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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.).
References
1 Walport MJ. Complement. First of two parts. N Engl J Med 2001;
344: 1058–66.
2 Zipfel PF. Hemolytic uremic syndrome: how do factor H mutants
mediate endothelial damage? Trends Immunol 2001; 22: 345–8.
3 Blackmore TK, Hellwage J, Sadlon TA, Higgs N, Zipfel PF,
Ward HM, Gordon DL. Identification of the second heparin-binding
domain in human complement factor H. J Immunol 1998; 160:
3342–8.
4 Hellwage J, Jokiranta TS, Friese MA, Wolk TU, Kampen E, Zipfel
PF, Meri S. Complement C3b/C3d and cell surface polyanions are
recognized by overlapping binding sites on the most carboxy-terminal
domain of complement factor H. J Immunol 2002; 169: 6935–44.
5 Rodriguez de Cordoba S, Lublin DM, Rubinstein P, Atkinson JP.
Human genes for three complement components that regulate the
activation of C3 are tightly linked. J Exp Med 1985; 161: 1189–95.
6 Friese MA, Hellwage J, Jokiranta TS, Meri S, Peter HH, Eibel H,
Zipfel PF. FHL-1/reconectin and factor H: two human complement
regulators which are encoded by the same gene are differently
expressed and regulated. Mol Immunol 1999; 36: 809–18.
7 Ault BH. Factor H and the pathogenesis of renal diseases. Pediatr
Nephrol 2000; 14: 1045–53.
8 Richards A, Buddles MR, Donne RL, Kaplan BS, Kirk E, Venning
MC, Tielemans CL, Goodship JA, Goodship THJ. Factor H muta-
tions in hemolytic uremic syndrome cluster in exons 18–20, a domain
important for host cell recognition. Am J Hum Genet 2001; 68: 485–
90.
9 Caprioli J, Bettinaglio P, Zipfel PF, Amadei B, Daina E, Gamba S,
Skerka C,MarzilianoN,RemuzziG,NorisM. Themolecular basis of
familial hemolytic uremic syndrome: mutation analysis of factor H
gene reveals a hot spot in short consensus repeat 20. J AmSoc Nephrol
2001; 12: 297–307.
10 Ying L, Katz Y, Schlesinger M, Carmi R, Shalev H, Haider N, Beck
G, Sheffield VC, Landau D. Complement factor H gene mutation
associated with autosomal recessive atypical hemolytic uremic syn-
drome. Am J Hum Genet 1999; 65: 1538–46.
11 Noris M, Ruggenenti P, Perna A, Orisio S, Caprioli J, Skerka C,
Vasile B, Zipfel PF, Remuzzi G. Hypocomplementemia discloses
genetic predisposition to hemolytic uremic syndrome and thrombotic
thrombocytopenic purpura: role of factor H abnormalities. The Ital-
ian Registry of Familial and Recurrent Hemolytic Uremic Syndrome/
Thrombotic Thrombocytopenic Purpura. J Am Soc Nephrol 1999; 10:
281–93.
12 Warwicker P, Goodship TH, Donne RL, Pirson Y, Nicholls A,
Ward RM, Turnpenny P, Goodship JA. Genetic studies into
inherited and sporadic hemolytic uremic syndrome. Kidney Int 1998;
53: 836–44.
13 Karpman D. Haemolytic uremic syndrome and thrombotic thromb-
ocytopenic purpura. Current Paediatrics 2002; 12: 569–74.
14 Zoja C, Remuzzi G. The pivotal role of the endothelial cell in the
pathogenesis of HUS. In: Kaplan BS, Trompeter RS, Moake J, eds.
Hemolytic Uremic Syndrome and Thrombotic Thrombocytopenic Pur-
pura. New York: Marcel Dekker Inc., 1992: 389–404.
15 Hindmarsh EJ, Marks RM. Complement activation occurs on
subendothelial extracellular matrix in vitro and is initiated by retrac-
tion or removal of overlying endothelial cells. J Immunol 1998; 160:
6128–36.
16 Taylor CM. Complement factor H and the haemolytic uraemic syn-
drome. Lancet 2001; 358: 1200–2.
17 Manuelian T, Hellwage J, Meri S, Caprioli J, Noris M, Heinen S,
Jozsi M, Neumann HPH, Remuzzi G, Zipfel PF. Mutations in factor
H reduce binding affinity to C3b and heparin and surface attachment
to endothelial cells in hemolytic uremic syndrome. J Clin Invest 2003;
111: 1181–90.
18 Devine DV, Rosse WF. Regulation of the activity of platelet-bound
C3 convertase of the alternative pathway of complement by platelet
factor H. Proc Natl Acad Sci USA 1987; 84: 5873–7.
19 Devine DV, Siegel RS, Rosse WF. Interactions of the platelets in
paroxysmal nocturnal hemoglobinuria with complement. Relation-
ship to defects in the regulation of complement and to platelet survival
in vivo. J Clin Invest 1987; 79: 131–7.
20 Carron JA, Bates RC, Smith AI, Tetoz T, Arellano A, Gordon DL,
Burns GF. Factor H co-purifies with thrombospondin isolated from
platelet secretate. Biochim Biophys Acta 1996; 1289: 305–11.
21 Leung LL. Role of thrombospondin in platelet aggregation. J Clin
Invest 1984; 74: 1764–72.
Factor H binds to washed human platelets 161
� 2004 International Society on Thrombosis and Haemostasis
22 Kuhn S, Skerka C, Zipfel PF.Mapping of the complement regulatory
domains in the human factor H-like protein 1 and in factor H. J
Immunol 1995; 155: 5663–70.
23 Hellwage J, Skerka C, Zipfel PF. Biochemical and functional char-
acterization of the factor H-related protein 4 (FHR-4). Immuno-
pharmacology 1997; 38: 149–57.
24 Marz L, Altmann F, Staudacher E, Kubelka V. Protein glycosylation
in insects. In: Montreuli J, Schachter H, Vliegenhart JFG, eds. Gly-
coproteins. Amsterdam: Elsevier, 1995: 543–63.
25 Marchal I, Jarvis DL, Cacan R, Verbert A. Glycoproteins from insect
cells: sialylated or not? Biol Chem 2001; 382: 151–9.
26 Sharma AK, Pangburn MK. Biologically active recombinant human
complement factor H: synthesis and secretion by the baculovirus
system. Gene 1994; 143: 301–2.
27 Karpman D, Papadopoulou D, Nilsson K, Sjogren AC, Mikaelsson
C, Lethagen S. Platelet activation by Shiga toxin and circulatory
factors as a pathogenetic mechanism in the hemolytic uremic syn-
drome. Blood 2001; 97: 3100–8.
28 Tolentino AR, Bahou WF. Thrombin receptors. In: Michelson AD,
ed. Platelets. Orlando: Harcourt Inc., 2002: 117–38.
29 Coller BS. A new murine monoclonal antibody reports an activation-
dependent change in the conformation and/or microenvironment of
the platelet glycoprotein IIb/IIIa complex. J Clin Invest 1985; 76:
101–8.
30 Lindahl TL, Festin R, Larsson A. Studies of fibrinogen binding to
platelets by flow cytometry: an improved method for studies of
platelet activation. Thromb Haemost 1992; 68: 221–5.
31 Dodds AW. Small-scale preparation of complement components C3
and C4. Meth Enzymol 1993; 223: 46–61.
32 Hellwage J, Meri T, Heikkila T, Alitalo A, Panelius J, Lahdenne P,
Seppala IJT, Meri S. The complement regulator factor H binds to the
surface protein OspE of Borrelia burgdorferi. J Biol Chem 2001; 276:
8427–35.
33 Jarva H, Jokiranta TS, Hellwage J, Zipfel PF, Meri S. Regulation
of complement activation by C-reactive protein: targeting the
complement inhibitory activity of factor H by an interaction with
short consensus repeat domains 7 and 8–11. J Immunol 1999; 163:
3957–62.
34 Payrastre B,Missy K, Trumel C, Bodin S, PlantavidM, Chap H. The
integrin alpha IIb/beta 3 in human platelet signal transduction. Bio-
chem Pharmacol 2000; 60: 1069–74.
35 Pichette V, Querin S, Schurch W, Brun G, Lehner-Netsch G, Delage
JM. Familial hemolytic–uremic syndrome and homozygous factor H
deficiency. Am J Kidney Dis 1994; 24: 936–41.
36 Pangburn MK. Host recognition and target differentiation by factor
H, a regulator of the alternative pathway of complement. Immuno-
pharmacology 2000; 49: 149–57.
37 Parker CJ, Baker PJ, Rosse WF. Comparison of binding character-
istics of factors B and H to C3b on normal and paroxysmal nocturnal
hemoglobinuria erythrocytes. J Immunol 1983; 131: 2484–9.
38 Pangburn MK, Pangburn KL, Koistinen V, Meri S, Sharma AK.
Molecular mechanisms of target recognition in an innate immune
system: interactions among factor H, C3b, and target in the alter-
native pathway of human complement. J Immunol 2000; 164: 4742–
51.
39 Prodinger WM, Hellwage J, Spruth M, Dierich MP, Zipfel PF. The
C-terminus of factor H. monoclonal antibodies inhibit heparin
binding and identify epitopes common to factor H and factor
H-related proteins. Biochem J 1998; 331: 41–7.
40 Jarva H, Janulczyk R, Hellwage J, Zipfel PF, Bjorck L, Meri S.
Streptococcus pneumoniae evades complement attack and opsono-
phagocytosis by expressing the pspC locus-encoded Hic protein that
binds to short consensus repeats 8–11 of factor H. J Immunol 2002;
168: 1886–94.
41 Lawler J. The structural and functional properties of thrombospon-
din. Blood 1986; 67: 1197–209.
162 F. Vaziri-Sani et al
� 2004 International Society on Thrombosis and Haemostasis