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Blood, Vol. 91 No. 10 (May 15), 1998:
pp. 3792-3799
Platelet Aggregation Induced by a Monoclonal Antibody to the A1
Domain of von Willebrand Factor
By
Hilde Depraetere,
Nadine Ajzenberg,
Jean-Pierre Girma,
Catherine Lacombe,
Dominique Meyer,
Hans Deckmyn, and
Dominique Baruch
From INSERM U143, Paris, France; Laboratory for Thrombosis Research,
KU Leuven, Campus Kortrijk, Belgium; and Biorheology URA CNRS 343, Paris, France.
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ABSTRACT |
Shear-induced platelet aggregation (SIPA) involves von Willebrand
Factor (vWF) binding to platelet glycoprotein (GP)Ib at high shear
stress, followed by the activation of
 IIb 3. The purpose of this study was to
determine the vWF sequences involved in SIPA by using monoclonal
antibodies (MoAbs) to vWF known to interfere with its binding to GPIb
and to IIb3. Washed platelets were exposed to shear rates between 100 and 4,000 seconds 1 in
a rotational viscometer. SIPA was quantitated by flow cytometry as the
disappearance of single platelets (DSP) in the sheared sample in the
presence of vWF, relative to a control in the absence of shear and vWF.
At a shear rate of 4,000 seconds1, DSP was increased
from 5.9% ± 3.5% in the absence of vWF to 32.7% ± 6.3% in the
presence of vWF. This increase in SIPA was not associated with an
elevation of P-selectin expression. vWF-dependent SIPA was completely
abolished by MoAb 6D1 to GPIb and partially inhibited by MoAb 10E5 to
IIb3. Three MoAbs to vWF were compared for their effect on SIPA at 4,000 seconds1 in the
presence of vWF: MoAb 328, known to block vWF binding to GPIb in the
presence of ristocetin, MoAb 724 blocking vWF binding to GPIb in the
presence of botrocetin, and MoAb 9, an inhibitor of vWF binding to
IIb3. Similar to the effect of MoAb 6D1,
MoAb 328 completely inhibited the effect of vWF, whereas MoAb 9 had a
partial inhibitory effect, as MoAb 10E5 did. In contrast, MoAb 724, as
well as its F(ab )2 fragments, promoted
shear-dependent platelet aggregation (165% of the DSP value obtained
in the absence of MoAb 724), indicating that MoAb 724 was responsible
for an enhanced aggregation, which was independent of binding to the platelet Fc receptor. In addition, the enhancement of aggregation induced by MoAb 724 was abrogated by MoAb 6D1 or 10E5 to the level of
SIPA obtained in the presence of vWF incubated with a control MoAb to
vWF. Finally, the activating effect of MoAb 724 was also found under
static conditions at ristocetin concentrations too low to induce
platelet aggregation. Our results suggested that on binding to a
botrocetin-binding site on vWF, MoAb 724 mimics the effect of
botrocetin by inducing an active conformation of vWF that is more
sensitive to shear stress or to low ristocetin concentration.
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INTRODUCTION |
ELEVATED LEVELS of shear stress are found
associated with arteriotic stenosis, which may be secondary to
atherosclerosis or vascular spasm. Under these conditions, platelet
adhesion and aggregation can occur and induce thrombotic occlusion. In
this process, von Willebrand factor (vWF) is the main protein required for the interaction with platelet receptors glycoprotein (GP) Ib and
GPIIb-IIIa, the IIb3
integrin.1 Under static conditions, binding to GPIb has
been described in the presence of nonphysiological agents, such as the
antibiotic ristocetin or the snake venom protein botrocetin, and
involves distinct sequences localized within the first type A repeat
(A1 domain) extending from amino acids (aa) 497 to 716 and containing a
disulfide bond between Cys 509 and Cys 695.2-4 However, in
the absence of these agents, fluid-phase vWF is unable to bind to GPIb,
presumably because of the presence of inhibitory sequences that
cooperate to maintain an inactive conformation of vWF.5 In
physiological conditions, binding of vWF to the endothelial
extracellular matrix occurs through its A1 domain and allows subsequent
interaction with platelet GPIb under high shear rate
conditions.6-8 However, plastic-immobilized vWF has also
been reported to bind to GPIb under static conditions.9 Thus, the exact mechanism by which vWF binds to GPIb in vivo remains to
be determined.
An approach to determine the effect of high shear rates on fluid-phase
vWF interaction with platelets is the so-called shear-induced platelet
aggregation (SIPA), originally reported by Moake et al.10 This process differs from platelet aggregation observed under static or
low shear rate conditions, which involves binding of the Arg-Gly-Asp
(RGD) sequence of fibrinogen to adenine diphosphate (ADP)-
or thrombin-activated IIb3. In contrast,
two essential features are observed in SIPA: platelet activation by
exogenous agents is not a prerequisite for
IIb3 activation, and the main effector is
not fibrinogen, but vWF. Based on studies performed with viscometers,
in which uniform shear fields can be applied in the absence of
exogenous modulators, the following working model has been proposed: in
high shear rate conditions, fluid-phase vWF becomes able to bind to
GPIb. The IIb3 integrin becomes activated
through intracellular signals generated by the vWF-GPIb complex
formation. Activated IIb3 binds to the
RGD sequence of vWF thereby stabilizing platelet
aggregation.11 By using antibodies against GPIb or
IIb3 as well as platelet-rich plasma from
patients with von Willebrand disease (vWD), Glanzmann's
thrombasthenia, or Bernard-Soulier syndrome, the effect of vWF as the
main effector of platelet aggregation in high shear rate conditions has
been confirmed.12,13
The aim of our study is to investigate the involvement of different
sequences of vWF on platelet aggregation at high shear rates generated
by means of a rotational viscometer. To this end, we used monoclonal
antibodies (MoAbs) to vWF, which inhibit its binding to GPIb or to
IIb3. MoAb 328 has been previously
reported as an inhibitor of ristocetin-induced platelet aggregation and vWF binding to platelet GPIb.14,15 In addition, at a wall
shear rate of 1,600 seconds1, this MoAb completely
blocks platelet adhesion to immobilized vWF in a perfusion
chamber.6 MoAb 724 inhibits vWF binding to platelet GPIb in
the presence of botrocetin and binds to normal vWF with a higher
affinity than to vWF from type 2B vWD, which has an increased
reactivity for GPIb.16 MoAb 9 inhibits vWF binding to
activated platelet IIb3 in static
conditions.17 We show that in high shear rate conditions
and in the presence of vWF, MoAbs 328 and 9 are able to block platelet
aggregation, whereas MoAb 724 enhances platelet aggregation, via a
Fc-receptor independent mechanism. To our knowledge, this is the
first description of an antibody against the A1 domain of vWF that
increases platelet aggregation.
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MATERIALS AND METHODS |
Preparation of washed platelets.
Blood was obtained from healthy individuals who had not ingested any
medication for 2 weeks before donation. The blood was drawn into 15%
(vol/vol) acid citrate dextrose (ACD), pH 5.8. Platelet rich plasma
(PRP) was obtained by centrifugation at 100g for 20 minutes at
37°C. To the PRP, ACD (1 mL for 40 mL) and apyrase (2 U/mL; Sigma,
St Louis, MO) were added and platelets were isolated by centrifugation
(500g for 15 minutes at 37°C). Platelets were washed twice
with HEPES buffer, pH 6.7 (10 mmol/L HEPES;
N-[2-hydroxyethyl]piperazine-N'-[ethanesulfonic acid], 0.136 mol/L
NaCl, 2.7 mmol/L KCl, and 2 mmol/L MgCl2) and 0.35% bovine
serum albumin (BSA) in the presence of apyrase (2 U/mL) and ACD (1 mL
for 40 mL). Finally, the platelets were resuspended in HEPES buffer pH
7.5, containing 1 mmol/L CaCl2, and BSA 0.15%, and they
were used after 1 hour incubation at 37°C. Platelets were counted
with an electronic particle counter (Model Z1, Coulter Electronics,
Margency, France), and the concentration was adjusted such that the
final concentration was 1.5 × 108 platelets/mL.
Purifications and radiolabeling of vWF.
Human vWF was purified from outdated high purity vWF concentrates (a
gift from Dr C. Mazurier, LFB, Lille, France) as
described.6 In some experiments, vWF was labeled with
Na125I (Amersham, Les Ulis, France) and Iodogen (Pierce
Chemical Co, Rockford, IL) as described.6 Specific
radioactivity varied from 1 to 4 µCi/µg of protein. Labeled protein
was used within a week. Fibrinogen was purchased from Biogenic (Maurin,
France). Botrocetin was purified from Bothrops Jararaca (Sigma)
as described.14
Antibodies.
Different MoAbs to vWF have been previously reported: MoAb 9 inhibits
vWF binding to platelet IIb3 and its
epitope has been localized between aa 1704 and 1746 by screening
recombinant cDNA fragments of vWF expressed in Escherichia
coli17; MoAb 328 blocks vWF binding to GPIb in the
presence of ristocetin, but not botrocetin.14,15 MoAb 724 blocks binding of vWF to botrocetin and binding to GPIb in the presence
of botrocetin but not ristocetin.16 Comparative
immunoblotting analysis indicated that both MoAbs 328 and 724 recognize
the T116 dimeric tryptic fragment (aa 449-728), but that only MoAb 328 reacts with the III-T2 fragment containing aa 273 to 511 and 674 to 728 and lacking the central part (aa 512-673) of the A1 domain. Comparison
of functional studies and immunostaining analysis allowed mapping the
724 epitope to the central part of the A1 loop (aa 565-587) and the 328 epitope to the distal part of the loop in either one or both segments
proximal to the disulfide loop (aa 480-511 and/or 674-718).15,16 In some experiments, MoAb 418 to vWF, in
which the epitope is located between aa 2 and 53, was used as a
control.17 We also used MoAbs 6D1 and 10E5 (a kind gift
from Dr B.S. Coller, SUNY, Stony Brook, NY), which are directed against
vWF binding sites on platelet GPIb-IX and
IIb3, respectively.18 MoAb to P-selectin (S12) was obtained from Dr R.P. McEver (Department of
Medicine, University of Oklahoma, Oklahoma City, OK). MoAb PAC-1 to activated IIb3 was provided by
Dr Shattil (Scripps Research Institute, La Jolla, CA).19
Isotypic controls were from Immunotech (Marseille, France).
Preparation of F(ab)2 fragments of MoAb 724.
F(ab)2 fragments of MoAb 724 were obtained after
digestion with pepsin (Sigma) at 37°C for 1 hour by using a
pepsin/protein ratio of 1/50 (wt/wt). F(ab)2
fragments were further purified by chromatography on protein-A
Sepharose (Pharmacia, Uppsala, Sweden). Purity of the fragments was
assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis
under reducing and nonreducing conditions. F(ab)2
fragments of MoAb 724 were used at 10- or 20-µg/mL concentrations.
Shear experiments.
The rotating device is a Couette type viscometer used after some
modifications.20 The main part of the viscometer consists of two coaxial cylinders: an outer cylinder rotating at various angular
speed ( ) and an inner cylinder of a smaller diameter, which is kept
static. The shear rate () applied to the samples in the cylinder gap
(e) can be calculated as = R/e, where R is the radius of the
outer cylinder (10 mm) and e is set at 0.5 mm. The equipment can
generate steady state flow conditions by achieving shear rates varying
between 50 and 4,000 seconds1, within 5 seconds. The
rotating device is kept at a constant temperature of 20°C by a
water-bath thermostating unit (Bioblock, Illkirch,
France). Washed platelet suspensions (1.5 × 108/mL) were exposed for 5 minutes to a continuous shear
rate in the absence or presence of purified vWF (10 µg/mL) and
different MoAbs to vWF or platelet receptors (20 µg/mL) in a final
volume of 240 µL. Preincubation with antibodies was performed for 5 minutes at 20°C except for the antiplatelet MoAbs, which were
preincubated with platelets at 37°C. After exposure to shear,
samples were fixed with 1% paraformaldehyde by addition of a 10-fold
concentrated solution and mixed for 30 seconds. A control platelet
sample was obtained by incubating the platelet suspension in the
absence of vWF in the cylinder gap for 5 minutes without exposure to
shear, followed by fixation in the same way.
Quantitation of SIPA.
An aliquot (10 µL) of the sheared or control sample was diluted in 1 mL of fluorescence-activated cell sorting (FACS)-flow buffer (Becton
Dickinson, Le Pont-de Claix, France). SIPA was measured in a FACScan
flow cytometer (Becton Dickinson) by modification of a method reported
for whole blood.21 Data acquisition was performed by
counting the particle number during a constant time (30 seconds) to
measure identical volumes in different samples. Washed platelets were
analyzed by forward light scatter and side light scatter without prior
labeling. The population of single platelets was defined by gating the
control platelet sample (no vWF and no shear). SIPA in the sheared
samples was compared by counting the gated population of single
platelets and results were expressed as the percentage of disappearance
of single platelets (DSP): DSP = [(n0  n)
/n0] × 100, where n0 represents the
single platelet population of the nonsheared control sample and n
represents the one of the sheared sample.
Fluorescence analysis of platelet samples.
Platelets (20 µL of the fixed samples) were incubated for 30 minutes
at 4°C with an appropriate dilution of the primary antibody (control IgM or PAC1 at 20 µg/mL, control IgG or S12 at 10 µg/mL). After washing in 80 mmol/L Na2HPO4, 20 mmol/L
KH2PO4 buffer, pH 7.4 phosphate-buffered saline
(PBS), antibody binding was assessed by flow cytometry by incubating
the platelets for 30 minutes at 4°C in the dark with a 100-fold
dilution of fluorescein isothiocyanate (FITC)-conjugated goat
F(ab)2 fragments directed against mouse IgG or IgM
(Caltag Laboratories, South San Francisco, CA). Acquisition of a
constant number of particles (usually 10,000) was used for the samples,
and fluorescence was detected by using the 525-nm band pass filter of
the flow cytometer.
Shear-independent platelet aggregation.
All shear-independent aggregation experiments were performed with fresh
platelets. Washed platelets (108/mL) were incubated in the
presence of fibrinogen (200 µg/mL) and 1 µmol/L ADP (Stago,
Asnières, France) or 2 µg/mL collagen (Nycomed, Munich,
Germany), and aggregation was measured in a dual channel aggregometer
(Chrono-Log Corp, Coultronics, France). Ristocetin-induced aggregation
was performed in the presence of vWF (10 µg/mL) and varying
concentrations (0.5-1 mg/mL) of ristocetin (abp, New York, NY). In some
cases, F(ab)2 fragments of MoAb 724 (10 µg/mL)
were preincubated with vWF before aggregation studies. PRP aggregation
studies were also performed by using 0.7 mg/mL of ristocetin and
varying concentrations of MoAb 724 (0.8 to 10 µg/mL).
Binding of 125I-vWF to platelets.
Platelets were isolated from PRP and fixed with paraformaldehyde (2%)
in 0.15 mol/L NaCl, 25 mmol/L Tris-HCl buffer, pH 7.4, containing 0.1%
BSA.14 125I-vWF was preincubated with varying
concentrations of MoAb 418 or MoAb 724 (0 to 20 µg/mL) during 30 minutes at 20°C. The final mixture contained 108
platelets/mL, 125I-vWF (0.5 µg/mL), MoAb to vWF, and
ristocetin (0.6 mg/mL). After 1 hour incubation at 20°C, duplicate
aliquots (100 µL) were layered onto 200 µL of 25% sucrose in
microfuge tubes and centrifuged for 3 minutes at 10,000g. The
tube tip containing bound ligand was separated from supernatant. Bound
and free radioactivities were counted in a counter (LKB Instruments
SA, Bromma, Sweden). The percentage of total bound radioactivity was
calculated as bound/(free + bound) radioactivity. Specific binding was
obtained by subtracting nonspecific binding in the absence of
ristocetin from total binding.
Statistical analysis.
Means ± standard error of the means (SEM) were calculated from
three experiments performed in duplicate. Statistical significance of
differences between means was evaluated by using Student's t-test for paired samples.
| |
RESULTS |
Effect of shear rate and vWF on platelet aggregation.
To verify the dependency of SIPA on vWF, different shear rates (0, 200, 2,000, and 4,000 seconds1) were applied to two
series of samples, either in the absence or presence of purified vWF.
When DSP in the single platelet region was plotted as a function of
shear rate, clear differences were observed between the vWF-free and
the vWF-containing samples (Fig 1). In the
absence of vWF, the increase of DSP from the unsheared sample (0%) to
the sheared sample at 4,000 seconds1 (5.9% ± 3.5%) was not statistically significant. In contrast, in the
vWF-containing samples, SIPA increased significantly with shear as
shown by DSP of 20.9% ± 4.5% at 2,000 seconds1
and 32.7% ± 6.3% at 4,000 seconds1 (P < .01 and P < .005, respectively, relative to the unsheared sample). At these shear rates, DSP values were increased significantly in the presence of vWF (P < .01). In addition, in the
presence of vWF, the dot plots showed aggregated platelets at shear
rates of 2,000 and 4,000 seconds1. Therefore, our
data indicate that vWF is required for shear-dependent aggregation of
washed platelets at a shear rate above 2,000 seconds1.
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| Fig 1.
Effect of vWF on shear-induced aggregation of washed
platelets. Different shear rates were applied for 5 minutes at 20°C
to washed platelet suspensions (1.5 × 108/mL) either in
the absence of vWF ( ) or in the presence of 10 µg/mL purified vWF
( ). After exposure to shear, samples were fixed with 1%
paraformaldehyde. Forward and side light scatter dotplots were obtained
by counting a constant volume in a flow cytometer. The single platelets
region was determined in the buffer-containing unsheared sample and
used as the reference value for calculation of disappearance of single
platelets (DSP). Means ± SEM from three experiments
performed in duplicate were expressed as a function of shear rate. In
the absence of vWF, DSP of sheared platelets was hardly modified
compared with the unsheared sample. In the vWF-containing samples,
percent of DSP increased significantly with shear.
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Effect of shear rate and vWF on PAC1 and S12 epitope expression.
Washed platelets were assessed for their ability to express activation
epitopes for activated IIb3 (PAC-1) and
P-selectin (S12) in response to exogenous agonists. We found that
collagen-induced aggregation resulted in 60% positive platelets both
for activated IIb3 and for P-selectin,
whereas ADP-induced aggregation resulted in 25% positive platelets for
activated IIb3 and 5% for P-selectin (data not shown). These results were compared with the expression of
the PAC1 and S12 epitopes in platelet suspensions exposed to different
shear rates in the absence or presence of vWF
(Fig 2). We found a shear-dependent
increased expression of activated IIb3 (Fig 2A). At 4,000 seconds1, PAC-1 expression was
almost twofold higher in the vWF-containing samples (36.3% ± 4.9%) than in vWF-free samples (17.5% ± 6.1%, P < .001). However, in samples devoid of vWF, we found a slightly higher
PAC-1 expression at 4,000 seconds1 than in unsheared
samples (9.2% ± 3%). In contrast, up to 2,000 seconds1 no detectable expression of P-selectin was
seen (<1%) and there was no significant difference between the
control samples and the vWF-containing samples (Fig 2B). At the highest
shear rate of 4,000 seconds1, the samples exhibited
a slightly increased level of P-selectin expression (3.6% ± 1.3%
and 6% ± 2.6% in the absence and presence of vWF, respectively)
comparable with that found in ADP-activated platelets. This result
indicates that a significant contribution of -granule-secreted
proteins, such as fibrinogen, may be ruled out in these shear rate
conditions.
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| Fig 2.
Effect of shear rate and vWF on PAC-1 and S12 epitope
expression. Washed platelets exposed to different shear rates were
incubated with PAC-1 directed to activated
IIb3 or S12 to P-selectin. The percentage
of positive platelets was assessed by flow cytometry by incubating the
platelets with an FITC-conjugated secondary antibody. Means ± SEM
were calculated from three experiments performed in duplicate.
Platelets were exposed to different shear rates in the absence () or
in the presence of 10 µg/mL vWF (). (A) PAC-1 expression. (B) S12
expression. Note differences in the scale of the y-axis.
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Effect on SIPA of MoAbs to vWF interfering with platelet-vWF
interactions.
The influence of different MoAbs to vWF on SIPA was compared in sheared
samples (4,000 seconds1) in the presence of vWF. In
a particular set of experiments, relative to the DSP obtained in the
absence of antibody (24.6% ± 3.2%) a complete inhibition of SIPA
was seen in the presence of MoAb 328, which is known to block the
vWF-GPIb interaction (DSP of 4% ± 2.1%). Furthermore, MoAb 328 completely suppressed the formation of large as well as small
aggregates and restored the single platelet population count to the
value observed in the control vWF-free sheared sample. In contrast,
MoAb 9 that blocks the vWF-IIb3
interaction, was able to inhibit SIPA by 45% (11.5% ± 5.2%).
Surprisingly, we found a completely different effect of MoAb 724 to
vWF, which blocks vWF binding to GPIb in the presence of botrocetin, as
shown by the strongly enhanced SIPA at a shear rate of 4,000 seconds1 (Fig 3). This
effect was clearly mediated by vWF because it was not observed when
platelets were exposed to shear in the absence of vWF. In contrast, no
effect was observed in samples incubated with MoAb 724 versus control
MoAb 418, in the absence of shear, whether vWF was present or absent
(data not shown). To rule out an interaction with the Fc-receptor,
we also studied 724 F(ab)2 fragments for their
ability to modify SIPA at 4,000 seconds1. Both MoAb
724 and its F(ab)2 fragments were able to
significantly enhance SIPA because DSP reached 165% of the value
obtained in the absence of MoAb 724 (P < .05), indicating
that the effect was Fc-receptor independent (Fig 3). In keeping with
an enhancing effect by MoAb 724 of vWF-dependent platelet activation at
4,000 seconds1, we found a 150% increase of
P-selectin expression relative to control MoAb. Similar results were
obtained for PAC-1 expression. Interestingly, similar to the effect of
MoAb 328 and 9, we found that the effect of anti-platelet MoAbs 6D1
(anti-GPIb) and 10E5 (anti-IIb3) resulted
in a complete or partial inhibition, respectively. In addition, MoAb
6D1 on the one hand and MoAb 10E5 on the other hand were equally
effective in inhibiting SIPA in the presence of vWF and either the
control MoAb 418 or MoAb 724 (Table 1). Furthermore, at a physiological temperature of 37°C a similar increase of DSP was observed on addition of MoAb 724 compared with
20°C.
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| Fig 3.
Effect of MoAb 724 to vWF on SIPA. Washed platelet
suspensions (1.5 × 108/mL) and either buffer ( ) or
purified vWF (10 µg/mL, ) were exposed to a shear rate of 4,000 seconds1 for 5 minutes at 20°C in the presence of
IgG of MoAb 418 as control, IgG of MoAb 724, or
F(ab)2 fragments of MoAb 724 (20 µg/mL). DSP was
calculated as outlined in the legend to Fig 1. Means ± SEM were
calculated from three experiments performed in duplicate. In the
presence of MoAb 724 or its F(ab)2 fragments, a
significant enhancement of SIPA was observed in the vWF-containing
samples, whereas this effect was not seen in the vWF-free samples.
*P < .05, for the effect of MoAb 724 (IgG or
F(ab)2 fragments) versus MoAb 418 in the
vWF-containing samples.
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Effect of MoAb 724 on shear-independent platelet aggregation.
To determine whether the enhancing effect of MoAb 724 was specific to
shear, we studied its influence on shear-independent platelet
aggregation. To this end, we incubated washed platelets and vWF with
ristocetin at concentrations too low to induce a significant
aggregation. Interestingly, in this range (0.5 to 0.7 mg/mL),
aggregation was significantly increased on addition of
F(ab)2 of MoAb 724 (Fig
4). At higher ristocetin concentrations, aggregation values were only
slightly higher in the presence of MoAb 724 than in control samples,
indicating that the effect of MoAb 724 was bypassed by these relatively
high ristocetin concentrations. In addition, we found a similar effect
in PRP, because at a low ristocetin concentration of 0.7 mg/mL, MoAb
724 was able to enhance platelet aggregation in a dose-dependent manner
(Fig 5). Incubation of washed platelets or
PRP with MoAb 724 in the absence of ristocetin did not result in a
spontaneous aggregation. This suggests that, by binding to vWF, MoAb
724 enhances the effect of low concentrations of ristocetin and induces
platelet activation and aggregation.
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| Fig 4.
Effect of F(ab)2 fragments of MoAb 724 on shear-independent platelet aggregation induced by different
ristocetin concentrations. Washed platelets (108/mL) and
purified vWF (10 µg/mL) were incubated with different ristocetin
concentrations either in the absence () or in the presence of
F(ab)2 fragments (10 µg/mL) of MoAb 724 ().
Slopes of aggregation were measured and results were expressed relative to the value obtained with 1 mg/mL ristocetin concentration in the
absence of MoAb 724, which was arbitrarily set as the maximal aggregation. At low ristocetin concentrations (0.5 to 0.8 mg/mL), F(ab)2 fragments of 724 increased the aggregation.
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| Fig 5.
Effect of varying concentrations of MoAb 724 on
shear-independent platelet aggregation. PRP was incubated with 0.7 mg/mL of ristocetin, in the presence of MoAb 724. Results were
expressed as slopes of aggregation. MoAb 724 was able to increase
platelet aggregation in a dose-dependent manner.
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Effect of MoAb 724 on 125I-vWF binding to platelets.
To determine whether MoAb 724 binding to vWF allows increased binding
to GPIb, the effect of MoAb 724 on 125I-vWF binding to
fixed platelets was studied in the presence of a low ristocetin
concentration (0.6 mg/mL). Interestingly, binding of vWF was enhanced
in a dose-dependent manner by MoAb 724, whereas it was unchanged in the
presence of the control MoAb 418 (Fig 6).
In addition, this MoAb 724-dependent increase was significantly higher
at the lowest ristocetin concentrations (data not shown), thus
comparable with the effect of MoAb 724 on platelet aggregation. Furthermore, we found that F(ab)2 fragments of MoAb
724 dose-dependently inhibited botrocetin-induced binding of
125I-vWF to GPIb with an IC50 of 4 µg/mL (data not
shown), confirming previously reported data on the effect of MoAb 724 IgG.16 Finally, because MoAb 724 has been reported as an
inhibitor of botrocetin binding to vWF, conversely it was of interest
to investigate the ability of botrocetin to interact with MoAb 724 binding to vWF. At the highest concentration added (50 µg/mL),
botrocetin was able to completely inhibit the binding of MoAb 724 with
immobilized vWF, whereas it had no effect on the binding of a control
MoAb.
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| Fig 6.
Effect of MoAb 724 on 125I-vWF binding to
platelets. Binding of 125I-vWF (0.5 µg/mL) to fixed
platelets (108/mL) was performed in the presence of
ristocetin (0.6 mg/mL) and varying concentrations of either MoAb 418 () or MoAb 724 (). Results were expressed as specific binding to
platelets after subtraction of nonspecific binding in the absence of
ristocetin. Means ± SEM were calculated from three experiments in
duplicate. In the presence of MoAb 724, 125I-vWF binding to
platelets was significantly enhanced compared with MoAb 418 (*P < .01).
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| |
DISCUSSION |
Using a coaxial viscometer to apply shear rates ranging from 200 to
4,000 seconds1, we report on the activating effect
of a MoAb directed against the A1 domain of vWF on SIPA. We thereby
provide new information on the molecular mechanism of high
shear-dependent vWF interaction with platelets.
It has been reported that SIPA does not require exogenous agents such
as ristocetin or botrocetin. To clarify the vWF sequences involved in
this interaction, we have chosen the straightforward approach of a
purified system, consisting of washed fresh platelet suspensions
incubated with purified plasmatic vWF and exposed to varying shear
rates. We confirm that SIPA is dependent on vWF at 2,000 and 4,000 seconds1, whereas at lower shear rates no
significant aggregation is observed in the presence or absence of vWF.
Thus, our model grossly reproduces the situation observed in patients
with afibrinogenemia and indicates that, whereas fibrinogen is the main
effector in low shear conditions, vWF is primarily involved as a
mediator of platelet aggregation in high shear
conditions.12 A complete inhibition was obtained by
incubating platelets with MoAb 6D1 or MoAb 328, previously reported to
completely block vWF binding to GPIb in static and high shear
conditions.18,22 We found that they both completely block
SIPA, thereby confirming in a purified system the involvement of vWF in
SIPA.13 Interestingly, we found that MoAb 10E5, an anti-IIb3 MoAb that completely blocks vWF
binding to activated platelets in static conditions, inhibits SIPA by
only about 50%, as reported by another group in different shear
conditions.22 Moreover, MoAb 9 to vWF, which completely
inhibits vWF binding to activated IIb3 in
static conditions, was also found to have a similar partial inhibitory
effect on SIPA, indicating a different involvement of each vWF receptor
in SIPA. Interestingly, similar results were recently reported on the
partial inhibition of SIPA by MoAb GUR76-23 to vWF, recognizing a
different epitope from MoAb 9.23
Because normal washed platelets were used, we cannot exclude a role of
adhesive -granule proteins, in particular a role of endogenous vWF
or fibrinogen released from platelets. However, this is unlikely
because we showed that P-selectin, another -granule protein, is
hardly expressed on the platelet surface even after exposure to the
highest shear rate tested. This low expression (6% in the presence of
vWF exposed at 4,000 seconds1 for 5 minutes) can be
compared with the value of 10.5% obtained in whole blood samples
exposed to 10,000 seconds1 during 30 seconds.21 In addition, we find a fourfold higher PAC-1
expression in sheared vWF-containing samples compared with nonsheared
vWF-free samples, indicating a significant increase of activated
IIb3 expressed on the surface after
exposure to shear. This value compares very well with the value
reported under slightly different conditions of exposure to shear
(10,000 seconds1, 30 seconds).21 This
confirms the prevailing model that the vWF-GPIb interaction acts as a
platelet activator under high shear conditions and, as recently shown,
that activated IIb3, even occupied by
ligands, is not sufficient to mediate platelet aggregation under high
shear stress conditions.24
Interestingly, SIPA provides a different approach from binding studies
in static conditions to analyze the effect of MoAbs to vWF. We have
compared two MoAbs, MoAb 724 and 328, that block binding of vWF to GPIb
in the presence of botrocetin and ristocetin, respectively.14,16 MoAb 328 is a potent inhibitor of SIPA
in high shear conditions. In contrast, MoAb 724 increases SIPA up to
165% of the value obtained in the absence of MoAb 724. This enhancing
effect is completely abrogated when platelets are incubated with MoAbs
6D1 or 10E5, so that the remaining SIPA is similar to the value
obtained in the absence of MoAb 724. In addition, the effect of MoAb
724 requires an interaction with vWF, because no increase is obtained
in the platelet samples devoid of vWF. Similar results with
F(ab)2 fragments were obtained, which rules out an
effect mediated by the platelet Fc-receptor. Because MoAb 724 binds
to normal vWF with a higher affinity than to vWF from type 2B vWD,
which has an increased reactivity for GPIb,16 altogether our data suggest that the vWF-MoAb 724 complex acts through the same
pathway but with a higher affinity than vWF alone does.
Finally, this report shows that the enhancement of platelet aggregation
is not restricted to a shear-dependent effect, because MoAb 724 can
enhance platelet aggregation in the presence of ristocetin at low
concentrations, unable to induce a detectable increase in turbidity in
a classical aggregometer. In addition, we find a significant increase
of vWF binding to fixed platelets by MoAb 724 in the presence of low
ristocetin concentrations. In contrast, MoAb 724 has no enhancing
effect on platelet aggregation in the presence of botrocetin (data not
shown). This is in agreement with the fact that MoAb 724 competes with
botrocetin for binding to the same site(s) on vWF as shown by the
strong inhibitory effect of MoAb 724 on botrocetin-induced vWF binding
to GPIb.16 Conversely, highly purified botrocetin was able
to completely inhibit the binding of 125I-MoAb 724 to vWF,
strongly suggesting that this MoAb shares a common site with
botrocetin. It should be mentioned that in using high concentrations of
MoAb 724 (50 to 100 µg/mL), an inhibitory effect of
botrocetin-induced platelet aggregation has been
reported.16 This effect does not contradict the present
findings, because these high IgG concentrations are likely to act
through a steric hindrance mechanism. According to our hypothesis, the
enhancing effect of MoAb 724 on SIPA is secondary to its binding to a
regulatory site of vWF, which would be responsible for an increased
affinity for platelet GPIb. Once in this "active" conformation,
vWF may be more sensitive to high shear conditions explaining the
increased SIPA compared with the control. Thus, MoAb 724 may be
considered as a modulatory antibody, similar to the effect obtained
when botrocetin binds to vWF. This is confirmed by the additive effects of high shear rates and low botrocetin concentrations resulting in
extensive aggregate formation (data not shown). This does not exclude a
direct effect of shear on the conformation of vWF suggested by
others,25 indirectly established in comparing the
properties of shear- versus ristocetin-induced vWF binding to
platelets.26 Very few studies have attempted to compare the
effect of anti-vWF MoAbs in shear-dependent and shear-independent
assays. Our data with MoAb 724 may indirectly indicate that the binding
is not affected by shearing conditions. Recently, studies performed by atomic force microscopy suggested that elevated shear stress can directly induce conformational change of vWF, although evidence is
still missing for the involvement of the A1 domain.27 It would be interesting to use MoAb 724 as a tool to determine with certainty whether the vWF conformation can be affected by shear. However, there is some debate on a role of high shear rates on the
structure and function of GPIb so that the active conformation of vWF
may be more easily recognized by GPIb, which is itself modified by
shear.28
Finally the significance of a platelet-activating antibody directed
against an adhesive protein can be addressed. Another antibody directed
to vWF has been previously described by Tornai et al,29
which could increase vWF binding to both its platelet receptors. This
result is different from ours, because we did not find any enhancing
effect of MoAb 724 on vWF binding to activated IIb3. Interestingly, the epitope of that
MoAb has been localized in the amino terminal part of vWF, within the
factor VIII-binding region, thus suggesting different regulatory
elements distributed along the vWF subunit. However, our results are in
favor of a direct conformation-dependent change of the A1 domain, which
contains the GPIb-binding sequence, because the MoAb 724 epitope is
located inside the A1 domain.
In conclusion, we have confirmed that SIPA requires vWF and its two
platelet receptors, GPIb and activated
IIb3. The following working model for the
interaction between vWF and GPIb can be proposed based on opposite
effects of two MoAbs to the A1 domain of vWF on SIPA. MoAb 328, which
inhibits vWF binding to GPIb in the presence of ristocetin, recognizes
a site on vWF involved in the shear-dependent interaction with GPIb. In
contrast, MoAb 724 recognizes a modulatory site that is common to a
botrocetin binding site. High shear rates, as well as high ristocetin
concentrations, may bypass the effect of the binding of MoAb 724 to
this regulatory site. However, MoAb 724 may unmask the effect of this
modulation site when added in combination with lower ristocetin
concentrations or with shear rates that induce submaximal aggregation.
| |
FOOTNOTES |
Submitted October 20, 1997;
accepted January 8, 1998.
Supported by grants of EC Biomed, PL 93 1685 and the Belgian Royal
Academy for Medicine to H.D. while on a postdoctoral leave in INSERM
U143 and an INSERM fellowship to N.A.
Address reprint requests to Dominique Baruch, MD, PhD, INSERM U143, 84 rue du General Leclerc, 94276 Bicêtre Cedex, France.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
Drs Coller, McEver, and Shattil are thanked for providing antibodies.
We thank Paulette Legendre and Stephan Vauterin for expert technical
assistance.
| |
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Abstract
Introduction
Abstract