Whitlow, M. Structures of potent selective peptide mimetics

bound to carboxypeptidase B. Acta Crystallogr. D. Biol. Crystallogr., 2008, 64, 149-57.

43 Adler, M.; Bryant, J.; Buckman, B.; Islam, I.; Larsen, B.; Finster, S.; Kent, L.; May, K.; Mohan, R.;

Yuan, S.; Whitlow, M. Crystal structures of potent thiol-based inhibitors bound to

carboxypeptidase B. Biochemistry, 2005, 44, 9339-47.

44 Islam, I.; Bryant, J.; May, K.; Mohan, R.; Yuan, S.; Kent, L.; Morser, J.; Zhao, L.; Vergona, R.; White,

K.; Adler, M.; Whitlow, M.; Buckman, B. O. 3-Mercaptopropionic acids as efficacious inhibitors of

activated thrombin activatable fibrinolysis inhibitor (TAFIa). Bioorg. Med. Chem. Lett., 2007, 17,

1349-54.

45 Bunnage, M. E.; Owen, D. R. TAFIa inhibitors in the treatment of thrombosis. Curr. Opin. Drug

Discov. Devel., 2008, 11, 480-6.

46 Wang, Y. X.; da, C., V; Vincelette, J.; Zhao, L.; Nagashima, M.; Kawai, K.; Yuan, S.; Emayan, K.;

Islam, I.; Hosoya, J.; Sullivan, M. E.; Dole, W. P.; Morser, J.; Buckman, B. O.; Vergona, R. A novel

inhibitor of activated thrombin activatable fibrinolysis inhibitor (TAFIa) - part II: enhancement of

both exogenous and endogenous fibrinolysis in animal models of thrombosis. Thromb. Haemost.,

2007, 97, 54-61.

Chapter 2

38

47 Wang, Y. X.; Zhao, L.; Nagashima, M.; Vincelette, J.; Sukovich, D.; Li, W.; Subramanyam, B.; Yuan,

S.; Emayan, K.; Islam, I.; Hrvatin, P.; Bryant, J.; Light, D. R.; Vergona, R.; Morser, J.; Buckman, B. O.

A novel inhibitor of activated thrombin-activatable fibrinolysis inhibitor (TAFIa) - part I:

pharmacological characterization. Thromb. Haemost., 2007, 97, 45-53.

48 Wang, X.; Smith, P. L.; Hsu, M. Y.; Ogletree, M. L.; Schumacher, W. A. Murine model of ferric

chloride-induced vena cava thrombosis: evidence for effect of potato carboxypeptidase inhibitor.

J. Thromb. Haemost., 2006, 4, 403-10.

49 Gils, A.; Ceresa, E.; Macovei, A. M.; Marx, P. F.; Peeters, M.; Compernolle, G.; Declerck, P. J.

Modulation of TAFI function through different pathways--implications for the development of

TAFI inhibitors. J. Thromb. Haemost., 2005, 3, 2745-53.

50 Schneider, M.; Nesheim, M. Reversible inhibitors of TAFIa can both promote and inhibit

fibrinolysis. J. Throm. Haem., 2003, 1, 147-54.

51 Walker, J. B.; Hughes, B.; James, I.; Haddock, P.; Kluft, C.; Bajzar, L. Stabilization versus inhibition

of TAFIa by competitive inhibitors in vitro. J. Biol. Chem., 2003, 278, 8913-21.

52 Leurs, J.; Nerme, V.; Sim, Y.; Hendriks, D. Carboxypeptidase U (TAFIa) prevents lysis from

proceeding into the propagation phase through a threshold-dependent mechanism. J. Throm.

Haem., 2004, 2, 416-23.

53 Walker, J. B.; Bajzar, L. The intrinsic threshold of the fibriolytic system is modulated by basic

carboxypeptidases, but the magnitude of the antifibrinolytic effect of activated thrombin-

activable fibrinolysis inhibitor is masked by its instability. J. Biol. Chem., 2004, 279, 27896-904.

54 Brouwers, G. J.; Vos, H. L.; Leebeek, F. W.; Bulk, S.; Schneider, M.; Boffa, M.; Koschinsky, M. L.;

Van Tilburg, N. H.; Nesheim, M.; Bertina, R. M.; Gomez-Garcia, E. B. A novel, possibly functional,

single nucleotide polymorphism in the coding region of the thrombin-activatable fibrinolysis

inhibitor (TAFI) gene is also associated with TAFI levels. Blood, 2001, 98, 1992-3.

55 Schneider, M.; Boffa, M.; Stewart, R. J.; Rahman, N. L.; Koschinsky, M. L.; Nesheim, M. Two

naturally occurring variants of TAFI (Thr-325 and Ile-325) differ substantially with respect to

thermal stability and antifibrinolytic activity of the enzyme. J. Biol. Chem., 2002, 277, 1021-30.

56 Henry, M.; Aubert, H.; Morange, P. E.; Nanni, I.; Alessi, M. C.; Tiret, L.; Juhan-Vague, I.

Identification of polymorphisms in the promoter and the 3' region of the TAFI gene: evidence

that plasma TAFI antigen levels are strongly genetically controlled. Blood, 2001, 97, 2053-8.

57 Franco, R. F.; Fagundes, M. G.; Meijers, J. C. M.; Reitsma, P. H.; Lourenco, D.; Morelli, V.; Maffei,

F. H.; Ferrari, I. C.; Piccinato, C. E.; Silva, W. A. j.; Zago, M. A. Identification of polymorphisms in

the 5'-untranslated region of the TAFI gene: relationship with plasma TAFI levels and risk of

venous thrombosis. Haematologica, 2001, 86, 510-7.


39

Ch

apte

r 2

58 Koschinsky, M. L.; Boffa, M. B.; Nesheim, M. E.; Zinman, B.; Hanley, A. J.; Harris, S. B.; Cao, H.;

Hegele, R. A. Association of a single nucleotide polymorphism in CPB2 encoding the thrombin-

activable fibrinolysis inhibitor (TAF1) with blood pressure. Clin. Genet., 2001, 60, 345-9.

59 Morange, P. E.; Juhan-Vague, I.; Scarabin, P. Y.; Alessi, M. C.; Luc, G.; Arveiler, D.; Ferrieres, J.;

Amouyel, P.; Evans, A.; Ducimetiere, P. Association between TAFI antigen and Ala147Thr

polymorphism of the TAFI gene and the angina pectoris incidence. Thromb. Haemost., 2003, 89,

554-60.

60 Kremer Hovinga, J. A.; Franco, R. F.; Zago, M. A.; Ten, C. H.; Westendorp, R. G.; Reitsma, P. H. A

functional single nucleotide polymorphism in the thrombin-activatable fibrinolysis inhibitor

(TAFI) gene associates with outcome of meningococcal disease. J. Thromb. Haemost., 2004, 2,

54-7.

61 de Bruijne, E. L.; Murad, S. D.; de Maat, M. P.; Tanck, M. W.; Haagsma, E. B.; van, H. B.;

Rosendaal, F. R.; Janssen, H. L.; Leebeek, F. W. Genetic variation in thrombin-activatable

fibrinolysis inhibitor (TAFI) is associated with the risk of splanchnic vein thrombosis. Thromb.

Haemost., 2007, 97, 181-5.

62 Fernandez-Cadenas, I.; varez-Sabin, J.; Ribo, M.; Rubiera, M.; Mendioroz, M.; Molina, C. A.;

Rosell, A.; Montaner, J. Influence of thrombin-activatable fibrinolysis inhibitor and plasminogen

activator inhibitor-1 gene polymorphisms on tissue-type plasminogen activator-induced

recanalization in ischemic stroke patients. J. Thromb. Haemost., 2007, 5, 1862-8.

63 Masini, S.; Ticconi, C.; Gravina, P.; Tomassini, M.; Pietropolli, A.; Forte, V.; Federici, G.; Piccione,

E.; Bernardini, S. Thrombin-activatable fibrinolysis inhibitor polymorphisms and recurrent

pregnancy loss. Fertil. Steril., 2008,

64 Vanhoof, G.; Wauters, J.; Schatteman, K.; Hendriks, D.; Goossens, F.; Bossuyt, P.; Scharpé, S. The

gene for human carboxypeptidase U (CPU)- a proposed novel regulator of plasminogen

activation- maps to 13q14.11. Genomics, 1996, 38, 454-5.

65 Tsai, S. P.; Drayna, D. The gene encoding human plasma carboxypeptidase B (CPB2) resides on

chromosome 13. Genomics, 1992, 14, 549-50.

66 Boffa, M.; Reid, S. T.; Joo, E.; Nesheim, M.; Koschinsky, M. L. Characterization of the gene

encoding human TAFI (Thrombin-activable fibrinolysis inhibitor; plasma procarboxypeptidas B).

Biochem., 1999, 38, 6547-58.

67 Garand, M.; Bastajian, N.; Nesheim, M. E.; Boffa, M. B.; Koschinsky, M. L. Molecular analysis of

the human thrombin-activatable fibrinolysis inhibitor gene promoter. Br. J. Haematol., 2007,

138, 231-44.

Chapter 2

40

68 Marx, P. F.; Wagenaar, G. T. M.; Reijerkerk, A.; Tiekstra, M. J.; Van Rossum, A. G.; Gebbink, M. F.;

Meijers, J. C. Characterization of mouse thrombin-activatable fibrinolysis inhibitor. Thromb.

Haemost., 2000, 83, 297-303.

69 Mosnier, L. O.; Buijtenhuijs, P.; Marx, P. F.; Meijers, J. C. M.; Bouma, B. N. Identification of

Thrombin Activatable Fibrinolysis Inhibitor (TAFI) in human platelets. Blood, 2003, 101, 4844-6.

70 Bajzar, L.; Nesheim, M. E.; Tracy, P. B. The profibrinolytic effect of activated protein C in clots

formed from plasma is TAFI-dependent. Blood, 1996, 88, 2093-100.

71 Mosnier, L. O.; Von dem Borne, P. A.; Meijers, J. C.; Bouma, B. N. Plasma TAFI levels determine

the clot lysis time in healthy individuals in the presence of an intact intrinsic pathway of

coagulation. Thromb. Haemost., 1998, 80, 829-35.

72 Van Tilburg, N. H.; Rosendaal, F. R.; Bertina, R. M. Thrombin-activatable fibrinolysis inhibitor and

the risk for deep vein thrombosis. Blood, 2000, 95, 2855-9.

73 Strömqvist, M.; Schatteman, K.; Leurs, J.; Verkerk, R.; Andersson, J.; Johansson, T.; Scharpé, S.;

Hendriks, D. Immunological assay for the determination of procarboxypeptidase U antigen levels

in human plasma. Thromb. Haemost., 2001, 85, 12-7.

74 Frere, C.; Morange, P. E.; Saut, N.; Tregouet, D. A.; Grosley, M.; Beltran, J.; Juhan-Vague, I.; Alessi,

M. C. Quantification of thrombin activatable fibrinolysis inhibitor (TAFI) gene polymorphism

effects on plasma levels of TAFI measured with assays insensitive to isoform-dependent artefact.

Thromb. Haemost., 2005, 94, 373-9.

75 Boffa, M. B.; Hamill, J. D.; Maret, D.; Brown, D.; Scott, M. L.; Nesheim, M. E.; Koschinsky, M. L.

Acute Phase Mediators Modulate Thrombin-activable Fibrinolysis Inhibitor (TAFI) Gene

Expression in HepG2 Cells. J. Biol. Chem., 2003, 278, 9250-7.

76 Gils, A.; Alessi, M. C.; Brouwers, E.; Peeters, M.; Marx, P.; Leurs, J.; Bouma, B.; Hendriks, D.;

Juhan-Vague, I.; Declerck, P. J. Development of a genotype 325-specific proCPU/TAFI ELISA.

Arterioscler. Thromb. Vasc. Biol., 2003, 23, 1122-7.

77 Willemse, J. L.; Matus, V.; Heylen, E.; Mezzano, D.; Hendriks, D. F. Influence of the Thr325Ile

polymorphism on procarboxypeptidase U (thrombin-activable fibrinolysis inhibitor) activity-

based assays. J. Thromb. Haemost., 2007, 5, 872-5.

78 Willemse, J.; Leurs, J.; Verkerk, R.; Hendriks, D. Development of a fast kinetic method for the

determination of carboxypeptidase U (TAFIa) using C-terminal arginine containing peptides as

substrate. Anal. Biochem., 2005, 340, 106-12.

79 Willemse, J. L.; Leurs, J. R.; Hendriks, D. F. Fast kinetic assay for the determination of

procarboxypeptidase U (TAFI) in human plasma. J. Throm. Haem., 2005, 3, 2353-5.


41

Ch

apte

r 2

80 Kim, P. Y.; Foley, J.; Hsu, G.; Kim, P. Y.; Nesheim, M. E. An assay for measuring functional

activated thrombin-activatable fibrinolysis inhibitor in plasma. Anal. Biochem., 2008, 372, 32-40.

81 Ceresa, E.; Brouwers, E.; Peeters, M.; Jern, C.; Declerck, P. J.; Gils, A. Development of ELISAs

measuring the extent of TAFI activation. Arterioscler. Thromb. Vasc. Biol., 2006, 26, 423-8.

82 Hillmayer, K.; Macovei, A.; Pauwels, D.; Compernolle, G.; Declerck, P. J.; Gils, A. Characterization

of rat thrombin-activatable fibrinolysis inhibitor (TAFI)--a comparative study assessing the

biological equivalence of rat, murine and human TAFI. J. Thromb. Haemost., 2006, 4, 2470-7.

83 Hillmayer, K.; Brouwers, E.; Leon-Tamariz, F.; Meijers, J. C.; Marx, P. F.; Declerck, P. J.; Gils, A.

Development of sandwich-type ELISAs for the quantification of rat and murine thrombin

activatable fibrinolysis inhibitor in plasma. J. Thromb. Haemost., 2008, 6, 132-8.

84 Te Velde, E. A.; Wagenaar, G. T.; Reijerkerk, A.; Roose-Girma, M.; Borel, R., I; Voest, E. E.; Bouma,

B. N.; Gebbink, M. F.; Meijers, J. C. Impaired healing of cutaneous wounds and colonic

anastomoses in mice lacking thrombin-activatable fibrinolysis inhibitor. J. Thromb. Haemost.,

2003, 1, 2087-96.

85 Guimaraes, A. H.; Barrett-Bergshoeff, M. M.; Criscuoli, M.; Evangelista, S.; Rijken, D. C.

Fibrinolytic efficacy of Amediplase, Tenecteplase and scu-PA in different external plasma clot

lysis models: sensitivity to the inhibitory action of thrombin activatable fibrinolysis inhibitor

(TAFI). Thromb. Haemost., 2006, 96, 325-30.

86 Guimaraes, A. H.; Bertina, R. M.; Rijken, D. C. A new functional assay of thrombin activatable

fibrinolysis inhibitor. J. Thromb. Haemost., 2005, 3, 1284-92.

87 Rijken, D. C.; Hoegee-de, N. E.; Jie, A. F.; Atsma, D. E.; Schalij, M. J.; Nieuwenhuizen, W.

Development of a new test for the global fibrinolytic capacity in whole blood. J. Thromb.

Haemost., 2008, 6, 151-7.

88 Willemse, J. L.; Polla, M.; Olsson, T.; Hendriks, D. F. Comparative substrate specificity study of

carboxypeptidase U (TAFIa) and carboxypeptidase N: development of highly selective CPU

substrates as useful tools for assay development. Clin. Chim. Acta, 2008, 387, 158-60.

89 Hillmayer, K.; Ceresa, E.; Vancraenenbroeck, R.; Declerck, P. J.; Gils, A. Conformational

(in)stability of rat TAFIa versus human TAFIa. J. Thromb. Haemost., 2008, 6, 1426-8.

90 Schatteman, K. A.; Goossens, F. J.; Scharpe, S. S.; Hendriks, D. F. Activation of plasma

procarboxypeptidase U in different mammalian species points to a conserved pathway of

inhibition of fibrinolysis. Thromb. Haemost., 1999, 82, 1718-21.

Chapter 2

42

91 Schroeder, V.; Chatterjee, T.; Mehta, H.; Windecker, S.; Pham, T.; Devantay, N.; Meier, B.; Kohler,

H. P. Thrombin activatable fibrinolysis inhibitor (TAFI) levels in patients with coronary artery

disease investigated by angiography. Thromb. Haemost., 2002, 88, 1020-5.

92 Schroeder, V.; Wilmer, M.; Buehler, B.; Kohler, H. P. TAFI activity in coronary artery disease: a

contribution to the current discussion on TAFI assays. Thromb. Haemost., 2006, 96, 236-7.

93 Kitagawa, N.; Yano, Y.; Gabazza, E. C.; Bruno, N. E.; Araki, R.; Matsumoto, K.; Katsuki, A.; Hori, Y.;

Nakatani, K.; Taguchi, O.; Sumida, Y.; Suzuki, K.; Adachi, Y. Different metabolic correlations of

thrombin-activatable fibrinolysis inhibitor and plasminogen activator inhibitor-1 in non-obese

type 2 diabetic patients. Diabetes Res. Clin. Pract., 2006, 73, 150-7.

94 Yano, Y.; Kitagawa, N.; Gabazza, E. C.; Morioka, K.; Urakawa, H.; Tanaka, T.; Katsuki, A.; Araki-

Sasaki, R.; Hori, Y.; Nakatani, K.; Taguchi, O.; Sumida, Y.; Adachi, Y. Increased plasma thrombin-

activatable fibrinolysis inhibitor levels in normatensive type 2 diabetic patients with

microalbuminuria. J. Clin. Endocrinol. Metabolism, 2003, 88, 736-41.

95 Yano, Y.; Gabazza, E. C.; Hori, Y.; Kitagawa, N.; Katsuki, A.; raki-Sasaki, R.; Sumida, Y.; Adachi, Y.

Association between plasma thrombin-activatable fibrinolysis inhibitor levels and activated

protein C in normotensive type 2 diabetic patients. Diabetes Care, 2002, 25, 1245-6.

96 Hori, Y.; Gabazza, E. C.; Yano, Y.; Katsuki, A.; Suzuki, K.; Adachi, Y.; Sumida, Y. Insulin resistance is

associated with increased circulating level of thrombin-activatable fibrinolysis inhibitor in type 2

diabetic patients. J. Clin. Endocrinol. Metab, 2002, 87, 660-5.

97 Malyszko, J.; Tymcio, J. Thrombin activatable fibrinolysis inhibitor and other hemostatic

parameters in patients with essential arterial hypertension. Pol. Arch. Med. Wewn., 2008, 118,

36-41.

98 Malyszko, J.; Malyszko, J. S.; Hryszko, T.; Mysliwiec, M. Thrombin activatable fibrinolysis inhibitor

in hypertensive kidney transplant recipients. Transplant. Proc., 2006, 38, 105-7.

99 Guven, G. S.; Kilicaslan, A.; Oz, S. G.; Haznedaroglu, I. C.; Kirazli, S.; Aslan, D.; Sozen, T.

Decrements in the thrombin activatable fibrinolysis inhibitor (TAFI) levels in association with

orlistat treatment in obesity. Clin. Appl. Thromb. Hemost., 2006, 12, 364-8.

100 Rooth, E.; Wallen, H.; Antovic, A.; von, A. M.; Kaponides, G.; Wahlgren, N.; Blomback, M.;

Antovic, J. Thrombin activatable fibrinolysis inhibitor and its relationship to fibrinolysis and

inflammation during the acute and convalescent phase of ischemic stroke. Blood Coagul.

Fibrinolysis, 2007, 18, 365-70.

101 Ladenvall, C.; Gils, A.; Jood, K.; Blomstrand, C.; Declerck, P. J.; Jern, C. Thrombin activatable

fibrinolysis inhibitor activation peptide shows association with all major subtypes of ischemic

stroke and with TAFI gene variation. Arterioscler. Thromb. Vasc. Biol., 2007, 27, 955-62.


43

Ch

apte

r 2

102 Leebeek, F. W.; Goor, M. P.; Guimaraes, A. H.; Brouwers, G. J.; Maat, M. P.; Dippel, D. W.; Rijken,

D. C. High functional levels of thrombin-activatable fibrinolysis inhibitor are associated with an

increased risk of first ischemic stroke. J. Thromb. Haemost., 2005, 3, 2211-8.

103 Monasterio, J.; Bermudez, P.; Quiroga, D.; Francisco, E.; Meneses, B.; Montaner, J. Plasma

thrombin-activatable fibrinolytic inhibitor (TAFI) among healthy subjects and patients with

vascular diseases: a validation study. Pathophysiol. Haemost. Thromb., 2003, 33, 382-6.

104 Ribo, M.; Montaner, J.; Molina, C. A.; Arenillas, J. F.; Santamarina, E.; Quintana, M.; varez-Sabin,

J. Admission fibrinolytic profile is associated with symptomatic hemorrhagic transformation in

stroke patients treated with tissue plasminogen activator. Stroke, 2004, 35, 2123-7.

105 Santamaria, A.; Oliver, A.; Borrell, M.; Mateo, J.; Belvis, R.; Marti-Fabregas, J.; Ortin, R.; Tirado, I.;

Souto, J. C.; Fontcuberta, J. Risk of ischemic stroke associated with functional thrombin-

activatable fibrinolysis inhibitor plasma levels. Stroke, 2003, 34, 2387-91.

106 Montaner, J.; Ribo, M.; Monasterio, J.; Molina, C. A.; Alvarez-Sabin, J. Thrombin-Activable

Fibrinolysis Inhibitor Levels in the Acute Phase of Ischemic Stroke. Stroke, 2003, 1038-40.

107 Emonts, M.; de Bruijne, E. L.; Guimaraes, A. H.; Declerck, P. J.; Leebeek, F. W.; de Maat, M. P.;

Rijken, D. C.; Hazelzet, J. A.; Gils, A. Thrombin-activatable fibrinolysis inhibitor is associated with

severity and outcome of severe meningococcal infection in children. J. Thromb. Haemost., 2008,

6, 268-76.

108 Zeerleder, S.; Schroeder, V.; Hack, C. E.; Kohler, H. P.; Wuillemin, W. A. TAFI and PAI-1 levels in

human sepsis. Thromb. Res., 2006, 118, 205-12.

109 Watanabe, R.; Wada, H.; Watanabe, Y.; Sakakura, M.; Nakasaki, T.; Mori, Y.; Nishikawa, M.;

Gabazza, E. C.; Nobori, T.; Shiku, H. Activity and antigen levels of thrombin-activatable fibrinolysis

inhibitor in plasma of patients with disseminated intravascular coagulation. Thromb. Res., 2001,

104, 1-6.

110 Gresele, P.; Binetti, B. M.; Branca, G.; Clerici, C.; Asciutti, S.; Morelli, A.; Semeraro, N.; Colucci, M.

TAFI deficiency in liver cirrhosis: relation with plasma fibrinolysis and survival. Thromb. Res.,

2008, 121, 763-8.

111 Bruno, N. E.; Yano, Y.; Takei, Y.; Qin, L.; Suzuki, T.; Morser, J.; essandro-Gabazza, C. N.; Mizoguchi,

A.; Suzuki, K.; Taguchi, O.; Gabazza, E. C.; Sumida, Y. Immune complex-mediated

glomerulonephritis is ameliorated by thrombin-activatable fibrinolysis inhibitor deficiency.

Thromb. Haemost., 2008, 100, 90-100.

112 Rigla, M.; Wagner, A. M.; Borrell, M.; Mateo, J.; Foncuberta, J.; de, L. A.; Ordonez-Llanos, J.;

Perez, A. Postprandial thrombin activatable fibrinolysis inhibitor and markers of endothelial

dysfunction in type 2 diabetic patients. Metabolism, 2006, 55, 1437-42.

Chapter 2

44

113 Ribo, M.; Montaner, J.; Molina, C. A.; Arenillas, J. F.; Santamarina, E.; varez-Sabin, J. Admission

fibrinolytic profile predicts clot lysis resistance in stroke patients treated with tissue plasminogen

activator. Thromb. Haemost., 2004, 91, 1146-51.

114 Nagashima, M.; Yin, Z. F.; Zhao, L.; White, K.; Zhu, Y.; Lasky, N.; Halks-Miller, M.; Broze, G. J., Jr.;

Fay, W. P.; Morser, J. Thrombin-activatable fibrinolysis inhibitor (TAFI) deficiency is compatible

with murine life. J. Clin. Invest, 2002, 109, 101-10.

115 Asai, S.; Sato, T.; Tada, T.; Miyamoto, T.; Kimbara, N.; Motoyama, N.; Okada, H.; Okada, N.

Absence of procarboxypeptidase R induces complement-mediated lethal inflammation in

lipopolysaccharide-primed mice. J. Immunol., 2004, 173, 4669-74.

116 Wang, X.; Smith, P. L.; Hsu, M. Y.; Tamasi, J. A.; Bird, E.; Schumacher, W. A. Deficiency in

thrombin-activatable fibrinolysis inhibitor (TAFI) protected mice from ferric chloride-induced

vena cava thrombosis. J. Thromb. Thrombolysis., 2007, 23, 41-9.

117 Mao, S. S.; Holahan, M. A.; Bailey, C.; Wu, G.; Colussi, D.; Carroll, S. S.; Cook, J. J. Demonstration

of enhanced endogenous fibrinolysis in thrombin activatable fibrinolysis inhibitor-deficient mice.

Blood Coagul. Fibrinolysis, 2005, 16, 407-15.

118 Swaisgood, C. M.; Schmitt, D.; Eaton, D. L.; Plow, E. F. In vivo regulation of plasminogen function

by plasma carboxypeptidase B. J. Clin. Invest., 2002, 110, 1275-82.

119 Renckens, R.; Roelofs, J. J.; ter Horst, S. A.; van, '., V; Havik, S. R.; Florquin, S.; Wagenaar, G. T.;

Meijers, J. C.; van der, P. T. Absence of thrombin-activatable fibrinolysis inhibitor protects against

sepsis-induced liver injury in mice. J. Immunol., 2005, 175, 6764-71.

120 Pahlman, L. I.; Marx, P. F.; Morgelin, M.; Lukomski, S.; Meijers, J. C.; Herwald, H. Thrombin-

activatable fibrinolysis inhibitor binds to Streptococcus pyogenes by interacting with collagen-

like proteins A and B. J. Biol. Chem., 2007, 282, 24873-81.

121 Bengtson, S.; Sadén, C.; Mörgelin, M.; Marx, P. F.; Olin, A.; Leeb-Lundberg, L.; Meijers, J. C. M.;

Herwald, H. Activation of TAFI on the Surface of Streptococcus pyogenes Evokes Inflammatory

Reactions by Modulating the Kallikrein/Kinin System. J Innate Immun, 2009, 1, 18-28.

122 Guimaraes, A. H.; Laurens, N.; Weijers, E. M.; Koolwijk, P.; van, H., V; Rijken, D. C. TAFI and

pancreatic carboxypeptidase B modulate in vitro capillary tube formation by human

microvascular endothelial cells. Arterioscler. Thromb. Vasc. Biol., 2007, 27, 2157-62.

Thrombin-activatable fibrinolysis inhibitor is degraded by Salmonella enterica and Yersinia pestis Mercedes Valls Serón, Johanna Haiko, Philip G. de Groot, Timo K. Korhonen, and Joost C.M. Meijers Journal of Thrombosis and Haemostasis, 2010; 8: 2232-40

Chapter 3

46

Abstract

Pathogenic bacteria modulate the host coagulation system to evade immune responses or to

facilitate dissemination through extravascular tissues. In particular, the important bacterial

pathogens Salmonella enterica and Yersinia pestis intervene with the plasminogen/fibrinolytic

system. Thrombin-Activatable Fibrinolysis Inhibitor (TAFI) has anti-fibrinolytic properties as

the active enzyme (TAFIa) removes C-terminal lysine residues from fibrin, thereby attenuating

accelerated plasmin formation. Here, we demonstrate inactivation and cleavage of TAFI by

homologous surface proteases, the omptins Pla of Y. pestis and PgtE of S. enterica. We show

that omptin-expressing bacteria decrease TAFI activatability by thrombin-thrombomodulin

and that the anti-fibrinolytic potential of TAFIa was reduced by recombinant Escherichia coli

expressing Pla or PgtE. The functional impairment resulted from C-terminal cleavage of TAFI

by the omptins. Our results indicate that TAFI is degraded by the omptins PgtE of S. enterica

and Pla of Y. pestis. This may contribute to the ability of PgtE and Pla to damage tissue

barriers, such as fibrin, and thereby to enhance spread of S. enterica and Y. pestis during

infection.

S. enterica and Y. pestis proteases degrade TAFI

47

Ch

apte

r 3

Introduction

Several bacterial infectious diseases are associated with an imbalance of the mammalian

fibrinolytic/coagulation systems, however, the mechanistic details and pathogenetic roles of

these processes have remained largely unexplored. Fibrinogen-binding proteins have been

mainly identified in Gram-positive bacterial pathogens, such as Staphylococcus aureus,

Staphylococcus epidermis, and group A, C, and G Streptococci [1]. They interact with

fibrinogen and are thought to protect invading bacteria against host immune response and to

enhance survival of bacteria inside the host [2]. On the other hand, enhanced fibrinolytic

activity has been associated with several bacterial infectious diseases [3-6].

Plasmin is a key player in fibrinolysis, and bacterial pathogens have been observed to enhance

fibrinolysis either by directly activating plasminogen, by inactivating the main plasmin

inhibitor α2-antiplasmin, or by immobilizing plasminogen/plasmin on the bacterial surface [6].

The relationships between bacteria and the coagulation/fibrinolysis pathways appear

complex, as recent reports have identified that some serious bacterial pathogens, such as S.

aureus, Yersinia pestis and Salmonella enterica exhibit in vitro capacities which promote

either procoagulant or anticoagulant activities that lead to enhanced or decreased formation

of fibrin clots [5,7-10].

Y. pestis is the causative agent of plague, a highly fatal zoonotic infection transmitted to

humans through an intradermal bite by an infected flea in bubonic plague, or via respiratory

tract in aerosols from individuals with pneumonic plague. The importance of plasminogen

activation in pathogenesis of plague is well established. The plague bacterium harnesses the

plasminogen system for migration from intradermal infection sites at the early stages of

bubonic plague [5]. Altered fibrinolysis and importance of plasminogen activation have been

detected in both bubonic and pneumonic plague [10-12] and plasminogen-deficient mice are

more resistant than normal mice to infections by Y. pestis [5].

Plasminogen activation by Y. pestis is mediated by the transmembrane -barrel surface

protease Pla, which also inactivates α2-antiplasmin, thus creating uncontrolled plasmin

activity at the infection site [12,13]. Deletion or inactivation of the pla gene attenuates Y.

pestis both in bubonic [11,12] and pneumonic plague [10], and the pla gene is highly

transcribed by Y. pestis from buboes in infected rats [14].

Pla belongs to the omptin family of bacterial outer membrane proteases whose members

occur in several Gram-negative bacterial pathogens [15-17]. PgtE is the Pla ortholog in S.

enterica serovar Typhimurium, an intracellular pathogen that causes gastroenteritis. PgtE is

highly expressed by S. enterica inside infected mouse macrophages [4] and its genetic

deletion attenuates S. enterica in infected mice [18]. Fibrinolysis has not been earlier

associated with salmonellosis, and PgtE is a poor plasminogen activator [8] but it efficiently

inactivates α2-antiplasmin and activates procollagenases and progelatinases [4,18,19] which

can increase proteolysis and motility of macrophages and bacteria during salmonellosis [17].

Chapter 3

48

Thombin-activatable fibrinolysis inhibitor (TAFI) is a regulatory, anti-fibrinolytic protein linking

the coagulation and fibrinolytic systems. The protein is synthesized in the liver and secreted

into plasma as a 56-kDa procarboxypeptidase with 2 domains: the first 92 amino acids form

the activation peptide; the next 309 amino acids form the catalytic domain. During

coagulation, the proenzyme is activated to TAFIa by thrombin. TAFIa has anti-fibrinolytic

properties as it inhibits plasmin-mediated blood clot lysis by removing C-terminal lysine

residues from partially degraded fibrin that are required for positive feedback in tissue-type

plasminogen activator dependent plasmin generation. Apart from thrombin, the serine

proteases plasmin, trypsin and neutrophil elastase have been reported to function as TAFI

activators [20-22]. TAFI activation by thrombin is inefficient but the process can be stimulated

approximately 1250-fold by the cofactor thrombomodulin [23]. TAFIa is unstable at 37 °C and

upon activation by thrombin it is inactivated (TAFIai) by a conformational change in a

temperature-dependent manner [24]. In contrast, in the presence of plasmin, TAFIa is

inactivated by plasmin proteolysis [20].

No direct physiological inhibitors of TAFIa have been identified to date. The cleavage of TAFI

by human proteinases has been well described biochemically and functionally [20,25],

whereas its possible activation or inactivation by bacteria has not been described.

As TAFIa regulates plasmin formation, invasive microorganisms that use the plasminogen

system to spread may target TAFI to enhance fibrinolysis. Therefore the aim of this study was

to investigate the effects of Y. pestis and S. enterica proteases Pla and PgtE on TAFI in order to

provide novel insights in the complex relationships between bacteria, coagulation and

fibrinolysis.

Materials and methods

Reagents

Hippuryl-arginine and H-D-Phe-Pro-Arg-chloromethylketone (PPACK) were purchased from

Bachem (Bubendorf, Switzerland), and ε-amino-n-caproic acid was purchased from Sigma-

Aldrich (St Louis, MO, USA). Rabbit lung thrombomodulin was purchased from American

Diagnostica (Stamford, CT, USA). Phosphoenolpyruvate (PEP), ATP, NADH, pyruvate

kinase/dehydrogenase (PK/LDH), and recombinant tissue-type Plasminogen Activator (t-PA,

Actilyse) were purchased from Biopool AB (Umeå, Sweden). Thrombin was a generous gift

from Dr. W. Kisiel (University of New Mexico, Albuquerque, NM, USA). TAFI was purified as

previously described [25], except that proteins were eluted from the Nik-9H10-Sepharose

column with 0.1 M glycine (pH 4.0) and that the fractions were collected in 20 mM Tris, 200

mM NaCl (pH 7.4). TAFI was stored at -80 °C after addition of Tween-20 (0.01%). Fibrinogen

was purchased from Kordia (Leiden, The Netherlands). A polyclonal antibody specific for TAFI

was obtained in rabbits as described elsewhere [27]. Plasminogen was purified from frozen

human plasma as previously described [28]. Alkaline-phosphatase-conjugated anti-rabbit

immunoglobulin G (IgG) was purchased from Dako (Glostrup, Denmark).


49

Ch

apte

r 3

Bacteria and plasmids

The bacterial strains and plasmids used in this study are listed in Table 1. pla, pgtE and their

derivatives have been cloned in the inducible pSE380 vector in Escherichia coli XL1-Blue MRF’

and described before [13,29]. The recombinant E. coli strains were cultivated overnight at 37

°C in Luria broth (10 ml) supplemented with glucose (0.2% wt/vol), ampicillin (100 µg/ml) and

tetracycline (12.5 µg/ml). For protein expression, the culture was pelleted, suspended in 150

μl of phosphate-buffered saline (PBS, pH 7.1) and plated on Luria plates containing 5 µM

isopropyl-β-D-thiogalactopyranoside (IPTG) and antibiotics as above. Y. pestis strains were

cultivated over two nights at 37 °C on brain-heart infusion (BHI) plates and then inoculated in

10 ml BHI broth supplemented with hemin (40 µg/ml) and cultivated twice over two nights at

37 °C. S. enterica 14028R and 14028R-1 were cultivated overnight at 37 °C in 10 ml PhoP/Q-

inducing medium (N-minimal medium, pH 7.4, supplemented with 38 mM glycerol, 0.1%

casamino acids, 2 mg/ml thiamine, and 8 mM MgCl2). S. enterica 14028R-1 complemented

with pSE380 or pMRK3 were cultivated overnight at 37 °C in 10 ml Luria broth supplemented

with 5 µM IPTG and ampicillin (100 µg/ml). For the assays, bacteria were collected in PBS or in

Tris-buffered saline (TBS, pH 7.4), pelleted, and adjusted to OD600 value of 2.0 (corresponding

to c. 2 × 109 cells/ml).

TAFIa activation and activity assay

TAFI (54 nM for Y. pestis and S. enterica; for E. coli, 179 nM or 714 nM in dose- and time-

dependent assays; all concentrations are final concentrations) was incubated with the

bacteria (2 × 107

or 4 × 107 in TBS) in 100 mM Hepes/0.01% Tween-20, pH 8.0. In some

experiments, ε-ACA (2.5 mM) was added. Incubations were performed at 37 °C shaking. After

incubation, the samples were centrifuged, and the supernatants were further analyzed. TAFIa

activity was measured as previously described [26,30]. TAFI (40 nM) was added to a premix of

thrombin (8 nM) and thrombomodulin (16 nM) in the presence of CaCl2 (5 mM) in 100 mM

Hepes/0.01% Tween-20, pH 8.0 for 15 min at room temperature. Subsequently, a reaction

mixture containing MgSO4 (2.7 mM), KCl (10.9 mM), PPACK (30 µM), PEP (2.4 mM), NADH (0.5

mM), ATP (2.7 mM), hippuryl-arginine (6 mM), PK (45 µg/ml), LDH (15 µg/ml), and excess of

arginine kinase was added in 100 mM Hepes/0.01% Tween-20, pH 8.0.

When plasma was used, 20 µl TAFI-deficient plasma (three times diluted) was reconstituted

with TAFI to a final concentration of 165 nM and incubated with 20 µl bacteria (4 × 109).

Incubations and TAFIa activity measurement were performed as described above. Prewarmed

reaction mixture (90 μl) was transferred to a prewarmed microtiter plate, and 10 μl of TAFI

activated as described above was added.

TAFI activation was followed over time as a loss of NADH absorbance at 340 nm in a

Thermomax microplate reader (MolecularDevices Corp., Menlo Park, CA, USA) or in a

Multiskan EX reader (Thermo, Waltham, MA, USA).

Chapter 3

50

Table 1. Bacterial strains and plasmids used in this study

Bacterial strain or plasmid Description Reference

Escherichia coli XL1 Blue MRF’ Δ(mcrA) 183 Δ(mcrCB-hsdSMR-mrr) 173 endA1

supe44 thi-1 recA1 gyrA96 relA1 lac *F’ proAB

lacIqZΔ M15 Tn 10 (tet)]

Stratagene

Yersinia pestis KIM D27 pPCP1+ Δpgm pYV

+ derivative of Y. pestis KIM-10 [52,53]

Yersinia pestis KIM D34 pPCP1-, otherwise identical to KIM D27 [52,53]

Salmonella enterica

serovar Typhimurium 14028R

Rough lipopolysaccharide derivative of 14028 [54]

Salmonella enterica

serovar Typhimurium 14028R-1

ΔpgtE derivative of 14028R [4]

pSE380 Expression vector, trc promoter, lacO operator,

lacI, bla

Invitrogen

pMRK1 pla in pSE380 [13]

pMRK3 pgtE in pSE380 [29]

pMRK111 Pla D206A [13]

pMRK31 PgtE D206A [29]

Clot-lysis assay

TAFI (179 nM or 714 nM in dose- and time-dependent assays) was incubated with the

bacteria (2 × 107

or 4 × 107 in TBS) in 100 mM Hepes/0.01% Tween-20, pH 8.0, at 37 °C with

continuous shaking. After incubation, the samples were centrifuged and the supernatants

were further analysed. The clot-lysis times were determined in a purified system. Briefly, 10

nM thrombin, 0.3 μg/ml recombinant t-PA (Actilyse), 20 mM CaCl2 and 5 nM thrombomodulin

were mixed with 40 nM preincubated TAFI (all concentrations are final concentrations in the

assay) in a 96-well microtiter plate. The volumes were adjusted to 30 μl with HBS (25 mM

Hepes, 137 mM NaCl, 3.5 mM KCl, pH 7.4) containing 0.1% (wt/vol) bovine serum albumin. A

mixture (20 μl) of fibrinogen (4.5 µM), plasminogen (90 nM) in HBS/0.1% bovine serum

albumin was added and turbidity was measured in time at 37 °C at 405 nm in a Thermomax

microplate reader. The clot-lysis time was defined as the time difference between half-

maximal lysis and half-maximal clotting.

Degradation of TAFI

For detecting the degradation of TAFI with anti-TAFI antibody, TAFI (820 nM) was incubated

with the bacteria (4 × 107 in PBS) in a volume of 12.5 µl for 2 h at 37 °C shaking. The bacteria

were pelleted, and the supernatant was boiled with sodium dodecylsulfate polyacrylamide gel

electrophoresis (SDS-PAGE) sample buffer containing β-mercaptoethanol. The samples were

run in a 12% (wt/vol) SDS-PAGE gel, transferred onto a nitrocellulose membrane and detected


51

Ch

apte

r 3

with polyclonal anti-TAFI antibody (1:2000), followed by alkaline-phosphatase-conjugated

anti-rabbit IgG (1:1000), and phosphatase substrate.

To detect the degradation of TAFI by SDS-PAGE, TAFI (1.9 µM) was incubated with bacteria

(4×107 in TBS) in 100 mM Hepes/0.01% Tween-20, pH 8.0. In some experiments, ε-ACA (5

mM) was added. The samples were prepared as above, subjected to SDS-PAGE (10%) and

stained with Coomassie Brilliant Blue.

Determination of TAFI cleavage site

Degraded TAFI samples with E. coli expressing Pla or PgtE were prepared as for degradation

assay by Western blotting with the exception that Pla-expressing bacteria were incubated for

5 h. For N-terminal sequencing, the samples were run in a 12 % SDS-PAGE, blotted and

sequenced by Edman degradation. For MALDI-TOF-MS, the samples were run on a 12 % SDS-

PAGE, cut from the gel, digested with trypsin, and exposed to matrix-assisted laser desorption

ionization - time of flight mass spectrometry (MALDI-TOF-MS). The obtained peptides were

analyzed with Mascot search engine (http://www.matrixscience.com).

Results

Decreased activatability of TAFI by bacteria expressing PgtE or Pla.

To determine the effect of omptins on TAFIa activity, TAFI was pre-incubated with S. enterica

and Y. pestis strains differing in possession of pgtE or pla genes, and subsequently activated

with the thrombin-thrombomodulin complex. The PgtE-positive S. enterica 14028R and the

Pla-positive Y. pestis KIM D27 decreased the activatability of TAFI to TAFIa (by 85% and 97%,

respectively) (Fig. 1A). In contrast, the pgtE deletion mutant S. enterica 14028R-1 had no

detectable effect on TAFIa activity, and Y. pestis KIM D34, cured of the pla-encoding plasmid

pPCP1, reduced TAFIa activity by 59% (Fig. 1A). Inhibition of TAFIa activity was seen with S.

enterica 14028R-1 (pMRK3) complemented with the pgtE-encoding plasmid but not after

complementation with the expression vector, S. enterica 14028R-1 (pSE380), further

indicating that reduction of TAFIa activity by 14028R resulted from the expression of PgtE.

To further confirm the role of PgtE and Pla on TAFI activity, we studied the activatability of

TAFI with recombinant E. coli XL1 expressing PgtE or Pla. E. coli XL1 (pMRK3) with PgtE and E.

coli XL1 (pMRK1) with Pla reduced TAFI activatability (by 80% and 61%, respectively). E. coli

expressing PgtE or Pla with the catalytic site substitution D206A (pMRK31 or pMRK111,

respectively) decreased TAFI activatability by approximately 32% and 31%, respectively (Fig.

1B).

The recombinant E. coli expressing Pla or PgtE attenuated TAFIa activity in a dose-dependent

manner (Fig. 1C). Moreover, when the bacteria were pre-incubated with TAFI for different

time periods (0.5-2 h), we observed a progressive reduction in TAFIa activity (Fig. 1D).

http://www.matrixscience.com/

Chapter 3

52

Figure 1. Effect of PgtE- or Pla-expressing bacteria on TAFIa activity. (A, B) Loss of TAFIa activity with S.

enterica, Y. pestis (A) and recombinant E. coli (B). Purified TAFI was incubated for 2 hours with the

bacterial strains indicated below the columns. After incubation, TAFI was activated by the thrombin-

thrombomodulin complex and TAFIa activity was measured. (C, D) Dose-dependent (C) and time-

dependent (D) reduction in TAFIa activity. TAFI was incubated in buffer (▲) or with recombinant E. coli

XL1 expressing PgtE (pMRK3) (■), Pla (pMRK1) (●), or the catalytic-site mutants PgtE D206A (pMRK31)

(□) and Pla D206A (pMRK111) (○).Data are expressed as a percentage of the maximal TAFIa activity in

buffer. For S. enterica and Y. pestis strains, a representative experiment is shown, and for recombinant

bacteria, mean ± SD, n = 3 is shown.

Next, we investigated the effect of recombinant E. coli XL1 expressing PgtE or Pla on TAFI in

plasma. TAFI was added to TAFI-depleted plasma and incubated for 2 hours at 37°C with

recombinant E. coli XL1 expressing PgtE, Pla or the catalytic-site mutants PgtE D206A or Pla

D206A . After incubation, TAFI was activated with the thrombin-thrombomodulin complex

and TAFIa activity was measured. Our results indicated that E. coli XL1 (pMRK3) with PgtE and

E. coli XL1 (pMRK1) with Pla reduced TAFI activatability by 49% and 60%, respectively. E. coli

expressing PgtE or Pla with the catalytic site substitution D206A (pMRK31 or pMRK111,

respectively) both decreased TAFI activatability by 31%. The omptins did not activate TAFI

directly. Also, the omptins had no effect on the half-life of activated TAFI (data not shown).


53

Ch

apte

r 3

The reduction in TAFIa activity by PgtE and Pla expressing bacteria could be due to an effect of

omptins on the TAFI activator thrombin-thrombomodulin. However, we could exclude this

possibility since the activation of another substrate of thrombin-thrombomodulin, protein C,

was not affected by the omptins (data not shown).

Taken together, these results indicated that PgtE of S. enterica and Pla of Y. pestis prevented

TAFI to become activated by thrombin-thrombomodulin and that the proteolytic activity of

these omptins was essential for the inhibition.

Reduction of the anti-fibrinolytic potential of TAFIa by recombinant E. coli.

The anti-fibrinolytic function of TAFIa was studied in a clot-lysis assay using purified

components and recombinant E. coli. TAFI-dependent prolongation of clot-lysis time was

reduced when TAFI was incubated with PgtE- or Pla-expressing E. coli XL1, in comparison to

bacteria expressing proteolytically inactive PgtE or Pla (Fig. 2A). These data indicate that

proteolytically active PgtE and Pla are responsible for functional inactivation of TAFI.

Incubation of TAFI with E. coli XL1 (pMRK3) or E. coli XL1 (pMRK1) showed that the TAFI-

dependent prolongation of clot-lysis time was dependent on bacterial numbers (Fig. 2B) and

on the incubation time of TAFI with the bacteria (Fig. 2C). Thus, PgtE- and Pla-mediated

proteolysis had negative effect on TAFIa function.

Proteolytic cleavage of TAFI by PgtE of S.enterica and by Pla of Y. pestis.

To determine why the activatability of TAFI was diminished by PgtE- or Pla-expressing

bacteria, we incubated TAFI with the bacteria and analyzed the supernatants by Western

blotting with an anti-TAFI antibody (Fig. 3A) and SDS-PAGE (Fig. 3B).

TAFI was degraded by PgtE- or Pla-expressing S. enterica and Y. pestis but not by bacteria

lacking the omptins (Fig. 3A). E. coli XL1 (pMRK3) with PgtE and XL1 (pMRK1) with Pla

degraded TAFI into smaller molecular weight peptides in a 2-h incubation, whereas XL1

(pMRK31) and XL1 (pMRK111) with mutated omptins were much weaker in TAFI degradation

(Fig. 3B). These results suggest that the decreased activatability of TAFI after its incubation

with omptin-expressing bacteria is a result of proteolytic degradation of TAFI by these

bacterial proteases.

Chapter 3

54

Figure 2. Effect of PgtE- or Pla-expressing bacteria on the anti-fibrinolytic potential of TAFIa. (A) Loss of

TAFIa anti-fibrinolytic potential. TAFI was incubated for 2 hours with the E. coli XL1 strains indicated

below the columns. (B, C) Dose-dependent (B) and time-dependent (C) reduction in the fibrinolytic

potential of TAFIa. TAFI was incubated in the presence or absence of recombinant bacteria, after

incubation, bacteria were removed and the anti-fibrinolytic potential of TAFIa was determined in a clot-

lysis assay using purified components. TAFI was incubated in buffer (▲) or in the presence of XL1

(pMRK3) (■), XL1 (pMRK1) (●), XL1 (pMRK31) (□) or XL1 (p MRK111) (○). The TAFIa-dependent

prolongation was defined as the difference in clot-lysis time in the presence or absence of TAFI. Data are

expressed as a percentage (mean ± SD, n = 3) of TAFIa dependent prolongation of TAFIa in buffer.


55

Ch

apte

r 3

Figure 3. Degradation of TAFI by bacteria expressing PgtE or Pla. TAFI degradation by PgtE-expressing S.

enterica and Pla-expressing Y. pestis (A) and PgtE- or Pla-expressing E. coli XL1 (B) is shown. TAFI was

incubated for two hours with the bacterial strains indicated above the lanes. Samples were analyzed by

Western blot with anti-TAFI antibody (A) or subjected to SDS-PAGE and Coomassie staining (B).

Migration distance of the molecular weight standard (in kilodaltons) is indicated on the left. For plasmid

constructs, see Table 1.

The fuzzy migration of the TAFI-degradation product suggested that the activation peptide,

where all the four N-linked glycosylation sites are located, was still present [31], suggesting a

C-terminal cleavage of TAFI. N-terminal sequencing and peptide mass fingerprint analysis of

trypsin-digested omptin-cleaved TAFI confirmed C-terminal cleavage of TAFI (data not

shown).

The lysine analogue ε-ACA is considered a TAFIa inhibitor [32], but it also stabilizes TAFIa

activity and prevents the formation of the no longer activatable 44.3-kDa TAFI fragment

generated after C-terminal cleavage by plasmin [20]. In order to assess if the C-terminal TAFI

cleavage by omptins was prevented by ε-ACA, the lysine analogue was added during

incubation of TAFI with recombinant E. coli XL1 expressing PgtE or Pla. Addition of ε-ACA

prevented the proteolytic cleavage of TAFI (Fig. 4A) and led to full TAFIa activity even in the

presence of proteolytically active PgtE and Pla (Fig. 4B). TAFIa activity in the presence of ε-

ACA was increased by < 10% (data not shown). This indicated that TAFI cleavage by omptins is

dependent on lysine residues, since ε-ACA acted as a competitive inhibitor of TAFI

degradation by omptins.

Chapter 3

56

Figure 4. Influence of ε-ACA on TAFI proteolysis and on TAFIa activity by PgtE-expressing (A) or Pla-

expressing (B) E. coli XL1. TAFI was incubated with recombinant E. coli XL1 in the presence or absence of

ε-ACA. After incubation, bacteria were removed and samples subjected to SDS-PAGE and Coomassie

staining. Migration distance of the molecular weight standards (in kilodaltons) is indicated on the left.

TAFI incubated with the bacteria was activated by the thrombin-thrombomodulin complex and TAFIa

activity was measured (lower panel). Data are expressed as a percentage (mean ± SD, n = 2) of maximal

TAFIa activity of TAFI incubated without bacteria. For plasmid constructs, see Table 1.

Discussion

TAFI is an important regulator that participates in maintaining the balance of coagulation and

fibrinolysis. Activated TAFI removes lysine residues from partially degraded fibrin, thus

reducing the binding of t-PA and plasminogen to the fibrin clot and subsequently diminishing

plasmin generation and fibrinolysis. TAFIa is regulated by its intrinsic, temperature-dependent

instability but no physiological inhibitors are known to date. Several inhibitory molecules for

TAFIa have however been characterized: the carboxypeptidase inhibitor from potato tubers

(CPI) [33], chelating and reducing agents [22,23], arginine and lysine analogues [34,35],

synthetic inhibitors [36-41], monoclonal antibodies [42-44] and nanobodies [45] against TAFI.

This is the first report in which two bacterial proteases, PgtE of S. enterica serovar

Typhimurium and Pla of Y. pestis, interfere with the activation of TAFI to TAFIa. Both bacterial

species are highly invasive pathogens, and PgtE and Pla have been identified as virulence

factors [5,10-12,18] Here we show that PgtE and Pla degrade TAFI via proteolytic breakdown.

Degraded TAFI can no longer be activated by the thrombin-thrombomodulin complex and


57

Ch

apte

r 3

therefore the ability of TAFIa to remove C-teminal lysine residues from partially degraded

fibrin is abrogated.

The biological significance of the TAFI degradation remains to be elucidated but it is

important to note that decrease in TAFIa activity by PgtE and Pla also occurred in a plasma

environment where several putative omptin substrates, such as plasminogen, serpins, and

complement proteins, are present [13,46,47].

Plasmin can activate TAFI and subsequently inactivate TAFIa. However, plasmin can also

cleave TAFI to a form that cannot be activated anymore, and the TAFIa activity in the

presence of plasmin is low. Besides the direct effect of the omptins on TAFI degradation, the

plasmin generated by Pla or PgtE may indirectly cause TAFI degradation. The functional

heterogeneity of omptins also concerns TAFI degradation as we have observed that OmpT, an

omptin of E. coli, does not interact with TAFI (M. Valls Serón, J. Haiko, unpublished

observations). As a pathogen, E. coli is less invasive than Y. pestis and S. enterica.

The observation that the S. enterica pgtE-deletion mutant was completely inactive in

decreasing TAFIa activity whereas KIM D34 caused an approximately 59% inhibition, indicate

that Y. pestis might have other so far unidentified protease(s) involved in the TAFI

degradation. The recombinant E. coli XL1 expressing the catalytic-site substitutions D206A in

PgtE or Pla were clearly weaker in reducing TAFIa activity but still a low degree of TAFI

degradation was detectable with E. coli XL1 expressing the substituted omptins, which

indicates that omptin proteolysis is critical for the observed decrease in TAFI activatability.

Single substitutions rarely abolish the proteolytic activity completely, which may explain the

activity remaining with the substituted Pla and PgtE. On the other hand, our results do not

rule out the possibility that additional proteases in E.coli may target TAFI.

We observed that lysine analogue ε-ACA also inhibits the C-terminal cleavage of TAFI by PgtE

and Pla, thus indicating that lysine residues are critical for the cleavage of TAFI by these

omptins. However, it is also possible that ε-ACA prevents the interaction of the bacterial

proteases with TAFI. OmpT preferentially cleaves its substrates after arginine or lysine [48],

and Pla prefers Arg-Val bond present in its plasminogen substrate, so it is possible that the

cleavage occurs at arginine or lysine residues.

Many invasive bacterial pathogens interact with the haemostatic system in order to

disseminate within the host or avoid the host inflammatory immune response. In particular,

the Gram positive Streptococcus pyogenes have developed a variety of strategies to

circumvent the host defense such as the expression of streptokinase, a plasminogen-

activating protein. The streptokinase-plasmin(ogen) complex is protected against enzymatic

inactivation by the plasmin inhibitor α2-antiplasmin [49]. Recent studies have shown that TAFI

binds to the bacterial surface by interacting with the streptococcal collagen-like surface

proteins A and B (SclA and SclB) [50]. Bacteria-bound TAFI was activated by its natural

activators, plasmin and thrombin, which are also recruited to the streptococcal surface. Other

studies revealed that TAFI was used to redirect inflammation from a transient to a chronic

state by modulation of the kallikrein/kinin system [51]. Our results thus show, for the first

Chapter 3

58

time, an interaction of TAFI with Gram negative bacteria, indicating that TAFI might be a

general target for bacteria.

Pla, PgtE, and OmpT have been recently shown to inhibit tissue-factor pathway inhibitor

(TFPI), which inhibits coagulation [9]. This function is seemingly contrary to our present and

other previously described results [4,8,12,13] showing enhanced plasminogen activation and

fibrinolysis in vitro and in vivo. The in vivo significance of the procoagulation effects of

omptins remain to be determined, these observations however indicate that the interactions

of omptin proteases with the haemostatic system are complex and perhaps lead to opposite

directions at different stages of the infectious processes. S. enterica and Y. pestis employ

omptins to advance fibrinolysis by plasminogen activation and α2-antiplasmin degradation,

and inhibition of TAFIa function is still another way for these pathogens to influence

fibrinolysis and inflammation and promote their spread during infection.


59

Ch

apte

r 3

Reference List

1 Rivera J, Vannakambadi G, Hook M, Speziale P. Fibrinogen-binding proteins of Gram-positive

bacteria. Thromb Haemost 2007; 98: 503-11.

2 Johnson LL, Berggren KN, Szaba FM, Chen W, Smiley ST. Fibrin-mediated protection against

infection-stimulated immunopathology. J Exp Med 2003; 197: 801-6.

3 Bergmann S, Hammerschmidt S. Fibrinolysis and host response in bacterial infections. Thromb

Haemost 2007; 98: 512-20.

4 Lähteenmäki K, Kyllönen P, Partanen L, Korhonen TK. Antiprotease inactivation by Salmonella

enterica released from infected macrophages. Cell Microbiol 2005; 7: 529-38.

5 Degen JL, Bugge TH, Goguen JD. Fibrin and fibrinolysis in infection and host defense. J Thromb

Haemost 2007; 5 Suppl 1: 24-31.

6 Sun H. The interaction between pathogens and the host coagulation system. Physiology

(Bethesda ) 2006; 21: 281-8.

7 Beaufort N, Wojciechowski P, Sommerhoff CP, Szmyd G, Dubin G, Eick S, Kellermann J, Schmitt

M, Potempa J, Magdolen V. The human fibrinolytic system is a target for the staphylococcal

metalloprotease aureolysin. Biochem J 2008; 410: 157-65.

8 Haiko J, Kukkonen M, Ravantti JJ, Westerlund-Wikström B, Korhonen TK. The single substitution

I259T, conserved in the plasminogen activator Pla of pandemic Yersinia pestis branches,

enhances fibrinolytic activity. J Bacteriol 2009; 191: 4758-66.

9 Yun TH, Cott JE, Tapping RI, Slauch JM, Morrissey JH. Proteolytic inactivation of tissue factor

pathway inhibitor by bacterial omptins. Blood 2009; 113: 1139-48.

10 Lathem WW, Price PA, Miller VL, Goldman WE. A plasminogen-activating protease specifically

controls the development of primary pneumonic plague. Science 2007; 315: 509-13.

11 Sebbane F, Jarrett CO, Gardner D, Long D, Hinnebusch BJ. Role of the Yersinia pestis plasminogen

activator in the incidence of distinct septicemic and bubonic forms of flea-borne plague. Proc

Natl Acad Sci U S A 2006; 103: 5526-30.

12 Sodeinde OA, Subrahmanyam YV, Stark K, Quan T, Bao Y, Goguen JD. A surface protease and the

invasive character of plague. Science 1992; 258: 1004-7.

13 Kukkonen M, Lähteenmäki K, Suomalainen M, Kalkkinen N, Emody L, Lang H, Korhonen TK.

Protein regions important for plasminogen activation and inactivation of alpha2-antiplasmin in

the surface protease Pla of Yersinia pestis. Mol Microbiol 2001; 40: 1097-111.

Chapter 3

60

14 Sebbane F, Lemaitre N, Sturdevant DE, Rebeil R, Virtaneva K, Porcella SF, Hinnebusch BJ.

Adaptive response of Yersinia pestis to extracellular effectors of innate immunity during bubonic

plague. Proc Natl Acad Sci U S A 2006; 103: 11766-71.

15 Rawlings ND, Morton FR, Kok CY, Kong J, Barrett AJ. MEROPS: the peptidase database. Nucleic

Acids Res 2008; 36: D320-D325.

16 Haiko J, Suomalainen M, Ojala T, Lähteenmäki K, Korhonen TK. Invited review: Breaking barriers-

-attack on innate immune defences by omptin surface proteases of enterobacterial pathogens.

Innate Immun 2009; 15: 67-80.

17 Hritonenko V, Stathopoulos C. Omptin proteins: an expanding family of outer membrane

proteases in Gram-negative Enterobacteriaceae. Mol Membr Biol 2007; 24: 395-406.

18 Ramu P, Lobo LA, Kukkonen M, Bjur E, Suomalainen M, Raukola H, Miettinen M, Julkunen I, Holst

O, Rhen M, Korhonen TK, Lähteenmäki K. Activation of pro-matrix metalloproteinase-9 and

degradation of gelatin by the surface protease PgtE of Salmonella enterica serovar Typhimurium.

Int J Med Microbiol 2008; 298: 263-78.

19 Handley SA, Miller VL. General and specific host responses to bacterial infection in Peyer's

patches: a role for stromelysin-1 (matrix metalloproteinase-3) during Salmonella enterica

infection. Mol Microbiol 2007; 64: 94-110.

20 Marx PF, Dawson PE, Bouma BN, Meijers JC. Plasmin-mediated activation and inactivation of

thrombin-activatable fibrinolysis inhibitor. Biochemistry 2002; 41: 6688-96.

21 Kawamura T, Okada N, Okada H. Elastase from activated human neutrophils activates

procarboxypeptidase R. Microbiol Immunol 2002; 46: 225-30.

22 Eaton DL, Malloy BE, Tsai SP, Henzel W, Drayna D. Isolation, molecular cloning, and partial

characterization of a novel carboxypeptidase B from human plasma. J Biol Chem 1991; 266:

21833-8.

23 Bajzar L, Morser J, Nesheim M. TAFI, or plasma procarboxypeptidase B, couples the coagulation

and fibrinolytic cascades through the thrombin-thrombomodulin complex. J Biol Chem 1996;

271: 16603-8.

24 Marx PF, Brondijk TH, Plug T, Romijn RA, Hemrika W, Meijers JC, Huizinga EG. Crystal structures

of TAFI elucidate the inactivation mechanism of activated TAFI: a novel mechanism for enzyme

autoregulation. Blood 2008; 112: 2803-9.

25 Marx PF, Hackeng TM, Dawson PE, Griffin JH, Meijers JC, Bouma BN. Inactivation of active

thrombin-activable fibrinolysis inhibitor takes place by a process that involves conformational

instability rather than proteolytic cleavage. J Biol Chem 2000; 275: 12410-5.


61

Ch

apte

r 3

26 Marx PF, Wagenaar GT, Reijerkerk A, Tiekstra MJ, van Rossum AG, Gebbink MF, Meijers JC.

Characterization of mouse thrombin-activatable fibrinolysis inhibitor. Thromb Haemost 2000; 83:

297-303.

27 Mosnier LO, von dem Borne PA, Meijers JC, Bouma BN. Plasma TAFI levels influence the clot lysis

time in healthy individuals in the presence of an intact intrinsic pathway of coagulation. Thromb

Haemost 1998; 80: 829-35.

28 Sangrar W, Gabel BR, Boffa MB, Walker JB, Hancock MA, Marcovina SM, Horrevoets AJ, Nesheim

ME, Koschinsky ML. The solution phase interaction between apolipoprotein(a) and plasminogen

inhibits the binding of plasminogen to a plasmin-modified fibrinogen surface. Biochemistry 1997;

36: 10353-63.

29 Kukkonen M, Suomalainen M, Kyllönen P, Lähteenmäki K, Lang H, Virkola R, Helander IM, Holst

O, Korhonen TK. Lack of O-antigen is essential for plasminogen activation by Yersinia pestis and

Salmonella enterica. Mol Microbiol 2004; 51: 215-25.

30 Willemse J, Leurs J, Verkerk R, Hendriks D. Development of a fast kinetic method for the

determination of carboxypeptidase U (TAFIa) using C-terminal arginine containing peptides as

substrate. Anal Biochem 2005; 340: 106-12.

31 Guimaraes AH, Barrett-Bergshoeff MM, Gils A, Declerck PJ, Rijken DC. Migration of the activation

peptide of thrombin-activatable fibrinolysis inhibitor (TAFI) during SDS-polyacrylamide gel

electrophoresis. J Thromb Haemost 2004; 2: 780-4.

32 Boffa MB, Wang W, Bajzar L, Nesheim ME. Plasma and recombinant thrombin-activable

fibrinolysis inhibitor (TAFI) and activated TAFI compared with respect to glycosylation,

thrombin/thrombomodulin-dependent activation, thermal stability, and enzymatic properties. J

Biol Chem 1998; 273: 2127-35.

33 Wang W, Boffa MB, Bajzar L, Walker JB, Nesheim ME. A study of the mechanism of inhibition of

fibrinolysis by activated thrombin-activable fibrinolysis inhibitor. J Biol Chem 1998; 273: 27176-

81.

34 Wang W, Hendriks DF, Scharpe SS. Carboxypeptidase U, a plasma carboxypeptidase with high

affinity for plasminogen. J Biol Chem 1994; 269: 15937-44.

35 Mao SS, Colussi D, Bailey CM, Bosserman M, Burlein C, Gardell SJ, Carroll SS.

Electrochemiluminescence assay for basic carboxypeptidases: inhibition of basic

carboxypeptidases and activation of thrombin-activatable fibrinolysis inhibitor. Anal Biochem

2003; 319: 159-70.

36 Wang YX, da Cunha V, Vincelette J, Zhao L, Nagashima M, Kawai K, Yuan S, Emayan K, Islam I,

Hosoya J, Sullivan ME, Dole WP, Morser J, Buckman BO, Vergona R. A novel inhibitor of activated

Chapter 3

62

thrombin activatable fibrinolysis inhibitor (TAFIa) - part II: enhancement of both exogenous and

endogenous fibrinolysis in animal models of thrombosis. Thromb Haemost 2007; 97: 54-61.

37 Lazoura E, Campbell W, Yamaguchi Y, Kato K, Okada N, Okada H. Rational structure-based design

of a novel carboxypeptidase R inhibitor. Chem Biol 2002; 9: 1129-39.

38 Barrow JC, Nantermet PG, Stauffer SR, Ngo PL, Steinbeiser MA, Mao SS, Carroll SS, Bailey C,

Colussi D, Bosserman M, Burlein C, Cook JJ, Sitko G, Tiller PR, Miller-Stein CM, Rose M,

McMasters DR, Vacca JP, Selnick HG. Synthesis and evaluation of imidazole acetic acid inhibitors

of activated thrombin-activatable fibrinolysis inhibitor as novel antithrombotics. J Med Chem

2003; 46: 5294-7.

39 Polla MO, Tottie L, Norden C, Linschoten M, Musil D, Trumpp-Kallmeyer S, Aukrust IR, Ringom R,

Holm KH, Neset SM, Sandberg M, Thurmond J, Yu P, Hategan G, Anderson H. Design and

synthesis of potent, orally active, inhibitors of carboxypeptidase U (TAFIa). Bioorg Med Chem

2004; 12: 1151-75.

40 Suzuki K, Muto Y, Fushihara K, Kanemoto K, Iida H, Sato E, Kikuchi C, Matsushima T, Kato E,

Nomoto M, Yoshioka S, Ishii H. Enhancement of fibrinolysis by EF6265 [(S)-7-amino-2-[[[(R)-2-

methyl-1-(3-phenylpropanoylamino)propyl]hydroxypho sphinoyl] methyl]heptanoic acid], a

specific inhibitor of plasma carboxypeptidase B. J Pharmacol Exp Ther 2004; 309: 607-15.

41 Bunnage ME, Blagg J, Steele J, Owen DR, Allerton C, McElroy AB, Miller D, Ringer T, Butcher K,

Beaumont K, Evans K, Gray AJ, Holland SJ, Feeder N, Moore RS, Brown DG. Discovery of potent &

selective inhibitors of activated thrombin-activatable fibrinolysis inhibitor for the treatment of

thrombosis. J Med Chem 2007; 50: 6095-103.

42 Gils A, Ceresa E, Macovei AM, Marx PF, Peeters M, Compernolle G, Declerck PJ. Modulation of

TAFI function through different pathways--implications for the development of TAFI inhibitors. J

Thromb Haemost 2005; 3: 2745-53.

43 Bajzar L, Nesheim ME, Tracy PB. The profibrinolytic effect of activated protein C in clots formed

from plasma is TAFI-dependent. Blood 1996; 88: 2093-100.

44 Hillmayer K, Vancraenenbroeck R, De MM, Compernolle G, Declerck PJ, Gils A. Discovery of novel

mechanisms and molecular targets for the inhibition of activated thrombin activatable

fibrinolysis inhibitor. J Thromb Haemost 2008; 6: 1892-9.

45 Buelens K, Hassanzadeh-Ghassabeh G, Muyldermans S, Gils A, Declerck PJ. Generation and

characterization of inhibitory nanobodies towards thrombin activatable fibrinolysis inhibitor. J

Thromb Haemost 2010; 6: 1302-12.


63

Ch

apte

r 3

46 Ramu P, Tanskanen R, Holmberg M, Lähteenmäki K, Korhonen TK, Meri S. The surface protease

PgtE of Salmonella enterica affects complement activity by proteolytically cleaving C3b, C4b and

C5. FEBS Lett 2007; 581: 1716-20.

47 Haiko J, Laakkonen L, Juuti K, Kalkkinen N, Korhonen TK. The omptins of Yersinia pestis and

Salmonella enterica cleave the reactive center loop of plasminogen activator inhibitor 1. J

Bacteriol 2010. Doi 10.1128/JB.00458-10

48 Dekker N, Cox RC, Kramer RA, Egmond MR. Substrate specificity of the integral membrane

protease OmpT determined by spatially addressed peptide libraries. Biochemistry 2001; 40:

1694-701.

49 Wiman B. On the reaction of plasmin or plasmin-streptokinase complex with aprotinin or alpha

2-antiplasmin. Thromb Res 1980; 17: 143-52.



and B. J Biol Chem 2007; 282: 24873-81.

51 Bengtson SH, Sanden C, Morgelin M, Marx PF, Olin AI, Leeb-Lundberg LM, Meijers JC, Herwald H.

Activation of TAFI on the surface of Streptococcus pyogenes evokes inflammatory reactions by

modulating the kallikrein/kinin system. J Innate Immun 2008; 1: 18-28.

52 Finegold MJ, Petery JJ, Berendt RF, Adams HR. Studies on the pathogenesis of plague. Blood

coagulation and tissue responses of Macaca mulatta following exposure to aerosols of

Pasteurella pestis. Am J Pathol 1968; 53: 99-114.

53 Une T, Brubaker RR. In vivo comparison of avirulent Vwa- and Pgm- or Pstr phenotypes of

yersiniae. Infect Immun 1984; 43: 895-900.

54 Wick MJ, Harding CV, Normark SJ, Pfeifer JD. Parameters that influence the efficiency of

processing antigenic epitopes expressed in Salmonella typhimurium. Infect Immun 1994; 62:

4542-8.

Binding characteristics of Thrombin-Activatable Fibrinolysis Inhibitor to streptococcal surface collagen-like proteins A and B Mercedes Valls Serón, Tom Plug, J. Arnoud Marquart, Pauline F. Marx, Heiko Herwald, Philip G. de Groot and Joost C.M. Meijers Thrombosis and Haemostasis, 2011; Sep 27; 106(4): 609-16

Chapter 4

66

Abstract

Streptococcus pyogenes is the causative agent in a wide range of diseases in humans.

Thrombin-Activatable Fibrinolysis inhibitor (TAFI) binds to collagen-like proteins SclA and SclB

at the surface of S. pyogenes. Activation of TAFI at this surface redirects inflammation from a

transient to chronic state by modulation of the kallikrein/kinin system. We investigated TAFI

binding characteristics to SclA/SclB. 34 overlapping TAFI peptides of approximately 20 amino

acids were generated. Two of these peptides (P18: residues G205-S221, and P19: R214-D232)

specifically bound to SclA/SclB with high affinity, and competed in a dose-dependent manner

with TAFI binding to SclA/SclB. In addition, the glycosaminoglycan consensus repeats in P18

and P19 were critical for the interaction of the TAFI peptides with SclA/SclB. In another series

of experiments, the binding properties of activated TAFI (TAFIa) to SclA/SclB were studied

with a quadruple TAFI mutant (TAFI-IIYQ) that after activation is a 70-fold more stable enzyme

than wild-type TAFIa. TAFI and TAFI-IIYQ bound to the bacterial proteins with similar affinities.

The rate of dissociation was different between the proenzyme (both TAFI and TAFI-IIYQ) and

the stable enzyme TAFIa-IIYQ. TAFIa-IIYQ bound to SclA/SclB, but dissociated faster than TAFI-

IIYQ. In conclusion, the bacterial proteins SclA and SclB bind to a TAFI fragment encompassing

residues G205-D232. Binding of TAFI to the bacteria may allow activation of TAFI, whereafter

the enzyme easily dissociates.

TAFI binding site to SclA and SclB

67

Ch

apte

r 4

Introduction

Streptococcus pyogenes is an important human Gram-positive pathogen that mainly causes

throat and skin infections, such us pharyngitis, impetigo and cellulitis. Although the majority

of streptococcal infections are superficial, some cases may progress into invasive and life

threatening diseases with an extremely rapid progression, such as streptococcal septic shock

and necrotizing fasciitis [1]. In order to infect the human host, S. pyogenes expresses a

number of virulence factors that mediate adhesion to host tissues, enable dissemination of

bacteria, and/or that modulate the immune response [2-4].

The streptococcal collagen-like surface proteins A and B (SclA and SclB), also known as Scl1

and Scl2, are two related proteins with a similar structure motif, including a C-terminal region

that is attached to the cell wall-membrane (CWM) via a LPATG anchor. This is followed by a

central region composed of a collagen-like domain (CL) with long segments of repeated GXY

amino acids, a sequence considered a defining feature of collagens. Next to the CL domain, an

amino terminal variable domain (V) is located. In addition, SclA, but not SclB, contains a linker

(L) region between the CWM and the CL domain. Both proteins are organized into a “lollipop-

like” structure, where the CL domain forms a triple helical-stalk and the V domain folds into a

globular head. The genes encoding SclA and SclB are located at different sites of the bacterial

chromosome and are differently regulated. While SclA is up-regulated by the Mga regulon,

the transcription of SclB is down-regulated by the same protein [5-10].

Recently, we reported the binding of Thrombin-Activatable Fibrinolysis Inhibitor (TAFI) to the

surface of a group A streptococci (M41 serotype) and its subsequent activation at the

bacterial surface via plasmin and thrombin-thrombomodulin [11]. Furthermore, activation of

TAFI on the surface of S. pyogenes evoked inflammatory reactions by modulating the

kallikrein/kinin systems [12]. Identifying the molecular determinants of Scls-TAFI interaction

may well offer possibilities for prevention of diseases caused by inflammatory reactions

induced by S. pyogenes.

TAFI is a zinc-dependent procarboxypeptidase that is synthesized in the liver [13]. It is

thereafter released into the bloodstream, where it circulates at a concentration in the range

of 70-275 nM [14]. Cleavage of the TAFI zymogen by enzymes such as thrombin or plasmin

results in the formation of activated TAFI (TAFIa), which attenuates fibrinolysis. TAFIa

prevents accelerated plasmin formation by removing C-terminal lysine residues from partially

degraded fibrin that augment the efficacy of plasminogen activation. Besides a function in

fibrinolysis, TAFIa also plays a role in inflammatory processes by hydrolysis of bradykinin,

osteopontin and the anaphylotoxins C3a and C5a [15].

The present study was undertaken to 1) identify the TAFI binding site involved in the

interaction with the SclA, SclB expressed by S. pyogenes and 2) establish the binding

properties of TAFIa to SclA and SclB. Using synthetic TAFI peptides, we demonstrated that

TAFI binds to both Scl proteins via residues G205 to D232. In addition, we determined that

TAFIa is able to bind SclA and SclB but that it rapidly dissociates from the bacterial proteins.

Chapter 4

68

Matherials and methods

Proteins

34 overlapping TAFI peptides and the TAFI mutant peptides were synthesized by Peptide 2.0

Inc. (Chantilly, VA). Surface Plasmon Resonance (SPR) experiments with all 34 TAFI peptides

and with P18 and P19 mutants (P18.1, P18.2, P18.3, P18.4; P19.1, P19.2) were performed

with crude peptides. In SPR experiments with peptide 18 (P18), and peptide 19 (P19) and in

solid-phase binding assays, peptides were 95 % pure as determined by high-pressure liquid

chromatography analysis, and their identity was confirmed by mass spectrometry.

Recombinant SclA and SclB from the S. pyogenes AP41 strain were produced and purified as

described elsewhere [11]. TAFI was purified as previously described [16]. The rTAFI-IIYQ

mutant, which harbours the Thr325Ile, Thr329Ile, His333Tyr and His335Gln

mutations, and the active form of the mutant (rTAFIa-IIYQ) were generated and expressed as

described previously [17,18]. A polyclonal antibody specific for TAFI was obtained in rabbits as

described elsewhere [19]. HRP (horseradish peroxidise)-labeled swine anti-rabbit

immunoglobulin G (IgG) was purchased from Dako.

SPR binding assays

All SPR measurements were performed at 25°C using a BIAcore 2000 biosensor System (GE

Healthcare). Recombinant AP41 SclA and SclB were immobilized to a CM5 sensor chip using

the amine coupling kit according to the supplier’s recommendation (GE Healthcare). SclA and

SclB were applied in 10 mM NaAc (pH 3.1). Immobilization of SclA on the chip resulted in an

increase of the resonance signal by ~400 RU (resonance units) and with SclB of ~440 RU.

Binding studies were done in 10 mM Hepes, 150 mM NaCl, 0.005% Sulfactant P20 (pH 7.4), at

a flow rate of 30 µl/min. Different concentrations of rTAFI-IIYQ, rTAFIa-IIYQ (0-200 nM) or 5

µM TAFI peptides were injected for 2 or 3 minutes. The dissociation was followed for a period

of 1 or 10 minutes and the ligand surface was regenerated with a 30-s injection of 1/3 or 1/5

ionic buffer (92 mM KSCN, 366 mM MgCl2, 184 mM urea, 366 mM guanidine) followed by

equilibration with flow buffer at the end of each binding cycle. The data sets were fit to a

simple interaction model to obtain rate constants. When appropriate, data sets were also fit

to a heterogeneous analyte model. The association (ka) and dissociation (kd) rate constants

were determined using the BIAEvaluation Software (version 4.1; Biacore). The equilibrium

dissociation constants (KD) were calculated from the ratio of the measured kinetic rate

constants kd/ka. To generate the KD in binding studies with P18, P19 and P18 and P19 mutants,

the responses at equilibrium were fitted by non-linear regression using the Scrubber software

( version 2.0; Biologic Software).

Solid-phase binding assays

Ninety-six-well NUNC MaxiSorpTM

plates (Nalge Nunc International) were coated overnight

with 100 µl of recombinant SclA or SclB (46 nM) in NaHCO3 (pH 9.6) at 4°C. Following three


69

Ch

apte

r 4

washes with Tris-buffered saline supplemented with Tween [50 mM Tris, 150 mM NaCl, 0.1%

Tween-20 (pH 7.4)], plates were blocked with 150 µl of blocking solution [1.5 % w/v bovine

serum albumin (BSA) in Tris-buffered saline] for 1 hour at 37ºC. Wells were washed as

described above, and 100 nM TAFI in the presence or absence of P18, P19 or P29 (0 to 150

µM) diluted in blocking buffer was applied to the wells. The assay mixture was incubated for 1

hour at room temperature. Following the competition step, microtiter plates were washed

three times, and 100 µl of anti-TAFI antibody was incubated in blocking buffer for 1 hour at

room temperature. Next, the plates were washed three times and incubated with HRP-

labeled swine anti-rabbit IgG diluted 1:5000 with blocking buffer. After four washes, the

reactions were developed by the addition of 100 µl o-phenylenediamine (Sigma-Aldrich)

substrate [8 mM Na2HPO4 (pH 5.0) 2.2 mM o-phenylenediamine, 3% H2O2]. Colour

development was stopped by the addition of 50 µl of 1 M H2SO4, and the plates were read at

490 nm using a Thermomax microplate reader (Molecular Devices Corp.). Data were

corrected for binding to empty microtiter wells.

Results

Characterization of TAFI binding to SclA and SclB

We have previously shown that TAFI binds to SclA and SclB [11]. In the present study, we

examined the TAFI region involved in SclA and SclB binding. To this end, 34 overlapping TAFI

peptides of approximately 20 amino acids comprising the complete TAFI molecule were

generated (Table 1).

SPR measurements were carried out to determine binding between TAFI peptides and the

Scls. The recombinant bacterial proteins, and BSA as a negative control, were immobilized on

the surface of the biosensor chip in three separate flow cells. As shown in Figure 1, only two

synthetic peptides (Gly205

-Ser221

, P18, and Arg214

-Asp232

, P19) were able to bind immobilized

Scls. The other synthetic peptides did not show appreciable binding to the bacterial proteins.

Chapter 4

70

Table 1. Overlapping synthetic peptides of TAFI.

The one-letter code for amino acid residues is used for the sequence. The positions of the amino acids in

TAFI protein are shown.

Peptide Sequence Position

1 FQSGQVLAALPRTSRQVQ F1 - Q18

2 RTSRQVQVLQNLTTTYE R12 - E26

3 TYEIVLWQPVTADLIVKKKQ T26 - Q45

4 TADLIVKKKQVHFFVNAS T36 - S53

5 HFFVNASDVDNVKAHLN H47 - N63

6 DNVKAHLNVSGIPCSVLLA D56 - A74

7 IPCSVLLADVEDLIQQQISN I67 - N86

8 DVEDLIQQQISNDTVSPR D75 - R92

9 DTVSPRASASYYEQYHSLNE D87 - E106

10 EQYHSLNEIYSWIEFITERH E99 - H118

11 TERHPDMLTKIHIGS T115 - S129

12 SFEKYPLYVLKVSGKEQTAK S130 - K149

13 GKEQTAKNAIWIDCGIHARE G143 - E162

14 HAREWISPAFCLWFIGH H159 - H175

15 GHITQFYGIIGQYTN G174 - N188

16 GQYTNLLRLVDFYVM G184 - M198

17 DFYVMPVVNVDGYDYSWKKN D194 - N213

18 GYDYSWKKNRMWRKNRS G205 - S221

19 RMWRKNRSFYANNHCIGTD R214 - D232

20 ANNHCIGTDLNRNFASKHW A224 - W242

21 DLNRNFASKHWCEEGASSSS D232 - S251

22 SETYCGLYPESEPEVKAVA S253 - A271

23 ESEPEVKAVASFLRRNINQ E262 - Q280

24 RRNINQIKAYISMHSYSQH R275 - H293

25 HSYSQHIVFPYSYTRSKSKD H288 - D307

26 PYSYTRSKSKDHEELSLVAS P297 - S316

27 KDHEELSLVASEAVRAIEKT K306 - T325

28 EAVRAIEKTSKNTRYTHGHG E317 - G336

29 SKNTRYTHGHGSETLYLAPG S326 - G345

30 SETLYLAPGGGDDWIYDLGI S337 - I356

31 GGDDWIYDLGIKYSFTIELR G346 - R365

32 KYSFTIELRDTGTYGFLLPE K357 - E376

33 DTGTYGFLLPERYIKPTCRE D366 - E385

34 CREAFAAVSKIAWHVIRNV C383 - V401


71

Ch

apte

r 4

Figure 1. Binding of TAFI peptides to immobilized SclA and SclB. 34 overlapping TAFI peptides were

used to assess the binding to immobilized SclA and SclB by SPR-based assay. The bars represent the

responses in resonance units (RU) corrected for non-specific binding to BSA.

To further analyze the relative affinity of TAFI for SclA and SclB compared to the synthetic

peptides, we tested P18 and P19 for their ability to interfere with the interactions between

TAFI and the immobilized Scls. TAFI binding to SclA and SclB was competed with various

concentrations of P18 or P19. P18 and P19 displayed a dose-dependent inhibition of TAFI

binding to immobilized SclA (Fig. 2A) and SclB (Fig. 2B).

Figure 2. Competitive binding of TAFI to immobilized recombinant SclA and SclB with TAFI peptides

P18 and P19. Binding of TAFI to SclA (A) or SclB (B) onto 96-well plate was measured in the presence of

various concentrations of P18, P19 and P29. The assays were repeated three times, and results of a

representative experiment with duplicate samples are shown.

Chapter 4

72

In addition, P18 and P19 bind to SclA and SclB in a dose-dependent fashion (Fig. 3). Next, we

evaluated the affinity of P18 and P19 towards the Scls. For SclA we calculated a KD of 230 nM

(P18) and 520 nM (P19) and for SclB we calculated a KD of 230 nM (P18) and 530 nM (P19).

These results suggest that the TAFI sequence Gly205

- Asp232

contains important residues

involved in the interactions with SclA and SclB.

These data imply that P18 and P19 have a similar binding region as TAFI for binding to SclA

and SclB.

Peptides 18 and P19 contain a number of positively charged amino acids (Table 1), that could

contribute to binding. In fact, P18 and P19 contain respectively two and one

glycosaminoglycan consensus repeats [20] that have been shown in many other proteins to

be involved in binding to proteins and heparin [21]. The consensus sequence of such a repeat

is XBBXBX, where B stands for a basic residue and X is a non-basic residue. We next

investigated if the glycosaminoglycan consensus motif is involved in the interaction of the

peptides with SclA and B: peptide mutants were generated in which several basic amino acids

were changed to glutamine (Table 2).

Table 2. Binding of peptides 18, 19 and mutants thereof to SclA and SclB. The one-letter code for

amino acid residues is used for the sequence. The equilibrium responses of SPR were fitted by non-

linear regression to generate KD values. Values are means ± S.D. ND, no detectable binding (KD > 50,000

nM).

Peptide Sequence KD (nM) for

binding to SclA

KD (nM) for

binding to SclB

18 GYDYSWKKNRMWRKNRS 230 ± 20 230 ± 20

18.1 GYDYSWQQNRMWRKNRS 1220 ± 5 1280 ± 5

18.2 GYDYSWKKNRMWQQNRS 1530 ± 4 1530 ± 8

18.3 GYDYSWQQNRMWQQNRS ND ND

18.4 GYDYSWQQNQMWQQNQS ND ND

19 RMWNRRSFYANNHCIGTD 520 ± 10 530 ± 10

19.1 RMWQQNRSFYANNHCIGTD 1090 ± 3 1030 ± 3

19.2 RMWQQNQSFYANNHCIGTD ND ND

These peptide mutants were tested for binding to immobilized SclA and B using surface

plasmon resonance (Fig. 3). Our results indicated that replacement of basic residues in one of

the two glycosaminoglycan consensus repeats of P18 (P18.1 and P18.2) resulted in lower

binding affinity for both bacterial proteins. In addition, when basic residues in both


73

Ch

apte

r 4

glycosaminoglycan consensus repeats were replaced, the peptides did not bind anymore to

the bacterial proteins. In analogy, replacement of some of the basic residues in the consensus

repeat of P19 (P19.1) resulted in decreased binding to SclA and SclB and replacement of all

the basic residues (P19.2) resulted in no binding to the bacterial proteins. KD values for the

binding of the peptides to the bacterial proteins are shown in Table 2.

Together, these data suggested that at least one intact glycosaminoglycan consensus motif

XBBXBX is critical for the interaction of the TAFI peptides with SclA or B.

Figure 3. Binding of TAFI peptides 18 and 19 and mutants thereof to immobilized SclA and SclB.

Peptides 18 (A, C), 19 (B, C) and mutants thereof were used to assess the binding to immobilized SclA (A,

B) and SclB (C, D) by SPR-based assay. Values at equilibrium were plotted as a function of the TAFI

peptides.

Chapter 4

74

Binding analysis of TAFIa to SclA and SclB

Next we used SPR to investigate the binding characteristics of TAFIa to SclA and SclB. Because

TAFIa is a labile enzyme that is inactivated by a conformational change in a temperature-

dependent way [16] we used a TAFI quadruple mutant, rTAFI-IIYQ, which has a 70-fold more

stable active form (rTAFIa-IIYQ) than the wild type.

Both SclA and SclB were immobilized onto the sensor chip surface, and binding curves were

recorded for both rTAFI-IIYQ and rTAFIa-IIYQ variants at 6 different concentrations between 0

and 200 nM. Representative curves for rTAFI-IIYQ and rTAFIa-IIYQ binding to SclA and B are

shown in Figure 4.

Figure 4. Binding of TAFI-IIYQ and TAFIa-IIYQ to immobilized SclA and SclB. SPR analysis of binding of

TAFI-IIYQ to SclA (A) and SclB (B) and binding of TAFIa-IIYQ to SclA (C) and SclB (D). SclA and SclB (~400

RU for SclA, 440 RU for SclB) were immobilized to a CM5 sensor chip and increasing concentrations (0, 5,

20, 50, 100, and 200 nM) of TAFI-IIYQ or TAFIa-IIYQ were applied to the chip.


75

Ch

apte

r 4

Fitting the SPR profiles to interaction models yielded kinetic and affinity information for the

different TAFI-Scls interactions. The responses for rTAFI-IIYQ were well described by a 1:1

interaction model, however, the responses for rTAFIa-IIYQ were not. Instead, the profiles for

the active form could be better described by a heterogeneous analyte model, which assumed

two or more different classes of binding sites. We found similar association rates of rTAFI-IIYQ

and rTAFIa-IIYQ towards the bacterial proteins. In contrast, the dissociation curves for rTAFI-

IIYQ and rTAFIa-IIYQ were different. rTAFIa-IIYQ displayed ~ 3 to 58 fold faster dissociation

phase towards SclA (kd1 = 4.3 ± 0.2 x 10-4

; kd2 = 9.8 ± 0.1 x 10-3

), and a ~ 2 to 46 fold faster

dissociation phase towards SclB (kd1 = 3.0 ± 0.0 x 10-4

; kd2 = 7.3 ± 0.1 x 10-3

).

Based on the best data fit, rTAFI-IIYQ bound the Scls with affinities in the nanomolar range

(rTAFI-IIYQ-SclA: KD = 3.5 nM. rTAFI-IIYQ-SclB: KD = 4.0 nM). These values were lower than

previously established for plasma TAFI [11]. We also investigated binding of plasma TAFI to

SclA and B and found KD’s of 5.4-6.6 nM respectively (data not shown). This indicated that

rTAFI-IIYQ and plasma TAFI had similar binding kinetics. However, it is unknown why the

values for plasma TAFI were lower than in our earlier study.

Compared to rTAFI-IIYQ, a lower affinity was measured for rTAFIa-IIYQ, suggesting that the

conformation of the Scl-recognition domain had been slightly changed upon TAFI activation.

Kinetic and affinity values obtained for the bacterial proteins with both TAFI variants are

shown in Table 3. Taken together, the results imply that rTAFIa-IIYQ dissociates faster from

SclA and SclB compared to rTAFI-IIYQ.

Table 3. Kinetic and affinity parameters for interactions between TAFI-IIYQ, TAFIa-IIYQ and SclA, SclB.

The parameters were determined by surface plasmon resonance measurements using immobilized SclA

and SclB as the ligand, and TAFI-IIYQ or TAFIa-IIYQ as the analyte. ka, association rate constant; kd,

dissociation constant; KD, equilibrium dissociation constant. Values are means ± S.D.

Analyte/ligand ka1 (M-1

s-1

) kd1 (s-1

) KD1 (nM) ka2 (M-1

s-1

) kd2 (s-1

) KD2 (nM)

TAFI-IIYQ/SclA 4.5±0.0x104 1.6±0.1x10

-4 3.5±0.3

TAFI-IIYQ/SclB 4.0±0.1x104 1.6±0.0x 0

-4 4.0±0.0

TAFIa-IIYQ/SclA 3.4±0.1x104 4.3±0.2x10

-4 12.6±0.3 2.0±0.0x10

6 9.8±0.1x10

-3 4.9±0.1

TAFIa-IIYQ/SclB 2.7±0.0x104 3.0±0.0x10

-4 11.1±0.2 1.1±0.0x10

6 7.3±0.1x10

-3 6.6±0.0

Chapter 4

76

Discussion

In the present study we investigated the binding interactions of TAFI and activated TAFI to

streptococcal collagen-like surface protein A and B. By using TAFI peptides we identified the

binding region involved in the interaction with SclA and B within amino acids 205 to 232

(partially overlapping peptides 18 and 19) of TAFI. P18 bound to the same extent to SclA (KD =

230 nM) and SclB (KD = 230 nM). Affinity of P19 to SclA and SclB was similar (KD = 520 nM and

KD = 530 nM, respectively). In addition, SclA/B-TAFI interaction was mediated by the

glycosaminoglycan-binding site suggesting that this consensus motif may play an important

role in the binding of TAFI to other proteins.

The region of Gly205

to Asp232

is located distally from the TAFI catalytic site (Fig. 5) within helix

α-4 and is surface exposed and does not interfere with the region known to influence TAFIa

stability (Arg302

, Arg320

, Arg330

, and Thr/Ile325

) [22-24], neither with the residues involved in

substrate binding (Gly336

, Tyr341

, and Glu363

) [25].

Figure 5. P18 and P19 shown in the overall structure of TAFI. Ribbon drawing (A) and space-filling

representation (B) of TAFI with the activation peptide shown in blue, the catalytic domain in green, and

residues 205-232 representing the partially overlapping peptides 18 and 19 in red.

Although different binding partners have been identified for TAFI, such as plasminogen and

fibrinogen [26], the region described here as involved in SclA and SclB binding has not been

shown to be overlapping with another TAFI-protein interaction.

We demonstrated that P18 and P19 have the potential to compete with TAFI for binding to

the streptococcal proteins. TAFI binding to SclA was inhibited 55% and 76% by P18 and P19

respectively. In contrast, TAFI binding to SclB was inhibited 44% and 26% by P18 and P19

respectively. The partial contribution of P18 and P19 to inhibit TAFI-SclB interaction may be

attributed to the fact that SclA and SclB contain different sizes of the variable region. In


77

Ch

apte

r 4

addition, it is likely that the recognition between the bacterial proteins and TAFI is dependent

on the three-dimensional conformation of the protein, which is not optimally represented by

the linear peptides.

Recent findings that the physiological activity of TAFIa is not inhibited by SclA or SclB [11], and

the ability of activated TAFI on the surface of S. pyogenes to evoke inflammatory reactions by

modulating the kallikrein/kinin system [12], prompted us to investigate the TAFIa binding

properties to SclA and SclB. Here we provide evidence that activated TAFI binds to SclA and

SclB and, in contrast to the TAFI proenzyme, is rapidly dissociated from the bacterial proteins.

It is tempting to speculate that this constitutes a mechanism whereby the bacteria attract

TAFI to their surface and localize it there. After activation however, the activated TAFI can

dissociate and act on substrates elsewhere. These findings suggested that the activation

peptide may play a role in the TAFI binding to SclA and SclB. However, we found that the TAFI

binding to SclA and SclB was mediated by a region within the TAFI catalytic domain. The

crystal structure of TAFI showed that TAFIa stability is directly related to the dynamics of a 55-

residue segment (residues 296-350) of the active site [17, 27]. Release of the activation

peptide increases dynamic flap mobility and in time this leads to conformational changes that

expose the cleavage site at Arg302

. It cannot be ruled out that binding of TAFIa to SclA and SclB

is influenced by the conformational change in TAFIa.

In summary, this study demonstrates that binding of TAFI to SclA and SclB can be inhibited by

TAFI peptides. We have identified the region on TAFI, encompassing residues 205-232, that

bind to the bacterial proteins. In addition, it was shown that TAFIa rapidly dissociates from

SclA and SclB. A better understanding of the molecular mechanisms behind host/bacteria

interactions has the potential to discover important targets in the human host and ultimately

new therapeutic approaches for treatment of severe infectious diseases.

Chapter 4

78

Reference List


Med Biol 2008; 609: 29-42.

2 Hertzen E, Johansson L, Wallin R, et al. M1 protein-dependent intracellular trafficking promotes

persistence and replication of Streptococcus pyogenes in macrophages. J Innate Immun 2010; 2:

534-545.

3 Maamary PG, Sanderson-Smith ML, Aziz RK, et al. Parameters governing invasive disease

propensity of non- M1 serotype group A streptococci. J Innate Immun 2010; 2: 596-606.

4 Pence MA, Rooijakkers SH, Cogen AL, et al. Streptococcal inhibitor of complement promotes

innate immune resistance phenotypes of invasive M1T1 group A Streptococcus. J Innate Immun

2010; 2: 587-595.

5 Lukomski S, Nakashima K, Abdi I, et al. Identification and characterization of the scl gene

encoding a group A Streptococcus extracellular protein virulence factor with similarity to human

collagen. Infect Immun 2000; 68: 6542-6553.

6 Lukomski S, Nakashima K, Abdi I, et al. Identification and characterization of a second

extracellular collagen-like protein made by group A Streptococcus: control of production at the

level of translation. Infect Immun 2001; 69: 1729-1738.

7 Rasmussen M, Bjorck L. Unique regulation of SclB - a novel collagen-like surface protein of

Streptococcus pyogenes. Mol Microbiol 2001; 40: 1427-1438.

8 Rasmussen M, Eden A, Bjorck, L. SclA, a novel collagen-like surface protein of Streptococcus

pyogenes. Infect Immun 2000; 68: 6370-6377.

9 Whatmore AM. Streptococcus pyogenes sclB encodes a putative hypervariable surface protein

with a collagen-like repetitive structure. Microbiology 2001; 147: 419-429.

10 Xu Y, Keene DR, Bujnicki JM, et al. Streptococcal Scl1 and Scl2 proteins form collagen-like triple

helices. J Biol Chem 2002; 277: 27312-27318.

11 Pahlman LI, Marx PF, Morgelin M, et al. Thrombin-activatable fibrinolysis inhibitor binds to

Streptococcus pyogenes by interacting with collagen-like proteins A and B. J Biol Chem 2007;

282: 24873-24881.

12 Bengtson SH, Sanden C, Morgelin M, et al. Activation of TAFI on the surface of Streptococcus

pyogenes evokes inflammatory reactions by modulating the kallikrein/kinin system. J Innate

Immun 2008; 1: 18-28.


79

Ch

apte

r 4

13 Marx PF, Bouma BN, Meijers JC. Role of zinc ions in activation and inactivation of thrombin-

activatable fibrinolysis inhibitor. Biochemistry 2002; 41: 1211-1216.

14 Bouma BN, Meijers JC. Thrombin-activatable fibrinolysis inhibitor (TAFI, plasma

procarboxypeptidase B, procarboxypeptidase R, procarboxypeptidase U). J Thromb Haemost

2003; 1: 1566-1574.

15 Leung LL, Nishimura T, Myles T. Regulation of tissue inflammation by thrombin-activatable

carboxypeptidase B (or TAFI). Adv Exp Med Biol 2008; 632: 61-69.

16 Valls Serón M, Haiko J, de Groot PG, et al. Thrombin-activatable fibrinolysis inhibitor is degraded

by Salmonella enterica and Yersinia pestis. J Thromb Haemost 2010; 8: 2232-2240.

17 Marx PF, Brondijk TH, Plug T, et al. Crystal structures of TAFI elucidate the inactivation

mechanism of activated TAFI: a novel mechanism for enzyme autoregulation. Blood 2008; 112:

2803-2809.

18 Marx PF, Plug T, Havik SR, et al. The activation peptide of thrombin-activatable fibrinolysis

inhibitor: a role in activity and stability of the enzyme? J Thromb Haemost 2009; 7: 445-452.

19 Mosnier LO, von dem Borne PA, Meijers JC, et al. Plasma TAFI levels influence the clot lysis time

in healthy individuals in the presence of an intact intrinsic pathway of coagulation. Thromb

Haemost 1998; 80: 829-835.

20 Cardin AD, Weintraub HJ. Molecular modeling of protein-glycosaminoglycan interactions.

Arteriosclerosis 1989; 9: 21-32.

21 Hileman RE, Fromm JR, Weiler JM, et al. Glycosaminoglycan-protein interactions: definition of

consensus sites in glycosaminoglycan binding proteins. Bioessays. 1998; 20: 156-67.

22 Boffa MB, Bell R, Stevens WK, et al. Roles of thermal instability and proteolytic cleavage in

regulation of activated thrombin-activable fibrinolysis inhibitor. J Biol Chem 2000; 275: 12868-

12878.

23 Marx PF, Hackeng TM, Dawson PE, et al. Inactivation of active thrombin-activable fibrinolysis

inhibitor takes place by a process that involves conformational instability rather than proteolytic

cleavage. J Biol Chem 2000; 275: 12410-12415.

24 Schneider M, Boffa M, Stewart R, et al. Two naturally occurring variants of TAFI (Thr-325 and Ile-

325) differ substantially with respect to thermal stability and antifibrinolytic activity of the

enzyme. J Biol Chem 2002; 277: 1021-1030.

25 Eaton DL, Malloy BE, Tsai SP, et al. Isolation, molecular cloning, and partial characterization of a

novel carboxypeptidase B from human plasma. J Biol Chem 1991; 266: 21833-21838.

http://www.ncbi.nlm.nih.gov/pubmed?term=%22Cardin%20AD%22%5BAuthor%5D

http://www.ncbi.nlm.nih.gov/pubmed?term=%22Weintraub%20HJ%22%5BAuthor%5D

http://www.ncbi.nlm.nih.gov/pubmed?term=%22Hileman%20RE%22%5BAuthor%5D

http://www.ncbi.nlm.nih.gov/pubmed?term=%22Fromm%20JR%22%5BAuthor%5D

http://www.ncbi.nlm.nih.gov/pubmed?term=%22Weiler%20JM%22%5BAuthor%5D

Chapter 4

80

26 Marx PF, Havik SR, Marquart JA, et al. Generation and characterization of a highly stable form of

activated thrombin-activable fibrinolysis inhibitor. J Biol Chem 2004; 279: 6620-6628.

27 Anand K, Pallares I, Valnickova Z, et al. The crystal structure of thrombin-activable fibrinolysis

inhibitor (TAFI) provides the structural basis for its intrinsic activity and the short half-life of

TAFIa. J Biol Chem 2008; 283: 29416-29423.

Susceptibility of humanized TAFI-transgenic mice to Streptococcus pyogenes Mercedes Valls Serón, Stefan R. Havik, Heiko Herwald Philip G. de Groot, and Joost C.M. Meijers Submitted for publication

Chapter 5

82

Abstract

Streptococcus pyogenes mainly causes throat and skin infections. Although the majority of

streptococcal infections are superficial, some cases may progress into invasive and life

threatening diseases such as sepsis and necrotizing fasciitis. S. pyogenes is highly host-specific

and normally only infects humans. Thrombin-Activatable Fibrinolysis inhibitor (TAFI) binds to

the collagen-like proteins SclA and SclB found at the surface of S. pyogenes. To test the

relative contribution of human TAFI to the outcome of S. pyogenes, we generated TAFI

knockout mice expressing human TAFI (humanized-TAFI). Wild-type and humanized-TAFI

transgenic mice were infected with S. pyogenes using a subcutaneous air-pouch model. Mice

were sacrificed after 24 or 48 h for determination of bacterial load, coagulation and

fibrinolysis activation, histopathology, inflammatory parameters, or were observed in a

survival study. Bacterial outgrowth was equal between groups. Levels of thrombin-

antithrombin complexes in spleen and lung were increased in humanized-TAFI transgenic

mice at 24 h and D-dimer was elevated in liver tissue in humanized-TAFI transgenic mice at 24

h after injection. Expression of human TAFI in mice had no effect on lung, liver, spleen and

kidney histopathology or cytokine levels. Remarkably, S. pyogenes inoculation led to a 65%

mortality rate in humanized-TAFI mice 120 h postinfection, in contrast to a 20% mortality rate

in wild-type mice (p=0.006). These findings suggest that introduction of human TAFI in mice

leads to an increased susceptibility to S. pyogenes infection.

Susceptibility of humanized TAFI mice to S. pyogenes

83

Ch

apte

r 5

Introduction

Streptococcus pyogenes (group A streptococcus, GAS) is an important gram-positive pathogen

that preferably colonizes humans. For the past two decades, several studies have reported an

increase in the severity and incidence of GAS infections [1]. S. pyogenes is responsible for a

wide variety of uncomplicated but also life-threatening infections in skin and throat.

Erysipelas, impetigo, pharyngitis and tonsillitis are considered rather harmless conditions

[2,3], whereas streptococcal toxic shock and necrotizing fasciitis are associated with invasive

and systemic infections [4]. Two serious complications are acute rheumatic fever and acute

glomerulonephritis [5,6].

S. pyogenes infection involves interactions with several components of the host hemostatic

system. The bacteria have been shown to bind and modulate the function of several

important factors involved in coagulation and fibrinolysis. Fibrinogen, factor V, XI, XII and

high-molecular-weight-kininogen are assembled at the surface of S. pyogenes through specific

interactions with bacterial surface proteins to promote formation of the fibrin network [7,8].

Plasminogen binding and its activation to the serine protease plasmin has been implicated as

a contributing mechanism of invasion for S. pyogenes and a variety of other bacterial species

[9,10]. Another hemostatic protein that interacts with S. pyogenes is TAFI [11].

TAFI is synthesized in the liver and secreted into plasma as a 56-kDa procarboxypeptidase.

During coagulation, the proenzyme is activated to TAFIa by thrombin and the thrombin-

thrombomodulin complex. TAFIa has anti-fibrinolytic properties as it inhibits plasmin-

mediated blood clot lysis by removing C-terminal lysine residues from partially degraded

fibrin that are required for positive feedback in tissue-type plasminogen activator dependent

plasmin generation. Apart from thrombin, the serine proteases plasmin, trypsin and

neutrophil elastase have been reported to function as TAFI activators [12-14]. TAFIa is

unstable at 37 ºC and upon activation by thrombin it is inactivated (TAFIai) by a

conformational change in a temperature-dependent manner [15].

TAFI binds to the surface of S. pyogenes (M41 serotype) and is subsequently activated at the

bacterial surface via plasmin and thrombin-thrombomodulin [11]. Furthermore, activation of

TAFI on the surface of Streptococcus pyogenes evoked inflammatory reactions by modulating

the kallikrein/kinin system [16].

The highly host-specificity nature of GAS in combination with the binding of TAFI to SclA and

SclB and subsequent activation at the bacterial surface, prompted us to investigate the

susceptibility of mice expressing human TAFI to S. pyogenes infection. To this end, mice

expressing only human TAFI were generated and infected with S. pyogenes serotype AP41.

The study suggests that introduction of human TAFI in mice leads to an increased

susceptibility for S. pyogenes infection.

Chapter 5

84

Materials and Methods

Animals

C57BL/6 mice were supplied by Harlan (Venray, The Netherlands). TAFI-transgenic mice were

generated by introduction of the human TAFI cDNA. The mouse albumin enhancer/promoter

was chosen to drive liver-specific expression of the human TAFI cDNA. The transgenic

construct containing the human TAFI cDNA was micro injected into fertilized oocytes of

FVB/NAU strain mice. Founders were bred with FVB/NAU strain mice. Transgenic mice were

identified by PCR analysis, and back-crossed to C57BL/6 background. TAFI-humanized

transgenic mice were generated by crossing TAFI-transgenic mice with TAFI- deficient mice.

Mice exclusively expressing human TAFI were selected. TAFI-deficient mice on a C57BL/6

background have been characterized previously [17-20]. Animals were bred in the animal

facility at the Academic Medical Center, The Netherlands and kept on a controlled 12 h

light/dark cycle and food and water were provided ad libitum. Experiments were carried with

8-12 weeks old male mice and all procedures were approved by the Institutional Animal Care

and Use Committee of the Academic Medical Center.

Experimental model of S. pyogenes infection

The S. pyogenes strain AP 41 (M41 type) was obtained from the Institute of Hygiene and

Epidemiology (Prague, Czech Republic). Bacteria were grown in Todd-Hewitt broth (BD

Bioscience) at 37°C, an aliquot of these cells were added to fresh media and grown up to the

exponential phase of growth (OD620=0.5). The cells were washed three times with saline (0.9

% NaCl) and diluted to 1 to 3.5 x 108/ml in saline. Wild-type and TAFI-humanized transgenic

mice were subjected to an air-pouch infection. Briefly, 0.8 ml of air and 0.2 ml of S. pyogenes

diluted were injected with a 25-gauge needle subcutaneously on the neck of the mouse. Mice

were sacrificed 24 and 48 hours after infection or were observed in a survival study for 5 days.

During the survival experiment, the animals were daily monitored for signs and symptoms of

infection.

Sample harvesting

At the time of sacrifice (24 and 48 hours), mice were first anesthetized by inhalation of

isoflurane (Abbott Laboratories). Blood was drawn from the vena cava inferior into a sterile

syringe containing 3.2% sodium citrate (1/10 vol) and immediately placed on ice. Thereafter,

spleen, kidney, lung and liver were harvested and processed for measurements of CFU,

histology, cytokines.


85

Ch

apte

r 5

Determination of bacterial outgrowth

After 24 and 48 hours postinfection, spleen, kidney, lung and liver were homogenized at 4°C

in 4 volumes of sterile isotonic saline with a tissue homogenizer (Biospect Products) which

was carefully cleaned and disinfected with 70% ethanol after each homogenization. Serial 10-

fold dilutions in sterile saline were made from these homogenates and blood. Thereafter, 50-

µl volumes were plated onto sheep-blood agar plates and incubated at 37°C. CFU were

counted after 16 h.

Histology

Organs collected from both mice groups 24 and 48 hours after infection were fixed in formalin

for 24 h and then embedded in paraffin. Sections 5- µm thick were cut, stained with

hematoxylin and eosin following standard procedures and then examined by light microscopy.

Assays

Interferon-γ, TNF-α, IL-6, IL-12, IL-10 and MCP-1 were measured by cytometric bead array

multiplex assay (BD Biosciences, San Jose, CA, USA) in accordance with the manufacturer’s

recommendations. For these cytokine measurements, harvested organs were homogenized

as described above. Sample homogenates were lysed in 1.25 volumes lysis buffer (300 mM

NaCl, 15 mM Tris [tris(hydroxymethyl) aminomethane], 2 mM MgCl2, 1% Triton X-100,

pepstatin A, leupeptin, and aprotinin (each at 20 ng/ml) (pH 7.4)) on ice for 20 min and spun

at 3600 rpm at 4°C for 10 min. The supernatant was frozen at -80°C until assayed. Thrombin-

antithrombin complexes (TATc; Siemens Healthcare Diagnostics, Marburg, Germany) and D-

dimer (Diagnostica Stago, Asnieres, France) were measured by ELISA.

Statistical analysis

Differences between groups were calculated by using the Mann-Whitney U test. Values were

expressed as means ±SE. A p value of < 0.05 was considered statistically significant. Survival

curves were analyzed by the Kaplan-Meyer log-rank test. All statistics were performed using

GraphPad Prism, version 5.01 (GraphPad Software, San Diego, CA, USA).

Results

Bacterial outgrowth

To investigate if human TAFI influences S. pyogenes dissemination, we compared the bacterial

load in spleen, kidney, lung, liver and blood of TAFI humanized and wild-type mice after 24

and 48 h of initiation of infection (Fig. 1). At 24 h, there were no differences in bacterial

outgrowth in spleen, lung or kidney between mouse strains. However, bacterial loads in liver

and blood from wild-type mice were slightly higher. After 48 h, bacterial loads in spleen, lung,

and blood from TAFI humanized and wild-type mice were equal, whereas the bacterial loads

in kidney and liver tended to be higher but not significant in TAFI-humanized transgenic mice.

Chapter 5

86

Figure 1. Bacterial dissemination. Wild-type (●) and humanized-TAFI mice (○) were injected with S.

pyogenes AP41 (3.5 x108/ml) administrated by subcutaneous injection. After 24 h or 48 h mice were

sacrificed and the bacterial load in the spleen, kidney, liver, lung and blood was determinated. Data are

expressed as CFU/ml of organ homogenate or blood from each animal with lines drawn at the median

for each group. The dotted horizontal line represents the detection limit. There were no statistically

significant differences between groups (Mann-Whitney test).

Activation of coagulation and fibrinolysis

To determine whether human TAFI impacts on local or systemic activation of coagulation

and/or fibrinolysis we determined levels of TATc and D-dimer after 24 and 48 h S. pyogenes

AP41 infection in organs and plasma.

At 24 h TATc levels were significantly elevated in spleen and lung of TAFI-humanized

transgenic (Fig. 2A, D), however after 48 h, TATc levels were not different between mouse

strains. In addition, TATc concentrations in kidney, liver, and plasma were not different

between the groups at both time points (Fig. 2B, C, E). To investigate if the introduction of

human TAFI influenced the fibrinolytic activity, we measured D-dimer (Fig. 3). D-dimer

concentrations were significantly increased in liver of TAFI-humanized transgenic mice after

24 h compared to wild-type and tended to be elevated (but not significant) after 48 h S.

pyogenes infection (Fig. 3C). D-dimer levels in spleen, kidney, and lung were not different

between the groups at both time points (Fig. 3A, B, D). Plasma D-dimer decreased after 48 h

but levels between strains remained similar at both time points (Fig. 3E).


87

Ch

apte

r 5

Figure 2. Activation of coagulationq. Levels of TATc in spleen (A), kidney (B), liver (C), lung (D) and

plasma (E) at 24 and 48 hours after inoculation of S. pyogenes in wild-type mice (black bars) and

humanized-TAFI mice (white bars). Data are expressed as means ± SE of eight mice per time point

(except at 48 h for all organs where n=7 and at 48 h for blood where n=6 in the wild-type group or n=4

in the humanized-TAFI transgenic group). Differences between groups were calculated using Mann-

Whitney U test. p values of humanized-TAFI mice vs wild-type mice of the same time point.

Local and systemic inflammation

Levels of various cytokines and chemokines were analyzed in organ homogenates and plasma

from TAFI-humanized transgenic and wild-type mice after 24 and 48 h infection. Levels of

interferon-γ, TNF-α, IL-6, IL-12, IL-10 and MCP-1 in organs and in plasma were below

detection in this model (data not shown). Histological examination of the organs showed no

differences between groups at both time points (data not shown).

Survival

To determine if introduction of human TAFI impacts on mortality during S. pyogenes infection

we performed a survival study. Despite the absence of clear differences in bacterial loads,

coagulation, fibrinolysis and inflammation, remarkably, introduction of human TAFI resulted

in significantly increased mortality in response to S. pyogenes compared to wild-type mice

(Fig. 4; p = 0.006). The mortality rates of the 5-day observation period were 20% for wild-type

and 65% for humanized-TAFI transgenic mice

Chapter 5

88

.

Figure 3. Activation of fibrinolysis. Levels of D-dimer in spleen (A), kidney (B), liver (C), lung (D) and

plasma (E) at 24 and 48 hours after inoculation of S. pyogenes in wild-type mice (black bars) and

humanized-TAFI mice (white bars). Data are expressed as means ± SE of eight mice per time point

(except at 48 h for all organs where n=7 and at 48 h for blood where n=6 in the wild-type group or n=4

in the humanized-TAFI transgenic group). Differences between groups were calculated using Mann-

Whitney U test. p values of humanized-TAFI mice vs wild-type mice of the same time point.

Discussion

In vitro experiments revealed that TAFI binds to S. pyogenes via SclA and SclB and that it can

be converted into its active fragment by recruitment of the natural activators, plasmin and

thrombin [11]. Analysis of the in vivo role of TAFI in S. pyogenes infection is complicated by

the fact that S. pyogenes is a human pathogen, limiting the use of animal models. Although ex

Figure 3. Survival rates. Wild-type (●) and

humanized-TAFI mice (○) were infected with 1

or 3 x108 CFU/ml of S. pyogenes AP41

subcutaneously. Data were pooled from two

independent experiments, each of which was

conducted with 10 mice per group. Survival

data are presented as a Kaplan-Meier plot.


89

Ch

apte

r 5

vivo [21] and primate models [22] are available, both have limitations and the mouse remains

the system of choice.

Fibrinogen is the major clotting protein of human plasma that is converted to fibrin by

thrombin cleavage upon vascular injury. Fibrin(ogen) plays multiple roles in the GAS/host

interaction. During infection, the host generates fibrin at the local site of infection that can be

used to wall off the site of infection and limit pathogen invasion and spread. However,

bacteria within a fibrin network could be protected from the host defense machinery. To be

able to circumvent the thrombotic host defense, S. pyogenes expresses a number of

molecules [23-27] which confer the bacterium ability to dissolve formed fibrin clots to

facilitate bacterial spread. Thus, at different stages of the infectious process, S. pyogenes may

recruit either thrombotic or thrombolytic factors to meet the demand for bacterial survival

and proliferation.

In this study, we examined the contribution of human TAFI to virulence in a murine model of

S. pyogenes infection. Using S. pyogenes M41 serotype strain at a dose of 1 to 3 x108 CFU/ml,

we observed 20% mortality in wild-type mice after 5 days. However, introduction of human

TAFI markedly increased mortality to 65%. Our results suggest that S. pyogenes may gain

additional advantages by binding and activating TAFI. For instance, S. pyogenes can use TAFI

to prevent fibrinolysis of the surrounding fibrin network, which may protect against

phagocytosis. Furthermore, activated TAFI is able to inactivate inflammatory peptides such as

complement factors C3a and C5a [28], which should lead to an impaired chemotactic activity

of these peptides.

Altered haemostasis and massive inflammation are common features in severe S. pyogenes

infection [29]. Our results demonstrated local activation of coagulation and fibrinolysis as

shown by increased TATc in spleen and lung and elevated d-dimer in liver after 24 h infection

in humanized-TAFI transgenic mice compared to wild-type mice. However, we were unable to

demonstrate an altered local or systemic inflammatory response in humanized-TAFI mice, as

evidenced by unaltered cytokine levels after 24 and 48 h S. pyogenes infection. In addition,

after 24 and 48 h S. pyogenes infection bacterial loads were not consistently altered. By the

design of our study we can not underline the mechanism(s) responsible for the increased

susceptibility of humanized-TAFI mice. It seems likely that acceleration of the disease

progression may have occurred later than for instance 48 h after S. pyogenes infection.

S. pyogenes has a repertoire of pathogenic mechanisms that assure its success as colonizing

and invading microorganism. This paper reports the first application of humanized-TAFI mice

in a model of infection. Although our data can not establish the etiology of the infection, it is

striking that mice expressing human TAFI are more susceptible to S. pyogenes infection. More

research is warranted to investigate the mechanisms by which TAFI contributes to S.

pyogenes infection.

Chapter 5

90

Reference List

1 Lamagni TL, Efstratiou A, Vuopio-Varkila J, Jasir A, Schalen C. The epidemiology of severe

Streptococcus pyogenes associated disease in Europe. Euro Surveill 2005; 10: 179-84.


Med Biol 2008; 609: 29-42.

3 Cappelletty D. Microbiology of bacterial respiratory infections. Pediatr Infect Dis J 1998; 17: S55-

S61.

4 Stevens DL. Streptococcal toxic shock syndrome associated with necrotizing fasciitis. Annu Rev

Med 2000; 51: 271-88.

5 Carapetis JR, Steer AC, Mulholland EK, Weber M. The global burden of group A streptococcal

diseases. Lancet Infect Dis 2005; 5: 685-94.

6 Johnson DR, Stevens DL, Kaplan EL. Epidemiologic analysis of group A streptococcal serotypes

associated with severe systemic infections, rheumatic fever, or uncomplicated pharyngitis. J

Infect Dis 1992; 166: 374-82.

7 Herwald H, Morgelin M, Dahlback B, Bjorck L. Interactions between surface proteins of

Streptococcus pyogenes and coagulation factors modulate clotting of human plasma. J Thromb

Haemost 2003; 1: 284-91.

8 Kantor FS. Fibrinogen precipitation by streptococcal M protein. I. Identity of the reactants, and

stoichiometry of the reaction. J Exp Med 1965; 121: 849-59.

9 Lahteenmaki K, Kuusela P, Korhonen TK. Bacterial plasminogen activators and receptors. FEMS

Microbiol Rev 2001; 25: 531-52.

10 Coleman JL, Benach JL. Use of the plasminogen activation system by microorganisms. J Lab Clin

Med 1999; 134: 567-76.



and B. J Biol Chem 2007; 282: 24873-81.






91

Ch

apte

r 5



21833-8.




16 Bengtson SH, Sanden C, Morgelin M, Marx PF, Olin AI, Leeb-Lundberg LM, Meijers JC, Herwald H.

Activation of TAFI on the surface of Streptococcus pyogenes evokes inflammatory reactions by

modulating the kallikrein/kinin system. J Innate Immun 2008; 1: 18-28.

17 te Velde EA, Wagenaar GT, Reijerkerk A, Roose-Girma M, Borel Rinkes IH, Voest EE, Bouma BN,

Gebbink MF, Meijers JC. Impaired healing of cutaneous wounds and colonic anastomoses in mice

lacking thrombin-activatable fibrinolysis inhibitor. J Thromb Haemost 2003; 1: 2087-96.

18 Morser J, Gabazza EC, Myles T, Leung LL. What has been learnt from the thrombin-activatable

fibrinolysis inhibitor-deficient mouse? J Thromb Haemost 2010; 8: 868-76.

19 Nagashima M, Yin ZF, Broze GJ, Jr., Morser J. Thrombin-activatable fibrinolysis inhibitor (TAFI)

deficient mice. Front Biosci 2002; 7: d556-d568.

20 Nagashima M, Yin ZF, Zhao L, White K, Zhu Y, Lasky N, et al. Thrombin-activatable fibrinolysis

inhibitor (TAFI) deficiency is compatible with murine life. J Clin Invest 2002; 109: 101-10.

21 Virgin HW. In vivo veritas: pathogenesis of infection as it actually happens. Nat Immunol 2007; 8:

1143-7.

22 Taylor FBJr, Bryant AE, Blick KE, Hack E, Jansen PM, Kosanke SD, Stevens DL. Staging of the

baboon response to group A streptococci administered intramuscularly: a descriptive study of

the clinical symptoms and clinical chemical response patterns. Clin Infect Dis 1999; 29: 167-77.

23 Broder CC, Lottenberg R, von Mering GO, Johnston KH, Boyle MD. Isolation of a prokaryotic

plasmin receptor. Relationship to a plasminogen activator produced by the same micro-

organism. J Biol Chem 1991; 266: 4922-8.

24 Pancholi V, Fischetti VA. A major surface protein on group A streptococci is a glyceraldehyde-3-

phosphate-dehydrogenase with multiple binding activity. J Exp Med 1992; 176: 415-26.

25 Pancholi V, Fischetti VA. alpha-enolase, a novel strong plasmin(ogen) binding protein on the

surface of pathogenic streptococci. J Biol Chem 1998; 273: 14503-15.

26 Berge A, Sjobring U. PAM, a novel plasminogen-binding protein from Streptococcus pyogenes. J

Biol Chem 1993; 268: 25417-24.

Chapter 5

92

27 Sanderson-Smith ML, Dowton M, Ranson M, Walker MJ. The plasminogen-binding group A

streptococcal M protein-related protein Prp binds plasminogen via arginine and histidine

residues. J Bacteriol 2007; 189: 1435-40.

28 Campbell WD, Lazoura E, Okada N, Okada H. Inactivation of C3a and C5a octapeptides by

carboxypeptidase R and carboxypeptidase N. Microbiol Immunol 2002; 46: 131-4.

29 Cunningham MW. Pathogenesis of group A streptococcal infections. Clin Microbiol Rev 2000; 13:

470-511.

Murine TAFI improves survival against Streptococcus pyogenes Mercedes Valls Serón, Stefan R. Havik, Joris J.T.H. Roelofs, Charlotte Spaendonk, Heiko Herwald, Philip G. de Groot and Joost C.M. Meijers

Chapter 6

94

Abstract

Thrombin-activatable fibrinolysis inhibitor (TAFI) is synthesized in the liver and secreted into

the bloodstream in a zymogen form. TAFI is activated by thrombin, either free or in complex

with thrombomodulin. Active TAFI (TAFIa) is involved in down regulation of plasmin formation

and plays a role in inflammatory processes by inactivation of inflammatory mediators. We

investigated the role of TAFI in the haemostatic and immune response during infection of

mice with Streptococcus pyogenes. Wild-type and TAFI-knockout mice were infected using a

subcutaneous air-pouch model with S. pyogenes. Mice were terminated after 24 or 48 hours

or observed in a 5-day survival study. Our results showed that absence of TAFI did not have a

consistent effect on bacterial dissemination into systemic organs in the first 48 hours. In

addition, wild-type and TAFI-knockout presented no differences in activation of coagulation

or fibrinolysis, as reflected by comparable plasma levels of thrombin-antithrombin complexes

and D-dimers. TAFI-deficiency had no effect on lung, liver, spleen or kidney histopathology, or

cytokine levels in plasma. Remarkably, TAFI-knockout mice had increased susceptibility to

infection, with a 35% survival of TAFI-knockout compared to 87% of wild-type mice.

Immunohistochemistry revealed that both wild-type and TAFI-knockout mice accumulated

megakaryocytes in spleen during the 5-day infection. Megakaryocyte levels were significantly

lower in TAFI-knockout compared to wild-type mice, which was associated with mortality.

These data suggest that murine TAFI is necessary for survival after S. pyogenes infection and

that TAFI plays a role in megakaryopoiesis in spleen. In addition, megakaryocytes in spleen

may contribute to host defense towards bacteria.

mTAFI improves survival against S. pyogenes

95

Ch

apte

r 6

Introduction

Thrombin-activatable fibrinolysis inhibitor (TAFI) is a 56-kDa metallo-carboxypeptidase that is

predominantly synthesized in the liver and circulates in plasma as a zymogen. Upon activation

during coagulation by thrombin or the complex of thrombin with thrombomodulin, TAFI

becomes active (TAFIa) and modulates fibrinolysis by cleaving off the C-terminal lysine

residues from partially degraded fibrin. Plasminogen and tissue tissue-type plasminogen

activator (t-PA) bind to those lysine residues [1] and therefore removal of the lysines

attenuates the fibrin cofactor function of t-PA-mediated plasminogen activation, resulting in

less plasmin formation and reduced fibrinolysis [2]. TAFI can also be activated by plasmin,

trypsin, neutrophil elastase and meizothrombin [3-6].

TAFI activation occurs after proteolytic cleavage of the Arg92

-Ala93

bond. TAFIa is

characterized by a temperature-dependent instability, showing a spontaneous decay with a

half-life less than 10 minutes at 37°C or several hours at 22°C [7]. TAFIa’s self-destruction

mechanism was elucidated by the crystal structure [8,9], in which it was shown that a highly

dynamic structure is responsible for its own regulation.

Besides fibrin, a number of other TAFIa substrates have been described. TAFIa inactivates the

pro-inflammatory mediators bradykinin (BK), complement factors C3a and C5a, thrombin-

cleaved osteopontin and fibrinopeptide B [10-13].

TAFIa can therefore have two different effects during infection: regulating plasmin

generation, and inactivating inflammatory mediators. This dual function makes TAFI an

interesting protein to study in relation to infection.

Streptococcus pyogenes is a common bacterial human pathogen that causes a variety of

diseases affecting the skin or the upper respiratory tract [14,14]. These pathological

conditions are relatively mild such as impetigo and pharyngitis [14,15]. However, S. pyogenes

can cause invasive diseases such as toxic shock syndrome and necrotizing fasciitis which over

the last twenty years have been reported to increase world-wide [16]. Serious sequeale

following S. pyogenes infections are rheumatic fever and glomerulonephritis.

Although several studies have investigated the role of TAFI after LPS challenge [17,18] or

during sepsis induced by Gram-negative intact bacteria [19], the in vivo role of TAFI after

Gram-positive bacterial infection has not been investigated. In the present study, we analyzed

the role of TAFI during infection caused by S. pyogenes in TAFI-deficient mice.

Materials and Methods

Animals

C57BL/6 mice (wild-type) were supplied by Harlan (Venray, The Netherlands). TAFI-KO mice

(backcrossed at least 10 times to a C57BL/6 background) [20-22] were bred in the animal

facility at the Academic Medical Center, The Netherlands. Animals were kept on a controlled

12 h light/dark cycle and food and water were provided ad libitum. Experiments were carried

Chapter 6

96

with 8-12 weeks old male mice and all procedures were approved by the Institutional Animal

Care and Use Committee of the Academic Medical Center.

Experimental model of S. pyogenes infection

The S. pyogenes strain AP 41 (M41 type) was obtained from the Institute of Hygiene and

Epidemiology (Prague, Czech Republic). Bacteria were grown in Todd-Hewitt broth (BD

Bioscience, San Jose, CA, USA) at 37°C and an aliquot of these cells were added to fresh media

and grown up to the exponential phase of growth (OD620=0.5). The cells were washed three

times with saline and diluted to 0.5 to 3.7 x 108/ml in saline.

Wild-type and TAFI-KO mice were subjected to an air-pouch infection model. Briefly, 0.8 ml of

air and 0.2 ml of S. pyogenes in saline were injected with a 25-gauge needle subcutaneously

on the back of the mouse.

Mice were sacrificed 24 and 48 hours (16 per group) after infection or were observed in a

survival study for 5 days (40 per group). During the 5 days, animal survival was recorded in 40

animals per group; animals were monitored (4-6 times a day) for signs of disease such as

hunched posture, ruffed fur, and decreased responsiveness to stimuli. When severe disease

signs were observed (moribund), mice were humanely euthanized. Animals (n=20) terminated

at day 5 (survivors) or those that were terminated within the 5 day observation (non-

survivors) were included for analysis of histopathology. Eight mice injected with saline were

used as controls.

Sample harvesting

At the time of sacrifice (24 hours, 48 hours or during the 5-days), mice were first anesthetized

by inhalation of isoflurane (2L/min O2/2% volume isoflurane, Abbott Laboratories). Blood was

drawn after cardiac puncture into a sterile syringe containing citrate and immediately placed

on ice. Blood was centrifuged for 20 minutes at 2000 g. Plasma was stored at -80°C until use.

Thereafter, spleen, kidney, lung and liver were harvested and processed for measurements of

CFU, histology.

Determination of bacterial outgrowth

Spleen, kidney, lung and liver were homogenized at 4°C in 4 volumes of sterile isotonic saline

with a tissue homogenizer (Biospec Products, Bartlesville, OK, USA) which was carefully

cleaned and disinfected with 70% ethanol after each homogenization. Serial 10-fold dilutions

in sterile saline were made from these homogenates and blood. Thereafter, 50-µl volumes

were plated onto sheep-blood agar plates and incubated at 37°C. CFU were counted after

16 h.


97

Ch

apte

r 6

Assays

Interferon-γ, TNF-α, IL-6, IL-12, IL-10 and MCP-1 were measured by cytometric bead array

multiplex assay (BD Biosciences, San Jose, CA, USA) in accordance with the manufacturer’s

recommendations. For these cytokine measurements, harvested organs were homogenized

as described above. Sample homogenates were lysed in 1.25 volumes lysis buffer (300 mM

NaCl, 15 mM Tris [tris(hydroxymethyl) aminomethane], 2 mM MgCl2, 1% Triton X-100,

pepstatin A, leupeptin, and aprotinin (each at 20 ng/ml) (pH 7.4)) on ice for 20 min and spun

at 3600 g at 4°C for 10 min. The supernatant was frozen at -80°C until assayed. Thrombin-

antithrombin complexes (TATc; Siemens Healthcare Diagnostics, Marburg, Germany) and D-

dimer (Diagnostica Stago, Asnieres, France) were measured by ELISA.

Histopathology of mouse tissue samples

Directly after sacrifice spleen, kidney, liver and lung were fixed in 4% formalin. Tissue samples

were embedded in paraffin, sectioned (5-µm) and stained with haematoxylin and eosin (H-E)

using standard methods. Megakaryocytes were detected in spleen by immunohistochemistry

staining using an antibody against human von Willebrand factor (anti-vWF; Dakocytomation,

Glostrup, Denmark) as primary antibody. Briefly, sections were deparaffined and then

immersed in 3% hydrogen peroxide in methanol to block endogenous peroxidase for 20

minutes. Sections were then rinsed with PBS, blocked with 10% normal goat serum in PBS for

30 minutes and incubated overnight with the primary antibody in PBS.

After the overnight incubation, the sections were incubated with a secondary antibody (HRP

conjugated). Immune complexes were revealed via incubation with 3, 3’-diaminobenzidine

(DAB) and sections were counterstained with haematoxylin. Sections were examined using an

Olympus BX51 microscope and microscope fields were photographed with an Olympus DP70

camera. To quantify the number of megakaryocytes in spleen from wild-type and TAFI-KO

mice, vWF positive megakaryocytes were counted under the microscope in 10 randomly

chosen X 4 observation fields. For image analysis the freeware ImageJ v1.45a was used.

Statistical analysis

Differences between groups were calculated using the Mann-Whitney U test. Values were

expressed as means ±SE. A p value of < 0.05 was considered statistically significant. Survival

curves were analyzed by the Kaplan-Meyer log-rank test. All statistics were performed using

GraphPad Prism, version 5.01 (GraphPad Software, San Diego, CA, USA).

Chapter 6

98

Results

Bacterial outgrowth

To determine whether TAFI deficiency influences S. pyogenes dissemination, we compared

the bacterial load in spleen, kidney, lung, liver and blood of TAFI-KO and wild-type mice after

24 and 48 h induction of infection (Fig.1). After 24 and 48 h there were no differences in

bacterial loads in organs or blood between mouse strains, suggesting that at early time of

infection TAFI deficiency does not influence S. pyogenes AP41 spread.

Figure 1. Bacterial dissemination. Wild-type (●) and TAFI-KO mice (○) were injected with S. pyogenes

AP41 (1.4 to 3.7 x108/ml) administrated by subcutaneous injection. After 24 or 48 h mice were sacrificed

and the bacterial load in the spleen (A), kidney (B), liver (C), lung (D) and blood (E) was determined. Data

are expressed as CFU/ml of homogenated organ or blood from each animal with lines drawn at the

median for each group (n=16). The dotted horizontal line represents the detection limit. There were no

statistically significant differences between groups (Mann-Whitney test).

Activation of coagulation and fibrinolysis

To determine whether TAFI plays a role in the coagulant and fibrinolytic host responses, we

determined levels of TATc and D-dimer after 24 and 48 h S. pyogenes AP41 infection in

plasma. At 24 h TATc levels in TAFI-KO mice tended to be higher, although not significant,

compared to wild-type mice. After 48 h, TATc levels were not different between the mouse

strains (Fig.2A). D-dimer concentrations were increased after 24 h and 48 h compared to the

saline control, but were not different between wild-type and knockout animals (Fig. 2B).


99

Ch

apte

r 6

Fiure 2. Activation of coagulation and fibrinolysis. Levels of TATc (A) or D-dimer (B) in plasma at 24 and

48 hours after inoculation of S. pyogenes in wild-type mice (black bars) and TAFI-KO mice (white bars).

Data are expressed as means ± SE of n= 8 (saline group) or n=16 (infected group). There were no

statistically significant differences between groups (Mann-Whitney test).

Survival

To determine if TAFI has an impact on mortality during S. pyogenes infection, we performed a

survival study. The survival of the mice in the two groups over time is illustrated in Figure 3.

Despite having similar responses to infection at earlier time points (24, 48 h) as shown by

analysis of bacterial loads, coagulation, fibrinolysis and inflammation, TAFI-KO mice exhibited

significantly increased mortality towards S. pyogenes (p < 0.0001). At day 5 post-infection,

35% TAFI-KO animals had survived infection in contrast to 87% of wild-type mice.

Histopathology and inflammation

We examined the influence of TAFI deficiency on tissue damage after 24 h, 48 h and during a

5-day survival experiment where survivors and non-survivors were included for analysis.

Histopathology examination of organs from infected wild-type and TAFI-KO mice after 24 and

48 h showed no relevant inflammation (data not shown). In addition, cytokines and

Figure 3. Survival rates. Wild-type (●) and TAFI-KO

mice (○) were infected with 0.5 to 3 x108 CFU/ml

of S. pyogenes AP41 subcutaneously. Data are

pooled from three independent experiments, each

of which was conducted using 20, 10 and 10 mice

per group. Survival data are presented as a Kaplan-

Meier plot.

Chapter 6

100

chemokines in plasma were measured. Levels of interferon-γ, TNF-α and IL-12 were below the

detection limit in this model, while levels of IL-6, IL-10 and MCP-1 did not differ between wild-

type and TAFI-KO mice (data not shown).

Histopathology examination of kidney, liver and lung from infected wild-type and TAFI-KO

mice during the 5-day infection showed no relevant changes compared to the saline control

group (data not shown). In contrast, as shown by haematoxylin and eosin staining of paraffin

sections, the spleens from S. pyogenes AP 41 infected wild-type and TAFI-KO mice showed

evidence of an infiltration with large, multinucleated cells (Fig. 4), that were present in

occasional clusters within the red pulpa in both groups and morphologically appeared to be

megakaryocytes.

Figure 4. Histopathology of wild-type and TAFI-KO mice. Representative H-E stained sections of spleen

tissue from S. pyogenes-infected wild-type (A, B) and TAFI-KO spleen tissue (C, D). Infected animals were

injected with 1 to 3 x108 CFU/ml and followed for 5 days as indicated in methods. Magnifications are

indicated in each panel. Data were from two independent experiments, each of which was conducted

with 10 mice per group.

Immunohistochemical staining with antibodies against von Willebrand factor confirmed these

cells to be megakaryocytes (Fig. 5). The presence of megakaryocytes in spleen was suggestive

for extramedullary hematopoiesis. Spleens from TAFI-KO mice showed decreased numbers of

megakaryocytes (Fig. 5A,B), in comparison to spleens of wild-type mice (Fig. 5C,D). In spleens

from wild-type and TAFI-KO mice injected with saline (controls), megakaryocytes were not

observed (data not shown). Quantification of the megakaryocytes in spleen (Fig. 5E)

confirmed that TAFI-KO have lower MK numbers in spleen after S. pyogenes infection,


101

Ch

apte

r 6

suggesting that TAFI could play a role in extramedullary hematopoiesis. Furthermore, non-

survivors from both infected groups had lower megakaryocyte numbers in spleen compared

to survivors (Fig. 5E), indicating that extramedullary hematopoiesis may play an important

role in survival against S. pyogenes.

Figure 5. Immunohistochemical staining of spleens of wild-type and TAFI-KO mice. Representative

immunohistochemical staining for vWF of sections from S. pyogenes-infected wild-type spleen tissue (A,

B) and TAFI-KO mice (C, D). Infected animals were injected with 1 to 3 x108 CFU/ml and followed for 5

days. Original Magnifications are indicated in each panel. Arrows indicate megakaryocytes. (E)

Quantification of vWF positive megakaryocytes in spleen from wild-type (●) and TAFI-KO (■) mice after

S. pyogenes AP41 injection. Horizontal lines in the graphs represent the medians of the groups.

Representative images of two independent experiments, each of which was conducted with 10 mice per

group. Non survivors (open symbols) are indicated for both groups.

Discussion

Upon infection, many complex processes such as inflammation and activation of both the

coagulation and the fibrinolytic systems are compromised. TAFI represents a link between

coagulation and fibrinolysis and inactivates pro-inflammatory mediators, therefore it can be

expected that TAFI is involved in the response to infection. The present study was performed

to elucidate the role of endogenous TAFI during infection of the Gram-positive bacteria, S.

pyogenes.

Creation of TAFI-KO mice has enabled the possibility of studying the relevance of endogenous

TAFI in different experimental settings. Compared to wild type animals, the TAFI-KO mice

were normal in many respects, including survival, development, and fertility. In addition,

TAFI-KO displayed no bleeding disorders but as expected prolonged clot lysis time [20,23].

Chapter 6

102

Using S. pyogenes M41 serotype strain at a dose of 0.5 to 3 x108 CFU/ml, we observed 87%

survival in wild-type mice after 5 days. TAFI deficiency markedly decreased survival to 35%.

Our results suggested that the increased mortality observed in TAFI-KO might be due to

impaired regulation of the inflammatory mediators C3a and C5a leading to uncontrolled

inflammation. C5a has been suggested to play a role in the increased mortality when TAFI-KO

mice were challenged with both LPS and cobra venom factor [17]. However, we were unable

to demonstrate an altered local or systemic inflammatory response in TAFI-KO mice, as

evidenced by unchanged cytokine levels after 24 and 48 h S. pyogenes infection. In addition,

bacterial loads were similar after 24 and 48 h S. pyogenes infection between wild-type and

TAFI-KO mice. It seems likely that acceleration of the disease progression may have occurred

later than 48 h after S. pyogenes infection.

Upon infection, various hematopoietic factors promote hematopoiesis mainly in liver and

spleen (extramedullary hematopoiesis) [24]. It has been suggested that inadequate

extramedullary hematopoiesis leads to insufficient production or maturation of blood cells

[24]. Extramedullary hematopoiesis can occur as long as there are local production of

hematopoietic factors that maintain and induce differentiation of the hematopoietic stem

and progenitor cells (HSPCs). In our mouse model, megakaryocytes appearance correlated

with reduced mortality in both wild-type and TAFI-KO mice. Surprisingly, in the 20 animals in

which immunohistopathology was performed (Fig. 5), the TAFI-KO survivors only represented

25% compared to 80% in the wild-type mice, suggesting that TAFI may be involved in

extramedullary hematopoiesis leading to megakaryocyte migration and/or differentiation. To

our knowledge, a role for TAFI in these processes has never been described. A potential target

for TAFI in this process may be stromal cell-derived factor-1α (SDF-1α). SDF-1α also known as

C-X-C type chemokine ligand 12 (Cxcl12) is produced by various cell types including bone

marrow stromal cells, inflammatory cells, endothelial cells and osteoblasts [25]. SDF-1α plays

a role in regulating the retention of progenitor cells in hematopoietic tissues [26]. It has been

suggested that perturbation of the SDF-1α chemoattractant gradient by cleavage of the C-

terminal lysine residue of SDF-1 α by carboxypeptidase M can lead to mobilization of HSPCs

from bone marrow to peripheral blood [27]. This might facilitate entrance of megakaryocyte

precursors in peripheral organs such as the liver and spleen leading to megakaryocyte

differentiation. Therefore, TAFI activation during S. pyogenes infection, additionally to other

carboxypeptidases, could allow colonization of the spleen with bone marrow-derived stromal

cells by cleaving the C-terminal lysine of SDF-1 α. Enhanced TAFI activation could be the result

of increased TAFI levels upon infection. Both, TAFI mRNA and TAFI protein have been

reported to be upregulated during Escherichia coli-induced abdominal sepsis [19]. Moreover,

a local increased level of TAFI in the bone marrow can be achieved by a pool of TAFI present

in megakaryocytes [28,29].

The spleens of survivors from TAFI-KO and wild-type mice contained higher numbers of

megakaryocytes, suggesting that they are important for host defense against S. pyogenes. A

role for toll-like receptors (TLR) in megakaryocytes has recently been demonstrated. The TLR2


103

Ch

apte

r 6

that recognizes the lipoteichoic acid of gram-positive bacteria has been detected in human

megakaryocytes and lately, platelets which derive from megakaryocytes were shown to bind

and internalize pathogens and release microbial proteins [30,31]. In addition, many studies

have identified TLRs on platelets [32-36]. In line with our observation, extramedullary

hematopoiesis has been reported before in direct association with infection [37,38].

Recently, we reported the binding of human TAFI to two surface proteins of S. pyogenes, and

its subsequent activation at the bacterial surface via plasmin and the thrombin-

thrombomodulin complex [39]. The TAFI region involved in binding to the streptococcal

collagen-like surface proteins A and B (SclA and SclB) is Gly205

to Asp232

which is 99%

conserved in mouse TAFI compared to human TAFI (chapter 4). In addition, mouse TAFI also

binds the S. pyogenes serotype used in this study (data not shown). Because both mouse and

human TAFI interact with S. pyogenes, it is tempting to speculate that mouse TAFI can also be

used by S. pyogenes to its own benefit. However, TAFI-KO mice but not wild-type mice were

susceptible to S. pyogenes AP41 infection, suggesting that mouse TAFI is of importance for

defense during S. pyogenes infection.

In conclusion, we have shown that TAFI-KO mice are susceptible to S. pyogenes infection.

However, the etiology of the disease could not be clarified. It seems likely that TAFI could

contribute to protection of infection by facilitating extramedullary hematopoiesis that might

be used to counteract the infection.

Chapter 6

104

Reference List

1 Suenson E, Lutzen O, Thorsen S. Initial plasmin-degradation of fibrin as the basis of a positive

feed-back mechanism in fibrinolysis. Eur J Biochem 1984; 140: 513-22.

2 Redlitz A, Tan AK, Eaton DL, Plow EF. Plasma carboxypeptidases as regulators of the plasminogen

system. J Clin Invest 1995; 96: 2534-8.





21833-8.



6 Cote HC, Bajzar L, Stevens WK, Samis JA, Morser J, MacGillivray RT, Nesheim ME. Functional

characterization of recombinant human meizothrombin and Meizothrombin(desF1).

Thrombomodulin-dependent activation of protein C and thrombin-activatable fibrinolysis

inhibitor (TAFI), platelet aggregation, antithrombin-III inhibition. J Biol Chem 1997; 272: 6194-

200.

7 Boffa MB, Wang W, Bajzar L, Nesheim ME. Plasma and recombinant thrombin-activable

fibrinolysis inhibitor (TAFI) and activated TAFI compared with respect to glycosylation,

thrombin/thrombomodulin-dependent activation, thermal stability, and enzymatic properties. J

Biol Chem 1998; 273: 2127-35.




9 Anand K, Pallares I, Valnickova Z, Christensen T, Vendrell J, Wendt KU, Schreuder HA, Enghild JJ,

Aviles FX. The crystal structure of thrombin-activable fibrinolysis inhibitor (TAFI) provides the

structural basis for its intrinsic activity and the short half-life of TAFIa. J Biol Chem 2008; 283:

29416-23.

10 Shinohara T, Sakurada C, Suzuki T, Takeuchi O, Campbell W, Ikeda S, Okada N, Okada H. Pro-

carboxypeptidase R cleaves bradykinin following activation. Int Arch Allergy Immunol 1994; 103:

400-4.

11 Campbell WD, Lazoura E, Okada N, Okada H. Inactivation of C3a and C5a octapeptides by

carboxypeptidase R and carboxypeptidase N. Microbiol Immunol 2002; 46: 131-4.


105

Ch

apte

r 6

12 Myles T, Leung LL. Thrombin hydrolysis of human osteopontin is dependent on thrombin anion-

binding exosites. J Biol Chem 2008; 283: 17789-96.

13 Brummel KE, Butenas S, Mann KG. An integrated study of fibrinogen during blood coagulation. J

Biol Chem 1999; 274: 22862-70.

14 Cunningham MW. Pathogenesis of group A streptococcal infections. Clin Microbiol Rev 2000; 13:

470-511.

15 Cappelletty D. Microbiology of bacterial respiratory infections. Pediatr Infect Dis J 1998; 17: S55-

S61.

16 Stevens DL. Streptococcal toxic shock syndrome associated with necrotizing fasciitis. Annu Rev

Med 2000; 51: 271-88.

17 Asai S, Sato T, Tada T, Miyamoto T, Kimbara N, Motoyama N, Okada H, Okada N. Absence of

procarboxypeptidase R induces complement-mediated lethal inflammation in

lipopolysaccharide-primed mice. J Immunol 2004; 173: 4669-74.

18 Verbon A, Meijers JC, Spek CA, Hack CE, Pribble JP, Turner T, et al. Effects of IC14, an anti-CD14

antibody, on coagulation and fibrinolysis during low-grade endotoxemia in humans. J Infect Dis

2003; 187: 55-61.

19 Renckens R, Roelofs JJ, ter Horst SA, van ', V, Havik SR, Florquin S, Wagenaar GT, Meijers JC, van

der Poll T. Absence of thrombin-activatable fibrinolysis inhibitor protects against sepsis-induced

liver injury in mice. J Immunol 2005; 175: 6764-71.

20 te Velde EA, Wagenaar GT, Reijerkerk A, Roose-Girma M, Borel Rinkes IH, Voest EE, Bouma BN,

Gebbink MF, Meijers JC. Impaired healing of cutaneous wounds and colonic anastomoses in

mice lacking thrombin-activatable fibrinolysis inhibitor. J Thromb Haemost 2003; 1: 2087-96.

21 Nagashima M, Yin ZF, Broze GJ, Jr., Morser J. Thrombin-activatable fibrinolysis inhibitor (TAFI)

deficient mice. Front Biosci 2002; 7: d556-d568.

22 Nagashima M, Yin ZF, Zhao L, White K, Zhu Y, Lasky N, et al. Thrombin-activatable fibrinolysis

inhibitor (TAFI) deficiency is compatible with murine life. J Clin Invest 2002; 109: 101-10.

23 Morser J, Gabazza EC, Myles T, Leung LL. What has been learnt from the thrombin-activatable

fibrinolysis inhibitor-deficient mouse? J Thromb Haemost 2010; 8: 868-76.

24 Chang H Kim. Homeostatic and pathogenic extramedullary hematopoiesis. [1]. 2010. Journal of

Blood Medicine.

25 Tashiro K, Tada H, Heilker R, Shirozu M, Nakano T, Honjo T. Signal sequence trap: a cloning

strategy for secreted proteins and type I membrane proteins. Science 1993; 261: 600-3.

Chapter 6

106

26 Peled A, Grabovsky V, Habler L, Sandbank J, Arenzana-Seisdedos F, Petit I, Ben-Hur H, Lapidot T,

Alon R. The chemokine SDF-1 stimulates integrin-mediated arrest of CD34(+) cells on vascular

endothelium under shear flow. J Clin Invest 1999; 104: 1199-211.

27 Marquez-Curtis L, Jalili A, Deiteren K, Shirvaikar N, Lambeir AM, Janowska-Wieczorek A.

Carboxypeptidase M expressed by human bone marrow cells cleaves the C-terminal lysine of

stromal cell-derived factor-1alpha: another player in hematopoietic stem/progenitor cell

mobilization? Stem Cells 2008; 26: 1211-20.

28 Mosnier LO, Buijtenhuijs P, Marx PF, Meijers JC, Bouma BN. Identification of thrombin

activatable fibrinolysis inhibitor (TAFI) in human platelets. Blood 2003; 101: 4844-6.

29 Schadinger SL, Lin JH, Garand M, Boffa MB. Secretion and antifibrinolytic function of thrombin-

activatable fibrinolysis inhibitor from human platelets. J Thromb Haemost 2010; 8: 2523-9.

30 Tang YQ, Yeaman MR, Selsted ME. Antimicrobial peptides from human platelets. Infect Immun

2002; 70: 6524-33.

31 Klinger MH, Jelkmann W. Role of blood platelets in infection and inflammation. J Interferon

Cytokine Res 2002; 22: 913-22.

32 Montrucchio G, Bosco O, Del SL, Fascio PP, Lupia E, Goffi A, Omede P, Emanuelli G, Camussi G.

Mechanisms of the priming effect of low doses of lipopoly-saccharides on leukocyte-dependent

platelet aggregation in whole blood. Thromb Haemost 2003; 90: 872-81.

33 Cognasse F, Hamzeh H, Chavarin P, Acquart S, Genin C, Garraud O. Evidence of Toll-like receptor

molecules on human platelets. Immunol Cell Biol 2005; 83: 196-8.

34 Aslam R, Speck ER, Kim M, Crow AR, Bang KW, Nestel FP, et al. Platelet Toll-like receptor

expression modulates lipopolysaccharide-induced thrombocytopenia and tumor necrosis factor-

alpha production in vivo. Blood 2006; 107: 637-41.

35 Shiraki R, Inoue N, Kawasaki S, Takei A, Kadotani M, Ohnishi Y, et al. Expression of Toll-like

receptors on human platelets. Thromb Res 2004; 113: 379-85.

36 Ward JR, Bingle L, Judge HM, Brown SB, Storey RF, Whyte MK, Dower SK, Buttle DJ, Sabroe I.

Agonists of toll-like receptor (TLR)2 and TLR4 are unable to modulate platelet activation by

adenosine diphosphate and platelet activating factor. Thromb Haemost 2005; 94: 831-8.

37 Chiavolini D, Alroy J, King CA, Jorth P, Weir S, Madico G, Murphy JR, Wetzler LM. Identification of

immunologic and pathologic parameters of death versus survival in respiratory tularemia. Infect

Immun 2008; 76: 486-96.

38 Murray PJ, Young RA, Daley GQ. Hematopoietic remodeling in interferon-gamma-deficient mice

infected with mycobacteria. Blood 1998; 91: 2914-24.


107

Ch

apte

r 6



and B. J Biol Chem 2007; 282: 24873-81.

Summary and general discussion

Chapter 7

110

The coagulation and fibrinolytic systems are tightly regulated and protected against

dysfunction by various activators and inhibitors. During bacterial infections, both systems in

cooperation with the inflammatory system act in a very efficient way to contain and eliminate

the infection. However, bacteria have developed mechanisms during evolution to target such

systems in a specific manner to protect themselves against the host hemostatic and immune

response. In addition, bacteria are capable to benefit from components of the hemostatic

system in order to promote invasion.

In this thesis we have investigated novel interactions between TAFI and Gram-negative

bacteria, and we elaborated in molecular detail on how TAFI binds to the Gram-positive

Streptococcus pyogenes and how these interactions could influence the outcome of infection.

As an introduction to the thesis, in chapter 1 we describe how the pathogenic bacteria S.

pyogenes, Yersinia pestis and Salmonella enterica interact with the coagulation and the

fibrinolytic systems in order to confer the bacteria ability to establish major infection. Next, in

chapter 2 we extensively review one component of the fibrinolytic system, thrombin-

activatable fibrinolysis inhibitor (TAFI). The glycoprotein TAFI circulates in plasma as zymogen

and once activated acts as a carboxypeptidase B-like enzyme and cleaves C-terminal lysine

and arginine residues. Activated TAFI plays a role in down-regulating fibrinolysis but also has a

function in regulating inflammatory processes.

Interaction of TAFI with Y. pestis and S. enterica

Proteolysis plays an important role in the pathogenesis of bacterial infections. Bacterial

proteases target several host factors during infection to gain pathogenesis. Both, Y. pestis and

S. enterica express outer membrane proteases, omptins such as Pla and PgtE, on their

surface. Both bacterial species are highly invasive pathogens, and Pla and PgtE have been

identified as virulence factors [1-4]. In chapter 3 we investigated the effect of the omptins Pla

of Y. pestis and PgtE of S. enterica on TAFI.

We observed that adding recombinant E.coli expressing the omptins or live Y. pestis and S.

enterica bacteria to purified TAFI or plasma, resulted in reduced TAFI antifibrinolytic activity.

This prompted us to investigate the underlying mechanism. We showed that Pla and PgtE

degrade TAFI via proteolytic breakdown. TAFI was degraded at the C-terminal region and the

lysine analogue ε-ACA prevented cleavage suggesting that lysine residues are critical for the

cleavage of TAFI by these omptins. OmpT, an omptin of Escherichia coli, preferentially cleaves

its substrates after arginine or lysine thus it is possible that the cleavage occurs at lysine

residues.

Besides TAFI, Y. pestis and S. enterica target other components of the fibrinolytic system to

increase plasmin formation. Pla and PgtE activate plasminogen, degrade α2-antiplasmin and

inactivate PAI-1, and this may be beneficial for the bacteria to penetrate through tissues and

spread to distant organs.

Summary and discussion

111

Ch

apte

r 7

This is the first report demonstrating that bacterial proteases degrade TAFI and thus interfere

with TAFI activation. In line with our findings, it was shown recently that the protease InhA

secreted by Bacillus anthracis degraded TAFI in vitro. Injection of InhA to mice reduced TAFIa

activity in plasma [5].

Interaction of TAFI with S. pyogenes

TAFI binds to the surface of group A streptococci of an M41 serotype [6]. The interaction is

mediated by the streptococcal collagen-like surface proteins A and B (SclA and SclB), and the

streptococcal-associated TAFI can then be activated at the bacterial surface by plasmin or

thrombin-thrombomodulin. In chapter 4 we further investigated the TAFI binding

characteristics to SclA and SclB.

Using TAFI peptides, we identified the binding region involved in the interaction with SclA and

B between amino acids 205 to 232 of TAFI which are located distally from the TAFI catalytic

site. The Gly205 to Asp232 region is surface exposed and does not interfere with the region

known to influence TAFIa stability (Arg302, Arg320, Arg330, and Thr/Ile325), neither with the

residues involved in substrate binding (Gly336, Tyr341, and Glu363).

The amino acids in the Gly205 to Asp232 region responsible for the interaction were those

belonging to the so called glycosaminoglycan consensus motifs. The consensus sequence of

such a repeat is XBBXBX, where B stands for a basic residue and X is a non-basic residue.

The glycosaminoglycan consensus repeats (XBBBXXBX and XBBXBX) are found in some

proteins that bind glycosaminoglycans such as vitronectin, laminin and protein C inhibitor.

Therefore, it is reasonable to propose that S. pyogenes might target proteins with

glycosaminoglycan-binding sites as a conserved mechanism to recruit proteins for its own

benefit.

Additionally, we demonstrated that not only the TAFI zymogen bound to bacterial proteins

but also the active enzyme. Interestingly, TAFIa is easily dissociated from the bacterial

proteins. These results suggested that the conformation of the Scl-recognition domain had

been slightly changed upon TAFI activation. It is tempting to speculate that this constitutes a

mechanism whereby the bacteria attract TAFI to their surface, localize it there and

subsequently may allow activation of TAFI, whereafter the active enzyme easily dissociates.

Role of TAFI during S. pyogenes infection

In chapter 5 and chapter 6 we investigated the role of TAFI during S. pyogenes infection.

The highly host-specific nature of S. pyogenes in combination with the binding of TAFI to SclA

and SclB and subsequent activation at the bacterial surface, prompted us to investigate the

susceptibility to S. pyogenes infection of mice expressing human TAFI (chapter 5). To this end,

mice expressing only human TAFI were generated and infected with S. pyogenes serotype

M41. Introduction of human TAFI resulted in significantly increased mortality in response to S.

Chapter 7

112

pyogenes compared to wild-type mice. Our results demonstrated increased local activation of

coagulation and fibrinolysis in some organs after 24 h infection in TAFI-humanized transgenic

mice compared to wild-type mice. However, we were unable to demonstrate an altered local

or systemic inflammatory response in humanized-TAFI mice, as evidenced by unaltered

cytokine levels after 24 and 48 h S. pyogenes infection. In addition, after 24 and 48 h S.

pyogenes infection bacterial loads were not consistently altered.

Although our data cannot establish the etiology of the infection, it is striking that mice

expressing human TAFI are more susceptible to S. pyogenes infection. More research is

warranted to investigate the mechanisms by which TAFI contributes to S. pyogenes infection.

The study in chapter 6 was performed to elucidate the role of endogenous TAFI during

infection of S. pyogenes. Upon induction of infection, the mortality of TAFI deficiency

markedly increased compared to wild-type mice. However, there were no clear differences in

bacterial loads, coagulation, fibrinolysis and inflammation at early phase of infection. These

data indicate that endogenous TAFI did not modify host defense at early stage, but is of

importance for the final outcome of infection.

Interestingly, immunohistochemistry revealed that both wild-type and TAFI-knockout mice

accumulated megakaryocytes in spleen during a 5-day infection. Megakaryocyte levels were

significantly lower in TAFI-knockout compared to wild-type mice, suggesting that TAFI could

play a role in promoting hematopoiesis in spleen (extramedullary hematopoiesis).

Furthermore, non-survivors from both infected groups had lower megakaryocyte numbers in

spleen compared to survivors, indicating that megakaryocytes may play an important role in

survival against S. pyogenes.

It is unclear why we failed to observe clear differences in host defense after S. pyogenes

infection in both TAFI-humanized transgenic and TAFI-KO mice. A reason may include that

acceleration of the disease progression may have occurred later than for instance 48 h after S.

pyogenes infection.

Discussion

In this thesis we studied TAFI interactions with Gram-positive and Gram-negative bacteria.

Here we show that S. pyogenes targets TAFI through binding to glycosaminoglycan consensus

repeats. Whether this interaction is crucial for the increased susceptibility of TAFI-humanized

mice compared to wild-type mice is unclear since murine TAFI which has 85% homology with

human TAFI was found to bind S. pyogenes AP41. In addition, the glycosaminoglycan

consensus motifs involved in binding to SclA and SclB are 100% conserved in mouse TAFI

compared to human TAFI. Having established that both mouse and human TAFI interact with

S. pyogenes, it was to our surprise that TAFI-KO mice have reduced survival towards S.

pyogenes infection. An explanation could be that mouse TAFI is not as well activated as

human TAFI at the bacterial surface. This would also explain why transgenic mice expressing

both mouse and human TAFI had similar susceptibility to S. pyogenes during a 5-day survival


113

Ch

apte

r 7

experiment than TAFI-humanized mice (Figure 1). In this experiment we analyzed survival of

TAFI-transgenic mice expressing both mouse and human TAFI. The mortality rates of the 5-

day observation period were 5% for wild-type and 55% for TAFI transgenic mice. Therefore

we suggest that contribution of human TAFI to infection was predominant to the protective

effects of mouse TAFI (chapter 6) during S. pyogenes infection.

In chapter 5 we observed that human TAFI contributes to S. pyogenes infection. In contrast, in

chapter 6 we showed that murine TAFI protects against S. pyogenes infection. Our results

suggested that human and murine TAFI contribute differently to the infection. S. pyogenes

might gain survival advantage by interacting with human TAFI. Contrary, murine TAFI could

contribute to protection of infection by facilitating extramedullary hematopoiesis that might

be used to counteract against infection.

One of the most striking findings in Chapter 6 was that TAFI deficiency can impair survival in

mice with S. pyogenes. Recently, it was shown that TAFI-KO mice exhibited significantly

reduced survival following inoculation with the human bacterium Yersinia enterocolotica,

which is responsible of self-limiting enterocolitis but can also cause extraintestinal disorders,

including sepsis [7]. Bacterial counts in liver on days 3 and 5 were similar between wild-type

and TAFI-KO mice, but were increased on day 7 in spleen from TAFI-KO compared to wild-type

mice. In addition, levels of plasma D-dimer did not increase significantly in wild-type or TAFI-

KO mice infected with Y. enterocolotica. Interestingly, mice lacking both plasminogen

activator inhibitor-1 (PAI-1) and TAFI displayed the greatest susceptibility, followed

sequentially by PAI-1-KO and TAFI-KO mice. These findings suggested that both TAFI and PAI-1

deficiency can impair host defense against Gam-negative bacteria.

Similar to many other procarboxypeptidases, the TAFI zymogen is activated through cleavage

of an activation peptide to form activated TAFI (TAFIa), which is involved in downregulation of

plasmin formation. However, TAFIa is unique among carboxypeptidases in that it

spontaneously inactivates with a short half-life, a property that is essential for its function in

controlling blood clot lysis. The recent determination of the TAFI crystal structure revealed

Figure 1. Survival rates. Wild-type (■) and

TAFI-transgenic mice (□), expressing both

mouse and human TAFI were infected with

0.5 x108 CFU/ml of S. pyogenes AP41

subcutaneously. Survival data (n=20) are

presented as a Kaplan-Meier plot.

Chapter 7

114

the mechanism by which TAFI is able to autoregulate its activity [8,9]. The crystal structures

showed that TAFIa stability is directly related to the dynamics of a 55-residue segment

(dynamic flap). The dynamic flap is stabilized in the TAFI zymogen by interactions with the

activation peptide. Upon activation, release of the activation peptide increases dynamic flap

mobility and in time this leads to conformational changes that disrupt the catalytic site and

make TAFIa prone to inactivation and aggregation. This novel mechanism for enzyme

regulation may be an advantage to S. pyogenes which could benefit from both TAFI activity

and inactivation.

If SclA and SclB do indeed bind to and facilitate TAFI activation in vivo, this would seem to

counteract the pro-fibrinolytic effects of plasminogen activation by streptokinase. How can

these two opposite functions be reconciled? Perhaps S. pyogenes employs a strategy whereby

it initially promotes activation of the clotting system [10] to generate a protective fibrin

barrier around the infected area, and uses active TAFI to prevent fibrinolysis of the

surrounding fibrin network, thereby decreasing the chance of elimination by host

inflammatory cells at the site of infection (and possibly preventing premature dissemination

of the bacteria into the bloodstream). This period of safety from immune attack could then be

used by S. pyogenes to allow for multiplication of bacterial numbers and upregulation of

genes to overcome subsequent immune attack, followed by generation of a sufficiently high

local TAFI concentration. A similar argument could be made for Y. pestis where Pla activates

factor VII and degrades TFPI in vitro [11] leading to clot formation but on the other hand, in

this thesis we showed that Pla degrades TAFI which together with other Pla targets such as

plasminogen activation, α2-antiplasmin inactivation and PAI-1 degradation may promote

uncontrolled fibrinolysis. This scenario is supported by a recent study where B. anthracis, the

anthrax-causing pathogen, targets the host fibrinolytic system [5]. Such effects were achieved

by the bacterial proteases InhA and NprB which respectively degraded TAFI in vitro and in

mice, and activated human pro-urokinase plasminogen activator. The activation of fibrinolysis

by NprB and InhS may contribute to bleeding seen in anthrax disease. In contrast, another

study demonstrated that B. anthracis is able to initiate coagulation [12], and clustering of B.

anthacis was capable of directly activating prothrombin and factor X, thus bypassing the

initiators of the intrinsic (FXII) or extrinsic (TF-FVII) pathways. The molecular component

responsible for activating prothrombin and FX coagulation factors was InhA.

Taken all together, pathogenic bacteria may exploit pro-coagulant and pro-fibrinolytic factors

for its convenience to meet the demand for bacterial survival and proliferation. Importantly,

we underscored multiple ways that bacteria interact with the anti-fibrinolytic/anti-

inflammatory protein TAFI. However, we cannot exclude the possibility that other (unknown)

TAFI functions play a role during bacterial infection.


115

Ch

apte

r 7

Reference List

1 Degen JL, Bugge TH, Goguen JD. Fibrin and fibrinolysis in infection and host defense. J Thromb Haemost 2007; 5 Suppl 1: 24-31.

2 Lathem WW, Price PA, Miller VL, Goldman WE. A plasminogen-activating protease specifically controls the development of primary pneumonic plague. Science 2007; 315: 509-13.

3 Sodeinde OA, Subrahmanyam YV, Stark K, Quan T, Bao Y, Goguen JD. A surface protease and the invasive character of plague. Science 1992; 258: 1004-7.

4 Ramu P, Lobo LA, Kukkonen M, Bjur E, Suomalainen M, Raukola H, et al. Activation of pro-matrix metalloproteinase-9 and degradation of gelatin by the surface protease PgtE of Salmonella enterica serovar Typhimurium. Int J Med Microbiol 2008; 298: 263-78.

5 Chung MC, Jorgensen SC, Tonry JH, Kashanchi F, Bailey C, Popov S. Secreted Bacillus anthracis proteases target the host fibrinolytic system. FEMS Immunol Med Microbiol 2011; 62: 173-81.

6 Pahlman LI, Marx PF, Morgelin M, Lukomski S, Meijers JC, Herwald H. Thrombin-activatable fibrinolysis inhibitor binds to Streptococcus pyogenes by interacting with collagen-like proteins A and B. J Biol Chem 2007; 282: 24873-81.

7 Luo D, Szaba FM, Kummer LW, Plow EF, Mackman N, Gailani D, et al. Protective Roles for Fibrin, Tissue Factor, Plasminogen Activator Inhibitor-1, and Thrombin Activatable Fibrinolysis Inhibitor, but Not Factor XI, during Defense against the Gram-Negative Bacterium Yersinia enterocolitica. J Immunol 2011.

8 Marx PF, Brondijk TH, Plug T, Romijn RA, Hemrika W, Meijers JC, et al. Crystal structures of TAFI elucidate the inactivation mechanism of activated TAFI: a novel mechanism for enzyme autoregulation. Blood 2008; 112: 2803-9.

9 Anand K, Pallares I, Valnickova Z, Christensen T, Vendrell J, Wendt KU, et al. The crystal structure of thrombin-activable fibrinolysis inhibitor (TAFI) provides the structural basis for its intrinsic activity and the short half-life of TAFIa. J Biol Chem 2008; 283: 29416-23.

10 Herwald H, Morgelin M, Dahlback B, Bjorck L. Interactions between surface proteins of Streptococcus pyogenes and coagulation factors modulate clotting of human plasma. J Thromb Haemost 2003; 1: 284-91.

11 Yun TH, Cott JE, Tapping RI, Slauch JM, Morrissey JH. Proteolytic inactivation of tissue factor pathway inhibitor by bacterial omptins. Blood 2009; 113: 1139-48.

12 Kastrup CJ, Boedicker JQ, Pomerantsev AP, Moayeri M, Bian Y, Pompano RR, et al. Spatial localization of bacteria controls coagulation of human blood by 'quorum acting'. Nat Chem Biol 2008; 4: 742-50.

Nederlandse samenvatting

Acknowledgements



119

Ch

apte

r 8

Nederlandse samenvatting Na beschadiging van de vaatwand treedt een bloeding op. Voordat het wondgenezingsproces

kan starten moet het bloeden ophouden. Het mechanisme waardoor bloedingen worden

voorkomen is de bloedstolling, en de primaire functie van het stollingsproces is om stolsels te

vormen om zodoende verder bloeden te voorkomen. Het fibrinolytische systeem kan op zijn

beurt de stolsels weer oplossen zodat wondgenezing kan plaatsvinden. Zowel het

bloedstollingssysteem als het fibrinolytische systeem (samen worden ze ook wel de

hemostase genoemd) zijn krachtige enzymatische systemen en worden gereguleerd door

verschillende activatoren en remmers.

Tijdens bacteriële infecties worden beide systemen net zoals het immuunsysteem in werking

gesteld om de infectie op een efficiënte wijze te bestrijden. Echter, de bacteriën hebben

tijdens de evolutie allerlei mechanismen ontwikkeld die aangrijpen op deze systemen om zich

zodoende te beschermen tegen de hemostatische en immuunreactie van de gastheer. Sterker

nog, de bacterien zijn in staat om gebruik te maken van verschillende componenten van het

hemostatische systeem om verdere verspreiding te vergemakkelijken.

In dit proefschrift hebben we nieuwe interacties bestudeerd tussen een component van het

fibrinolytische systeem, de zogenaamde trombine-activeerbare fibrinolyse inhibitor of TAFI,

en bacteriën. De binding van TAFI aan een specifieke bacterie, Streptococcus pyogenes, werd

gekarakteriseerd, en vooral hoe deze interactie de uitkomst van infectie door de bacterie

beinvloedt.

Het proefschrift begint met een introductie: in hoofdstuk 1 wordt de interactie beschreven

tussen de stollings- en fibrinolytische sytemen en de bacteriën S. pyogenes, Yersinia pestis en

Salmonella enterica met de nadruk op hoe de bacteriën gebruik maken van de systemen van

de gastheer om infectie te bevorderen. Vervolgens wordt in hoofdstuk 2 een overzicht

gegeven over TAFI. TAFI is een zogenaamd proenzym, wat betekent dat het circuleert in

plasma in een inactieve vorm. Voordat het zijn functie kan utivoeren moet TAFI geactiveerd

worden door enzymen uit de stolling (trombine) of fibrinolyse (plasmine). Als TAFI eenmaal

geactiveerd is (TAFIa) dan speelt het een rol om fibrinolyse te verminderen, doordat het

eindstandige lysine en arginine residuen afknipt van fibrine. TAFIa heeft ook een regulerende

functie bij ontstekingen.

Interactie van TAFI met Y. pestis en S. enterica

Proteolyse of enzymatische activiteit speelt een belangrijke rol bij bacteriële infecties. De

bacteriën maken gebruik van verschillende gastheer eiwitten om verdere verspreiding

mogelijk te maken. Beide bacterien, Y. pestis en S. enterica, expresseren eiwitten op hun

membraan die omptins worden genoemd. Y. pestis heeft het Pla eiwit en S. enterica PgtE.

Beide soorten bacteriën zijn zeer infectieus en de Pla en PgtE eiwitten zijn belangrijke

virulentiefactoren [1-4]. In hoofdstuk 3 hebben we de effecten onderzocht van de omptins

Chapter 8

120

Pla en PgtE op TAFI. Door toevoeging van recombinante E. coli bacterien die de omptins op

hun oppervlak expresseren, of door gebruik te maken van levende Y. pestis en S. enterica

bacteriën werd duidelijk dat de omptins de TAFI activiteit en antifibrinolytische functie

remden doordat ze het TAFI eiwit degraderen. Dit was de eerste beschrijving in de literatuur

dat bacteriële eiwitten TAFI kunnen degraderen en op deze wijze konden interfereren met de

activatie en activiteit van TAFI.

Interactie van TAFI met S. pyogenes

TAFI bindt aan het oppervlak van groep A streptococcen van het M41 serotype [5]. Deze

interactie wordt bewerkstelligd door de streptococcal collageen-achtige oppervlakte eiwitten

SclA en SclB. Aan het oppervlak gebonden TAFI kan geactiveerd worden door plasmine of het

trombine-trombomoduline complex. In hoofdstuk 4 hebben we de binding van TAFI aan SclA

en SclB verder gekarakteriseerd.

Met behulp van peptiden van de TAFI sequentie hebben we de bindingsplaats voor de

interactie van SclA en SclB met TAFI vastgesteld tussen de aminozuren glycine 205 en

asparaginezuur 232. Deze bindingsplaats is op enige afstand van de actieve site van TAFIa.

Deze regio zit aan het oppervlak van het molecuul en heeft geen invloed op de aminozuren

die de stabiliteit van het geactiveerde TAFI bepalen (arginine 302, arginine 330, en

threonine/isoleucine 325) noch met de residuen die betrokken zijn bij substraat binding

(glycine 336, tyrosine 341 en glutaminezuur 363).

Interessant was dat TAFI niet alleen als proenzym aan het oppervlak van bacterien kan

binden, maar ook in de geactiveerde vorm. TAFIa dissocieerde wel makkelijker van de

bacteriële eiwitten vergeleken met TAFI. Deze resultaten suggereren dat de conformatie van

het domein wat aan de bacteriële eiwitten bindt iets veranderd bij activatie van TAFI.

Hierdoor ontstaat een aantrekkelijke hypothese dat de bacteriën TAFI aantrekken, binden en

activatie mogelijk maken van TAFI. Het actieve enzyme kan echter elders zijn functie gaan

uitoefenen omdat het ook weer makkelijk van de bacteriën loslaat.

Rol van TAFI tijdens S. pyogenes infectie

In hoofdstuk 5 en hoofdstuk 6 hebben we de rol van TAFI tijdens S. pyogenes infectie

onderzocht. Deze bacterie is nogal kieskeurig voor zijn gastheer en infecteert voornamelijk

mensen. Aangezien er binding is van eiwitten van de bacterie en het humane TAFI eiwit

hebben we onderzocht of het hebben van humaan TAFI in een muis model bijdraagt aan de

gevoeligheid voor infectie met S. pyogenes (hoofdstuk 5). Hiervoor werden muizen

gegenereerd die in plaats van het normale muis TAFI uitsluitend humaan TAFI expresseren.

Na infectie met het serotype M41 van S. pyogenes werd duidelijk dat de humaan TAFI

expresserende muizen in vergelijking met normale muizen veel gevoeliger waren voor infectie

wat leidde tot een verhoogde mortaliteit. Dit ging gepaard met een verhoogde locale


121

Ch

apte

r 8

activiteit van de stollings- en fibrinolytische systemen in sommige organen na 24 uur infectie.

Echter, we waren niet in staat om een veranderde locale of systemische ontstekingsresponse

te bepalen in de humaan TAFI expresserende muizen, en de cytokine niveaus waren na 24 en

48 uur vergelijkbaar aan normale muizen. Bovendien waren er geen verschillen in aantallen

bacteriën na 24 of 48 uur infectie.

Hoewel onze data niet kunnen aantonen waarom de muizen een verhoogde mortaliteit lieten

zien, is de bevinding van een verhoogde sterfte in humaan TAFI expresserende muizen zeker

interessant. Meer onderzoek is nodig om het precieze mechanisme te ontrafelen.

De studie die in hoofdstuk 6 is beschreven werd uitgevoerd om de rol van het muis TAFI

tijdens infectie door S. pyogenes vast te stellen. Hiervoor werden normale muizen vergeleken

met muizen waarin het TAFI gen was uitgeschakeld, zogenaamde TAFI knockout muizen. Na

infectie met S. pyogenes was de mortaliteit van TAFI knockout muizen hoger dan van normale

muizen. Echter, er waren geen duidelijke verschillen in aantallen bacteriën en stollings-,

fibrinolytische of ontstekingsparameters bij de vroege fase van infectie. Hieruit bleek dat het

normale muis TAFI geen rol speelt bij de bescherming van de gastheer bij vroege infectie,

maar wel van belang is bij de uiteindelijke uitkomst van infectie.

Een interessante bevinding tijdens het onderzoek kwam bij het bestuderen van de organen

van de muizen die geïnfecteerd waren. In de milt van geïnfecteerde muizen werd een

ophoping gezien van megakaryocyten. Deze cellen zijn normaal betrokken bij de productie

van bloedplaatjes in het beenmerg. De niveaus van megakaryocyten in de milt waren

significant lager in de TAFI knockout muizen in vergelijking met normale muizen. Dit

suggereert dat TAFI een rol kan spelen bij de bevordering van hematopoiese in de milt, ook

wel extramedullaire hematopoiese genoemd. Bovendien hadden de muizen die de infectie

niet overleefden lagere aantallen megakaryocyten in de milt in vergelijking met muizen de de

infectie wel overleefden. Hieruit blijkt dat megakaryocyten een belangrijke rol kunnen spelen

bij de overleving na een infectie met S. pyogenes.

Het is nog onduidelijk waarom er geen duidelijke verschillen in ontstekingsresponse werden

waargenomen na infectie van humaan TAFI expresserende muizen en TAFI knockout muizen.

Een mogelijke verklaring is dat de verslechtering van de gezondheid van de muizen vooral

optreedt na 2 dagen, het laatste tijdstip waarop de response op infectie werd beoordeeld.

Tenslotte, werden in hoofdstuk 7 de bevindingen in dit proefschrift samengevat en

bediscussieerd.

Chapter 8

122

Referenties

1 Degen JL, Bugge TH, Goguen JD. Fibrin and fibrinolysis in infection and host defense. J Thromb Haemost 2007; 5 Suppl 1: 24-31.

2 Lathem WW, Price PA, Miller VL, Goldman WE. A plasminogen-activating protease specifically controls the development of primary pneumonic plague. Science 2007; 315: 509-13.

3 Sodeinde OA, Subrahmanyam YV, Stark K, Quan T, Bao Y, Goguen JD. A surface protease and the invasive character of plague. Science 1992; 258: 1004-7.

4 Ramu P, Lobo LA, Kukkonen M, Bjur E, Suomalainen M, Raukola H, et al. Activation of pro-matrix metalloproteinase-9 and degradation of gelatin by the surface protease PgtE of Salmonella enterica serovar Typhimurium. Int J Med Microbiol 2008; 298: 263-78.

5 Pahlman LI, Marx PF, Morgelin M, Lukomski S, Meijers JC, Herwald H. Thrombin-activatable fibrinolysis inhibitor binds to Streptococcus pyogenes by interacting with collagen-like proteins A and B. J Biol Chem 2007; 282: 24873-81.

Ackowledgements

123

Ch

apte

r 8

Acknowledgements

Hereby, I would like to take the chance to thank everyone who contributed to the journey of

my PhD research. I am most lucky to have had enormous support along the way.

To my promotors: prof. dr. Joost Meijers and prof. dr. Philip de Groot. Beste Joost, thank you

for your guidance and for always being so enthusiastic and available for discussion of

(sometimes) challenging data. Beste Flip, I very much appreciate your supervision of this

thesis.

Bedank voor jullie (vele) hulp, steun bij het bedenken en opschrijven van dit proefschrift. Ik

heb veel van jullie geleerd!

My gratitude also goes to the members of the supervisory committee: Prof.dr. F. Leebeek,

Prof. dr. C. J. F van Noorden, Prof. dr. T. van der Poll, and Prof. dr. A. J. Verhoeven and in

particular to Prof. Erik Hack, for giving me the opportunity to start science in The Netherlands

(7 years ago).

To my colleagues at G1:

Thank you to Pauline who properly introduced me to the subject and has always been

available when I needed help and provided useful critique and guidance when it was most

necessary. Your depth of knowledge on both experimental and writing details is very much

appreciated.

Stefan, a big thank you for all your help but in particular for doing the animal experiments

with me. I enjoyed being “on vacation” at the AMC with you! 5 star room, delicious meals and

the latest movie at the cinema! What else did we want, ehh??

Tom, you always had an answer to ALL my questions. It was a pleasure to share our “TAFI-

issues”! Things were much easier thanks to your help. I wish you all the best!

Chantal, I enjoyed being your TAFI-PhD colleague and sharing all the conferences we attended

together!

Arnoud, I really appreciate your invaluable assistance in all laboratory related matters. Always

very enthusiastic to solve the unsolvable “challenges”!

Gwen and Çetin, we all started together and learned from each other. It was a pleasure to

share this journey with you.

A las latinas, Chiara y Joana. Muchas grácias (sobretodo) por esos momentos fuera del

laboratorio.

To Mirella, always up for a chat. Thank you for your support!

Jorge, Alinda, Han, Mauritz and Joram: thank you for all the fun in and outside the laboratory,

you created a great working atmosphere! And also to the rest of my colleagues from the EVG,

MEVG and DEVG, in particular to Wil who was always kind to help me with my experiments.

Chapter 8

124

Ad and Agnes: thank you for making the “non-lab work” easier. And in particular to Agnes at

the end, with all the forms and procedures linked to finishing my PhD, you were a great help!

I also thank the students (Joslyn Lardy, Sofie Verschueren and Charlotte van Spaendonck) for

their help.

Besides in the EVG department, my PhD has exposed me to so many other people that greatly

enriched the experience of working on my PhD thesis. Marcel Schouten, thank you for

dedicating your time to help me with setting up the animal experiments. From the

(Neuro)Pathology department: Rene Sersansie and Jasper Anik: you always found time to help

me. I learned a lot from you! Emanuele Zurolo and Anand Iyer: it was always fun to work

around you. To all the people from the University Medical Center in Utrecht: thank you for

your hospitality whenever I had to perform experiments there and for the fun times during

the conferences.

In addition, I would like to thank my collaborators and in particular to prof. dr. Heiko Herwald,

thank you for your hospitality and guidance throughout my doctoral project. Monica

Heidenholm: always so kind to help me. And I also would like to thank prof. dr. Artur

Schmidtchen and Martina Kalle for their hospitality and help. It was fun to work with you

Martina!

To Regina, Alida, Seema, Wing, Melissa and Ya-hui, thank you for being patient with my

schedules and adjusting always your times for our gezellige dinners. Thank you for

understanding!

To my paranimfs Stefi and Marta, thank you for being with me during these years but

specially for being with me during my thesis defense. Your help is very much appreciated!

A mis amigos Toni, Claudia, Douwe, Noelia, Olivier, Marta, Santi, Ester (y Sabina!), Rubén y

Yolanda MUCHAS GRÁCIAS por las cenas, los domingos “a la española” y por esas risas y

buenos momentos que me han hecho desconectar y empezar los lunes con más ganas de

trabajar! Vane, Marta y Noemi: muchas grácias por estar SIEMPRE a mi lado.

A mis herman@s, cuñad@s, cognati, suocero, mi schoonmoeder, tíos y primos, especialmente

a aquellos que han podido venir a mi presentación de tesis: muchas grácias por compartir este

día conmigo!

A mis padres, grácias por apoyarme siempre en mis decisions. Grácias a vosotros siempre he

tenido la voluntad de iniciar proyectos nuevos.

Y sobretodo muchas grácias a tí, Orlando, a pesar de no estar familiarizado con los términos

científicos siempre me has entendido y apoyado. Me allegro de tenerte día tras día a mi lado!


125

Ch

apte

r 8


Valls Serón M, Plug T, Marquart JA, Marx PF, Herwald H, de Groot PG, Meijers JCM.

Binding characteristics of Thrombin-Activatable Fibrinolysis Inhibitor to streptococcal

surface collagen-like proteins A and B. Thromb Haemost. 2011. Sep 27;106(4):609-16

Valls Serón M, Haiko J, de Groot PG, Korhonen TK, Meijers JC. Thrombin-activatable

fibrinolysis inhibitor is degraded by Salmonella enterica and Yersinia pestis. J Thromb

Haemost. 2010 Oct; 8 (10): 2232-40

Marx PF, Verkleij CJ, Valls Serón M, Meijers JMC. Recent developments in thrombin-

activatable fibrinolysis inhibitor research. Mini Rev Med Chem. 2009 Sep; 9 (10): 1165-73

Padilla ND, van Vliet AK, Schoots IG, Valls Serón M, Maas MA, Peltenburg EE, de Vries A,

Niessen HW, Hack CE, van Gulik TM. C-reactive protein and natural IgM antibodies are

activators of complement in a rat model of intestinal ischemia and reperfusion. Surgery.

2007 Nov; 142 (5): 722-33

http://www.ncbi.nlm.nih.gov/pubmed/10928463

http://www.ncbi.nlm.nih.gov/pubmed?term=%22Valls%20Ser%C3%B3n%20M%22%5BAuthor%5D

http://www.ncbi.nlm.nih.gov/pubmed?term=%22Haiko%20J%22%5BAuthor%5D

http://www.ncbi.nlm.nih.gov/pubmed?term=%22DE%20Groot%20PG%22%5BAuthor%5D

http://www.ncbi.nlm.nih.gov/pubmed?term=%22Korhonen%20TK%22%5BAuthor%5D

http://www.ncbi.nlm.nih.gov/pubmed?term=%22Meijers%20JC%22%5BAuthor%5D

http://www.ncbi.nlm.nih.gov/pubmed?term=%22Marx%20PF%22%5BAuthor%5D

http://www.ncbi.nlm.nih.gov/pubmed?term=%22Verkleij%20CJ%22%5BAuthor%5D

http://www.ncbi.nlm.nih.gov/pubmed?term=%22Valls%20Seron%20M%22%5BAuthor%5D

http://www.ncbi.nlm.nih.gov/pubmed?term=%22Meijers%20JC%22%5BAuthor%5D

http://www.ncbi.nlm.nih.gov/pubmed?term=%22Padilla%20ND%22%5BAuthor%5D

http://www.ncbi.nlm.nih.gov/pubmed?term=%22van%20Vliet%20AK%22%5BAuthor%5D

http://www.ncbi.nlm.nih.gov/pubmed?term=%22Schoots%20IG%22%5BAuthor%5D

http://www.ncbi.nlm.nih.gov/pubmed?term=%22Valls%20Seron%20M%22%5BAuthor%5D

http://www.ncbi.nlm.nih.gov/pubmed?term=%22Maas%20MA%22%5BAuthor%5D

http://www.ncbi.nlm.nih.gov/pubmed?term=%22Peltenburg%20EE%22%5BAuthor%5D

http://www.ncbi.nlm.nih.gov/pubmed?term=%22de%20Vries%20A%22%5BAuthor%5D

http://www.ncbi.nlm.nih.gov/pubmed?term=%22Niessen%20HW%22%5BAuthor%5D

http://www.ncbi.nlm.nih.gov/pubmed?term=%22Hack%20CE%22%5BAuthor%5D

http://www.ncbi.nlm.nih.gov/pubmed?term=%22van%20Gulik%20TM%22%5BAuthor%5D

Documents

UvA-DARE (Digital Academic Repository) Thrombin ... · Thrombin-activatable fibrinolysis inhibitor and bacterial infections General rights It is not permitted to download or to forward/distribute