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Factor H binds to washed human platelets.

Vaziri-Sani, F; Hellwage, J; Zipfel, P F; Sjöholm, Anders; Iancu, Ruxandra; Karpman, Diana

Published in:Journal of Thrombosis and Haemostasis

DOI:10.1111/j.1538-7836.2004.01010.x

2005

Link to publication

Citation for published version (APA):Vaziri-Sani, F., Hellwage, J., Zipfel, P. F., Sjöholm, A., Iancu, R., & Karpman, D. (2005). Factor H binds towashed human platelets. Journal of Thrombosis and Haemostasis, 3(1), 154-162. https://doi.org/10.1111/j.1538-7836.2004.01010.x

Total number of authors:6

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ORIGINAL ARTICLE

Factor H binds to washed human platelets

F . VAZ IR I - SAN I , J . HELLWAGE ,* P . F . Z I PFEL ,* A . G . S J OHOLM,� R . IANCU and D . KARPMANDepartment of Pediatrics, Lund University, Lund, Sweden; *Molecular Immunobiology Group and Department of Infection Biology, Hans Knoell

Institute for Natural Products Research, Jena, Germany; and �Institute of Laboratory Medicine, Section for Microbiology, Immunology and

Glycobiology, Lund University, Lund, Sweden

To cite this article: Vaziri-Sani F, Hellwage J, Zipfel PF, Sjoholm AG, Iancu R, Karpman D. Factor H binds to washed human platelets. J Thromb

Haemost 2005; 3: 154–62.

Summary. Background: Factor H regulates the alternative

pathway of complement. The protein has three heparin-binding

sites, is synthesized primarily in the liver and copurifies from

platelets with thrombospondin-1. Factor H mutations at the

C-terminus are associated with atypical hemolytic uremic

syndrome, a condition in which platelets are consumed.

Objectives The aim of this study was to investigate if factor H

interacts with platelets. Methods: Binding of factor H, recom-

binantC- orN-terminus constructs and aC-terminusmutant to

washed(plasmaandcomplement-free)plateletswasanalyzedby

flow cytometry. Binding of factor H and constructs to

thrombospondin-1 was measured by surface plasmon reson-

ance.Results: Factor H bound to platelets in a dose-dependent

manner. Themajor binding sitewas localized to theC-terminus.

The interaction was partially blocked by heparin. Inhibition

with anti-GPIIb/IIIa, or with fibrinogen, suggested that the

platelet GPIIb/IIIa receptor is involved in factor H binding.

Factor H binds to thrombospondin-1. Addition of thrombo-

spondin-1 increased factor H binding to platelets. Factor H

mutated at the C-terminus also bound to platelets, albeit to a

significantly lesser degree. Conclusions: This study reports a

novel property of factor H, i.e. binding to platelets, either

directly via the GPIIb/IIIa receptor or indirectly via thrombo-

spondin-1, in the absence of complement. Binding to platelets

was mostly mediated by the C-terminal region of factor H and

factor Hmutated at the C-terminus exhibited reduced binding.

Keywords: complement, factor H, hemolytic uremic syn-

drome, platelets.

Introduction

Factor H (FH) is a glycoprotein known to play a regulatory

role in the activation of the alternative pathway of complement.

This plasma protein prevents formation of the C3bBb conver-

tase by competing with factor B and destabilizes the formed

convertase by displacing factor B fromC3b. It also functions as

a cofactor for the proteolytic cleavage of C3b by factor I

resulting in the formation of iC3b [1]. FH consists of 20 short

consensus repeat (SCR) elements [2]. There are three heparin-

binding sites located within SCRs 7, 12–14 and 20 as well as

three C3b binding sites at SCRs 1–4, 10–15 and 20 [2–4]. FH

circulates in human plasma as a 150-kDa protein, at a

concentration of about 500 lg mL)1. The FH-like protein-1

(FHL-1) is a 42-kDa protein which consists of SCRs 1–7, and is

derived from the FH transcript by means of alternative

splicing. FH and FHL-1 are encoded by a single gene on

chromosome 1q32 [5] and both proteins have complement

regulatory functions [6].

Homozygous mutations of FH have been found in some

patients with membranoproliferative glomerulonephritis and

atypical hemolytic uremic syndrome (HUS) (reviewed in [2,7]).

Heterozygous FH mutations have been identified in a

subgroup of patients with atypical HUS [7–12] and a hot-spot

was recognized within SCR 20 [9]. HUS is characterized by a

triad of microangiopathic hemolytic anemia, thrombocytope-

nia and renal failure [13]. The typical form has been associated

with infections caused by Shiga toxin producing bacteria in

which HUS is usually preceded by a diarrheal prodrome,

whereas the atypical form has heterogeneous etiologies [13].

Endothelial cell injury is pivotal to the pathogenesis of HUS

[14]. Thrombocytopenia ensues due to platelet consumption in

microthrombi, presumably secondary to endothelial cell dam-

age and exposure of the subendothelium. The mechanisms by

which FHmutations may lead to HUS are poorly understood.

It has been postulated that complement activation may

propagate endothelial cell injury [15] and that alterations in

FH may thus lead to vascular damage [2,16,17]. A direct

interaction of FH with platelets has not been previously

studied.

FH is mainly synthesized in the liver, but also in monocytes,

fibroblasts, mesangial cells and endothelial cells as summarized

by Friese et al. [6]. FH has also been localized to the a-granulesof platelets from where it is released upon stimulation with

thrombin [18] or upon binding of C3 to the platelet surface [19].

Correspondence: D. Karpman, Department of Pediatrics, Lund

University, 22185 Lund, Sweden.

Tel.: +46 46 222 0747; fax: +46 46 222 0748; e-mail: diana.karpman@

pedi.lu.se

Received 24 March 2004, accepted 16 July 2004

Journal of Thrombosis and Haemostasis, 3: 154–162

� 2004 International Society on Thrombosis and Haemostasis

Furthermore, FH was found to copurify from platelets with

thrombospondin-1 (tsp-1) [20], a glycoprotein with a well-

characterized role in platelet aggregation [21]. Since FH

mutations have been associated with HUS, a condition in

which platelet activation is a main manifestation, and since FH

is released from platelets, we postulated that native FH

interacts directly with platelets. The aim of this study was

therefore to investigate binding of normal and mutated FH to

washed platelets in the absence of complement.

Materials and methods

FH, recombinant constructs and mutant

Wild-type FH was obtained from Calbiochem (La Jolla, CA,

USA). Recombinant histidine-tagged deletion constructs SCR

1–7 (FHL-1), SCR 8–11, SCR 8–20, SCR 15–20 were cloned

and expressed in the baculovirus system and purified as

described [22]. Recombinant fragments SCRs 1–7, 8–11, 8–

20, 15–20 and SCR 15–20mut, histidine-tagged and mutated in

SCR 20 at positions R1203E, R1206E, R1210S, K1230S and

R1231A, with reduced binding to C3b and heparin, have been

previously described [4]. The bioactivity of FH fragments,

which are to a certain extent glycosylated [23–25], is comparable

to the wild-type [17,23,26]. For certain experiments FH was

labeled with Alexa555 (Molecular Probes, Leiden, the Nether-

lands) or 125I. Purity of the proteins (> 95%)was confirmed by

sodium dodecyl sulfate–polyacrylamide gel electrophoresis

(SDS–PAGE) and by silver staining and no contamination of

FHwith tsp-1 was foundwhen immunoblottingwas performed

with rabbit anti-tsp-1 antibody (1 : 100; Calbiochem).

Platelet-rich plasma and washed (plasma-free) platelets

Venous blood was collected from 44 healthy adult volunteers

(22 males and 22 females) not using any medications and

platelet-rich plasma (PRP) obtained as described [27]. Platelets

were washed of plasma components [27] and resuspended in

1% bovine serum albumin (BSA; ICN Biomedicals, Aurora,

OH, USA) in Hank’s balanced salt solution (HBSS without

phenol red; Invitrogen, Paisley, UK). The CaCl2 concentration

was adjusted to 2.5 mM. Platelets were diluted to a final

concentration of 1 · 107 mL)1 in 1% BSA–HBSS.

Thrombin activation of platelets leads to degranulation and

increases the expression of receptors and ligands on the platelet

surface such asGPIIb/IIIa and P-selectin [28].Washed platelets

were, in certain experiments, activated with thrombin (Sigma,

St Louis, MO, USA) at a final concentration of 1 U mL)1 for

2 min at 25 �C, washed and fixed as described [27]. CD62P

(P-selectin) expression was used as an indicator of platelet

activation. Thrombin induced a 14% increase in CD62P

expression on platelets as detected by flow cytometry (mono-

clonal anti-CD62P; Immunotech, Marseilles, France). As a

control platelets were even activated with adenosine 5¢-diphos-phate sodium salt (ADP; Sigma) at a final concentration of

1 · 10)4 M for 2 min at 25 �C.

In each experiment platelets were identified by positive

immunofluorescence using mouse antihuman CD41–phyco-

erythrin (PE) antibody (Immunotech) by flow cytometry.

Flow cytometry

Binding of FH, recombinant deletion andmutant constructs to

platelets was determined by flow cytometry.Washed thrombin-

activated platelets were incubated with or without FH (1, 10 or

100 lg mL)1; 10 lg mL)1 ¼ 0.07 lM) for 1 h at 37 �C and

gently mixed every 15 min. Cells were washed and incubated

with goat antihuman FH antibody (1 : 10; Calbiochem) or

goat serum as a negative control (1 : 10; Vector Laboratories,

Burlington, CA, USA) for 1 h at 37 �C, both diluted in 1%

BSA–HBSS. In separate experiments washed platelets were

incubated with mouse antihuman FH antibody (20 lg mL)1;

Serotec, Oxford, UK) or mouse IgG1 as an isotype control

(Dako, Glostrup, Denmark).

Saturability of binding was tested by incubating increasing

concentrations of FH (1, 10, 100, 250, 500, 750, 1000 lg mL)1)

with thrombin-activated washed platelets, using the mouse

antihuman FH antibody.

Secondary antibodies, rabbit antigoat IgG:FITC (1 : 400;

Calbiochem) or goat F(ab¢)2 antimouse Ig:FITC (1 : 20;

Dako), in HEPES buffer [27] were applied as appropriate.

Platelets were diluted in 500 lL ice-cold phosphate-buffered

saline (PBS; 0.140 M NaCl, 0.003 M KCl, 0.01 M phosphate,

pH 7.4; Medicago, Uppsala, Sweden) and binding detected by

flow cytometry using a FACS Calibur instrument (FACScan;

Becton Dickinson Immunocytometry Systems, San Jose, CA,

USA). Acquisition and processing of data from 5000 cells per

sample was carried out with the CELLQuest software. Percent

binding was calculated after subtraction of the background

fluorescence as determined by the binding of the control

antibody. Similarly, Alexa555-labeled FH at 50 lg mL)1 was

incubated with thrombin-activated platelets.

A F(ab¢)2 fragment was produced from the polyclonal goat

antihumanFHantibody by pepsin digestion (Pierce, Rockford,

IL,USA). Inhibition experiments were carried out in which FH

100 lg mL)1 was preincubated with this F(ab¢)2 fragment

(150 lg mL)1) at 37 �C for 1 h before addition to platelets. In

these experiments binding was detected with the mouse

antihuman FH antibody.

Similarly to experiments with FH, platelets were incubated

with 1 and 10 lg mL)1 of SCRs 1–7 (10 lg mL)1 ¼ 0.21 lM),8–11 (10 lg mL)1 ¼ 0.37 lM), 8–20 (10 lg mL)1 ¼ 0.11 lM),15–20 (10 lg mL)1 ¼ 0.24 lM) and SCR 15–20mut

(10 lg mL)1 ¼ 0.24 lM) and binding detected with the goat

antihuman FH antibody.

The importance of the heparin-binding sites was analyzed by

preincubation of FH 100 lg mL)1 and SCRs 1–7, 8–11, 8–20,

15–20 as well as SCR 15–20mut (10 lg mL)1) with heparin

1000 IU mL)1 (Leo Pharma,Malmo, Sweden) at 37 �C for 1 h

before addition to thrombin-activated washed platelets. Like-

wise, in separate experiments platelets were first preincubated

with heparin before FH was added.

Factor H binds to washed human platelets 155

� 2004 International Society on Thrombosis and Haemostasis

The role of the platelet receptor GPIIb/IIIa for FH binding

to platelets was studied by preincubation of thrombin-activated

washed platelets with Reopro� (abciximab 100–200 lg mL)1;

Eli Lilly, Indianapolis, IN, USA) [29], at room temperature for

15 min, after which platelets were incubated with FH 10 lg or100 lg mL)1, alternatively SCRs 1–7, 8–20, 15–20 as well as

SCR15–20mut 10 lg mL)1. Similarly,monoclonal antiplatelet

receptor GP1b (CD42b, 100 lg mL)1; Dako) was used as a

control. In certain experiments platelets were preincubatedwith

Reopro� and FH preincubated with heparin before these were

combined.

We investigated if FH competitively inhibits binding of

fibrinogen to the GPIIb/IIIa receptor. Platelets were not

thrombin-activated in these experiments. The method used for

detection of fibrinogen binding has been previously described

[27,30]. Washed platelets were incubated with or without FH

10 lg mL)1 or 100 lg mL)1 at 37 �C for 30 min, washed in

PBS and fixed. Cells were then washed and fibrinogen

1 mg mL)1 was added for 5 min at room temperature.

Platelets were washed and incubated with FITC-conjugated

chicken antihuman fibrinogen antibody (Diapensia, Gothen-

burg, Sweden) at a final concentration of 100 lg mL)1 or

FITC-conjugated chicken antihuman insulin antibody as a

negative control (100 lg mL)1; Diapensia), both diluted in

HEPES buffer, incubated for 30 min on ice and fluorescence

detected.

The possibility that FH binds to platelets via tsp-1 was

investigated by preincubation of platelets with rabbit antihu-

man tsp-1 antibody (1 : 400) for 1 h at 37 �C prior to addition

of FH (100 lg mL)1) and detection of FHbinding as described

above. In addition, in some experiments Reopro�was added to

the platelets for a further 15 min as described above. In other

experiments FH (100 lg mL)1) was combined with tsp-1

(2 lg mL)1; Sigma) and incubated with thrombin-activated

washed platelets. Binding was compared with binding in which

FH alone was used. The effect of added tsp-1 was evaluated by

1 h preincubation of tsp-1 with rabbit antihuman tsp-1.

The effect of thrombin activation on tsp-1 expression was

tested in washed platelets. Platelets were activated with

thrombin or not activated, fixed and incubated with a

polyclonal rabbit antihuman tsp-1 (1 : 1500) or rabbit serum

(1 : 1500) as a negative control for 1 h at 37 �C. Platelets werewashed and incubated with swine antirabbit IgG:FITC (1 : 20;

Dako).

The possible presence of C3 on washed platelets was tested.

Platelets were incubated with rabbit antihuman C3c (1 : 50;

Dako), or rabbit immunoglobulin fraction (Dako) as a

negative control, for 1 h at 37 �C. Platelets were washed and

incubated with swine antirabbit Ig:FITC (1 : 10). As a positive

control platelets were incubated for 30 min with 1 mg mL)1

purified C3 [31] at 37 �C.

Equilibrium and kinetics of FH binding to platelets

The Kd of FH binding to platelets was measured by a modified

Scatchard analysis. Washed platelets were incubated with a

combination of radiolabeled 125I-FH (10 lg mL)1) and

increasing concentrations of cold FH (0–100 lg mL)1) for

1 h at 37 �C. After washing with PBS, the radioactivity counts

were measured in a gamma counter and the Kd value

determined by a Scatchard equation and graph.

In order to establish the number of FHmolecules bound per

platelet, a state of equilibrium binding was achieved using

increasing concentrations of radiolabeled FH (1, 10, 25, 50,

75 lg mL)1) incubated for 1 h with 106 mL)1 platelets. When

equilibrium was attained the kinetics of binding was deter-

mined by measuring bound 125I-FH after 2, 10, 30 and 60 min.

FH, constructs and mutant binding to tsp-1 detected by

surface plasmon resonance

Interactions of FH and tsp-1 were analyzed by the surface

plasmon resonance technique using a Biacore 3000 instrument

(Biacore AB, Uppsala, Sweden) as described [4,32]. Tsp-1 was

coupled via a standard amine-coupling procedure to the

flowcell of a sensor chip (carboxylated dextran chip CM5;

Biacore) until an appropriate level of coupling for the binding

experiments (> 4000 resonance units) was reached. A control

flowcell was prepared without injecting protein. Native FH and

recombinant construct SCRs 1–7, 8–11, 8–20 or 15–20 as well

as SCR 15–20mut were dialyzed against the running buffer

(PBS, pH 7.4, 75 mM NaCl). Each ligand (1 lM) was injectedseparately into the flowcell coupled with tsp-1 and into the

control flowcell (without tsp-1). Each analysis was done at least

twice using independently prepared sensor chips. For kinetic

analysis of the interaction of FH with tsp-1, a concentration

series of FH (1–320 nM) was run at a flow rate of 30 lL min)1

in three independent experiments on a chip with a lower surface

density of tsp-1 (< 1000 resonance units) as described earlier

[4]. For the heparin inhibition series, increasing concentrations

of heparin (3–300 lg mL)1, lowmolecular weight; Sigma) were

added to the analyte, SCR 15–20 (50 lg mL)1), directly before

the injection.

Statistics

Differences between platelets incubated with or without FH,

with respect to binding, were assessed by the Mann–Whitney

U-test. A P-value of £0.05 was considered significant. Statis-

tical analyses were performed using SPSS version 11 (SPSS,

Chicago, IL, USA).

Results

FH binds to washed platelets

FH bound to non-activated platelets and to platelets activated

with either thrombin or ADP. Thrombin activation increased

FH (10 lg mL)1) binding by 22% (from 9 to 11% binding to

the platelet population, median of three flow cytometry

experiments) in comparison with non-activated platelets, and

was also documented using radiolabeled FH (Fig. 1A). The

156 F. Vaziri-Sani et al

� 2004 International Society on Thrombosis and Haemostasis

number of radiolabeled FH molecules bound to each throm-

bin-activated platelet was 3.8 · 105, whereas the number of

molecules bound to each resting platelet was 2.7 · 105. ADP

activation increased FH (10 lg mL)1) binding by 89% (from 9

to 17%, median of four flow cytometry experiments). Results

pertaining to thrombin-activated platelets are shown. Binding

was dose dependent as shown in Fig. 1B. A final concentration

of 100 lg mL)1 FH was incubated with platelets at

1 · 107 mL)1 in an attempt to correspond to the ratio of FH

to platelets in the human circulation (FH 500 lg mL)1;

platelets 1.4–4 · 108 mL)1). At this concentration a median

binding of 24% (polyclonal antibody) and 18% (monoclonal

antibody) was found (Fig. 1B). FH binding as detected by

fluorescence intensity using polyclonal and monoclonal

Fig. 1. Factor H (FH) binding to platelets. (A) A dose-dependent increase in binding of radiolabeled FH to thrombin-activated platelets ( ) vs. non-

activated ( ). The abscissa shows the total amount of FH incubated with platelets and the ordinate shows the amount of FH that bound. In these

experiments increasing concentrations of radiolabeled FH (1, 10, 25, 50, 75 lg mL)1) were incubated with 106 mL)1 platelets corresponding to the

concentration conditions used in flow cytometry experiments which were 10-fold higher for both FH and platelets. Data were corrected for volume and

are presented as amounts. (B) Thrombin-activated washed platelets incubated with or without FH. Dose-related binding detected by flow cytometry

is shown. Statistical significance of platelets incubated with or without FH is given above each category. The number of experiments is given in parentheses

below each category. The thin dark line in each box represents the median. The upper and lower limits of the box plot represent the interquartile

range; the lower and upper limits represent the range. (C) One experiment showing FH 100 lg mL)1 binding to 23.4% of platelets detected by the

polyclonal antibody (thick line) after subtraction of the control antibody (thin line). (D) One experiment showing FH 100 lg mL)1 binding to 28.9%

of platelets detected by the monoclonal antibody (thick line) after subtraction of the control antibody (thin line). (E) Saturability was obtained at a FH

concentration of approximately 500 lg mL)1 detected by flow cytometry using the monoclonal anti-FH antibody (median of four experiments). The

ordinate shows the percent of binding to the platelet population. (F) Kinetics of FH binding to platelets showing that maximal binding was achieved after

10 min incubation. The ordinate depicts the number of FH molecules bound per platelet.

Factor H binds to washed human platelets 157

� 2004 International Society on Thrombosis and Haemostasis

antibodies is shown in Fig. 1C and D, respectively. Alexa555-

labeled FH bound to 19.5% of platelets.

Saturability of FH binding to platelets (1 · 107 mL)1) was

obtained at a FH concentration of approximately 500 lg mL)1

(Fig. 1E). Maximal binding was noted after 10 min incubation

(Fig. 1F).

The Kd of FH binding to platelets, tested by combining

radiolabeled FH with cold FH and analyzed by a Scatchard

plot, was 2.39 lM (data not shown).

Inhibition experiments using a F(ab¢)2 fragment (goat

antihuman FH) reduced binding by a mean of 42% (from

9.7 to 5.6%, two experiments).

Mechanism by which FH binds to platelets

In order to identify the mechanisms by which FH binds to

platelets we localized the binding domain(s) within FH and

tried to identify the receptor(s) on platelets that mediate

binding.

FH binds to platelets mainly via the C-terminus domain

Recombinant constructs representing SCR 1–7, SCR 8–20 and

SCR 15–20 of FH bound to platelets in a dose-related manner

and all constructs that contained the C-terminus of FH (SCR

8–20 and SCR 15–20) showed strong binding (Table 1).

Involvement of heparin-binding sites in FH binding to

platelets

Preincubation of FH with heparin reduced binding of FH to

platelets by 36% (median of percentage reduction, from 32 to

18% binding, eight experiments). Preincubation of SCR 1–7,

8–20 and 15–20 with heparin reduced binding by 14, 23 and

22%, respectively. Preincubation of platelets with heparin

before addition of FH had no effect on FH binding, indicating

that the heparin-binding sites on FH and not on platelets are

essential for this interaction. These results were further

confirmed by incubating platelets with SCR 8–11, a construct

lacking heparin-binding sites [33]. As shown in Table 1,

binding of this construct was clearly reduced and no dose–

response was demonstrated. Furthermore, preincubation with

heparin did not reduce binding of this construct.

Platelet GPIIb/IIIa receptor mediates FH binding

The platelet GPIIb/IIIa receptor is essential for platelet

interactions with fibrinogen, von Willebrand factor and other

known platelet-activating peptides [34]. We therefore investi-

gated if this receptor was involved in FH binding to platelets.

This was achieved by blocking the receptor using an anti-

GPIIb/IIIa antibody (Reopro�) and by competitive inhibition

with fibrinogen.

Preincubation of platelets with Reopro� 100 lg mL)1

reduced binding of FH 100 lg mL)1 to platelets by 34%

(median of percentage reduction) from 32 to 21% binding

(seven experiments) (P < 0.02). Preincubation of platelets with

Reopro� reduced binding of FH 10 lg mL)1 to platelets by

45% (median) from 19 to 11% binding (six experiments)

(P < 0.03). As a control for platelet receptors, FH was

preincubated with anti-GPIb antibody, which did not reduce

binding.

Similar experiments were carried out using FH constructs to

determine binding via the GPIIb/IIIa receptor. Reopro�

100 lg mL)1 reduced binding of SCR 1–7 by 36% (from

21 to 14% binding, three experiments), of SCR 8–20 by 32%

(from 24 to 17%, three experiments) and of SCR 15–20 by

23% (from 32 to 25%, six experiments).

Washed platelets (not thrombin-activated) were incubated

with or without FH, after which fibrinogen was added.

Fibrinogen binding was detected on platelets not exposed to

FH at a median of 44% (four experiments). When FH

10 lg mL)1 was present binding of fibrinogen was reduced to a

median of 39% (11% reduction, four experiments). When FH

100 lg mL)1 was present binding of fibrinogen was reduced to

a median of 35% (21% reduction, four experiments). These

results indicate that the presence of the higher concentration of

FH partially inhibited fibrinogen binding to the GPIIb/IIIa

receptor and together with the above-mentioned Reopro�

experiments suggest that GPIIb/IIIa is, to a certain extent,

involved in FH binding to platelets via both the C- and

N-termini.

A combination of Reopro� and heparin reduced FH

binding to platelets from 32 to 13% (58% reduction, three

experiments).

FH binds to tsp-1 mainly via its C-terminus

Since FH is secreted from platelets together with tsp-1, and FH

binding to platelets was not totally abrogated by the

Table 1 Binding of factor H (FH) constructs and mutant to platelets as

determined by flow cytometry

FH construct

Concentration

(lg mL)1)

Binding (%)

median (range)

polyclonal anti-FH P-value

Platelets not

incubated with

FH constructs

0 6.5 (0.0–16.0)a

SCR 1–7 1 10.3 (9.7–13.6)b 0.13 NS

10 17.7 (16.4–33.3)c < 0.0001

SCR 8–11 1 8.7 (4.2–22.6)d 0.30 NS

10 8.5 (3.8–23.5)d 0.37 NS

SCR 8–20 1 20.2 (8.9–30.5)b 0.03

10 30.6 (21.7–34.9)b < 0.0001

SCR 15–20 1 20.3 (11.6–32.9)b 0.011

10 30.0 (15.2–54.3)e < 0.0001

SCR 15–20mut 10 15.7 (4.1–27.24)d 0.057 NS

P-values evaluated by comparison of platelets incubated with or

without FH constructs. Platelets that were not incubated with FH

constructs were washed and thrombin-activated, similarly to those

incubated with FH constructs. Median of: a21; b3; c5; d6; e12 experi-

ments. NS, Not significant.

158 F. Vaziri-Sani et al

� 2004 International Society on Thrombosis and Haemostasis

anti-GPIIb/IIIa antibody, we studied interactions between FH

and tsp-1 and their importance for FH binding to platelets. We

used the surface plasmon resonance technique to show binding

ofFHto immobilized tsp-1and todetermine thebindingaffinity

and kinetics (Fig. 2A). TheKd value for binding of FH to tsp-1

was 49 (± 4.4) nM at 25 �C.We localized the regions involved

in binding using recombinant deletion constructs SCRs 1–7,

8–11, 8–20 and 15–20 (Fig. 2B). SCR 8–20 and SCR 15–20

bound to tsp-1. In contrast, construct SCR 1–7, containing the

N-terminus, and SCR 8–11, did not bind to tsp-1.

The effect of heparin on the interaction between SCR 15–20

and tsp-1 was studied. The addition of heparin inhibited the

interaction of SCR 15–20 with tsp-1 dose-dependently

(Fig. 2C). This indicates that binding of SCR 15–20 to tsp-1

occurs at the heparin-binding site (SCR 20).

Tsp-1 and FH expression on thrombin-activated platelets

and FH binding to platelets via tsp-1

The above-mentioned experiments indicated that FH and tsp-1

interact with each other. We therefore investigated if these

proteins are expressed on the surface of thrombin-activated

platelets. Thrombin activation increased expression of tsp-1

4-fold (from 4 to 18% binding of the anti-tsp-1 antibody) and

of FH by 0.5-fold (from 4 to 6% binding of the anti-FH

antibody). This result suggests that FH may bind to activated

platelets via surface expression of tsp-1.

Preincubation of platelets with anti-tsp-1 before addition of

FH reduced fH binding by 36% (from 14 to 9%). Combination

of tsp-1 with FH increased FH binding to platelets by 28%

(median of percentage increase). This increase of FH binding

was totally inhibited by preincubation of tsp-1 with anti-tsp-1.

An attempt was made to inhibit FH binding by preincubation

of FH with anti-tsp-1 and Reopro�, but these experiments

could not be interpreted due to very high background

fluorescence.

Lack of C3 on washed platelets

No C3 was identified on the surface of washed platelets. This

indicates that FH does not bind to washed platelets via C3.

When C3 was added to washed platelets (as the positive

control) the anti-C3c antibody bound to 36%of the population

(three experiments, data not shown).

Fig. 2. Binding of factor H (FH), FH constructs and mutant to thrombospondin-1 (tsp-1) detected by surface plasmon resonance. (A) Strong binding

of FH to tsp-1 is shown. The inserted figure shows equilibrium binding of FH to tsp-1 expressed in resonance units at increasing concentrations of

the analyte (FH concentrations from 1 to 320 nM). The controls (binding to a flowcell without immobilized protein) were subtracted from the binding

curves. (B) FH constructs SCR 8–20 and SCR 15–20 bound to tsp-1 whereas SCR 8–11 and SCR 1–7 did not bind. (C) Heparin inhibited binding

of SCR 15–20 to tsp-1 in a dose-dependent manner. (D) SCR 15–20mut exhibited weaker binding to tsp-1 than SCR 15–20.

Factor H binds to washed human platelets 159

� 2004 International Society on Thrombosis and Haemostasis

Mutant FH binding to platelets and tsp-1

SCR 15–20mut bound to platelets significantly less than SCR

15–20 (P < 0.0001, Table 1). Binding was not inhibited by

heparin but was reduced by Reopro� 100 lg mL)1 by 50%

(from 16 to 8%, six experiments) and by Reopro�

200 lg mL)1 by 71% (from 16 to 5%, six experiments).

Furthermore, SCR 15–20mut exhibited considerably lower

binding to tsp-1 than SCR 15–20 as studied by surface plasmon

resonance (Fig. 2D).

Discussion

A novel property of FH is presented in this study that shows

binding of FH to washed platelets. FH mutated at the

C-terminus exhibited significantly lower binding. This interac-

tion occurred in the absence of complement and other plasma

factors. FH mutations have been identified in a subset of

patients with atypical HUS [7–12], a condition in which

platelets are activated and consumed, leading to thrombocy-

topenia [13]. In some of these patients the alternative comple-

ment pathway is activated [35]. Although this study did not

address the mechanisms by which platelets are activated in

HUS, we have shown that, in a plasma-free environment, FH

can interact with platelets and that mutated FH displays lesser

binding.

FH binding to platelets is multivalent and may engage more

than one site of the FH protein as well as various binding sites

and different proteins on the platelet surface (Fig. 3). Binding

seems to involve mainly the C-terminus of FH. The C-terminus

contains C3b and heparin-binding sites. Recent studies provide

evidence that this region is involved in binding to cell surfaces

containing glycosaminoglycans such as sialic acids [3,4] and

enables FH to differentiate between activating (foreign) and

non-activating (host) surfaces [36]. FH binds to platelets

directly via the GPIIb/IIIa receptor, which is exposed after

platelet activation, or indirectly via tsp-1, which is expressed on

the surface of activated platelets. Thus FH may bind via more

specific mechanisms such as receptor-mediated binding, which

may be inhibited by antibodies and competitive inhibition with

fibrinogen, as well as less-specific mechanisms involving the

heparin-binding sites of FH and glycosaminoglycans on cells.

The complexity of FH binding and ligand specificity were

previously documented for binding to C3b on erythrocytes

[37,38].

Binding of FH to platelets involves the heparin-binding sites.

The heparin-binding site at the C-terminus appears to be more

accessible than the other two heparin-binding sites since

heparin binding of the full protein can be inhibited by an

antibody directed to the C-terminus [39]. This may explain why

the N-terminus construct (SCR 1–7) binds to a lesser degree

than the C-terminus construct (SCR 15–20) though both have

one heparin-binding site. The SCR 8–11 construct, which lacks

a heparin-binding site, has a binding site for C-reactive protein

and binds to microbial ligands such as the pneumococcal Hic

protein [33,40]. This construct bound platelets weakly and no

binding to tsp-1 was detected. This further indicates that the

heparin-binding sites are involved in binding of FH to platelets.

In addition, our results suggest that binding of FH to GPIIb/

IIIa occurs both at the C- and N-termini, whereas binding via

tsp-1 occurs mostly via the C-terminus.

FH mutations in HUS patients have been mostly identified

at the C-terminus of the protein. Using purified mutant FH

from a HUS patient and a recombinant SCR 20-mutated

construct, a recent study showed reduced binding to endothel-

ial cells in comparison with the wild-type protein, suggesting

that the mutated protein may not be capable of protecting the

endothelial cell layer [17]. Mutated FH was capable of binding

SCR 1-717.7%

SCR 8-2030.6%

NH2 COOH

Regions on factor H involved in binding to washed platelets:Heparin-binding sites (more so at the C-terminus)Factors on washed platelets involved in binding to factor H:GPIIb/IIIa receptorThrombospondin-1 (binds mostly to the C-terminus of factor H)Factors not involved in binding of factor H to washed platelets:C3

wholeFactor H

24.4%

GPIIb/IIIaReceptor

Tsp-1

C3b binding C3b/C3c binding C3b/C3d bindingHeparin binding Heparin binding Heparin binding

1 20

Washed thrombin-activated platelet

Fig. 3. Schematic diagram of mechanisms by which factor H (FH) binds to thrombin-activated washed platelets. FH binds to platelets mostly via

the C-terminus and heparin-binding sites. On washed platelets it may bind both to the GPIIb/IIIa receptor and to surface-bound thrombospondin-1

(tsp-1). % refers to percent binding of whole FH or constructs to platelets.

160 F. Vaziri-Sani et al

� 2004 International Society on Thrombosis and Haemostasis

to platelets, although to a lesser degree. The reason for this loss

of function may be related to the fact that SCR 15–20mut does

not contain a heparin-binding site and does not bind to tsp-1.

Thus binding of the C-terminus mutant to platelets is primarily

mediated by GPIIb/IIIa and this binding could be almost

completely abrogated by anti-GPIIb/IIIa (Reopro�).

Tsp-1 is a glycoprotein stored in and secreted from the

a-granules of platelets andknown to induceplatelet aggregationvia integrin-associated protein (IAP,CD47) andfibrinogen [41].

It copurifies fromplateletswithFH [20] andwehave shown that

these two proteins interact with each other and localized the

binding site to the C-terminus. Binding at the C-terminus was

diminished by addition of heparin and the SCR 15–20mut

exhibited reduced binding, indicating that the C-terminus

heparin-binding site in SCR 20 of FH is important for the

interaction. As the two proteins are secreted simultaneously

fromactivatedplateletswe suggest that theymay interact in vivo.

Current theories regarding the pathogenesis of HUS have

focused on endothelial cell damage, with exposure of the

subendothelium leading to deposition and consumption of

platelets. We show that FH binds to platelets and suggest that

this interaction may regulate complement activation on the

platelet membrane. Reduced binding of mutated FH to

platelets may promote uninhibited complement activation.

Further studies are ongoing to address the interactions of FH

with platelets in the presence of complement.

Acknowledgements

Presented in part at the XIXComplementWorkshop, Palermo

Sicily, 22–26 September 2002. This study was supported by

grants from The Swedish Research Council (06X-14008), Knut

andAliceWallenberg Foundation, SwedishRenal Foundation,

Ake Wiberg Foundation, Anna-Lisa and Sven-Eric Lundgren

FoundationforMedicalResearch,RonaldMcDonaldPediatric

Fund, Greta and Johan Kock Foundation, Swedish Society of

Medicine, Crafoord Foundation, Inga and John Hains Foun-

dation, The Blood and Defence Network at Lund University,

Royal Physiographic Society in Lund, Alfred Osterlund

Foundation, Crown Princess Lovisa’s Society for Child Care,

Thelma Zoegas Foundation, The Swedish Society of Nephrol-

ogy, Skanska Provinsiallogens Welfare Fund and the Lund

University Hospital Funds (all to D.K.). The Sven Jerring

Foundation (to F.V-S. and D.K.). The Deutsche Forschungsg-

emeinschaft and Foundation for Children with Atypical HUS

(to P.F.Z.). King Gustaf V’s 80th Birthday Fund (to A.G.S.).

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