(Received for publication, June 7, 1995; and in revised form, July 13, 1995)
From the
We have studied the mechanism of interaction between soluble von
Willebrand factor (vWF), labeled with fluorescein isothiocyanate
(FITC), and platelets exposed to shear in a cone-and-plate viscometer.
A flow cytometer calibrated with fluorescent bead standards was used to
calculate the number of molecules associated with each platelet in
suspension. To validate the methods and reagents used, binding of the
same labeled vWF was assessed in the presence of ristocetin or
-thrombin and found to be saturable, with a narrow and symmetric
distribution on >90% of the platelets. As expected, essentially all
bound ligand interacted exclusively with platelet membrane glycoprotein
(GP) Ib
in the presence of ristocetin and with GP IIb-IIIa after
stimulation with
-thrombin. In contrast, only a minor proportion
(<20%) of the platelets exposed to shear were found to bind vWF,
with no evidence for saturation and markedly decreased interaction when
the platelet count was below 100,000 µl. Moreover, shear-induced
vWF binding was blocked equally effectively by selected monoclonal
antibodies against either GP Ib
or GP IIb-IIIa or against the
respective binding sites in vWF. Thus, both receptors are involved in
the process, possibly through initial transient interactions mediated
by GP Ib
that lead to platelet activation and subsequent
irreversible binding supported by GP IIb-IIIa. While the levels of
shear stress theoretically applied to platelets in these experiments
are above those thought to occur in the normal circulation, our
findings demonstrate a unique vWF binding mechanism that is not
mimicked by other known modulators and correlates with platelet
aggregation. Similar processes may occur in response to lower shear
stress when platelets are exposed to thrombogenic surfaces and agonists
generated at sites of vascular injury during thrombus formation.
The role of von Willebrand factor (vWF) ()in platelet
thrombus formation, particularly under conditions of high shear, is
well established and supported by experimental evidence as well as
clinical observations (1) . It is known that platelets have two
distinct binding sites for vWF, glycoprotein (GP) Ib
in the GP
Ib-IX-V complex and the integrin
(GP IIb-IIIa complex)(2) . Binding of soluble normal vWF
to GP Ib
in the absence of flow requires the presence of exogenous
modulators, like ristocetin (3) or botrocetin (4) ,
while interaction with GP IIb-IIIa can only occur after platelet
activation(5) . In addition to vWF, the GP IIb-IIIa receptor
can also bind fibrinogen, fibronectin(6) , and
vitronectin(7) , but the respective role of these proteins in
thrombogenesis is still a topic for investigation.
Exposure of
platelets to levels of shear higher than thought to occur in the normal
circulation results in aggregation if soluble vWF is
present(8) ; no other adhesive ligand can support this
process(9) . Shear-induced platelet aggregation may represent
an important pathophysiological function of vWF, but the underlying
mechanism is still poorly understood. It has been proposed that vWF
binds to GP Ib under the effect of shear, causing an increase in
intracytoplasmic calcium ion levels and, consequently, GP IIb-IIIa
activation; vWF interaction with activated GP IIb-IIIa would then
mediate aggregation(10, 11) . There is, however, no
evidence that shear can promote the binding of vWF to GP Ib
;
rather, there is only the indirect observation that anti-GP Ib
antibodies capable of inhibiting vWF binding mediated by other
modulators can block shear-induced
aggregation(9, 12) . Thus, it is not known whether the
characteristics of vWF binding to GP Ib
induced by shear are
comparable to those defined in the presence of exogenous modulators
like ristocetin or botrocetin.
We have performed studies to clarify
the process of vWF interaction with platelets under shear. Our results
demonstrate the existence of a unique mechanism that involves both GP
Ib and GP IIb-IIIa in supporting vWF binding and is not mimicked
by the effect of exogenous modulators nor platelet agonists inducing
activation. The observed binding appears to correlate well with the
occurrence of aggregation.
Fibrinogen was purified from frozen plasma by glycine
precipitation and characterized as previously reported(16) . It
was labeled with FITC as described above for vWF, except that the pH of
the labeling mixture was kept at 8.5 to avoid denaturation of the
molecule and the F/P weight ratio was 1:10. A final molar F/P ratio
between 1 and 3 was found compatible with preservation of FITC-labeled
fibrinogen function. The extinction coefficient (A) used to calculate the fibrinogen
concentration was 1.5. Bovine serum albumin was labeled with FITC as
described above for vWF; the extinction coefficient (A
) used to calculate its concentration was 0.5.
Figure 1: Flow cytometer calibration using standard fluorescent beads. These measurements were performed using a gain setting identical to that used for the evaluation of platelet-bound fluorescence. One drop of each bead standard was mixed with 200 µl of modified HEPES-Tyrode buffer, pH 7.4, and the mixture was kept on ice, protected from light, until tested. The distribution of measured fluorescence is shown for each standard identified with the stated number of MESF bound/bead; the correlation between the two parameters is shown in the inset.
Figure 2:
Binding of FITC-labeled vWF to platelets
in the presence of ristocetin or after activation with -thrombin.
For ristocetin-mediated binding (upper panel), 40 µl of
2.4
10
platelets/µl washed platelet suspension
in modified HEPES-Tyrode buffer, pH 7.4, containing 6.25 mg/ml BSA and
1.25 mM Ca
, was mixed with 35 µl of the
same buffer, 20 µl of FITC-labeled vWF in HEPES buffer, pH 7.4, at
the concentration necessary to achieve the desired final concentration
in the mixture, and 5 µl of a 25 mg/ml solution of ristocetin in
HEPES buffer; or, for binding induced by
-thrombin (lower
panel), 40 µl of the platelet suspension was mixed with 35
µl of modified HEPES-Tyrode buffer and 2.5 µl of a 20 NIH
units/ml solution of
-thrombin in HEPES buffer followed, after a
15-min incubation at room temperature (22-25 °C), by 2.5
µl of a 200 unit/ml solution of hirudin in HEPES buffer followed,
after 5 min, by 20 µl of FITC-labeled vWF solution at the
concentration necessary to achieve the desired final concentration in
the mixture. At this point, the mixtures were incubated at room
temperature for 15 min, then diluted with 400 µl of modified
HEPES-Tyrode solution (but with no BSA nor cation), and stored on ice
with protection from light until tested in the flow cytometer. The
calculated number of vWF subunit molecules bound/platelet (median of
the distribution shown in the inset) is shown as a function of
added FITC-labeled vWF concentrations, demonstrating near saturation of
binding in both instances. The insets in each panel display
the actual fluorescence distribution of the population of platelets
analyzed in the flow cytometer, which is narrow and symmetric in both
cases. Error bars indicate the standard error calculated from
three (ristocetin) and five (thrombin) different
experiments.
Figure 3:
Effect of monoclonal antibodies on
ristocetin and thrombin-induced vWF binding to platelets. These assays
were performed as described in the legend to Fig. 2, except that
a fixed concentration (15 µg/ml) of FITC-labeled vWF was used, and
10 µl of the appropriate monoclonal antibody solution (purified IgG
or Fab) was added to platelets, replacing an equivalent volume of
buffer, before adding the ligand (in the case of experiments with
thrombin, antibodies were added during the first activation step). Anti
GP Ib IgG were used at a final concentration of 150 µg/ml;
anti-GP IIb-IIIa IgG at 50 µg/ml; anti vWF Fab at 500 µg/ml.
The number of vWF molecules bound/platelet in the presence of
antibodies is expressed as percent of the value measured in control
mixtures without antibody. Data are the mean ± S.E. of four to
eight experiments. Upper panel, ristocetin-mediated binding;
note that antibody NMC-4 inhibits completely. Lower panel,
binding induced by activation with
-thrombin.
Figure 4:
Flow cytometric analysis of shear-induced
vWF binding to platelets. The two upper panels show the
distribution of forward and side light scattering (representing
essentially particle size and granularity) measured in a mixture of
platelets (400 µl of the suspension described in the legend to Fig. 3; final count 1.92 10
/µl) and
FITC-vWF (100 µl of a solution in HEPES buffer; final concentration
15 µg/ml) before and after exposure to shear (10,800 dynes/cm
for 6 min at 24 °C). After rotation in the cone-and-plate
viscometer (or incubation without agitation at the same temperature for
the samples not exposed to shear), 100 µl of the suspension was
mixed with 400 µl of HEPES-Tyrode buffer, pH 7.4 (with no BSA nor
divalent cation), and kept on ice protected from light until analyzed
in the flow cytometer. Particles both larger (R1) and smaller (R3) than single platelets (R2) appeared after
exposure to shear, representing aggregated platelets and
platelet-derived microparticles, respectively. The two lower panels show the fluorescence distribution in the same experimental
mixtures. Analysis of the whole population of particles in suspension
without gating for size (lower left) demonstrated a small
number of highly fluorescent elements appearing in the sample exposed
to shear as compared to the distribution seen before exposure to shear.
After gating for size (lower right), it became apparent that
the fluorescent particles seen after exposure to shear correspond to
aggregated platelets (R1).
Figure 5:
Flow cytometric analysis of FITC-vWF
binding to platelets exposed to shear. These experiments were conducted
in a manner similar to that described in the legend to Fig. 4,
exposing platelets to shear in the presence of FITC-labeled vWF (400
µl of platelet suspension and 100 µl of vWF solution; 1.92
10
/µl and 15 µg/ml, respectively) with or
without the addition of the monoclonal antibodies LJ-CP8 (50 µg/ml)
or LJ-Ib1 (150 µg/ml), added in a volume of 10 to 20 µl
replacing HEPES buffer in the FITC-vWF solution; all indicated
concentrations are final. The six panels on the left show the distribution of particles in suspension with respect to
side and forward light scattering, indicating the gate chosen for the
analysis of single platelets (R2); the three panels on the right show the fluorescence distribution of the R2
population (single platelets). All different samples gave similar
results when not exposed to shear (Before). After exposure to
shear, aggregated platelets appeared in the absence of antibody LJ-CP8
but the fluorescence distribution of the single platelet population did
not change (Control, upper row). In the presence of
the anti-GP IIb-IIIa antibody LJ-CP8, aggregated platelets essentially
disappeared, and the fluorescence of a limited portion of the single
platelet population increased (middle row). The latter change
was abolished when the anti-GP Ib
antibody LJ-Ib1 was added to
LJ-CP8 (lower row).
Figure 6:
Flow cytometric analysis of
FITC-fibrinogen binding to platelets exposed to shear. These control
experiments were performed as described in the legend to Fig. 5,
except that nonlabeled vWF was used in the mixtures, and FITC-labeled
fibrinogen was added at the concentration of 18 µg/ml (to give the
same molar concentration as vWF subunit). The panels in the upper row show the distribution of particles in suspension
with respect to side and forward light scattering, indicating the gates
chosen for the analysis of particles of different size set exactly as
for the experiments presented in Fig. 5; left panel,
control experiment; middle panel, in the presence of the
anti-GP IIb-IIIa antibody, LJ-CP8 (50 µg/ml); right panel,
in the presence of antibody LJ-CP8 and the anti-GP Ib antibody,
LJ-Ib1 (150 µg/ml). Note that aggregation occurs due to the
presence of vWF (R1 particles on the left) and is markedly reduced in
the presence of the antibodies. The panels in the bottom
row show the fluorescence distribution after exposure to shear in
the three gated populations (left); the comparison between
fluorescence distribution before and after exposure to shear in the R2
(single platelet) population in the presence of the antibody LJ-CP8 (middle); and the effect of the antibody LJ-Ib1 added to
LJ-CP8 on the fluorescence distribution in the R2 (single platelet)
population exposed to shear (right). Note that the population
of aggregates (R1) contains some highly fluorescent particles,
but much less so than in the experiment performed with FITC-labeled vWF
(compare with Fig. 4). Note also that the R2 population shows a
slight increase in fluorescence after shear but this is not affected by
the addition of antibody LJ-Ib1 (compare with the results shown in Fig. 5).
Figure 7:
Flow cytometric analysis of the
dose-dependent binding of FITC-labeled vWF to platelets exposed to
shear. All experiments presented here were performed as described in
the legend to Fig. 5, in the presence of antibody LJ-CP8 (50
µg/ml) to block aggregation, with or without addition of antibody
LJ-Ib1 (150 µg/ml) to block GP Ib function (as indicated by
two different lines) and in the presence of the indicated
concentrations of FITC-labeled vWF. The figure shows the distribution
of particles in the R2 population (single platelets as shown in Fig. 5) as a function of the calculated number of vWF molecules
bound (see ``Experimental Procedures'' for the method used
for calculation). The insets show the distribution obtained
after subtracting the values of bound vWF measured in the presence of
the anti-GP Ib
antibody, LJ-Ib1, from those measured in its
absence; thus, the observed distribution corresponds to bound vWF that
can be inhibited by the anti-GP Ib
antibody. Each curve represents
the mean from four different experiments.
Figure 8:
Dose-response analysis of shear-induced
vWF binding to platelets and correlation with aggregation. The upper panel shows the dose-response curve of specific
(inhibited by anti-GP Ib antibody) shear-induced vWF binding
calculated from the median of the distribution observed for each
experimental point (see Fig. 7). Each point is the mean ±
S.E. of two to six different experiments. The lower panel shows the relationship between shear-induced vWF binding and
platelet aggregation (based on single platelet count, i.e. the
values shown represent the percent decrease in the number of single
platelets in suspension, or 100 -% residual single platelets,
after shear versus before shear). One to four experiments were
performed for each point, and the mean is shown when more than one
experiment was performed. Regression analysis demonstrated the positive
correlation between the two parameters. Aggregation was measured in
mixtures prepared as described for measuring binding, but without the
monoclonal antibodies.
The binding of vWF under
shear was greatly influenced by the number of platelets in suspension
and was minimal at a count below 50,000/µl (Fig. 9). This is
in contrast to the fact that the binding mediated by ristocetin or
induced by activation with -thrombin is essentially the same with
platelet count between 50,000 and 600,000/µl (not shown).
Shear-induced aggregation was similarly influenced by the count of
platelets in suspension and was diminished considerably at
50,000/µl (not shown).
Figure 9:
Effect of platelet count on shear-induced
vWF binding. These experiments were performed as described in the
legend to Fig. 5, adjusting the count of platelets in suspension
to obtain the indicated values, with a constant amount of FITC vWF
present (15 µg/ml) as well as antibody LJ-CP 8 to inhibit
aggregation, with or without the addition of antibody LJ-Ib1 to block
GP Ib function (shown by different lines). The figure shows the
distribution of particles in the R2 population (single platelets as
shown in Fig. 5) as a function of the calculated number of vWF
molecules bound (see ``Experimental Procedures'' for the
method used for calculation). The insets show the distribution
obtained after subtracting the values of bound vWF measured in the
presence of the anti-GP Ib
antibody, LJ-Ib1, from those measured
in its absence; thus, the observed distribution corresponds to bound
vWF that can be inhibited by the anti-GP Ib
antibody. Each curve
represents the mean of three different experiments. Note the
progressive decrease in binding with decreasing platelet
count.
The level of shear rate to which
platelets were exposed was also a crucial determinant of vWF binding. A
positive population of single platelets with bound vWF was clearly
detectable after shearing at 10,800 s, but binding
was barely above background level after shearing at 7,200
s
for the same period of time (Fig. 10).
Figure 10:
Effect of shear rate on vWF binding to
platelets. These experiments were performed as described in the legend
to Fig. 5, with a constant amount of FITC vWF present (15
µg/ml) as well as antibody LJ-CP 8 to inhibit aggregation, with or
without the addition of antibody LJ-Ib1 to block GP Ib function
(shown by different lines), but varying the rotational speed of the
cone in the viscometer to achieve the indicated shear rate values. The
figure shows the distribution of particles in the R2 population (single
platelets as shown in Fig. 5) as a function of the calculated
number of vWF molecules bound (see ``Experimental
Procedures'' for the method used for calculation). The insets show the distribution obtained after subtracting the values of
bound vWF measured in the presence of the anti-GP Ib
antibody
LJ-Ib1 from those measured in its absence; thus, the observed
distribution corresponds to bound vWF that can be inhibited by the
anti-GP Ib
antibody. Each curve represents the mean of three
different experiments. Note the progressive decrease in binding with
the decreasing shear rate.
Another unique and important feature of vWF binding to platelets
exposed to shear was highlighted by the use of well characterized
monoclonal antibodies (Table 1). As expected, antibodies known to
interfere with the ristocetin-dependent vWF-GP Ib interaction, one
against the receptor (LJ-Ib1) and the other against the corresponding
binding domain in the ligand (NMC-4), also inhibited shear-dependent
binding. Control antibodies defined as noninhibitory in the assay
modulated by ristocetin were also without effect on shear-dependent
binding. The unexpected result was that two antibodies known to block
the interaction of vWF with GP IIb-IIIa, one against the receptor
(LJ-P5) and the other against the domain of vWF containing the
Arg-Gly-Asp integrin recognition sequence (LJ-152B/6), consistently
inhibited vWF binding to platelets exposed to shear (Table 1).
Thus, unlike ristocetin- or thrombin-induced binding, the stable
interaction of vWF with platelets under shear appears to require ligand
binding to both GP Ib and GP IIb-IIIa, either in a sequential or
concurrent fashion. Of note, all antibodies known to inhibit the
ristocetin-mediated vWF-GP Ib interaction also prevented shear-induced
binding and aggregation; however, not all those interfering with vWF
binding to GP IIb-IIIa on platelets stimulated by -thrombin had
the same effect on platelets exposed to shear, although they all
inhibited aggregation. Like the latter group of antibodies, the
synthetic peptide GRGDSP, containing the Arg-Gly-Asp integrin
recognition sequence, was effective in blocking shear-induced
aggregation but not vWF binding (not shown).
These studies provide experimental evidence that shear can
modulate the binding of vWF to platelets eliciting a unique mechanism
of interaction that requires two platelet receptors, the GP Ib-IX-V
complex and the integrin (GP
IIb-IIIa). While there is no other example of a dual receptor
requirement for soluble vWF binding to platelets, our findings are
reminiscent of conditions previously established for the adhesion of
nonstimulated platelets to surface-bound vWF, a process that depends on
both GP Ib and GP IIb-IIIa to become irreversible(36) . There
are obvious limitations in the methodology we have used to perform
these studies, essentially dictated by the nature of the phenomena
being evaluated. Since aggregation always occurs when platelets are
exposed to high levels of shear in the presence of vWF, it was
necessary to use a blocking anti-GP IIb-IIIa antibody in order to study
vWF binding to single platelets. We cannot exclude the possibility that
the extent of measured vWF binding was substantially less than would
have otherwise been detected in the absence of antibody. Other factors
that may have influenced the results are the need to stop shear in
order to measure vWF binding and the need to manipulate platelets for
flow cytometric analysis, making it impossible to measure transient
interactions that may only occur under shear. Unfortunately, such
concerns cannot be addressed satisfactorily with presently available
technology. It is possible, therefore, that all the numerical estimates
obtained with these experiments are an approximation by defect.
Nevertheless, the important conclusion that, unlike in the presence of
exogenous modulators, GP Ib by itself cannot support irreversible vWF
binding to platelets exposed to shear, and yet is indispensable for
such binding to occur, cannot have been influenced by the methodology
used.
A proposed model for the sequence of events leading to
shear-dependent vWF binding to platelets, and then aggregation, is
presented in Fig. 11. The model considers that, under flow, vWF
and GP Ib interact through the respective recognition sites (20, 30) in a transient and reversible manner. When
shear exceeds a certain level, GP IIb-IIIa is activated, interacts with
the Arg-Gly-Asp (RGD) sequence in vWF (28) and the ligand
becomes irreversibly bound to the platelet surface through both
receptors. Elongated vWF multimers can then bridge multiple platelets
and cause aggregation. Of interest is the fact, not documented here,
that inhibitors of platelet activation, like prostaglandin
E
, can indeed prevent shear-induced vWF binding, (
)providing additional evidence in support of a dual
receptor mechanisms where the function of one is linked to a response
initiated by the other. This is also consistent with the observation
that vWF interaction with GP Ib under shear leads to a transmembrane
flux of calcium ions that precedes and is independent of an interaction
with GP IIb-IIIa(10, 11) . This hypothetical mechanism
of shear-dependent vWF binding to platelets poses some key questions
that our present results begin to address.
Figure 11:
Schematic model of the mechanism of
shear-induced vWF binding to platelets. See ``Discussion''
for a justification of the hypothetical processes shown here. The double-headed arrows (figure on top) indicate the reversible
interaction between A1 domain of vWF and GP Ib. The asterisks (figure on bottom) indicate GP IIb-IIIa molecules after
activation, when they can interact with soluble vWF. RGD indicates the
Arg-Gly-Asp sequence recognized by GP
IIb-IIIa.
One problem is to
understand how shear modulates the vWF-GP Ib interaction. Many
have hypothesized, but without supporting experimental evidence, that
shear forces cause a conformational change in either the ligand or the
receptor, or both, thus allowing the binding to occur. Our data cannot
formally exclude this possibility, but they do not favor it. First, it
seems clear that vWF cannot bind irreversibly to GP Ib under shear, as
it does in the presence of exogenous modulators, since GP IIb-IIIa
function is also required for binding to become manifest. This may be
because the off rate of the interaction is inherently high, as
suggested by studies performed with surface-bound vWF to be reported
elsewhere. (
)Those experiments demonstrate a GP
Ib-dependent, transient platelet contact with immobilized vWF that
occurs at any shear rate tested between 50 and 1,500
s
. Indeed, evidence for reversible GP Ib interaction
with immobilized vWF can also be obtained without imposition of
flow(36) , clearly indicating that vWF binding to GP Ib in the
absence of exogenous modulators is not necessarily or exclusively
regulated by shear forces. Moreover, the characteristics of
shear-induced binding shown here are not congruent with an interaction
regulated by diffusion rate, since no saturation is apparent at
concentrations greatly in excess of those required for saturation in
the presence of modulators, and the extent of binding is markedly
reduced when the platelet count is below a threshold level.
Another important consideration is that, at present, there is no clear understanding of what responses are evoked when platelets in suspension are exposed to a shear field. According to fluid dynamics theory, the surface of a body moving freely in a streaming fluid is not subject to tangential forces imposed by the flow(37) ; thus, it is unlikely that platelets are subjected to a significant shear stress when moving freely with the liquid as single particles. We hypothesize that if more than one platelet is transiently interacting with a vWF multimer (Fig. 11), greater shear stress may be exerted on the larger, more irregular ``particle.'' Increased shear stress on the membrane may combine with the consequences of proximity, established during the transient binding of several platelets to a vWF multimer, and lead to activation (38, 39, 40, 41) . Such a mechanism could favor the local availability of greater concentrations of released ADP, a synergistic agonist that has been shown to be required for shear-induced aggregation(42) , and could also account for the greater efficiency of larger vWF multimers in mediating shear-induced activation and aggregation. Limiting factors in a process like this would be a low platelet count, in agreement with our experimental results, and the relatively low concentration of soluble large vWF multimers present, thus explaining why only a small proportion of platelets in suspension appear to bind vWF. These hypotheses will have to be tested by future experiments.
Our results
indicate that the interaction of vWF with GP IIb-IIIa is absolutely
required to achieve irreversible shear-induced binding to the platelet
surface, but this cannot be prevented by blocking the pool of GP
IIb-IIIa molecules accessible to inhibitory antibodies and peptides on
the membrane of resting platelets. As a possible explanation of this
observation, we suggest that vWF interacts with GP IIb-IIIa molecules,
initially not accessible to antibodies, that become exposed on the
platelet surface only after GP Ib-mediated activation (Fig. 11).
The surface expression and functional integrity of a previously
internal pool of GP IIb-IIIa molecules has been well documented after
platelet stimulation(16) . These newly exposed molecules may be
more easily occupied by vWF already interacting with GP Ib on the
platelet surface than by competing antibodies or peptides in solution.
If this concept is correct, it is not immediately apparent why an
anti-GP IIb-IIIa antibody like LJ-P5 can inhibit shear-induced vWF
binding when other antibodies with well established inhibitory activity
on GP IIb-IIIa function (like LJ-CP8) cannot. The unique effect of
LJ-P5 may be due to its epitope specificity, since this is the only
antibody available to us that has been well documented to inhibit
selectively soluble vWF binding to activated GP IIb-IIIa (27) .
This property, coupled with the high affinity of the antibody (and
presumably fast on rate) may allow more effective blockade of newly
exposed GP IIb-IIIa in competition with vWF. It is consistent with the
proposed model that blocking the Arg-Gly-Asp sequence in vWF (as with
the antibody LJ-152B/6) is effective in preventing a stable interaction
with platelets under shear. It is also relevant that antibodies like
LJ-CP8 and RGD peptides, that block GP IIb-IIIa function in most other
situations but are not inhibitors of shear-dependent vWF binding, do
nevertheless interfere with shear-induced aggregation. This implies
that, once bound irreversibly to single platelets, a vWF multimer
interacts with GP IIb-IIIa on other platelets that have also bound vWF
and have become activated, thus mediating aggregation (Fig. 11).
It follows that the pools of GP IIb-IIIa molecules that mediate
shear-dependent vWF binding and aggregation are not necessarily the
same, explaining why we could measure binding after blocking
aggregation with an anti-GP IIb-IIIa antibody. At present, it is not
known whether vWF binding to GP Ib becomes stable after GP IIb-IIIa is
also engaged in the interaction (concurrent binding) or whether only GP
IIb-IIIa supports vWF binding to platelets after activation mediated by
the transient interactions with GP Ib (sequential binding).
In
conclusion, our studies provide evidence for a unique mechanism of
interaction between soluble vWF and platelets that is not mimicked by
other known modulators of vWF binding to GP Ib. These findings are
likely to be relevant for normal hemostasis as well as pathological
thrombosis even though the levels of shear stress theoretically applied
to platelets in these experiments are above those thought to occur in
the normal circulation of man and other mammals, for which estimates of
maximal shear rate vary from 800 to 2,000
s(43) . In fact, it is conceivable that
similar processes occur even in response to lower shear when platelets
are exposed to thrombogenic surfaces and agonists generated at sites of
vascular injury during thrombus formation. For example, some key
aspects of the mechanism described here, notably the sequential
requirement for two distinct receptors, appear to be similar if not
identical to those involved with platelet adhesion to surface-bound vWF
even in the absence of flow(36) . Further progress in this
important area of platelet physiopathology should come from a better
understanding of the nature of the vWF-GP Ib bond and the subsequent
mechanisms of platelet activation.