ADAMTS13, TTP, and Beyond

X. Long Zheng, M.D., Ph.D.

The University of Alabama at Birmingham, Birmingham, AL

ASFA (April 21, 2017)

Disclosure of Conflict of Interests

• Consultant: Ablynx and BioMedica

Diagnostics

• Speaker’s Bureau: Alexion

• Research collaboration: Lee’s

Pharmaceuticals, Alexion and

Synapse

Outline

• Discovery of ADAMTS13

• Pathogenesis and potential triggers of

TTP

• Mechanism of autoantibody-mediated

inhibition of ADAMTS13 activity

• Diagnostic dilemma and potential novel

therapeutics

• Prospective

• 42 Y AAF presented with lethargy, blurred vision, and

disorientation.

• Thrombotic thrombocytopenic purpura

(TTP)

• Blood smear: >10 schistocytes

• Platelet 24,000 and Hb 9.6

• LDH 1,157, haptoglobin 0.09, creatinine: 1.6, PT and

PTT normal, and DAT (-)

• CT scan: multiple cerebral infarcts.

My first case

1924

A16-year-old adolescent girl

Abrupt onset of petechiae

Hemolytic anemia, followed by paralysis, coma, and

death

Autopsy showed widespread hyaline thrombi in the

terminal arterioles and capillaries of various organs.

Eli Moschcowitz (1924)

Moschcowitz’s disease, now

known as TTP

Moake et al. NEJM, 1982:307. 23:1432-35

Ultra large VWF multimers in patient

plasma with chronic relapsing TTP

Endo

thelia

l

TT

P a

ctive

TT

P R

em

Norm

al

UL

VWF

-500 KDa

-2000 KDa Dr. Joe Moake

- 1982 N

orm

al

TT

P

Lack of a VWF-depolymerase may be the cause of TTP.

Hypothesis:

Deficiency of a plasma VWF–cleaving protease

in patients with chronic relapsing TTP

Dr. Bernhard Lämmle

-1997

Furlan M et al. Blood 1997; 89:3097-3103

Asymptomatic

family members

Chronic

Relapsing TTP

Sample 1 2 1 2 1 2 1 2 1 2

NHP

Urea or

guanidine

Shear

VWF-cleaving

protease

IgG autoantibodies against VWF-cleaving

protease are responsible for acquired TTP

Tsai and Lian. NEJM, 1998

Pro

tease a

ctivity (

%)

Resid

ual a

ctivity (

%)

Acute Rem Acute Rem

Dr. Han-Mou Tsai

- 1998

IgG fraction isolated from acquired TTP patients inhibited

VWF-cleaving protease activity.

200-

150-

100-

50-

0-

200-

150-

100-

50-

0-

Plasma VWF-cleaving protease is essential for

maintaining normal hemostasis

VWF-cleaving

protease

LM Morning Report

Washington University, 1999

Deficiency of VWF-cleaving activity results in

disseminated microvascular thrombosis

Hereditary

(<5%)

Blood Flow

LM morning report

Washington University, 1999

Autoantibodies

(>95%)

Microvascular

thrombosis

Exposure of

subendothelial matrix

What is VWF-cleaving protease?

Structure of von Willebrand Factor-cleaving

Protease (ADAMTS13), a Metalloprotease Involved

in Thrombotic Thrombocytopenic Purpura*

Xinglong Zheng‡§, Dominic Chung§¶, Thomas K. Takayama‖, Elaine M.

Majerus**, J. Evan Sadler**‡§§ and Kazuo Fujikawa¶

Departments of ‡Pathology, **Medicine, and §§Biochemistry and Molecular

Biophysics and Howard Hughes Medical Institute, Washington University

School of Medicine, St. Louis, Missouri 63110 and the Departments of ¶Biochemistry and ‖IUrology, University of Washington, Seattle, Washington

98195

J Biol Chem. 2001 Nov 2;276(44):41059-63. Epub 2001 Sep 13.

A year of breakthrough in

TTP research

Gerritsen HE et al. Partial amino acid sequence of purified

von Willebrand factor-cleaving protease. Blood. 2001; Sep

15.

Soejima K, et al. A novel human metalloprotease

synthesized in the liver and secreted into the blood: possibly,

the von Willebrand factor-cleaving protease? J Biochem.

2001, Oct 1.

Levy GG, et al. Mutations in a member of the ADAMTS gene

family cause thrombotic thrombocytopenic purpura. Nature.

2001; Oct 4.

1 3 6 7 9 12 15 17 20 22 24 28 29

5kb

A Disintegrin And Metalloprotease with ThromboSpondin

type 1 repeats (ADAMTS), 13

Zheng et al. J. Biol. Chem. Sept. 13, 2001

ADAMTS13

CUB1 S P Metallo- protease Dis 1 Cys Spacer 2 3 4 5 6 7 8 CUB2

RQRR ★★ ★★★★ ★ ★ ★ ★

O-glycosylation ★ N-glycosylation

1,427 amino acids 145 -190 kDa

Summary of the discovery of ADAMTS13

-19

24

1st case

description

Moschcowitz

-20

01

Identification

& cloning of

ADAMTS13

Zheng

Gerritsen

Fujikawa

Soejima

Levy

TTP

-19

82

Ultra large

VWF

Moake

-19

78

-19

60

Plasma

factor

deficiency

Schulman

Upshaw -1

99

7

-19

98

VWF-cp &

autoantibody

Furlan

Lammle

Tsai

Pathogenesis of TTP

Hereditary TTP: - ADAMTS13 mutations (>200)

Acquired TTP: - Autoantibodies against ADAMTS13

• 59% missense • 13% nonsense • 13% deletions • 6% insertions • 9% alternative splicing

Age of onset for the 1st

thrombocytopenia in patients

with hereditary deficiency of ADAMTS13 varies greatly

Fujimura et al. ADAMTS13 Biology and Disease

ed. By G.M. Rodgers, Springer 2015

Early onset (75%)

Late onset (25%)

Other genetic or environmental factors may contribute to the

pathogenesis of TTP

All patients except for one have ADAMTS13 activity < 0.5%

Anti-ADAMTS13 IgG is present in all

patients with acquired TTP at UAB

Cao W, Pham HP, Williams III LA, McDaniel JK, Siniard RC, Lorenz RG,

Marques MB, and Zheng XL. Haematologica. 2016;101:1319-1326

In 52 patients with ADAMTS13<5%, 46 patients had inhibitor >0.4 U/ml (positive), but 6 patients <0.4 U/ml (negative).

However, inhibitors are not always demonstrated with functional assays.

0

2,000

4,000

6,000

8,000

10,000

12,000

14,000

16,000

0.0 1.0 2.0 3.0 4.0 5.0 6.0

An

ti-A

DA

MT

S13 I

gG

(U

/ml)

<0.4 0.4-1.2 1.3-2.4 2.5—5.0 >5.0

Titer (U/ml)

Anti-ADAMTS13 IgG was detected in TTP

patients with inhibitor<0.4 U/ml

Cao et al. Haematologica, 2016

Normal

How do IgG autoantibodies inhibit

ADAMTS13 activity?

Casina et al, PNAS, 2015, Aug 4;112:9620-5

Five small flexible loops in the spacer domain are

specifically targeted by autoantibodies from TTP patients.

Anti-A13

v v v v v v v CUB TSP1 2-8

Veronica Casina Ph,D

Potential triggers

• Infection or system inflammation

• Complement amplification conditions

• Pregnancy

• Certain medications

• Additional genetic factors

These are the same triggers for hemolytic

uremic syndrome (HUS)

I have low ADAMTS13

“Second Hit”

Human neutrophil peptides (HNPs) are

dramatically elevated in acquired TTP

HNPs

MPO

Histone-DNA

Cao W, Pham HP, Williams III LA, McDaniel JK, Siniard RC, Lorenz RG,

Marques MB, and Zheng XL. Haematologica. 2016;101:1319-1326

Fuchs et al. Blood 2012:120:1157-64

Plasma nucleosomes and DNA are

markers of acute TMAs

Plasma levels of complement

factor Bb increase in acquired TTP

C3

C5

FH FI MCP

FB FD

C5b6789

Cao W, Pham HP, Williams III LA, McDaniel JK, Siniard RC,

Lorenz RG, Marques MB, and Zheng XL. Haematologica.

2016;101:1319-1326

How to distinguish TTP from aHUS

clinically?

• Platelets < 30,000/μl

• Normal or near normal

renal function

• Response to plasma

exchange

• ADAMTS13 activity

(<10%)*

• Less severe

thrombocytopenia

• Significant renal

dysfunction

• Lack of complete response

to plasma exchange

• Normal ADAMTS13

(>10%)

*ADAMTS13 activity is the single most important test to

distinguish TTP from aHUS at the present time.

Bentley. ADAMTS13 Biology and Disease

ed. By G.M. Rodgers, Springer 2015

How is ADAMTS13 test performed?

Tyr-Met

Met

Plasma (or ADAMTS13)

Normal

TTP Patient

Time (min)

Rat

e o

f fl

uo

resc

ence

ge

ner

atio

n (

AU

/se

c)

FRETS-VWF73

Kokame K et al. Br J Haematol. 2005, 129:93-100.

ADAMTS13 activity measured by an ELISA

10

63

10

Current & future therapies

• Hereditary TTP

Plasma infusion vs.

Recombinant ADAMTS13 or

AAV gene therapy.

• Acquired TTP

Plasma exchange & immunotherapy

Other potential novel therapies

Dr. Paul Coppo

Plasma infusion resulted in an intermittent

recovery of platelet counts in hereditary TTP

Furlan & Lammle. Best Pract. Res. Clin. Haematol. 2001, 14, 437-454

AAV8-mediated liver expression of rADAMTS13

results in cure of hereditary TTP in mice

B

Jin et al. Blood. 2013,121(19):3825-9

0 14

Shigatoxin

i.v.

CBC

(Days) Adamts13-/-

ADAMTS13

activity AAV8-

mMDTCS

i.v.

7 21

Sheng-yu, MD, PhD

Kaplan-Meir Survival Curve

Jin et al. Blood. 2013,121(19):3825-9

AAV8-hAAT-LacZ 2.6x1011 vg/kg

Surv

ival

rat

e (%

)

AAV8-hAAT-mMDTCS

2.6x1011 vg/kg

Stx-2

Antibody-resistant rADAMTS13

T633

R636

P590

L591

T669 Y661F R660K

Y665F

β1

β10

β3

β8 β7 β6

β7-β8

loop

β9-β10

loop R568K

F592Y

Motif A

Molecular Surgery to ADAMTS13

Cleavage of FRETS-VWF73 by WT and rADAMTS13 variants

rADAMTS13: M4 and M5 exhibit increased activity by 4-5 fold

Jian et al, Blood, 2012; 119:3836-43.

Cui Jian

rADAMTS13-M4 and M5 were more resistant than WT to

autoantibody from acquired TTP

rADAMTS13

+ normal or patient

plasma

37C, 60 min

Residual activity N: normal human plasma

P: TTP patient plasma

ADAMTS13 variants are resistant to ≈85% TTP patient plasma IgGs.

Jian et al, Blood, 2012; 119:3836-43.

The autoantibody-resistant (ar)

ADAMTS13 may be used to treat acquired

TTP without plasma exchange.

arADAMTS13

VS.

Apheresis

Anti-ADAMTS13

IgG

Thrombin

Quiescent

Platelets

VWF

multimers

ADAMTS13

Cleaved VWF

& platelets

ADP

Collagen

Hypothesis:

Shear

Activated

Platelets

Delivery of rADAMTS13 via platelets

Can human platelets uptake

rADAMTS13 in vitro?

25

rADAMTS13 (ng)

0 20 40 80

rA13 (μg/ml)

230-

180-

116-

66-

40-

12-

(kDa) 0 20 40 80

rA13 (μg/ml)

37 °C 25 °C

50 100 12.5 0

Donor platelets + rADAMTS13

A13 VWF

Abdelgawwad, et al. unpublished

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

0 25 50 75 100 125 150 175

Pla

tele

t C

ove

rag

e

Time (Seconds)

Control

Platelets

0

0.2

0.4

0.6

0.8

1

0 25 50 75 100 125 150 175

Pla

tele

t C

ove

rag

e

Time (Seconds)

Control

Platelets

N=3

N=3

PBS

Plts

w/rA13

50 μg/ml

PBS

Plts

w/rA13

100 μg/ml

Platelet-delivered rADAMTS13 is efficacious in

reducing the rate of thrombus formation under shear

Abdelgawwad, et al. unpublished

Moh

Transgenic mice expressing rADAMTS13

in megakaryocytes and platelets in mice

TGPlt+A13

Adamts13-/-

Pickens et al. Blood, 2015,125(21):3326-34

Brandy Pickens, PhD

Genotype Number Platelet count (×109/L)

TG 20 896 ± 156**

KO 20 631 ± 93

WT 16 708 ± 179

The data presented are the mean ± SD.

TG, Adamts13−/−PltA13; KO, Adamts13−/−; WT, wild-type.

** P < .001 when compared with KO and WT mice, respectively.

Higher baseline platelet counts in

transgenic mice than in Adamts13-/- and

wild- type mice

Pickens et al. Blood, 2015,125(21):3326-34

Platelet-delivered rADAMTS13 reduces the rate

of thrombus formation in mesenteric arteriole

after FeCl3 injury

0

2

4

6

8

10

12

14

01:22.9 06:22.9 11:25.5 16:25.5 21:26.3 26:26.8 31:26.8

Th

rom

bu

s F

orm

ati

on

(C

han

ge i

n f

lou

rescen

ce)

KO

WT

TG

Time (min)

Pickens et al. Blood, 2015,125(21):3326-34

Platelet-delivered rADAMTS13 significantly reduced

thrombocytopenia and mortality rate in the presence

of anti-ADAMTS13 IgG

B 10 μg/gram rVWF

*****

Pickens et al. Blood, 2015,125:3326-34

C

0 14

rVWF (i.v.)

CBC

(Hours)

scFv4-20 (i.p.)

7 24

ADAMTS13 activity

WT vs. TGPtA13

A

Take home…...

• Identification of ADAMTS13 provides a new avenue for studying the mechanism, diagnosis, and treatment of TTP.

• Mapping studies let us identify the critical

region in ADAMTS13 where autoantibody

bind, which shed new light on the

mechanism of autoantibody-mediated

inhibition of ADAMTS13 activity.

• rADAMTS13 and AAV8-based gene therapy

may become the treatment of choice for

hereditary TTP.

• arADAMTS13 variants, platelet-delivery of

rADAMTS13, as well as antagonists for

VWF-platelet interaction may be further

developed as novel therapeutics for

acquired TTP.

• Whether anti-complement therapy is needed

or not for a subset of acquired TTP patients

is yet to be determined.

Prospective

Low ADAMTS13 is associated with many thrombotic

and inflammatory diseases:

Ischemic cerebral infarction

Myocardial infarction

Preeclampsia/eclampsia

Malignant malaria

Septic DIC and ICU patients with renal failure

Trauma-associated mortality.

Acknowledgement

Marisa Marques, M.D.

Lance Williams, M.D.

Huy P. Pham, M.D., M.P.H.

Robin Lorenz, M.D., Ph.D.

Bruce Sachais, MD, PhD.

Don Siegel, M.D., Ph.D.

Douglas Cines, M.D.

Walter Englander, Ph.D.

Adam Miszta, PhD, MSc. Eng.

Bas de Laat, Ph.D.

Current Wendy Cao, M.D., Ph.D.

Vikram Pillai, Ph.D.

Catherine Zander, Ph.D.

Liang Zheng, Ph.D.

Jenny McDaniel, M.D.

Jay Li, Ph.D.

Nicole Kocher, B.S.

Moh Abdelgawwad, M.D., M.S.

05.07.2016

Grant supports: NBF, AHA, and NIH-R01s

Collaborators