© 2000 American Heart Association, Inc.
Thrombosis |
Platelet Thrombus Formation on Collagen at High Shear Rates Is Mediated by von Willebrand Factor–Glycoprotein Ib Interaction and Inhibited by von Willebrand Factor–Glycoprotein IIb/IIIa Interaction
Presented in part at a meeting of the American Society of Hematology, Orlando, Fla, December, 1996 (Blood. 1996;88:29a) and a meeting of the European Haematology Association, Paris, France, June, 1996 (Br J Haematol. 1996;93:180).
From the Department of Hematology, Graduate School of Biomembranes, University Medical Center, Utrecht, the Netherlands.
Correspondence to Dr J.J. Sixma, University Medical Center Utrecht, Department of Haematology (G03.647), PO Box 85.500/Heidelberglaan 100, 3508 GA Utrecht/3584 CX Utrecht, Netherlands. E-mail jsixma{at}lab.azu.nl
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Abstract |
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Abstract—We studied the role of von Willebrand Factor (vWF) in platelet thrombus formation in flowing blood by using a perfusion system and mutant forms of vWF lacking either interaction with glycoprotein Ib (GpIb) or with glycoprotein IIb/IIIa (
IIb-ß3). These
mutants were added to the blood of patients with severe von
Willebrand’s disease (vWD) or to normal blood reconstituted
with a human albumin solution instead of plasma. This blood was
then perfused over collagen type III spray-coated on a glass surface
and preincubated for 2 hours with 20 µg/mL plasma vWF. In this way,
the adhesion step was mediated by the preincubated plasma vWF bound to
collagen type III, whereas thrombus formation was mediated by mutant
vWF added to the perfusate. Thrombus formation was absent at
all 3 shear rates studied (300, 800, and 2600 s-1) when
A1-vWF, lacking interaction with GpIb, was added to the
perfusate, indicating the importance of GpIb-vWF interaction
for thrombus formation. The interaction of vWF and GpIb is currently
thought to be possible under physiological
conditions in which the conformation of vWF has been changed by
adsorption to a surface. Our results regarding the role of GpIb-vWF
interaction in thrombus formation suggest that a second mechanism may
operate by which a change may occur in GpIb on the surface of adhered
platelets either by activation of the molecule or as a consequence
of shear stress. Increased thrombus formation was observed when the
Arg-Gly-Gly-Ser–vWF, which does not interact with IIb-ß3,
was added to vWD blood and perfused at 2600 s-1. This
increase was not observed in vWD blood at lower shear rates or after
addition of Arg-Gly-Gly-Ser–vWF to reconstituted normal blood.
Thrombus formation at a high shear rate was largest when either vWF or
fibrinogen was present as a single ligand for IIb-ß3 at a high
shear rate. When both were present, thrombus formation was
decreased. We postulate that thrombus formation is less efficient
because of incomplete bridge formation when vWF and fibrinogen are both
present as ligands for IIb-ß3.
Key Words: von Willebrand factor • thrombus formation • GpIb • collagen
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Introduction |
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Platelet adhesion to collagen in flow is followed by thrombus formation, which is, in many respects, similar to the aggregate formation in plasma that occurs when platelets are activated and stirred in a cuvette. This aggregation is dependent on the integrin glycoprotein IIb/IIIa (
IIb-ß3). Fibrinogen (and perhaps also fibronectin) serves as a
bridge between adjacent platelets.1 A role for von
Willebrand factor (vWF) has not been shown for aggregation in a
cuvette; in this respect, aggregation differs from thrombus formation
in flow, which is dependent on vWF at high shear.2 3
Exposure of platelets to high shear stress in a cone/plate
viscometer causes platelet aggregation4 5 as
consequence of interaction between vWF and glycoprotein Ib
(GpIb). This interaction leads to platelet activation and
subsequent exposure of IIb-ß36 7 8 ; this is then
followed by binding to IIb-ß3 of ligands, which form a firm bridge
between aggregating platelets. At the highest shear rates, vWF was
found to be this ligand.9
We investigated whether platelet thrombus formation on collagen
under flow (at shear rates that occur in the circulation) is dependent
on GpIb-vWF interaction as well; we also investigated whether
IIb-ß3–vWF interaction occurs at all in whole blood. To study
this, we used vWF mutants defective in interaction with either GpIb or
IIb-ß3.
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Methods |
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Human collagen type III from placental origin was obtained from Sigma Chemical Co. Low molecular weight heparin (Fragmin) was from Kabi Pharmacia. Plasma-vWF was purified from cryoprecipitate by gel filtration on Biogel A15-m (Bio-Rad) as described.10 vWF in the void volume was stored at -20°C. Human fibrinogen (98% clottable, American Diagnostica) was further purified by size exclusion chromatography on Sepharose 4B. Peak fractions were lyophilized, dissolved in Tyrode’s buffer (18 mg/mL), and stored at -20°C. LJ-P5 (courtesy of Dr Z.M. Ruggeri, Scripps Clinic & Research Foundation, La Jolla, Calif) is a monoclonal antibody against
IIb-ß3, inhibiting the binding of vWF but not of
fibrinogen or fibronectin.11 Fab fragments of this
antibody were prepared by using standard techniques.
Coating of the Coverslips
Glass coverslips (18x18 mm, Menzel Gläser) were
cleaned overnight by a chromosulfuric acid (2% chromium trioxide)
solution and rinsed with distilled water before spraying. Monomeric
collagen type III was solubilized in 50 mmol/L acetic acid and
sprayed with a density of 30 µg/cm2 on glass
coverslips with a retouching airbrush (Badger model 100, Badger Brush
Co).12 13 After the spraying procedure, the collagen
surface was blocked for 1 hour with 1% human albumin in PBS
(10 mmol/L phosphate buffer, pH 7.4, and 0.15 mol/L NaCl) to
prevent nonspecific protein binding during the subsequent perfusion and
then incubated with 20 µg/mL vWF in PBS for 2 hours.
Perfusion Studies
Perfusion studies over collagen type III were carried out in a
specially devised small parallel-plate perfusion chamber with
well-defined rheological characteristics accommodating a glass
coverslip as described previously.14 Whole blood was
obtained by venipuncture from 3 patients with no vWF in
either platelets or plasma (2 patients with severe von
Willebrand’s Disease [vWD] and 1 patient with type-1
platelet-low vWD).15 The blood was anticoagulated with
1/10 vol of 200 U/mL low molecular weight heparin. In a separate series
of experiments, reconstituted blood, prepared as described
previously,16 was used.
Mutant vWF
The role of the platelet receptors GpIb and IIb-ß3 was
studied by using mutant vWF. A1-vWF lacks the Al repeat of vWF and
is defective for the interaction with GpIb, under static and flow
conditions.17 Arg-Gly-Gly-Ser (RGGS)-vWF, in which Asp
(D)-1746 has been changed to Gly (G), has virtually no binding to
thrombin-stimulated platelets, indicating that the interaction of
this vWF with IIb-ß3 is defective. RGGS-vWF does not support
platelet adhesion in flow when coated to a surface, indicating that
this defect is also present under flow conditions. The construction
of these mutants has been described.16 17 For the
present study, we used wild-type and mutant vWF without propeptide.
This was achieved by transfection of a stable BHK cell line
overexpressing furin, as published previously.18
Recombinant vWF was purified by immunoaffinity purification as
described.18 Plasma vWF (pvWF), wild-type recombinant vWF
(rvWF), A1-vWF, and RGGS-vWF were added to the blood of the patients
with vWD to a concentration of 10 µg/mL plasma. As a control, we also
used A3-vWF, a mutant vWF in which the A3 repeat of vWF has been
deleted and which lacks the ability to bind to collagen type I and
III.18 Experiments with this mutant indicate that the
preincubated vWF was sufficient to support the adhesion step.
Evaluation of Platelet Deposition and Thrombus
Formation
Platelet deposition was evaluated with a light microscope
equipped with a JAI-CCD camera coupled to a Matrox frame grabber
(Matrox Electronic Systems Ltd) with the use of Optimas 6.0 software
(DVS) for image analysis. Platelet deposition was expressed
as percentage of the surface covered with platelets. Thrombus size
en face was evaluated by using the Watershed program provided by the
Optimas software package. The Watershed technique separates confluent
or overlapping objects on the basis of color intensity levels. This
technique was reproducible with a variation coefficient of 9.8%.
The height of thrombi was determined by confocal laser scanning microscopy (CLSM) in 1 series of experiments. For this determination, coverslips were fixed for 30 minutes in 3% paraformaldehyde/0.025% glutaraldehyde in PBS, blocked by 0.15 mol/L glycine and 3% BSA in PBS (30 minutes), and permeabilized with 0.5% Triton X-100 in PBS (5 minutes). The coverslips were then incubated with 100 µL Bodipy FL-phallacidin (0.13 µmol/L, Molecular Probes Inc) in PBS with 3% BSA. After each step, the coverslips were washed 3 times with PBS. The coverslips were mounted in Mowiol 40-88 (Aldrich-Chemie) and 2.5% diazobicyclo-octane (Sigma) on concave slides with a depth of 0.79 mm. The thrombus height was measured by CLSM (Leica TCS 4D). For each slide, 10 fields of 0.25x0.25 mm2 were selected to measure the maximal height of each thrombus. Calibration was performed by using 5.5- and 7.4-µm FITC-conjugated standard beads (FCS Corp)
Scanning Electron Microscopy
For scanning electron microscopy, coverslips with platelets
fixed for 1 hour with 2% glutaraldehyde/PBS at room
temperature were postfixed in 1% osmium tetroxide for 1 hour,
dehydrated in a graded series of ethanol, and dried by the
critical-point procedure with use of CO2 as a
transitional fluid. The samples were sputter-coated with a thin layer
of gold and viewed in a scanning electron microscope (Philips
XL30).
Statistics
All perfusions were performed in triplicate for each condition.
The results are presented as the mean±SEM from experiments
with blood from 3 different patients or 3 separate series of
experiments with reconstituted blood, unless indicated otherwise. The
difference between the various conditions was tested by a general
linear model for repeated measurements as described in the SPSS
package. The data on thrombus size obtained with the Watershed
technique were tested by the 
2 test.
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Results |
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Studies in vWD Blood
To study the role of vWF in thrombus formation independent from its role in platelet adhesion, we first created a good adhesive surface by precoating collagen type III with pvWF and subsequently varied the perfusate by adding different mutant forms of vWF to the whole blood of patients with severe vWD. These patients have no vWF in either their plasma or platelets. The morphology of the platelet deposition at shear rate of 2600 s-1 under different conditions as shown by scanning electron microscopy is presented in Figure 1
; the surface coverage was quantified by
image analysis after May-Grünwald-Giemsa staining and is
summarized in Table 1.
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The following results were obtained. With no vWF in the system (no
preincubation of collagen with vWF and no addition of vWF to the
blood), deposition of only a few single platelets (surface coverage
1%) was observed. When collagen type III was preincubated with vWF but
no vWF added to the blood, platelet deposition was still small
(7.1±0.9% [mean±SEM]) and consisted of single platelets. After
preincubation with and the addition of vWF, platelet deposition
increased to a surface coverage of 30%. The actual values were similar
for pvWF (31.4±4.3%) and rvWF (29.5±3%). Platelet deposition
with RGGS-vWF was higher (39.2±1.9%). Platelet deposition after
the addition of A1-vWF was low (3.3±0.2%), lower even than that
without added vWF.
Figure 1
shows that platelet deposition after the addition
of pvWF, rvWF, and RGGS-vWF consisted of thrombi. The size of these
thrombi en face was evaluated by the Watershed technique. The results
are presented in Table 2.
Platelet thrombi were largest after the addition of RGGS-vWF.
The addition of pvWF yielded larger thrombi than those found after the
addition of rvWF. Platelet deposition after the addition of
A1-vWF consisted completely of single platelets.
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Data on thrombus height as measured by CLSM, with the use of
fluorescent phallacidin as a platelet label, are
presented in Table 1. Mean thrombus height was higher
with RGGS-vWF than with rvWF and pvWF.
Platelet deposition was also studied at shear rates of 300
and 800 s-1 in a single triplicate experiment
with vWD blood. The results are summarized in Table 3. Single platelets were found after
the addition of A1-vWF. Addition of other forms of vWF yielded
thrombi. Platelet deposition with RGGS-vWF was not increased, in
contrast to deposition found at 2600 s-1.
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Studies With Reconstituted Blood
The role of the interaction of vWF with GpIb and IIb-ß3 was
further studied in reconstituted blood. The results are summarized in
Table 4 for shear rates of 2600
s-1 and 300 s-1. Thrombi
were absent when no vWF or A1-vWF was added. The deposition was
slightly, but not significantly, lower after the addition of RGGS-vWF
than after rvWF. Analysis of thrombus size with the Watershed
technique showed smaller thrombi with RGGS-vWF than with rvWF or pvWF
This difference was more pronounced at 2600 s-1
than at 300 s-1 (Table 5).
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Role of IIb-ß3
The experiments in which RGGS-vWF was present as a single
ligand in reconstituted blood (Table 4) may suggest that
IIb-ß3 is not involved in thrombus formation. RGGS-vWF was unable
to interact with IIb-ß3, and there was no other ligand
present. Previous studies involving Glanzmann’s disease have
demonstrated the complete absence of thrombus formation in
flow.20 21 We confirmed the importance of
IIb-ß3 by adding an antibody to ß3 to the perfusate
(C17, courtesy of Dr A.E.G.K. von dem Borne, Central Laboratory of
Blood Transfusion Service, Amsterdam, the Netherlands). This blocked
thrombus formation completely (results not shown).
Ligands secreted from platelet granules (fibrinogen,
fibronectin, and vWF) have to be responsible for thrombus
formation. We investigated the role of vWF by using Fab
fragments of LJ-P5, an antibody directed against IIb-ß3, which
blocks the interaction with vWF but not with fibrinogen or fibronectin.
We first tested the specificity of the Fab fragments. They completely
blocked platelet adhesion in flow to surface-coated vWF and had no
effect on adhesion in flow to surface-coated fibrinogen (results not
shown). The Fab fragments of LJ-P5 reduced platelet deposition to
vWF-precoated collagen type III when rvWF had been added from 45.8% to
29% (Table 6). Doubling the dose to 40
µg/mL gave no further inhibition. When RGGS-vWF had been added
instead of rvWF, the Fab fragments had no effect on platelet
thrombus formation. These data indicate that vWF is important as a
ligand for IIb-ß3 when it is present in the perfusate
but not when platelet thrombus formation relies on secreted ligands
from platelet granules.
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We also studied the relative importance of the IIb-ß3–vWF
interaction under more physiological conditions by
adding LJ-P5 Fab fragments (20 µg/mL SC) to whole blood before
perfusion. Coverage decreased from 23.1±3.1 to 16.5±0.9 at 300
s-1, from 24.9±0.2 to 14.9±1.1 at 800
s-1, and from 40.2±0.5 to 30.3±0.45 at 2000
s-1. These data demonstrate that
IIb-ß3–vWF interaction contributes to platelet thrombus
formation at all shear rates.
In vWD blood, we found that the addition of RGGS caused larger thrombi
than did the addition of rvWF at 2600 s-1. In
reconstituted blood, this was not the case. The difference may be that
fibrinogen and vWF are both present in vWD blood, whereas vWF is
the single initial ligand in reconstituted blood. To investigate this
further, we added fibrinogen to reconstituted blood (Table 6).
This caused inhibition in the case of rvWF but no inhibition in the
case of RGGS-vWF. The maximal effect was at fibrinogen concentrations
of 
1 mg/mL (dose response not shown).
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Platelet adhesion and platelet thrombus formation are generally viewed as completely different processes with their own special ligands and adhesion receptors. Adhesion concerns nonactivated platelets and involves vessel wall molecules and some plasma proteins binding to it as ligands and GpIb and integrins of the ß1 family as receptors. Adhesion to fibrinogen/fibrin is an exception: the ß3 integrin
IIb-ß3 is the
adhesion receptor for this ligand. Thrombus formation, ie,
aggregation, starts from a first layer of adhered and activated
platelets. It has been thought to be mediated exclusively by the
ß3 integrin IIb-ß3, with fibrinogen, fibronectin
(vitronectin), and vWF used as ligands. Previous studies
involving the binding of vWF to IIb-ß3 of activated
platelets showed strong competition by fibrinogen: this competition
was so powerful that no vWF binding occurred at plasma
concentrations.19 Thus, it was unexpected that perfusion
studies in which the blood of patients with vWD was circulated over
rabbit subendothelium showed a defect in thrombus
formation at a shear rate of 2600
s-1.2 The same authors suggested in
a later study that thrombus formation at this relatively high shear
rate was mediated by the vWF–IIb-ß3
interaction.3 A role for GpIb-vWF interaction in platelet aggregate formation was found in experiments in a cone-plate viscometer.4 5 This role is difficult to investigate under flow conditions, because inhibition of GpIb affects adhesion and the subsequent thrombus formation. Goto et al9 approached this by introducing a 2-step procedure in which adhesion was achieved in a short perfusion of 1 minute. Thrombus formation was then studied in the presence or absence of relevant antibodies by perfusing for another 4 minutes. This procedure has the drawback that the antibodies may still have an effect on the platelets that had adhered during the first minute.
We approached this issue by using vWF mutants. Adhesion was supported by pvWF that had been preadsorbed to collagen type III by preincubation. The role of vWF in thrombus formation was then studied by adding various mutant forms of vWF to the vWF-free blood.
The experiments showed unequivocally that the interaction of vWF with
GpIb is absolutely required for thrombus formation. This is based on
the observation that no thrombus formation occurred at all when
A1-vWF was added to whole blood (Figure 1 and Tables 1 through 3), whereas thrombus formation occurred when either pvWF or
rvWF was added.
Earlier studies involving Glanzmann’s thrombasthenia have
demonstrated without any doubt that IIb-ß3 is also absolutely
required for thrombus formation.20 21 Our data suggest
that adhesion and thrombus formation are analogous processes. In
adhesion, platelet GpIb interacts with vWF in a rolling
interaction.22 Firm attachment is then caused by
interaction with an integrin receptor, in this case 2ß1. Direct
studies of platelet thrombus formation in real time (Y.-P.W. et al,
unpublished data, 1999) show that platelets also roll on top
of the thrombi that form on collagen fibers and that this rolling is
then followed by firm attachment. We postulate that the rolling is
mediated by GpIb-vWF and that the firm attachment is mediated by an
integrin (in this case IIb-ß3).
An interesting consequence of our observation is the different mechanism that may lead to vWF-GpIb interaction in the adhesive situation versus the thrombotic situation. According to our present insights, the GpIb-vWF interaction in adhesion is mediated by a conformational change in vWF caused by binding to a surface.23 In the case of the thrombotic situation, one has to postulate that a change in conformation occurs in GpIb present on an adhered platelet. This conformational change may be due to an activation process caused by adhesion or may be due to the effect of shear stress of the passing plasma on GpIb. The spontaneous binding of vWF to GpIb in platelet-type vWD, caused by a point mutation in GpIb, indicates that conformational changes in GpIb may indeed cause binding of vWF.
The studies performed at low shear rates indicate that in contrast to
the original idea, GpIb-vWF interaction is of importance for thrombus
buildup at all the shear conditions existing in arteries. This is of
interest for the potential use of the GpIb-vWF interaction as a target
in drug development. In contrast to the use of IIb-ß3
inhibitors, GpIb inhibitors may be expected to
inhibit thrombus formation only under flow conditions and not under
static conditions. It would be interesting if one could separate the
effect of inhibitors on platelet adhesion from the
effect of inhibitors on platelet thrombus formation. In
principle, this may be possible. The difference in the protein
undergoing the conformational change, in vWF in adhesion or in GpIb in
thrombus formation, may be used to generate inhibitors that
would block one interaction and not the other.
The preferred ligand for IIb-ß3 remains to be determined. The data
on RGGS-vWF addition in vWD blood as presented in Tables 1 and 2 show increased thrombus formation with this
ligand, and this may mean that vWF/IIb-ß3 may be less effective
than fibrinogen–IIb-ß3 interaction. On the other hand, we find in
Table 6 that the addition of fibrinogen to reconstituted blood
containing rvWF yields a decrease in thrombus formation. This would
indicate that vWF–IIb-ß3 interaction is more effective than
fibrinogen–IIb-ß3 interaction. This discrepancy becomes better
understandable when we see that fibrinogen does not inhibit thrombus
formation when RGGS-vWF is added to reconstituted blood. RGGS-vWF does
not interact with IIb-ß3, and ligands secreted from platelet
granules have to act as a bridge between platelets. LJ-P5 Fab
fragments did not inhibit thrombus formation in this case, which
indicates that it is fibrinogen rather than vWF from granules that
is responsible for thrombus formation. We now begin to see that
whenever a single ligand is present, thrombus formation is higher
than when vWF and fibrinogen are both present. This leads us to the
hypothesis shown in Figure 2. When a
single ligand for IIb-ß3 is present (Figure 2A and 2B),
bridge formation will be efficient and complete. When 2 ligands are
present (Figure 2C), part of the bridges will be incomplete.
The ligand bound to IIb-ß3 on one of the platelets cannot
interact with the adjacent IIb-ß3 on the other platelet
because this is occupied by the other ligand. Whether it can displace
this will depend on the affinity and local concentration of the 2
ligands. In the case of homotypic interaction of fibrinogen or vWF with
the IIb-ß3-molecules on adjacent platelets, bridge formation
will be easier, because the affinity for the bound versus the free
ligand will at least be equal, which will lead to easier
displacement.
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In developing this hypothesis, we have only considered fibrinogen and vWF as ligands. We believe that this is justified. Fibrinogen is present at a 10-fold higher concentration than fibronectin, and vWF is already present on the platelet surface, interacting with GpIb. The negative effect of the presence of 2 ligands was observed only at 2600 s-1 but not at 800 and 300 s-1. This may be caused by the higher shear stress.
Recently, Ruggeri et al24 published a study in
which they also focused on the role of GpIb versus IIb-ß3 and vWF
versus fibrinogen in thrombus formation in collagen in flow. There are
a number of methodological differences, which may have an effect on the
results, but the overall conclusion was similar. GpIb was important for
thrombus formation at shear rates between 300 and 1500
s-1. They also found that vWF may act as single
ligand in thrombus formation at 1500 s-1.
Addition of fibrinogen caused lower initial thrombus formation but
stabilized thrombus formation at later times. They could investigate a
situation in which fibrinogen was the only ligand for IIb-ß3
because they did not use mutant vWF, and they needed vWF for the GpIb.
Their observation of reversible thrombus size with vWF at later times
and of the stabilizing effect of fibrinogen will require further
study.
The different roles of vWF and fibrinogen as ligands for IIb-ß3
may have clinical consequences. Preliminary data in the blood of a
patient with afibrinogenemia showed that the large thrombi that formed
on collagen became smaller after the addition of fibrinogen.
Further studies will be required to look more precisely at shear rate
and time effects. Real time perfusion studies as performed by Ruggeri
et al24 and use of mutants as performed by us may be
required to get the full picture.
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Acknowledgments |
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Dr Y.-P. Wu and Dr G.H. van Zanten were supported by the Netherlands Heart Foundation (grant 93.112). Dr T. Vink was supported by grant 43.051 of the Netherlands Heart Foundation. We thank Dr G.J.A.M. Strous and Dr G. Posthuma for advice concerning the CLSM studies and P. van Maurik for his assistance in the scanning electron microscopy studies. We thank Dr Z. Ruggeri for his generous gift of the LJ-P5 monoclonal antibody.
Received July 12, 1999; accepted November 26, 1999.
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