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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 93 No. 2 (January 15), 1999:
pp. 564-570
Fibrin-Dependent Platelet Procoagulant Activity Requires GPIb
Receptors and von Willebrand Factor
By
S. Béguin,
R. Kumar,
I. Keularts,
U. Seligsohn,
B.S. Coller, and
H.C. Hemker
From the Department of Biochemistry, Cardiovascular Research
Institute Maastricht (CARIM) and Medical Faculty University of
Maastricht, Maastricht, The Netherlands; the Department of Hematology,
Sheba Medical Center, Tel Hashomer and Sackler Faculty of Medicine,
Tel-Aviv University, Tel-Aviv, Israel; and the Department of Medicine,
Mount Sinai School of Medicine, New York, NY.
 |
ABSTRACT |
Thrombin generation in platelet-rich plasma (PRP) involves complex
interactions between platelets and coagulation proteins. We previously
reported that the addition of fibrin to PRP enhances tissue-factor
initiated thrombin generation by  40%, and the current
studies were designed to assess the mechanism(s) underlying thrombin
generation in the absence and presence of fibrin. Blocking platelet
GPIIb/IIIa +  v 3 receptors with a monoclonal antibody (MoAb)
inhibited basal thrombin generation, but did not affect the enhancement
produced by fibrin. In contrast, blocking GPIb with any of three
different MoAbs had no effect on basal thrombin generation, but
essentially eliminated fibrin enhancement of thrombin generation. When
thrombin generation was tested in PRP deficient in von Willebrand
factor (vWF), both basal and fibrin-enhanced thrombin generation were
markedly reduced, and the addition of factor VIII did not normalize
thrombin generation. Botrocetin, which induces the binding of vWF to
GPIb, enhanced thrombin generation. In all studies, the ability of PRP
to support thrombin generation correlated with the production of
platelet-derived microparticles and serum platelet-derived procoagulant
activity. Thus, two separate mechanisms, both of which depend on vWF,
appear to contribute to platelet-derived procoagulant activity: one is
independent of fibrin and relies primarily on GPIIb/IIIa, but with a
minor contribution from v3; and the other is fibrin-dependent and relies on GPIb. These data may have implications for understanding the
mechanisms of the abnormalities in serum prothrombin times reported in
Bernard-Soulier syndrome, hemorrhage in von Willebrand disease (vWD),
and the increased risk of thrombosis associated with elevated vWF
levels.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
THE FORMATION OF AN arterial thrombus
involves platelet deposition, activation of coagulation, and fibrin
formation. There is abundant evidence that platelets can facilitate
thrombin generation by a number of different mechanisms.1
We recently showed that in a fibrin-free system consisting of
gel-filtered platelets and defibrinated plasma, inhibition of platelet
GPIIb/IIIa + v3 receptors with monoclonal antibody (MoAb), 7E3,
inhibits ex vivo thrombin generation induced by tissue factor by 47%1; similar inhibition by 7E3 was observed using
platelet-rich plasma (PRP),1 a system in which fibrin
formation occurs late in the course of the experiment. The inhibition
of thrombin generation correlated with decreased platelet microparticle
formation, offering a possible mechanistic explanation, as platelet
microparticle formation results in loss of the normal asymmetry of
platelet membrane phospholipids leading to surface exposure of
negatively-charged phospholipids, which are highly active in supporting
thrombin generation.2-4
In separate experiments, we found that fibrin itself can enhance
platelet membrane procoagulant activity (PMPA) when added to PRP, even
when fibrin is formed in a way that it does not contain thrombin.5,6 Thus, the explosive generation of thrombin
that occurs in recalcified PRP after a lag-phase (see eg, Fig 1) is probably the result of a composite resonance loop in which (1) the
generation of small amounts of thrombin converts fibrinogen to fibrin,
(2) both thrombin and fibrin activate platelet to produce PMPA, and (3)
the enhanced PMPA facilitates the generation of more thrombin and
fibrin. Thus, fibrin appears to play an active role in amplifying
thrombin generation and further fibrin formation.
The current studies were designed to assess the relative contributions
of thrombin and fibrin to PMPA production under different experimental
conditions and to identify the platelet membrane receptors and adhesive
ligands that are responsible for the fibrin-platelet interactions that
result in enhanced thrombin generation. Thus, we investigated the roles
of GPIIb/IIIa, v3, GPIb, GPIa/IIa (21), and von Willebrand
factor (vWF).
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MATERIALS AND METHODS |
Reagents.
The chromogenic substrate used for measuring thrombin was S2238:
H-D-Phe-Pip-Arg-pNA.2HCl. Buffer A: 20 mmol/L HEPES, 150 mmol/L NaCl,
0.5 g/L bovine serum albumin (BSA; Lot A-7030, Sigma, St Louis,
MO), pH 7.35. Buffer B: same as buffer A with 20 mmol/L EDTA, pH = 7.9. Antibody binding buffer (FACScan): 10 mmol/L HEPES, 0.15 mol/L NaCl, 5 mmol/L KCl, 1 mmol/L MgCl2, 2.5 mmol/L
CaCl2 (pH 7.4). Synthetic
d-arginyl-L-glycyl-L-aspartyl-L-tryptophan (d-RGDW) (Mr 530) was
obtained from Rhone-Poulenc Rorer Antony (Paris, France).
Botrocetin was from Kordia (Leiden, The Netherlands). Phospholipid
vesicles were prepared from a mixture of 20% brain phosphatidyl serine
(PS) and 80% egg phosphatidyl choline (PC) sonicated into a buffer
containing 50 mmol/L Tris HCl (pH 7.35 ) and 100 mmol/L NaCl. All other
reagents were of the highest grade commercially available.
Proteins and antibodies.
Bovine factors Xa and Va and human prothrombin were kindly provided by
Dr R Wagenvoord (Maastricht University, Maastricht, The
Netherlands). Recombinant tissue factor was obtained from Dade (Düdingen, Switzerland). Agihal, purified fraction of
Agkistrodon halys halys snake venom, which splits fibrinopeptide A from
fibrinogen, was obtained from Prof L. Yukelson (Tashent, Uzbekistan).
Staphylocoagulase was prepared as described.7 Recombinant
factor VIII (rFVIII; Recombinate, Baxter, Deerfield, IL) contains only
traces of vWF (<2 ng per unit of factor VIII). Fluorescein
isothiocyanate (FITC)-labeled annexin V (Apoptest, Oregon Green) was
from NeXius Research BV (Hoeven, The Netherlands).
Murine MoAbs, 7E3 (anti-GPIIb/IIIa + v3)8, 6D1
(anti-GPIb),9 and 6F1 (anti-GPIa/IIa)10 have
been previously described in detail. MoAb CD42b (anti-GPIb) was from
Immuntech (Marseille, France) and MoAb AP-1 (anti-GPIb) was a kind gift
of Dr Thomas Kunicki (Scripps Institute, La Jolla, CA).
R-phycoerythrin-conjugated MoAbs to human GPIIb/IIIa (5B12) and GPIb
(AN51) were obtained from Dako (Glostrup, Denmark). An
affinity-purified polyclonal antibody to vWF was obtained from the
Central Laboratory of the Red Cross (CLB) in Amsterdam, The
Netherlands.
Preparation of plasma.
PRP was obtained by centrifuging fresh citrated blood (9 parts of blood
to one part of 0.13 mol/L trisodium citrate) at 250g, 15°C
for 10 minutes. The platelet count was adjusted to 3 × 108/mL using autologous platelet-poor
plasma (PPP).
The study included four patients with Glanzmann thrombasthenia of
Iraqi-Jewish descent (GPIIIa defect leading to loss of both GPIIb/IIIa
and v3) and three of Arab descent (GPIIb defect leading to loss
of GPIIb/IIIa, but normal or increased v3) residing in
Israel.11 By courtesy of Dr Karly Hamulyak (Academic
Hospital, Maastricht), blood was obtained from one patient with
Glanzmann thrombasthenia, and one with von Willebrand disease (vWD)
type IIa. By courtesy of Dr J. Eikelboom12 (Academic
Hospital Leiden), blood was obtained from a patient with type III vWD
(described in Weiss et al13).
Preparation of clots.
Fibrin I clots (noncross-linked, des AA fibrin) were prepared as
previously described using the snake venom protease Agihal. Addition of
clots to PPP did not cause coagulation within 2 hours and did not
influence either the clotting time or thrombin generation in
recalcified PPP. Three clots were added to PRP in the thrombin generation experiments, representing 3 times the potential fibrin content of the PRP.
Measurement of thrombin generation.
Thrombin generation in plasma was performed as described
previously.14,15 In short, for thrombin generation in PPP,
20 µL of a kaolin suspension and 20 µL of Buffer A were added to 240 µL of PPP. At 4 minutes, 20 µL of PS/PC (20 µmol/L)
was added and at 5 minutes, coagulation was triggered by adding 60 µL
of 0.1 mol/L CaCl2. For thrombin generation in PRP, 240 µL of PRP was incubated with 60 µL of Buffer A or buffer containing
the antibody or other additions to be tested for 10 minutes at
37°C. Fibrin clots were added just before coagulation was initiated by adding 60 µL of 0.1 mol/L CaCl2, 1.8 fmol/L tissue factor.
One minute after initiating coagulation, 10 µL-samples of the
reaction mixture were taken at 1-minute intervals and added to
prewarmed (37°C) cuvettes containing 490 µL of 200 µmol/L S2238 in buffer B. The reaction was stopped after 2 minutes by adding 300 µL of 1 mol/L citric acid, and the optical density (OD) was measured
at 405 nm. Thrombin amidolytic activity was calculated by comparing the
OD/minute value of the test sample to a thrombin standard calibration
curve. Free thrombin was calculated from thrombin amidolytic activity
using our previously described computer program that takes into account
the contributions of free thrombin and 2-macroglobulin-bound
thrombin.15
The lag time of thrombin formation is defined as the time from addition
of the triggering solution to the time at which the thrombin
concentration increases above 10 nmol/L and the endogenous thrombin
potential (ETP) is defined as the area under the thrombin generation
curve.16,17 In normal PRP, the ETP is 411 ± 13 nmol/L × min (mean ± standard error of mean [SEM], n = 28).
Measurement of platelet-derived procoagulant activity and
microparticles in serum.
The platelet-membrane derived procoagulant phospholipid activity (PMPA)
was determined by diluting serum prepared from PRP after thrombin
generation experiments 3:20 in buffer A and adding a 50-µL aliquot of
the diluted serum to 100 µL of an assay mixture containing 0.45 nmol/L factor Xa, 10.5 nmol/L factor Va, 3 µmol/L prothrombin and 12 mmol/L Ca2+ in buffer A. At 4 minutes, a 10 µL subsample
was added to cuvettes containing 465 µL of buffer B. Thrombin
concentrations were then assayed with S2238 by determining the change
in absorbance over time. Normal serum gives values of 115 ± 5.5 nmol/L/minute (mean ± SEM, n = 22), equivalent to the
effect of 300 nmol/L PS/PC (20%/80%) vesicles. Normal PPP or the
serum left after a thrombin generation experiment in PPP gives
values < 12 nmol/L/minute.
To detect platelet-derived microparticles, 15 µL of serum was
incubated with R-phytoerythrin-conjugated mouse MoAbs against GPIIb/IIIa or GPIb. To assess the presence of anionic phospholipids on
the microparticles, 250 µL of 2 µg/mL FITC-labeled annexin V was
added. The analysis was performed in a flow cytometer (EPICS XL-MCL;
Becton Dickinson & Co, San Jose, CA). A total of 10,000 events was
recorded and the data were analyzed using the CellQuest software
program, version 1.2 (Becton Dickinson & Co). There was a
high correlation between annexin-V-positive and platelet
glycoprotein-positive particles, indicating that the particles that
exposed PS were derived from platelets.
Measurement of residual prothrombin in serum.
Residual prothrombin was assessed as previously
described.7,15,18
| |
RESULTS |
The effects of adding fibrin clots to normal PRP and of blocking
GPIIb/IIIa + v3, GPIb, and GPIa/IIa (21)
receptors.
Consistent with our earlier observations, adding fibrin clots to
normal PRP enhanced the ETP by 42%, peak thrombin generation by
64%, platelet-derived microparticles (PDMP) by
44%, and PMPA by 78%5
(Fig 1 and
Table 1). It also decreased
residual prothrombin by 50% (Table 1). Also consistent with our
earlier observations, blockade of GPIIb/IIIa + v3 receptors by
antibody 7E3 or the peptide d-RGDW (60 µmol/L) decreased ETP of PRP
to 42% and 60% of normal, respectively1 (Fig 1 and Table
1). The other parameters of thrombin generation were affected in a
manner consistent with the inhibitory effects of these agents on the
ETP (Table 1). What was most remarkable, however, was the ability of
fibrin clots to enhance thrombin generation in PRP in which GPIIb/IIIa + v3 receptors had been blocked by 7E3 or d-RGDW (Fig 1 and Table
1).
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| Fig 1.
Effect of blocking GPIIb/IIIa + v3 receptors on
thrombin generation in the absence and presence of fibrin clots.
Thrombin generation was triggered at t = 0 in PRP (adjusted to 3 × 108/mL) by recalcification and addition
of tissue factor. ( ) Control; ( ) three fibrin clots added at t
= 0; (*) PRP preincubated with antibody 7E3
(anti-GPIIb/IIIa + v3; 20 µg/mL); ( ) preincubation with
7E3 and fibrin clots added.
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Table 1.
Effect of GPIIb/IIIa + v3, GPIb, and GPIa/IIa
Blockade on Thrombin Generation in PRP in the Presence and Absence of
Fibrin Clots
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Blocking the vWF binding domain of GPIb with antibody 6D1 had the
mirror image effect of GPIIb/IIIa + v3 blockade; thus, it had no
effect on thrombin generation in the absence of added fibrin, but
prevented the procoagulant-enhancing effect of fibrin (Fig 2 and Table 1). Two other antibodies
against GPIb (AP-1 and CD 426) gave similar results (Table 1). When
antibodies 7E3 and 6D1 were used in combination, the results were
additive, with both a reduction in thrombin generation and near
elimination of the enhancement of thrombin generation by adding fibrin
(Table 1). The MoAb 6F1, which blocks GPIa/IIa, affected neither normal thrombin generation nor the enhanced thrombin generation in the presence of fibrin, and thus served as a control (Table 1).
Ionomycin-treated PRP supported thrombin generation to the same extent
as did normal PRP with added fibrin (Table 1). None of the antibodies
inhibited thrombin generation supported by ionomycin-treated platelets
(data not shown).
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| Fig 2.
Effect of blocking GPIb receptors on fibrin-enhanced
platelet procoagulant activity. Thrombin generation was measured as in
Fig 1. () Control; () three fibrin clots added at t = 0; (*)
PRP preincubated with antibody 6D1 (anti-GPIb, 20 µg/mL); ()
preincubation with 6D1 and fibrin clots added.
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Studies using PRP from patients with Glanzmann thrombasthenia.
Because antibody 7E3 blocks both platelet GPIIb/IIIa and v3, we
tried to assess the contributions of each of these receptors by
comparing the results using PRP from different patients with Glanzmann
thrombasthenia. Iraqi-Jewish patients have no detectable GPIIb/IIIa or
v3, whereas Israeli-Arab patients have virtually no GPIIb/IIIa,
but approximately twice the normal level of platelet v3.11,19 In both patient groups, thrombin generation
in PRP is decreased to about 60% of normal
(Table 2). Antibody 7E3 decreased thrombin
generation in PRP of two Arab patients tested (from 68% to 59% and
from 35% to 25%), but had virtually no effect on thrombin generation
in the PRP of Iraqi-Jewish patients (from 71% to 70% and from 63% to
65%) (Table 2). Addition of fibrin clots to the PRP of patients in
either group resulted in increased thrombin generation (increases of
18%, 44%, and 103% for Arab patients B, C, and D; and 28% for
Iraqi-Jewish patient A), supporting the conclusions derived from the
antibody studies, namely that the effect of fibrin does not require
either GPIIb/IIIa or v3 receptors.
Experiments with vWF.
Because vWF has been reported to bind to fibrin and support an
interaction with platelet GPIb20, we investigated the
effect of vWF on thrombin generation. Decreasing vWF activity of normal PRP with a neutralizing antibody not only prevented the enhancement of
thrombin generation produced by fibrin, but, unexpectedly it also
diminished baseline thrombin generation
(Fig 3). In contrast, thrombin generation
in PPP was unaffected by vWF neutralization (Fig 3, inset), indicating
that there was sufficient factor VIII coagulant activity in the
antibody-treated plasma to support thrombin generation. Addition of
ionomycin (Fig 3) or a frozen and thawed platelet lysate (not shown)
restored normal thrombin generation, indicating that the defect caused
by vWF could be overcome by activated platelets.
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| Fig 3.
Effect on thrombin generation of reducing vWF activity in
plasma. () Control (normal PRP with 10 µg/mL rabbit IgG); ( )
PRP preincubated with vWF antibody (10 µg/mL); (X) PRP preincubated
with anti-vWF (10 µg/mL), three fibrin clots added at t = 90 s;
() preincubated with vWF antibody and 10 µmol/L ionomycin added at
t = 10 s. (Inset) Thrombin generation in PPP. The reaction
was triggered with PS/PC and Ca2+. () Control PPP with
10 µg/mL rabbit IgG; () PPP preincubated with anti-vWF (10 µg/mL).
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Additional experiments were performed with PRP of patients with mild
and severe vWD. Thrombin generation in the PRP of a patient with mild
vWD (type IIa, 30% factor VIII and 4% vWF antigen) was 60% of
normal. Adding anti-vWF antibody reduced thrombin generation to 12%
of normal (Fig 4). The patient's factor
VIII coagulant activity was sufficient to support thrombin generation as demonstrated by normal thrombin generation using the patient's PPP
(Fig 4, inset), with or without the addition of the anti-vWF antibody.
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| Fig 4.
Thrombin generation in PRP of a patient with mild type
IIa vWF deficiency. The patient's plasma contained 30% factor VIII
and 4% of vWF antigen. () Control PRP (with 10 µg/mL rabbit
IgG); X, patient's PRP (rabbit IgG added); () patient's PRP
preincubated with anti-vWF antibody (10 µg/mL). Inset: thrombin
generation in PPP. () Normal control; (X) patient.
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Thrombin generation in PRP from a patient with type III, severe vWD
(< 1% of vWF antigen and factor VIII) was more severely diminished
than in the PRP of a patient with mild vWD
(Fig 5, upper frame). Addition of
sufficient rFVIII to increase the factor VIII coagulant activity to
100% normalized thrombin generation in PPP (Fig 5, lower frame) yet
only partially restored thrombin generation in PRP to 40% of normal
(Fig 5, upper frame). The addition of 2.5% of normal rFVIII was
without effect on thrombin generation in PRP, but the addition of 2.5%
of normal PPP, which provides both vWF and essentially the same amount
of factor VIII, restored thrombin generation
(Fig 6). Adding ionomycin or either normal or patient platelet lysate (not shown) restored thrombin generation, indicating that procoagulant phospholipids were indeed rate-limiting. To further assess whether the interaction of vWF and GPIb enhanced thrombin generation, we added botrocetin to PRP and found that it did,
indeed, increase thrombin generation (Fig
7). Ristocetin could not be tested because in control experiments it
inhibited thrombin generation in PPP.
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| Fig 5.
Thrombin generation in plasma of patient with type III
severe vWD. The patient has less than 1% of factor VIII and vWF
antigen and ristocetin cofactor activity. (Upper frame) () Normal
PRP; (X) patient's PRP; ( ) patient's PRP with 100% recombinant
factor VIII added. (Lower frame) Thrombin generation in intrinsically
triggered nondefibrinated PPP. () Normal PPP; (X) patient's PPP;
() patient's PPP with 100% recombinant FVIII added.
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| Fig 6.
Thrombin generation in PRP of a patient with severe (type
III) deficiency of vWF. () patient's PRP with 2.5% recombinant
FVIII added; ( ) patient's PRP with 2.5% normal PPP added; (+)
patient's PRP with 2.5% normal PPP and with a frozen and thawed
suspension of normal platelets added at t = 90 s; () patient's
PRP with 2.5% normal PPP and with a frozen and thawed suspension of
the patient's platelets. The final concentration of platelet material
was equivalent to 2 × 107 platelets/mL.
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| Fig 7.
Effect of botrocetin on thrombin generation in PRP. ()
Normal PRP; () 5 µg/mL botrocetin added; (X) 25 µg/mL; () 50 µg/mL.
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DISCUSSION |
We previously observed two different phenomena related to platelets and
thrombin generation: (1) in a fibrin-free system or in a system in
which fibrin is generated late in the reaction, blockade of GPIIb/IIIa
and to a lesser extent v3 decreases thrombin generation, and (2)
adding fibrin to PRP enhances thrombin generation. The present studies
were designed to obtain data on the receptors, ligand, and mechanisms
responsible for these phenomena. Despite the ability of GPIIb/IIIa to
bind fibrinogen, polymerizing fibrin, and clotted fibrin under static
or flow conditions,21-27 our current data indicate that the
enhancing effect of fibrin on thrombin generation cannot be attributed
to a GPIIb/IIIa-mediated mechanism because fibrin retains its
stimulating effect in the presence of a GPIIb/IIIa + v3 blocking
antibody or peptide, as well as when added to the PRP of Glanzmann
patients.
We next studied the interaction between GPIb and vWF because platelets
have been shown to bind to fibrin through the interaction between
fibrin-bound vWF and platelet GPIb.20 Although anti-GPIb MoAbs had no effect on thrombin generation in the absence of added fibrin clots, they essentially abolished the enhancing effect of the
added fibrin. Thus, the fibrin effect seems to be mediated via GPIb.
This observation provides a possible explanation for the abnormal
prothrombin consumption previously reported in patients with
Bernard-Soulier syndrome, whose platelets lack GPIb28-32
and the abnormal prothrombin consumption that we previously reported when antibody 6D1 was added to normal blood.9
In view of the important role of GPIb in the fibrin-dependent
enhancement of thrombin generation in PRP, it is perhaps surprising that in normal, recalcified PRP, thrombin generation initiated by
tissue factor is minimally inhibited by blocking GPIb. The most likely
explanation is that in these experiments, fibrin begins to form late in
the process, at the very beginning of the thrombin burst, and thus
there is insufficient time for it to affect the process. When fibrin in
the form of preformed clots is added before the thrombin burst occurs,
it enhances thrombin generation and shortens the lag-phase in a
GPIb-dependent mechanism. Taken together, our previous and current data
indicate that these are two different pathways for augmenting platelet
coagulant activity: (1) a GPIIb/IIIa- and perhaps v3dependent
pathway that operates independently of fibrin, and (2) a fibrin-and
GPIb-dependent pathway. Of note, based on data using neutralizing
antibody to vWF and plasmas of patients with vWD, as well as our data
using botrocetin, both pathways appear to depend on vWF, suggesting
that vWF binding to GPIIb/IIIa (and perhaps v3) is important in
the development of platelet coagulant activity.
The generation of PMPA and platelet-derived microparticles in serum and
the consumption of prothrombin followed the same pattern of inhibition
as did thrombin generation. This suggests that microparticle formation
is responsible for the PMPA and that GPIIb/IIIa (and perhaps v3),
GPIb, and vWF are all required for maximal microparticle formation in a
fibrin-containing system. The ability of ionomycin-treated platelets
and platelet lysates to overcome the abnormalities produced by the
antibodies further suggests that the defects result in decreased
microparticle formation.
From our results it appears that the fibrin in a clot or thrombus is
not merely an inert, mechanical component.33 It also is
clear that vWF, apart from its established function in platelet adherence and as a carrier of factor VIII, may also play an important role in the generation of thrombin through its effect on the generation of platelet microparticles and platelet coagulant activity. Because it
has been proposed that the thrombosis associated with heparin-induced thrombocytopenia is linked to platelet microparticle
formation,34,35 it is interesting to speculate that
variations in vWF levels may account for the interindividual
differences in thrombotic risk. Recently, platelet microparticles were
found to support transcellular metabolism of eicosanoids,36
leading to activation of platelets and endothelial cells, as well as
modulation of monocyte-endothelial interactions,37 and so
it is possible that the mechanisms we are studying have implications
for these phenomena as well. Finally, our observations have potential
implications for understanding better the pathophysiology of the
bleeding in vWD, as well as the association between elevated plasma vWF
activity and acute myocardial infarction,38,39 as well as
death after stroke.40
| |
ACKNOWLEDGMENT |
We are grateful to the patients who volunteered to give their blood and
to their doctors, Dr Karly Hamulyak and Dr J. Eikenboom, for arranging
the opportunity to study their plasma. Dr Ariella Zivelin has been a
great support in the experiments with the PRP from the Iraqi-Jewish and
Arab Glanzmann patients. Our thanks are due to Dr J.A. van Mourik for
providing us the vWF antibodies. We also thank Dr J. Heemskerk for
performing the Ca2+-influx experiments and Dr Hu Kai for
his method of measurement of microparticle procoagulant activity.
| |
FOOTNOTES |
Submitted May 26, 1998;
accepted September 8, 1998.
Supported in part by Program Grant No. 900-526-192 from the Dutch
Organization for Scientific Research (N.W.O.) and in part by Grants No.
19278 and 54469 from the National Heart, Lung and Blood Institute,
Bethesda, MD.
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.
Address reprint requests to B. S. Coller, MD, Box 1118, Mount Sinai
School of Medicine, One Gustave L. Levy Place, New York, NY 10029;
e-mail: bcoller{at}smtplink.mssm.edu.
| |
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Abstract
Introduction