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Blood, 15 November 2000, Vol. 96, No. 10, pp. 3480-3489
HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
Cytoskeletal regulation of the platelet glycoprotein Ib/V/IX-von
Willebrand factor interaction
Nayna Mistry,
Susan L. Cranmer,
Yuping Yuan,
Pierre Mangin,
Sacha M. Dopheide,
Ian Harper,
Simon Giuliano,
Dave E. Dunstan,
Francois Lanza,
Hatem H. Salem, and
Shaun P. Jackson
From the Department of Medicine, Australian Centre for
Blood Diseases, Monash Medical School, Victoria, Australia; Department
of Chemical Engineering, University of Melbourne, Victoria, Australia;
Etablissement de Transfusion Sanguine, Institut National de la Sante et
de la Recherche Medicale Unite 311, Strasbourg Cedex, France.
 |
Abstract |
Shear-induced binding of von Willebrand factor (vWf) to the
platelet glycoprotein (GP) Ib/V/IX complex plays a key role in initiating platelet adhesion and aggregation at sites of vascular injury. This study demonstrated that pretreating human platelets with
inhibitors of actin polymerization, cytochalasin D or latrunculin B,
dramatically enhances platelet aggregation induced by vWf. The effects
of these inhibitors were specific to the vWf-GPIb interaction
because they enhanced vWf-induced aggregation of Glanzmann thrombasthenic platelets and Chinese hamster ovary (CHO) cells transfected with GPIb/V/IX. Moreover, cytochalasin D enhanced the
extent of platelet aggregation induced by high shear stress (5000 s 1) and also lowered the shear threshold required
to induce aggregation from 3000 s1 to as low as 500 s1. Studies of CHO cells expressing GPIb cytoplasmic
tail truncation mutants that failed to bind actin-binding protein-280
(deletion of residues 569-610 or 535-568) demonstrated that the
linkage between GPIb and actin-binding protein-280 was not required for vWf-induced actin polymerization, but was critical for the enhancing effects of cytochalasin D on vWf-induced cell aggregation. Taken together, these studies suggest a fundamentally important role for the
cytoskeleton in regulating the adhesive function of
GPIb/V/IX.
(Blood. 2000;96:3480-3489)
© 2000 by The American Society of Hematology.
| |
Introduction |
The ability of platelets to adhere to sites of
blood vessel injury is essential for the arrest of bleeding and
subsequent vascular repair. A key adhesive protein initiating
platelet-vessel wall interactions is the large multimeric protein, von
Willebrand factor (vWf). Once immobilized at the site of vessel wall
injury, the A1 domain of vWf "captures" platelets from rapidly
flowing blood by a specific interaction with the platelet glycoprotein (GP) Ib/V/IX complex. This tethering mechanism decelerates platelet velocity relative to free-flowing blood as a prerequisite step for
integrin-mediated irreversible platelet adhesion.1-3
Growing evidence suggests that this multistep adhesion mechanism is not
only important for platelet-vessel wall interactions but also for
platelet aggregation, particularly at elevated shear rates. This was
initially demonstrated from studies of platelet aggregation using a
cone-plate viscometer. With this device, exposing platelets in
suspension to pathologic levels of shear stress ( 3000
s1) induces platelet aggregation independent of
the addition of an exogenous stimulus.4-7 Shear-induced
platelet aggregation is initiated by the binding of soluble vWf to
GPIb/V/IX. This interaction not only tethers platelets to one another
but also triggers platelet activation, converting the major platelet
integrin IIb 3 from a low-affinity to a
high-affinity receptor capable of binding fluid-phase adhesive proteins
such as soluble vWf or fibrinogen.8-10 More recent studies
using in vitro flow chambers, in which platelets in flowing blood
adhere to the surface of platelets immobilized on a reactive surface,
suggest that this dual-step aggregation mechanism also operates at
physiologically relevant shear stresses.11,12
Despite the fundamental importance of the vWf-GPIb interaction in
initiating both platelet adhesion and aggregation under flow, the
physiologic mechanisms regulating this adhesive event remain poorly
defined. In contrast to the integrins, which undergo affinity
modulation as a result of conformational changes or receptor clustering
in the plane of the plasma membrane,13-16 no current evidence suggests a similar mode of regulation for GPIb/V/IX. One
potential mechanism regulating the ligand-binding function of GPIb is
through its association with the membrane skeleton. In the resting
platelet, the GPIb/V/IX complex is anchored to the membrane skeleton
through a specific interaction between the cytoplasmic tail of GPIb
and actin-binding protein-280 (ABP-280).17-21 This
interaction may limit lateral mobility of the receptor complex in the
plane of the plasma membrane,22 thereby influencing the number of active bonds formed between GPIb and multimeric vWf. However,
to date, there is limited evidence that the cytoplasmic tail of GPIb
or the cytoskeleton plays a major role in regulating the vWf-GPIb
interaction particularly under physiologically relevant shear conditions.
In this study we have defined an important role for the cytoskeleton in
regulating the vWf-GPIb interaction. We demonstrate that pretreating
platelets with inhibitors of actin polymerization dramatically
increased the rate and extent of platelet aggregation induced by vWf.
Pretreating platelets with cytochalasin D (CD) also enhanced
shear-induced platelet aggregation and dramatically reduced the shear
threshold required to induce platelet aggregation from 3000 s1 down to as low as 500 s1.
Studies of GPIb cytoplasmic tail mutants expressed on the surface of
Chinese hamster ovary (CHO) cells demonstrated that the linkage between
GPIb and the membrane skeleton is not required for GPIb/vWf-induced actin polymerization, but is essential for the enhancing effects of CD
on GPIb/V/IX-mediated cell aggregation. These studies suggest that the
link between GPIb and the membrane skeleton plays a key role in
regulating the adhesive function of the GPIb/V/IX complex.
| |
Materials and methods |
Materials
Jasplakinolide and Alexa488-conjugated phalloidin were from
Molecular Probes (Eugene, OR). Apyrase was purified from potatoes according to the method of Molnar and Lorand.23 Human vWf
(HvWf), bovine vWf (BvWf), and fibrinogen were purified as previously described.24,25 Monoclonal antibody (mAb) ALMA12
against GPIb was generated and characterized as previously
reported.26 Full-length complementary DNAs (cDNAs)
for GPIb, Ib, and IX cloned into the pDX vector and CHO cells
expressing GPIb and GPIX (CHOIX) were generous gifts from Dr J. Lopez (Houston, TX). All other reagents and antibodies were from
sources described previously.27-29
Platelet preparation and aggregation studies
Blood was collected from healthy donors or individuals with
Glanzmann thrombasthenia (containing < 1%
IIb3) or type III von Willebrand disease
(vWD) (< 1% plasma and platelet vWf), and the platelet-rich
plasma (PRP) and washed platelets were prepared as described
previously.27 Platelet aggregation studies using BvWf or
HvWf and ristocetin were performed according to previously published
methods.29 Thrombin-induced aggregations were performed in
the presence of 1 mmol/L CaCl2; aggregation induced by
collagen or adenosine diphosphate (ADP) was performed in the presence
of 0.5 mg/mL fibrinogen and 1 mmol/L CaCl2. Shear-induced
platelet aggregation (SIPA) was performed in a cone-and-plate
viscometer (Carrimed rheometer, CSL100, Carri-Med, Dorking, United
Kingdom). In these studies, PRP or washed platelets in the
presence of 10 µg/mL HvWf were exposed to the indicated shear rate
for 2 minutes at room temperature (22°C). In some experiments,
platelets were pretreated for 10 minutes with the indicated
concentrations of cytochalasin D (CD), latrunculin B (LB),
jasplakinolide, apyrase, or prostaglandin E1
(PGE1). In control experiments, 0.5 U/mL apyrase completely
inhibited PRP or washed platelet aggregation induced by 10 µmol/L ADP
and preincubation of platelets with 0.5µg/mL PGE1 totally
blocked washed platelet aggregation induced by 1 U/mL thrombin.
Pretreatment of platelets with 5 µmol/L jasplakinolide, a
concentration that has been demonstrated to inhibit actin filament disassembly,30 completely inhibited platelet spreading on
a vWf matrix. In other studies, washed platelets were preincubated for
10 minutes with either 2 mmol/L EDTA or the
anti-IIb3 c7E3 Fab (20 µg/mL) to block
ligand binding to integrin IIb3.
Following aggregation, platelets were fixed for 60 minutes in 1%
paraformaldehyde, stained overnight with 1 µmol/L DiOC6,
and mounted in Permafluor. Platelet aggregates were imaged using
confocal fluorescence microscopy (Leica TCS SP Confocal microscope,
Leica Microsystems, Heidelberg, Germany), and volumetric
analysis of 3-dimensional reconstructed platelet aggregates was
performed using the Microcomputer Imaging Device (MCID, Imaging
Research, St Catherine's, Ontario, Canada) or Voxblast
(Vaytek, Fairfield, IA).
Generation of GPIb mutants and expression of the GPIb/V/IX
complex on the surface of Chinese hamster ovary cells
The GPIb 535-568 deletion mutant was generated as
described previously.31 GPIb569-610 was generated
using a similar protocol, with the following primers flanking the
designed deletion region: 5' GAA GAG GCT GGA GCG GAA AGA 3' (4718-4698)
and 5' TGA GGG TGG GAG GTT TGG GGA 3' (4947-4967) (Genbank accession
number M22403). Both GPIb535-568 and GPIb569-610 deletions
were confirmed by DNA sequence analysis. Transfection of CHO cells with
cDNAs for GPIb (CHO-Ib/IX), GPIb569-610 (CHO-Ib569), or
GPIb535-568 (CHO-Ib535), and their subsequent characterization
by FACS analysis, were performed as described
previously.31
Chinese hamster ovary cell aggregation studies
CHO-Ib/IX, CHO-Ib569, or CHO-Ib535 cells
(1 × 106 cells/mL) were resuspended in Tyrode buffer
containing 2 mmol/L EDTA. CHO cell aggregation was initiated using the
indicated concentrations of HvWf and 1 mg/mL ristocetin, or BvWf, at
37°C with constant stirring. Aggregation was monitored using a
Chronolog Dual Channel Aggrometer, Chromo-Log, Havertown, PA.
In some studies, CHO cells were preincubated with 5 µmol/L CD for 10 minutes prior to initiation of aggregation. Cells were processed and
subjected to confocal fluorescence microscopy and volumetric analysis,
as described above for platelets.
Estimation of filamentous-actin content
Washed platelets (1 × 109/mL) were stimulated
with either 10 µg/mL HvWf in the presence of 1 mg/mL ristocetin, or 1 U/mL thrombin for the indicated times, with stirring. Alternatively,
GPIb/V/IX-transfected CHO cells (3 × 106/mL) were
aggregated with 10 µg/mL BvWf for 20 minutes. Cells were lysed with
an equal volume of 2 × Triton X-100 lysis buffer (40 mmol/L Tris-HCl,
pH 7.4, 2% Triton X-100, 10 mmol/L EDTA, 2 mmol/L phenylmethylsulfonyl
fluoride, 2 mmol/L Na3VO4, 4 mmol/L benzamidine, and 0.1 µmol/L phallacidin). Filamentous
(F)-actin content was determined using the DNaseI inhibition
assay32,33 or by the sedimentation method described by
Fox.17
Chinese hamster ovary cell adhesion studies
Adhesion studies were performed using CHO-Ib/IX and CHO-Ib569
cells according to previously published methods.29 Where indicated, cells were fixed in suspension with 3.7% formaldehyde for
10 minutes, then allowed to adhere onto
poly-L-lysine-coated coverslips and imaged as previously
described.29
| |
Results |
We have recently demonstrated that vWf binding to GPIb/V/IX is
sufficient to induce actin polymerization and cytoskeletal reorganization in human platelets and GPIb/V/IX-transfected CHO cells.29 To investigate the possibility that these
cytoskeletal changes may influence the adhesive function of GPIb/V/IX,
we examined the effect of pretreating platelets with CD on vWf-induced
platelet aggregation. As demonstrated in Figure
1A, pretreating platelets with CD
dramatically enhanced the rate and extent of platelet aggregation
induced by HvWf in the presence of 1 mg/mL ristocetin. The extent of
enhancement of platelet aggregation induced by CD was most evident when
using low threshold concentrations of HvWf (0.25-1 µg/mL), although
even at high concentrations of HvWf (5-10 µg/mL) an increase in
aggregation was also observed. The enhancing effects of CD on
ristocetin-induced platelet aggregation were observed using either
washed platelets (Figure 1A) or PRP (Figure 1B). In control studies,
preincubating platelets with a blocking antibody against GPIb
completely eliminated platelet aggregation induced by HvWf, in the
presence or absence of CD (data not shown), demonstrating that
aggregation was GPIb dependent. Moreover, pretreating platelets with CD
alone or with CD in the presence of HvWf (without ristocetin) failed to
induce platelet aggregation, demonstrating that CD was unlikely to be
directly inducing vWf binding to GPIb (data not shown). Ristocetin
dose-response studies were performed to investigate whether CD could
enhance platelet aggregation at ristocetin concentrations less than 1 mg/mL, to produce a phenotype similar to that observed with
platelet-type vWD. As demonstrated in Figure 1B, CD enhanced platelet
aggregation when ristocetin was added to the reaction at 1 mg/mL, but
it failed to induce aggregation at lower ristocetin concentrations
(0.25-0.75 mg/mL). Studies using BvWf, which can bind GPIb and induce
aggregation independent of artificial modulators, demonstrated CD
enhancement of platelet aggregation induced by both low (1 µg/mL) and
high concentrations (10 µg/mL) of BvWf (Figure 1A). These latter
studies demonstrate that the effects of CD are not ristocetin
dependent.
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| Figure 1.
Cytochalasin D enhances vWf-induced platelet
aggregation.
(A) Washed platelets (3 × 108/mL) were incubated with
vehicle alone ( CD) or 5 µmol/L CD (+CD), then aggregated with the
indicated concentrations of HvWf in the presence of 1 mg/mL ristocetin
(i,ii) or BvWf alone (iii,iv). The aggregation tracings are from 1 experiment, representative of 10 independent experiments. (B) PRP was
incubated with 5 µmol/L CD for 10 minutes followed by the addition of
ristocetin at the indicated concentrations to initiate aggregation
under stirred conditions.
|
|
In further studies we confirmed that the enhancing effect of CD on
vWf-induced aggregation was due to inhibition of actin polymerization
rather than a direct effect on either vWf or GPIb/V/IX. As demonstrated
in Figure 2A, concentrations of CD
between 0.5 and 5 µmol/L enhanced platelet aggregation induced by
BvWf in a dose-dependent manner that correlated closely with the
inhibition of actin polymerization (Figure 2B). Moreover, treating
platelets with a structurally unrelated inhibitor of actin
polymerization, LB, also dramatically enhanced platelet aggregation
induced by HvWf/ristocetin (Figure 2C) or BvWf (data not shown). LB is
a potent marine toxin that sequesters actin monomers and prevents elongation of pre-existing actin filaments.34 As with CD,
the effects of LB on vWf-induced platelet aggregation were
dose-dependent and correlated closely with its ability to inhibit actin
polymerization (data not shown). The importance of metabolically active
platelets for the enhancing effects of CD or LB was demonstrated using
formalin-fixed platelets. In these studies, neither CD or LB enhanced
vWf-mediated agglutination of fixed platelets (data not shown).
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| Figure 2.
Inhibition of actin polymerization enhances vWf-induced
platelet aggregation.
(A) Washed platelets (3 × 108/mL) were treated with the
indicated concentration of CD prior to the performance of aggregation
studies using BvWf (1 µg/mL). The aggregation tracings are from 1 experiment, representative of 5. (B) Washed platelets were treated with
the indicated concentrations of CD prior to platelet stimulation with
thrombin (1 U/mL) for 10 minutes. Cells were then lysed and F-actin
content in the whole-cell lysates determined by actin filament
sedimentation assays as described under "Materials and methods."
Results are the mean ± SE from 4 independent experiments. (C)
Washed platelets were incubated with either vehicle alone (LB) or 200 ng/mL latrunculin B (+LB), prior to the initiation of platelet
aggregation with HvWf (0.5 µg/mL) in the presence of ristocetin (1 mg/mL). The aggregation tracings are from 1 experiment, representative
of 3 performed in duplicate.
|
|
A recent report has suggested that pretreating platelets with CD can
induce fibrinogen binding to integrin
IIb3,35 raising the
possibility that inhibiting actin polymerization may lead to a general
enhancement in platelet aggregation induced by threshold concentrations
of agonists. To investigate the specificity of the effects of CD on
vWf-induced platelet aggregation, aggregation studies were performed
with multiple physiologic agonists including thrombin, collagen, or
ADP. As demonstrated in Figure 3,
pretreating platelets with CD failed to enhance platelet aggregation
induced by either low or high concentrations of the indicated agonist. In general, inhibiting actin polymerization resulted in a reduction in
the rate and extent of platelet aggregation induced by each agonist.
Similar observations were made with platelets pretreated with LB (data
not shown), confirming that the enhancing effect of these inhibitors
was specific to vWf-mediated platelet aggregation.
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| Figure 3.
Cytochalasin D specifically enhances vWf-induced
platelet aggregation.
Washed platelets (3 × 108/mL) were incubated with either
vehicle alone () or 5 µmol/L CD (+) prior to the initiation of
platelet aggregation with the indicated concentrations of thrombin (A),
collagen (B), ADP (C), HvWf (0.5 µg/mL) and ristocetin (1 mg/mL), or
BvWf (1 µg/mL). These aggregation tracings are from 1 experiment,
representative of 5. The accompanying bar graph is a quantitative
representation of the effects of CD on the rate of platelet aggregation
induced by low concentrations of the indicated agonist. Results are
presented as the fold increase in the initial rate of aggregation
relative to Me2SO-treated platelets. Results are the
mean ± SE from 5 experiments.
|
|
To determine whether the enhancing effect of CD on platelet aggregation
was primarily due to effects on the vWf-GPIb interaction or involved
integrin IIb3, ligand binding to
IIb3 was prevented by preincubating
washed platelets with either the anti-3 integrin antibody c7E3 Fab or EDTA. As demonstrated in Figure
4A, inhibiting ligand binding to integrin
IIb3 did not inhibit the ability of CD to
enhance the rate and extent of platelet aggregation induced by
HvWf/ristocetin or BvWf (data not shown). Similarly, studies using
Glanzmann thrombasthenic platelets also demonstrated that the rate and
extent of vWf-induced platelet aggregation was enhanced by CD (Figure
4B), confirming that the effects of CD occurred independent of integrin
IIb3. To definitively establish that the
enhancing effects of CD were specific to the vWf-GPIb interaction and
did not involve other platelet adhesion receptors or endogenous platelet stimuli, studies were performed on CHO cells transfected with
the GPIb/V/IX complex. Consistent with previous reports,36 the addition of HvWf/ristocetin or BvWf to a stirred suspension of
CHO-Ib/IX cells induced the formation of macroscopic CHO cell aggregates. The formation of these aggregates could be analyzed in real
time on the basis of changes in light transmission using a platelet
aggregometer. Similar to platelets, pretreating CHO-Ib/IX cells with CD
enhanced both the rate and extent of CHO cell aggregation (Figure 4C).
Overall the extent of CHO cell aggregation was less than that observed
with platelets, presumably due to the lower GPIb/V/IX receptor density
on CHO cells. As with platelets, the enhancing effects of CD were most
obvious when using threshold concentrations of vWf to aggregate the
cells. Confocal imaging of CHO cell aggregates demonstrated that BvWf
alone induced small aggregates consisting of approximately 5 to 15 cells (Figure 4D), whereas the aggregates formed in the presence of CD
consisted of more than 50 to 100 cells (Figure 4D). In control studies, CHO-Ib/IX cells preincubated with the anti-GPIb antibody, AK2, or CHO
cells transfected with GP Ib/IX failed to aggregate in response to
BvWf, in the presence or absence of CD (Figure 4D and data not shown).
In all studies, CHO-Ib/IX cells were aggregated in the presence of EDTA
to exclude a role for endogenous CHO cell integrins in the aggregation
process. Taken together, these studies provide strong evidence that the
enhancing effect of CD on vWf-induced aggregation is due to modulation
of the vWf-GPIb/V/IX interaction.
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| Figure 4.
Cytochalasin D enhances vWf-induced platelet and
CHO-Ib/IX cell aggregation independent of integrin
IIb3.
(A) Washed platelets (3 × 108/mL) from healthy donors
were incubated with vehicle alone (0.25% Me2SO,  ) or CD
(5 µmol/L,  ). Platelet aggregation was induced with HvWf (0.5 µg/mL) and ristocetin (1 mg/mL), in the presence of buffer alone
(control), EDTA (2 mmol/L), or c7E3 Fab (20 µg/mL). Results are
presented as percent change in aggregation rate (percent relative to
Me2SO control, arbitrarily defined as 100%). (B) Glanzmann
thrombasthenic platelets were aggregated with HvWf (0.5 µg/mL) and
ristocetin, in the absence (CD) or presence of 5 µmol/L CD (+CD).
The aggregation tracings are from one experiment performed in
triplicate. (C) CHO-Ib/IX cells (1 × 106/assay) were
stirred for 6 minutes in the presence 5 µmol/L CD alone (CD), 10 µg/ml BvWf (BvWf), or BvWf and CD (BvWf + CD) in a platelet
aggregometer. The aggregation tracings are from 1 experiment
representative of 5. (D) CHO-Ib/IX cells were stirred in the presence
of vehicle alone (resting), 10 µg/mL BvWf (BvWf), BvWf and 5 µmol/L
CD (BvWf + CD), or BvWf in the presence of the anti-GPIb mAb, AK2
(BvWf + AK2). The cells were then fixed, stained with
DiOC6 (1 µmol/L), mounted onto glass slides and subjected
to confocal microscopy (10 × objective) as described under
"Materials and methods." The images of aggregated cells were
reconstructed in 3-dimension using Voxblast (Vaytek Inc). Results
presented are from 1 experiment, representative of 5.
|
|
To investigate further the mechanism by which CD enhances vWf-induced
platelet aggregation, in particular the role of actin filament
depolymerization in this process, we examined the effect of the actin
filament-stabilizing reagent, jasplakinolide. This reagent is a
membrane-permeable cyclic peptide derived from the marine sponge
Jaspis johnstoni, which binds to and stabilizes pre-existing
actin filaments, preventing filament depolymerization. Jasplakinolide
has recently been demonstrated to effectively inhibit CD-induced
integrin IIb3 activation on the surface
of human platelets.35 As demonstrated in Figure
5A, pretreating washed platelets with
jasplakinolide at concentrations that have previously been demonstrated
to inhibit actin filament disassembly and completely inhibit platelet
spreading (see "Materials and methods") did not prevent the ability
of CD to enhance vWf-induced platelet aggregation. Similar findings
were apparent using LB (data not shown). These findings suggest that
the primary mechanism by which CD and LB promote vWf-induced platelet
aggregation is through inhibition of actin polymerization, rather than
as a result of actin filament depolymerization. To investigate further
the relationship between inhibition of actin polymerization and
enhanced vWf-induced platelet aggregation, we examined the effect of
the platelet activation inhibitor, PGE1. This reagent is a
potent activator of the adenyl cyclase signaling pathway resulting in
down-regulation of multiple platelet functional responses, including
vWf-induced actin polymerization.29 As demonstrated in
Figure 5B, pretreating platelets with PGE1 dramatically
enhanced platelet aggregation induced by a threshold concentration of
vWf, similar in magnitude to that observed with CD or LB. CD did not
promote PGE1-enhanced aggregation further, indicating that
the effects of these reagents were not additive. The ability of
PGE1 to enhance vWf-induced platelet aggregation is
consistent with its ability to inhibit actin polymerization, because a
number of other signal transduction inhibitors, including inhibitors of
tyrosine kinases (genistein, tyrphostin, or erbstatin), phosphoinositide (PI) 3-kinase (wortmannin or LY294002), protein kinase
C (PKC) (calphostin C or bisindoylmaleimide), or prostaglandin metabolism (aspirin), that do not inhibit vWf-induced actin
polymerization,29 failed to enhance the aggregation
response (data not shown). These latter findings are consistent with
the jasplakinolide data, suggesting enhanced platelet aggregation is
due to inhibition of actin polymerization rather than disassembly of
pre-existing actin filaments.
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| Figure 5.
Cytochalasin D enhances vWf-induced platelet aggregation
independent of actin filament severing or ADP.
(A) Washed platelets (3 × 108/mL) were preincubated with
vehicle alone (control) or 5 µmol/L jasplakinolide (+ Jasp), and/or 5 µmol/L CD (+CD) for 10 minutes, prior to aggregation with 1 µg/mL
BvWf. All aggregations were performed in the presence of 2 mmol/L EDTA.
The aggregation tracings are from 1 experiment representative of 4 independent experiments performed in triplicate. (B) Washed platelets
were preincubated with vehicle alone (control), 0.5 µg/mL
PGE1 (+ PGE1) or PGE1 and 5 µmol/L CD (+ PGE1 + CD), then aggregated with BvWf
(1 µg/mL). The tracings shown are from 1 experiment, representative
of 5 independent experiments. (C) PRP was preincubated with either
vehicle alone (control) or 5 µmol/L CD (+CD) for 10 minutes in the
presence (+ apy) or absence of 0.5 U/mL apyrase. Aggregation of
platelets was initiated with 1 mg/mL ristocetin. All aggregations were
performed in the presence of anti-3 antibody, c7E3 Fab
(20 µg/mL), to prevent vWf binding to integrin
IIb3. The aggregation tracings are from 1 experiment representative of 4 individual experiments performed in
duplicate. (D) Washed platelets were preincubated with either vehicle
alone (control) or 5 µmol/L CD for 0, 30, or 60 seconds, then
aggregated with 1 µg/mL BvWf. All aggregations were performed in the
presence of 2 mmol/L EDTA to block ligand binding to integrin
IIb3. The aggregation tracings are from 1 experiment representative of 5 individual experiments performed in
duplicate.
|
|
Recent studies by Bennett and colleagues35 have suggested
that the cytoskeletal regulation of integrin
IIb3 requires a subthreshold
concentration of ADP to induce slow actin filament turnover. We
therefore examined the effects of the ADP scavenging enzyme, apyrase,
on vWf-induced platelet aggregation. As demonstrated in Figure 5C,
pretreating platelets with apyrase at concentrations that eliminate the
stimulatory effects of ADP (see "Materials and methods") did not
significantly inhibit platelet aggregation induced by vWf. Moreover,
apyrase did not affect the ability of CD to enhance vWf-induced
platelet aggregation (Figure 5C). Further evidence suggesting that slow
actin filament turnover was not responsible for the effects of the
cytoskeleton on GPIb/V/IX was derived from CD time-course experiments.
As demonstrated in Figure 5D, the enhancing effect of CD on platelet
aggregation was extremely rapid, occurring within seconds of CD
addition to the reaction mixture and reaching a maximum after 30 to 60 seconds of preincubation. Taken together, the results presented in
Figure 5 suggest that the mechanism of cytoskeletal regulation of
GPIb/V/IX is distinct from that reported for integrin
IIb3 (see "Discussion").
To investigate the significance of cytoskeletal regulation of GPIb/V/IX
under pathophysiologically relevant shear conditions, we examined the
effect of CD on SIPA. Consistent with previous reports,4-7
exposure of PRP, or washed platelets in the presence of soluble vWf (10 µg/mL), to pathologic levels of shear (3000-5000 s1) resulted in platelet aggregation. As
demonstrated in Figure 6, the size of
these aggregates increased as a function of shear (Figure 6Ai) and was
dependent on ligand binding to both GPIb or integrin
IIb3, because pretreating platelets with
blocking antibodies against either receptor inhibited SIPA (data not
shown). Pretreating washed platelets or PRP with CD greatly enhanced
the formation of platelet aggregates under high shear (Figure 6Aii,B) and also lowered the shear threshold required to induce platelet aggregation from pathologic (3000 s1) to physiologic (500 s1) levels of shear (Figure 6Aii). It is well
established that at lower shear rates soluble agonists can induce
platelet aggregation independent of vWf.11,37 To
investigate the requirement for vWf in CD-enhanced platelet
aggregation, studies were performed on platelets derived from an
individual with type III vWD. Washed platelets or PRP failed to
aggregate in response to intermediate (500-1000 s1) or
high shear (5000 s1) in the presence or absence
of CD (Figure 7A and data not shown). The
addition of soluble vWf (20 µg/mL) to vWD- washed platelets or PRP
restored SIPA, as well as their heightened responsiveness to shear in
the presence of CD (Figure 7A). Interestingly, these aggregates were
substantially smaller than aggregates from normal platelets, consistent
with the potentially important role for platelet vWf in promoting
SIPA.37 Moreover, under all shear conditions (500-5000 s1) CD-enhanced aggregation of platelets was
prevented by pretreating platelets with antibodies against GPIb or
integrin IIb3 (Figure 7B). Taken
together, these findings suggest that inhibiting actin polymerization
reduces the shear threshold by which the vWf-GPIb interaction induces
platelet activation and integrin
IIb3-mediated irreversible aggregation.
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| Figure 6.
Cytochalasin D enhances shear-induced platelet
aggregation.
(A) Washed platelets (1.5 × 108/mL) were incubated with
either vehicle alone (CD, i) or 5 µmol/L CD (+CD, ii) in the
presence of HvWf (10 µg/mL). Platelets were then exposed to the
indicated shear rates in a cone-and-plate viscometer as described under
"Materials and methods." Platelets were then fixed, stained with 1 µmol/L DiOC6, mounted onto glass slides, and subjected to
confocal microscopy (20 × objective). The 3-dimensional images of
aggregated platelets were reconstructed using Voxblast (Vaytek Inc).
Results presented are from 1 experiment, representative of 5 independent experiments. (B) Platelet aggregates from 8 random fields (10 × objective) were subjected to volumetric analysis,
and the extent of aggregation was expressed as the fold increase in the
volume of aggregates over that detected for Me2SO-treated
control platelets. Results are the mean ± SE from 5 experiments.
, buffer; , 5 µmol/L CD.
|
|
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| Figure 7.
An essential role for vWf, GPIb/V/IX, and integrin
IIb3 in mediating shear-induced platelet
aggregation in the presence or absence of cytochalasin D.
(A) Platelet-rich plasma (PRP) from an individual with type III vWD was
incubated with either vehicle alone (CD, i) or 5 µmol/L CD (+CD,
ii) prior to exposing platelets to a shear rate of 5000 s1 (top panel). Alternatively, purified HvWf (20 µg/mL) was added to vWD PRP (+ vWf) prior to their exposure to the
indicated shear rates. The size of platelet aggregates formed under the
various experimental conditions was determined as described in Figure
6. (B) Washed platelets from a normal donor were incubated with an
anti-GPIb (ALMA12) or anti-3 integrin mAb (c7E3
Fab) for 10 minutes in the presence of 5 µmol/L CD (+ CD) prior to
the exposure of platelets to shear (5000 s1).
Platelets were then imaged as described in Figure 6. (C) Platelet
aggregates from 8 random fields (10 × objective) were subjected to
volumetric analysis, and the extent of aggregation was expressed as the
fold increase in the volume of aggregates over that detected for
Me2SO-treated control platelets. Results for vWD platelets
are from a single vWD patient and results for normal platelets are the
mean ± SE from 5 experiments. , buffer; , 5 µmol/L.
|
|
To investigate the relationship between the cytoskeletal linkage of
GPIb/V/IX and the ability of CD to enhance cell aggregation, studies
were performed on CHO cells expressing mutant forms of the GPIb/V/IX
receptor complex. We initially generated a GPIb cytoplasmic tail
truncation mutant lacking the C-terminal 41 amino acids (CHO-Ib569),
which has previously been demonstrated not to associate with the
cytoskeleton.38 Our immunoprecipitation studies using
biotin-labeled CHO cells have confirmed that this deletion is
sufficient to disrupt the interaction between GPIb and ABP (data not
shown). Flow cytometric analysis of CHO cells expressing mutant
GPIb569 demonstrated efficient expression of the glycoprotein on the
surface of CHOIX cells (CHO-Ib569), similar to the level of
wild-type GPIb on CHO-Ib/IX (Figure
8A). We also confirmed that GPIb569
was able to bind vWf by the ability to tether and roll on a BvWf matrix
under flow conditions in a similar manner to CHO-Ib/IX (data not
shown). Previous studies have demonstrated that CHO cells expressing
GPIb569 extend filopodia and spread on a vWf matrix,38
suggesting that the linkage between GPIb and ABP-280 is uncoupled from
vWf-induced cytoskeletal reorganization. However, this conclusion is
complicated by the observations that cytoskeletal reorganization in
GPIb/V/IX-transfected CHO cells is not strictly dependent on vWf
binding to GPIb/V/IX but also involves activation of endogenous CHO
cell integrins.29,39 To investigate the ability of vWf to
induce actin polymerization in CHO cells expressing a truncated form of
GPIb (CHO-569), we performed DNaseI inhibition assays on
whole-cell lysates prepared from nonaggregated or vWf-aggregated
CHO-Ib569 cells (Figure 8B). As demonstrated, vWf-induced
aggregation of CHO-Ib569 was associated with a 60% increase in the
level of F-actin from a resting level of 27% up to a maximum of 45%.
This increase was similar to that observed with CHO-Ib/IX and was
abolished by preincubating these cells with the anti-GPIb mAb, AK2. A
similar increase in F-actin was observed in vWf-aggregated CHO-Ib/IX
and CHO569 cells by monitoring changes in F-actin through FACS
analysis of cells labeled with Alexa488-phalloidin (data not shown).
Moreover, adhesion of CHO-Ib569 cells to immobilized HvWf in the
presence of botrocetin was associated with cytoskeletal reorganization
leading to filopodial extension, similar to that observed with CHO
cells expressing the wild-type receptor (Figure 8C). It should be noted
that all experiments were performed in the presence of EDTA, excluding a role for endogenous integrins in mediating these cytoskeletal changes. Similar cytoskeletal changes were obtained with an additional GPIb cytoplasmic tail mutant that lacks the primary ABP-280 binding domain (deletion of residues 535-568, CHO-Ib535, data not shown), confirming that vWf-induced actin polymerization and cytoskeletal reorganization do not require physical linkage between GPIb and the
cytoskeleton.
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| Figure 8.
vWf induces actin polymerization in CHO-Ib/IX and
CHO-Ib569 cells.
(A) Surface expression of GPIb on CHO-Ib/IX and CHO-Ib569 cells
was examined by FACS analysis using the anti-GPIb mAb,
ALMA12 (filled histogram), as detailed under "Materials
and methods." (B) CHO-Ib/IX and CHO-Ib569 cells
(3 × 106/mL) were stirred in the presence of buffer
(resting) or BvWf (10 µg/mL) for 20 minutes. Where indicated, cells
were also incubated with an anti-GPIb mAb, AK2 (5 µg/mL), prior to
the initiation of aggregation. The cells were then lysed and F-actin
contents in the whole lysates determined using the DNaseI inhibition
assay. Results are the mean ± SE from 3 experiments, performed in
duplicate. (C) CHO-Ib/IX cells (1 × 106/mL) were fixed
in suspension (preadherent) with 3.7% formaldehyde for 10 minutes,
prior to adhesion to poly-L-lysine (100 µg/mL)-coated
coverslips. Alternatively, CHO-Ib/IX or CHO-Ib569 cells were adhered
to a HvWf matrix (10 µg/mL) in the presence of 1 µg/mL botrocetin
and 2 mmol/L EDTA for 60 minutes (adherent). Adherent cells were fixed,
permeabilized, stained with fluorescein isothiocyanate-conjugated
phalloidin and subjected to confocal fluorescence microscopy (100 ×
objective) as described under "Materials and methods." The images
presented were reconstructed using VoxBlast software and are
representative of 5 independent experiments.
|
|
To determine the effects of disrupting the link between GPIb and the
cytoskeleton on the ability of CD to enhance vWf-induced cell
aggregation, comparative aggregation studies were performed on
CHO-Ib569 and CHO-Ib/IX. As demonstrated in Figure
9A, CHO-Ib569 stirred in the presence
of BvWf aggregated more rapidly and to a much greater extent than
CHO-Ib/IX. In contrast to CHO-Ib/IX, pretreating CHO-Ib569 with CD
failed to enhance the aggregation process. It was unlikely that the
inability of CD to enhance CHO-Ib569 cell aggregation was because
these cells were maximally aggregated, because CD also failed to
enhance aggregation of these cells when using threshold concentrations
of BvWf (data not shown). Volumetric analysis of CHO-Ib569 cell
aggregates following confocal imaging demonstrated that these
aggregates were about 5-fold larger than CHO-Ib/IX in the absence of CD
(Figure 9C), but were of similar size to CHO-Ib/IX cell aggregates
formed in the presence of CD (Figure 9B,C). The increased aggregation
response and insensitivity to CD was not unique to CHO-Ib569 because
it was also observed with CHO-Ib535 (data not shown), confirming an
important role for the GPIb-ABP-280 linkage in enabling cytoskeletal
regulation of the vWf-GPIb interaction.
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| Figure 9.
Disrupting the physical link between GPIb and the
cytoskeleton abolishes the ability of CD to enhance vWf-induced cell
aggregation.
(A) CHO-Ib/IX and CHO-Ib569 cells (3 × 106/mL) were
incubated with vehicle alone () or 5 µmol/L CD (+). The cells were
then aggregated in a platelet aggregometer using BvWf (5 µg/mL). The
aggregation traces are from 1 experiment, representative of 5 independent experiments. (B) CHO-Ib/IX and CHO-Ib569 cells
(3 × 106/mL) were incubated with vehicle alone or 5 µmol/L CD, then stirred for 5 minutes in the presence of control
buffer (resting) or BvWf (5 µg/mL). Cells were then fixed, stained,
mounted onto glass slides, and subjected to confocal microscopy (10 ×
objective) as described for Figure 4D. Images were obtained from 1 experiment, representative of 5. (C) Cells from 8 random fields (10 ×
objective) were subjected to volumetric analysis, and the extent of
cell aggregation was expressed as the fold increase in the volume of
cell aggregates over that detected for Me2SO-treated
control cells. Results are the mean ± SE from 5 experiments.
|
|
| |
Discussion |
A unique feature of the vWf-GPIb interaction is its positive
regulation by high shear forces. Shear-induced binding of vWf to GPIb
is a key step in the normal hemostatic process and may also promote
pathologic thrombus formation in stenosed arteries and the
microcirculation. Despite the fundamental importance of the vWf-GPIb
interaction in hemostasis and thrombosis, the factors negatively
regulating this adhesion event, particularly under elevated shear
conditions, remain poorly defined. The studies presented here suggest
for the first time a potentially important role for the cytoskeleton in
regulating the adhesive function of GPIb/V/IX. Our studies have
demonstrated that pretreating platelets or GPIb/V/IX-transfected CHO
cells with inhibitors of actin polymerization selectively enhances cell
aggregation induced by vWf. Moreover, our studies of CHO cells
expressing mutant GPIb/V/IX complexes have demonstrated that the
physical linkage between GPIb and the membrane skeleton is not required
for vWf-induced actin polymerization but is essential for the enhancing
effect of actin polymerization inhibitors on the aggregation process.
Finally, studies of shear-induced platelet aggregation demonstrated
that inhibiting actin polymerization not only enhanced the extent of
platelet aggregation but also dramatically lowered the shear threshold
required to induce the aggregation process. These latter findings raise
the intriguing possibility that cytoskeletal regulation of the vWf-GPIb
interaction plays a key role in preventing shear-induced platelet
aggregation in the normal circulation.
Studies on Glanzmann thrombasthenic platelets and GPIb/V/IX-transfected
CHO cells have demonstrated that CD enhances vWf-induced cell
aggregation through regulation of GPIb/V/IX and does not require
integrin IIb3 or endogenous platelet
stimuli. The recent studies of Bennett and coworkers35
demonstrated that CD induces platelet integrin
IIb3 activation through a process
requiring ADP-dependent actin filament turnover. These studies
postulate that ADP-induced actin filament severing relieves
cytoskeletal constraints on integrin IIb3
in resting platelets, leading to receptor activation. In contrast, our
studies suggest that actin polymerization induced by vWf (as opposed to
depolymerization of pre-existing filaments), is the primary mechanism
by which the cytoskeleton regulates the adhesive function of GPIb/V/IX and is distinct from that observed with integrin
IIb3. For example, pretreating platelets
with jasplakinolide to stabilize actin filaments does not prevent CD
enhancement of vWf-induced aggregation. Moreover, the ability of
PGE1 to enhance vWf-induced aggregation suggests that actin
filament disassembly is not the primary mechanism by which CD and LB
enhance the adhesive function of GPIb/V/IX. Two other lines of evidence
indicate that slow actin filament turnover in resting platelets is not
the principal mechanism of cytoskeletal regulation of GPIb/V/IX. First,
removing the cellular effects of ADP did not inhibit CD enhancement of
aggregation, and second, the effects of CD on aggregation were very
rapid, occurring within seconds of its addition to the assay. Taken
together, these studies suggest that the adhesive function of GPIb/V/IX
is regulated by actin filament polymerization induced by vWf or other
agonists, rather than depolymerization of pre-existing actin filaments.
Our studies of CHO-Ib569 cells demonstrate an important role for the
cytoplasmic tail of GPIb in regulating the adhesive function of the
GPIb/V/IX receptor complex. We have demonstrated that CHO-Ib569
cells aggregate to a much greater extent in response to vWf than CHO
cells expressing the wild-type receptor. Moreover, the aggregation
response of CHO-Ib569 cells was not sensitive to the enhancing
effects of CD. The simplest interpretation of these findings is that
the mutant receptors are not subjected to the normal constraints
imposed by the cytoskeleton, presumably as a consequence of the
physical disruption of the link between GPIb and the membrane
skeleton. An alternative possibility is that mutating the GPIb
cytoplasmic tail alters the intrinsic ligand-binding properties of the
receptor complex. We do not, however, favor this latter possibility for
3 reasons. First, we have not detected a major difference in the
ability of CHO cells expressing GPIb569, GPIb535, or wild-type
GPIb/V/IX to tether and roll on a vWf matrix under shear conditions
(reference 31 and unpublished observations). Second, previous studies
have demonstrated normal vWf binding to CHO cells expressing
GPIb569.38 Third, changes in the intrinsic binding
characteristics of GPIb would not easily explain the inability of CD to
enhance the aggregation process.
The ability of GPIb569 (or GPIb535) to support vWf-induced actin
polymerization distinguishes GPIb/V/IX from other adhesion receptors,
such as the integrins, which require physical linkage with the
cytoskeleton to induce cytoskeletal remodelling.40 It
remains to be established which structural domains of the GPIb/V/IX receptor complex are required for vWf-induced cytoskeletal
reorganization and whether other surface receptors are involved in this
process. For example, there is evidence for colocalization of GPIb/V/IX with Fc RIIA,41 and a number of recent reports suggest a
contribution of this latter receptor to vWf-induced
signaling.42-44 Our studies with CHO cells indicate that
FcRIIA is not essential for vWf-induced cytoskeletal changes because
these cells totally lack Fc receptors.45 It should be
noted that the GPIb569-610 mutant lacks the 14-3-3 binding
site,46 raising the interesting possibility that
vWf-induced actin polymerization does not involve 14-3-3. There is,
however, evidence suggesting that 14-3-3 can associate with the tail
of GPIb47,48 and that this binding is regulated by
phosphorylation of serine-166 in the cytoplasmic tail of GPIb by
protein kinase A (PKA).49 Studies are currently
underway in our laboratory to examine the contribution of the
cytoplasmic tail of GPIb to vWf-induced cytoskeletal reorganization,
and in particular, the importance of phosphorylation of GPIb in this process.
A major finding from our studies is the ability of actin polymerization
inhibitors to reduce the shear threshold required to induce platelet
aggregation from about 3000 s1 down to shear rates as low
as 500 s1. By performing studies on vWD platelets
and using antibodies against GPIb and integrin
IIb3, we have established that CD-induced platelet aggregation at arterial shear rates (500-1000 s1) is mediated by vWf engagement of both
GPIb/V/IX and integrin IIb3. The
mechanism by which CD promotes vWf-induced platelet activation at
physiologic shear rates is an important issue for future investigation.
It is possible that pretreating platelets with CD alters the adhesive
properties of GPIb/V/IX such that it has a higher "affinity" for
soluble vWf at arterial shear rates. Alternatively, vWf binding to GPIb
may be a normal ongoing process in the arterial circulation and
inhibiting actin polymerization removes the inhibitory effects of the
cytoskeleton on the vWf-GPIb interaction, thereby promoting platelet
activation. Evidence supporting the hypothesis that vWf binding to GPIb
is a normal ongoing process in the arterial circulation has been
derived from studies of patients with elevated platelet
counts.50 These individuals typically have reduced levels
of high- molecular-weight vWf multimers in the circulation, presumably
as a consequence of vWf binding to the expanded pool of GPIb. The
ability of high-molecular-weight multimers to interact with GPIb under
normal physiologic flow conditions is also supported by the observation
that the plasma pool of vWf does not contain the very
high-molecular-weight multimers present in platelet
-granules51 and in the Weibel-Palade bodies of
endothelial cells.52 These multimers appear to be
subjected to proteolytic regulation by a plasma metalloproteinase
activity.53-56 Deficiency of this activity leads to
vWf-induced platelet aggregation throughout the microcirculation,
leading to organ failure and possibly death, highlighting the
pathologic significance of unchecked shear-induced platelet aggregation
in vivo.
A key issue for future investigation is to define the precise mechanism
by which the cytoskeleton regulates the vWf-GPIb interaction. It is
possible that actin polymerization induces a conformational change in
the receptor that changes the kinetics of the vWf-GPIb bond. This may
lead to a slower on-rate, rapid off-rate, or a combination of these
events, leading to a reduced "affinity" of interaction. However,
this seems unlikely for 2 reasons. First, pretreating platelets with CD
did not induce platelet aggregation by vWf at low concentrations of
ristocetin (< 1 mg/mL), as has been demonstrated for platelets
expressing the mutant forms of GPIb associated with platelet-type
vWD57 that exhibit increased affinity for vWf. Second,
previous vWf-binding studies on CHO cells expressing GPIb569 did not
reveal an increased affinity for vWf.38 An alternative
possibility is that actin polymerization regulates GPIb receptor
distribution on the cell surface, thereby influencing the number or
"avidity" of the bonds formed between GPIb and the A1 domains of
multimeric vWf. The anchorage of GPIb to the membrane skeleton is
thought to play an important role in regulating the mobility of the
receptor complex in the plane of the plasma membrane.22
Thus, it is conceivable that vWf-induced cytoskeletal changes may alter
receptor distribution on the cell surface such that a reduced number of
GPIb molecules engage vWf. This may not only reduce the overall
"avidity" of the vWf-GPIb adhesion interaction but also reduce the
ability of these receptors to transduce signals necessary for integrin
IIb3 activation and irreversible platelet
aggregation. A precedent for such cytoskeletal regulation of receptor
distribution has been established from the studies of leukocyte
integrin L2 (LFA-1), in which
inhibitors of actin polymerization induce receptor clustering leading
to an increase in receptor avidity.58,59 A third
possibility is that the cytoskeleton may mediate rapid GPIb/V/IX
internalization following engagement of vWf. It is well established
that platelet activation by physiologic agonists such as thrombin or
ADP can induce internalization of GPIb/V/IX60,61 after 30 to 60 seconds of agonist stimulation.62 This
internalization appears to require actin polymerization and
cytoskeletal reorganization because it is completely prevented by
pretreating platelets with CD.60,62 We have demonstrated
that the effects of CD occur rapidly (within seconds of addition to
platelets), suggesting that internalization of GPIb/V/IX is unlikely to
be the primary mechanism regulating the vWf-GPIb/V/IX interaction.
Studies are currently underway in our laboratory to address these
various possibilities.
The ability of the cytoskeleton to regulate the vWf-GPIb interaction
also has potentially important clinical considerations. A recent
study37 has demonstrated that platelets from patients suffering an acute myocardial infarction have heightened reactivity to
shear, leading to enhanced shear-induced platelet aggregation. This
increased shear sensitivity appears to be due, at least in part, to an
increase in the plasma level of vWf. Although purely speculative, our
studies raise the interesting possibility that defects in the
cytoskeletal regulation of GPIb/V/IX may represent a novel mechanism of
promoting platelet reactivity in vivo, leading to excessive platelet
adhesion and aggregation at sites of vascular injury. Unraveling the
molecular mechanism by which vWf induces actin polymerization may not
only improve our understanding of the mechanisms regulating
platelet-platelet and platelet-vessel wall interactions, but may also
have potentially important therapeutic implications, because
pharmacologic modulation of this event may represent a novel approach
to influence platelet reactivity in vivo.
| |
Acknowledgments |
We thank Prof Michael Berndt and Dr Robert Andrews for both their
helpful discussions and for their generous donations of botrocetin and
monoclonal antibodies. We would also like to thank Dr Jose Lopez for
the GPIb/V/IX receptor constructs and CHO/IX cell line, Dr Corinne
de la Salle for technical assistance, and Dr Simone Schoenwaelder for
helpful advice.
| |
Footnotes |
Submitted December 20, 1999; accepted July 24, 2000.
Supported by a grant from the National Health and Medical Research
Council of Australia. N.M. is a recipient of a Monash Graduate Scholarship.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
Reprints: Shaun P. Jackson, Australian Centre for
Blood Diseases, Department of Medicine, Monash Medical School, Box Hill
Hospital, Arnold St, Box Hill, Victoria 3128, Australia; e-mail:
Shaun.Jackson{at}med.monash.edu.au.
| |
References |
1.
Savage B, Saldivar E, Ruggeri ZM.
Initiation of platelet adhesion by arrest onto fibrinogen or translocation on von Willebrand factor.
Cell.
1996;84:289-297[Medline]
[Order article via Infotrieve].
2.
Savage B, Almus-Jacobs F, Ruggeri ZM.
Specific synergy of multiple substrate-receptor interactions in platelet thrombus formation under flow.
Cell.
1998;94:657-666[Medline]
[Order article via Infotrieve].
3.
Ruggeri ZM.
Mechanisms initiating platelet thrombus formation.
Thromb Haemost.
1997;78:611-616[Medline]
[Order article via Infotrieve].
4.
Moake JL, Turner NA, Stathopoulos NA, Nolasco LH, Hellums JD.
Involvement of large plasma von Willebrand factor (vWF) multimers and unusually large vWF forms derived from endothelial cells in shear stress-induced platelet aggregation.
J Clin Invest.
1986;78:1456-1461.
5.
Peterson DM, Stathopoulos NA, Giorgio TD, Hellums JD, Moake JL.
Shear-induced platelet aggregation requires von Willebrand factor and platelet membrane glycoproteins Ib and IIb-IIIa.
Blood.
1987;69:625-628[Abstract/Free Full Text].
6.
Ikeda Y, Handa M, Kawano K, et al.
The role of von Willebrand factor and fibrinogen in platelet aggregation under varying shear stress.
J Clin Invest.
1991;87:1234-1240.
7.
Ikeda Y, Handa M, Kamata T, et al.
Transmembrane calcium influx associated with von Willebrand factor binding to GP Ib in the initiation of shear-induced platelet aggregation.
Thromb Haemost.
1993;69:496-502[Medline]
[Order article via Infotrieve].
8.
Gralnick HR, Williams SB, Coller BS.
Asialo von Willebrand factor interactions with platelets. Interdependence of glycoproteins Ib and IIb/IIIa for binding and aggregation.
J Clin Invest.
1985;75:19-25.
9.
De Marco L, Girolami A, Russell S, Ruggeri ZM.
Interaction of asialo von Willebrand factor with glycoprotein Ib induces fibrinogen binding to the glycoprotein IIb/IIIa complex and mediates platelet aggregation.
J Clin Invest.
1985;75:1198-1203.
10.
Weiss HJ, Hawiger J, Ruggeri ZM, Turitto VT, Thiagarajan P, Hoffmann T.
Fibrinogen-independent platelet adhesion and thrombus formation on subendothelium mediated by glycoprotein IIb-IIIa complex at high shear rate.
J Clin Invest.
1989;83:288-297.
11.
Goto S, Ikeda Y, Saldivar E, Ruggeri ZM.
Distinct mechanisms of platelet aggregation as a consequence of different shearing flow conditions.
J Clin Invest.
1998;101:479-486[Medline]
[Order article via Infotrieve].
12.
Kulkarni S, Dopheide SM, Yap CL, et al.
A revised model of platelet aggregation.
J Clin Invest.
2000;105:783-791[Medline]
[Order article via Infotrieve].
13.
Diamond MS, Springer TA.
The dynamic regulation of integrin adhesiveness.
Curr Biol.
1994;4:506-517[Medline]
[Order article via Infotrieve].
14.
Phillips DR, Charo IF, Parise LV, Fitzgerald LA.
The platelet membrane glycoprotein IIb-IIIa complex.
Blood.
1988;71:831-843[Free Full Text].
15.
Shattil SJ, Kashiwagi H, Pampori N.
Integrin signaling: the platelet paradigm.
Blood.
1998;91:2645-2657[Free Full Text].
16.
Hato T, Pampori N, Shattil SJ.
Complementary roles for receptor clustering and conformational change in the adhesive and signaling functions of integrin alphaIIb beta3.
J Cell Biol.
1998;141:1685-1695[Abstract/Free Full Text].
17.
Fox JE.
Linkage of a membrane skeleton to integral membrane glycoproteins in human platelets. Identification of one of the glycoproteins as glycoprotein Ib.
J Clin Invest.
1985;76:1673-1683.
18.
Okita JR, Pidard D, Newman PJ, Montgomery RR, Kunicki TJ.
On the association of glycoprotein Ib and actin-binding protein in human platelets.
J Cell Biol.
1985;100:317-321[Abstract/Free Full Text].
19.
Ezzell RM, Kenney DM, Egan S, Stossel TP, Hartwig JH.
Localization of the domain of actin-binding protein that binds to membrane glycoprotein Ib and actin in human platelets.
J Biol Chem.
1988;263:13303-13309[Abstract/Free Full Text].
20.
Andrews RK, Fox JE.
Interaction of purified actin-binding protein with the platelet membrane glycoprotein Ib-IX complex.
J Biol Chem.
1991;266:7144-7147[Abstract/Free Full Text].
21.
Andrews RK, Fox JE.
Identification of a region in the cytoplasmic domain of the platelet membrane glycoprotein Ib-IX complex that binds to purified actin-binding protein.
J Biol Chem.
1992;267:18605-18611[Abstract/Free Full Text].
22.
Dong JF, Li CQ, Sae-Tung G, Hyun W, Afshar-Kharghan V, Lopez JA.
The cytoplasmic domain of glycoprotein (GP) Ibalpha constrains the lateral diffusion of the GP Ib-IX complex and modulates von Willebrand factor binding.
Biochemistry.
1997;36:12421-12427[Medline]
[Order article via Infotrieve].
23.
Molnar J, Lorand L.
Studies on apyrases.
Arch Biochem Biophys.
1961;93:353-363[Medline]
[Order article via Infotrieve].
24.
Montgomery RR, Zimmerman TS.
von Willebrand's disease antigen II. A new plasma and platelet antigen deficient in severe von Willebrand's disease.
J Clin Invest.
1978;61:1498-1507.
25.
Jakobsen E, Ly B, Kierulf P.
Incorporation of fibrinogen into soluble fibrin complexes.
Thromb Res.
1974;4:499-507[Medline]
[Order article via Infotrieve].
26.
Azorsa DO, Moog S, Cazenave JP, Lanza F.
Leucocyte Typing VI. London, England: Garland Publishing; 1997.
27.
Jackson SP, Schoenwaelder SM, Yuan Y, Rabinowitz I, Salem HH, Mitchell CA.
Adhesion receptor activation of phosphatidylinositol 3-kinase. von Willebrand factor stimulates the cytoskeletal association and activation of phosphatidylinositol 3-kinase and pp60c-src in human platelets.
J Biol Chem.
1994;269:27093-27099[Abstract/Free Full Text].
28.
Yuan Y, Dopheide SM, Ivanidis C, Salem HH, Jackson SP.
Calpain regulation of cytoskeletal signaling complexes in von Willebrand factor-stimulated platelets. Distinct roles for glycoprotein Ib-V-IX and glycoprotein IIb-IIIa (integrin alphaIIbbeta3) in von Willebrand factor-induced signal transduction.
J Biol Chem.
1997;272:21847-21854[Abstract/Free Full Text].
29.
Yuan Y, Kulkarni S, Ulsemer P, et al.
The von Willebrand factor-glycoprotein Ib/V/IX interaction induces actin polymerization and cytoskeletal reorganization in rolling platelets and glycoprotein Ib/V/IX-transfected cells.
J Biol Chem.
1999;274:36241-36251[Abstract/Free Full Text].
30.
Cramer LP.
Role of actin-filament disassembly in lamellipodium protrusion in motile cells revealed using the drug jasplakinolide.
Curr Biol.
1999;9:1095-1105[Medline]
[Order article via Infotrieve].
31.
Cranmer SL, Ulsemer P, Cooke BM, et al.
Glycoprotein (GP) Ib-IX-transfected cells roll on a von Willebrand factor matrix under flow. Importance of the GPib/actin-binding protein (ABP-280) interaction in maintaining adhesion under high shear.
J Biol Chem.
1999;274:6097-6106[Abstract/Free Full Text].
32.
Blikstad I, Markey F, Carlsson L, Persson T, Lindberg U.
Selective assay of monomeric and filamentous actin in cell extracts, using inhibition of deoxyribonuclease I.
Cell.
1978;15:935-943[Medline]
[Order article via Infotrieve].
33.
Fox JE, Dockter ME, Phillips DR.
An improved method for determining the actin filament content of nonmuscle cells by the DNase I inhibition assay.
Anal Biochem.
1981;117:170-177[Medline]
[Order article via Infotrieve].
34.
Spector I, Shochet NR, Blasberger D, Kashman Y.
Latrunculins novel marine macrolides that disrupt microfilament organization and affect cell growth, I: comparison with cytochalasin D.
Cell Motil Cytoskeleton.
1989;13:127-144[Medline]
[Order article via Infotrieve].
35.
Bennett JS, Zigmond S, Vilaire G, Cunningham ME, Bednar B.
The platelet cytoskeleton regulates the affinity of the integrin alpha(IIb)beta(3) for fibrinogen.
J Biol Chem.
1999;274:25301-25307[Abstract/Free Full Text].
36.
Dong JF, Hyun W, Lopez JA.
Aggregation of mammalian cells expressing the platelet glycoprotein (GP) Ib-IX complex and the requirement for tyrosine sulfation of GP Ib alpha.
Blood.
1995;86:4175-4183[Abstract/Free Full Text].
37.
Goto S, Sakai H, Goto M, et al.
Enhanced shear-induced platelet aggregation in acute myocardial infarction.
Circulation.
1999;99:608-613[Abstract/Free Full Text].
38.
Cunningham JG, Meyer SC, Fox JE.
The cytoplasmic domain of the alpha-subunit of glycoprotein (GP) Ib mediates attachment of the entire GP Ib-IX complex to the cytoskeleton and regulates von Willebrand factor-induced changes in cell morphology.
J Biol Chem.
1996;271:11581-11587[Abstract/Free Full Text].
39. Meyer SC, Lowry BR, Fox JEB. Role of the cytoplasmic domain of GPIb-IX
in regulating adhesion-induced cytoskeletal reorganizations
[abstract]. Thromb Haemost Suppl. 1997:167.
40.
Hughes PE, Pfaff M.
Integrin affinity modulation.
Trends Cell Biol.
1998;8:359-364[Medline]
[Order article via Infotrieve].
41.
Sullam PM, Hyun WC, Szollosi J, Dong J, Foss WM, Lopez JA.
Physical proximity and functional interplay of the glycoprotein Ib-IX-V complex and the Fc receptor FcgammaRIIA on the platelet plasma membrane.
J Biol Chem.
1998;273:5331-5336[Abstract/Free Full Text].
42.
Torti M, Bertoni A, Canobbio I, Sinigaglia F, Lapetina EG, Balduini C.
Rap1B and Rap2B translocation to the cytoskeleton by von Willebrand factor involves FcgammaII receptor-mediated protein tyrosine phosphorylation.
J Biol Chem.
1999;274:13690-13697[Abstract/Free Full Text].
43. Cauwenberghs N, Vauterin S, Schlammendinger A, Tornai I, Girma JP.
Anti-GPIb and anti-vWf monoclonal antibodies activate platelets by
crosslinking GPIb with FcgammaRII [abstract]. Thromb Haemost Suppl.
1999:46.
44. Falati S, Edmead C, Poole A. Glycoprotein Ib-V-IX couples to the Fc
receptor gamma chain, Fyn and Lyn, to activate human platelets
[abstract]. Thromb Haemost Suppl. 1999:208.
45.
Quilliam AL, Osman N, McKenzie IF, Hogarth PM.
Biochemical characterization of murine Fc gamma RI.
Immunology.
1993;78:358-363[Medline]
[Order article via Infotrieve].
46.
Du X, Fox JE, Pei S.
Identification of a binding sequence for the 14-3-3 protein within the cytoplasmic domain of the adhesion receptor, platelet glycoprotein Ib alpha.
J Biol Chem.
1996;271:7362-7367[Abstract/Free Full Text].
47.
Andrews RK, Harris SJ, McNally T, Berndt MC.
Binding of purified 14-3-3 zeta signaling protein to discrete amino acid sequences within the cytoplasmic domain of the platelet membrane glycoprotein Ib-IX-V complex.
Biochemistry.
1998;37:638-647[Medline]
[Order article via Infotrieve].
48.
Calverley DC, Kavanagh TJ, Roth GJ.
Human signaling protein 14-3-3zeta interacts with platelet glycoprotein Ib subunits Ibalpha and Ibbeta.
Blood.
1998;91:1295-1303[Abstract/Free Full Text].
49.
Fox JE, Berndt MC.
Cyclic AMP-dependent phosphorylation of glycoprotein Ib inhibits collagen-induced polymerization of actin in platelets.
J Biol Chem.
1989;264:9520-9526[Abstract/Free Full Text].
50.
Budde U, Scharf RE, Franke P, Hartmann-Budde K, Dent J, Ruggeri ZM.
Elevated platelet count as a cause of abnormal von Willebrand factor multimer distribution in plasma.
Blood.
1993;82:1749-1757[Abstract/Free Full Text].
51.
Williams SB, McKeown LP, Krutzsch H, Hansmann K, Gralnick HR.
Purification and characterization of human platelet von Willebrand factor.
Br J Haematol.
1994;88:582-591[Medline]
[Order article via Infotrieve].
52.
Sporn LA, Marder VJ, Wagner DD.
Inducible secretion of large, biologically potent von Willebrand factor multimers.
Cell.
1986;46:185-190[Medline]
[Order article via Infotrieve].
53.
Tsai HM.
Physiologic cleavage of von Willebrand factor by a plasma protease is dependent on its conformation and requires calcium ion.
Blood.
1996;87:4235-4244[Abstract/Free Full Text].
54.
Furlan M, Robles R, Lamie B.
Partial purification and characterization of a protease from human plasma cleaving von Willebrand factor to fragments produced by in vivo proteolysis.
Blood.
1996;87:4223-4234[Abstract/Free Full Text].
55.
Furlan M, Robles R, Galbusera M, et al.
von Willebrand factor-cleaving protease in thrombotic thrombocytopenic purpura and the hemolytic-uremic syndrome.
N Engl J Med.
1998;339:1578-1584[Abstract/Free Full Text].
56.
Tsai HM, Lian EC.
Antibodies to von Willebrand factor-cleaving protease in acute thrombotic thrombocytopenic purpura.
N Engl J Med.
1998;339:1585-1594[Abstract/Free Full Text].
57.
Miller JL, Castella A.
Platelet-type von Willebrand's disease: characterization of a new bleeding disorder.
Blood.
1982;60:790-794[Abstract/Free Full Text].
58.
Lub M, van Kooyk Y, van Vliet SJ, Figdor CG.
Dual role of the actin cytoskeleton in regulating cell adhesion mediated by the integrin lymphocyte function-associated molecule-1.
Mol Biol Cell.
1997;8:341-351[Abstract].
59.
van Kooyk Y, van Vliet SJ, Figdor CG.
The actin cytoskeleton regulates LFA-1 ligand binding through avidity rather than affinity changes.
J Biol Chem.
1999;274:26869-26877[Abstract/Free Full Text].
60.
Kovacsovics TJ, Hartwig JH.
Thrombin-induced GPIb-IX centralization on the platelet surface requires actin assembly and myosin II activation.
Blood.
1996;87:618-629[Abstract/Free Full Text].
61.
Hourdille P, Heilmann E, Combrie R, Winckler J, Clemetson KJ, Nurden AT.
Thrombin induces a rapid redistribution of glycoprotein Ib-IX complexes within the membrane systems of activated human platelets.
Blood.
1990;76:1503-1513[Abstract/Free Full Text].
62.
Michelson AD, Ellis PA, Barnard MR, Matic GB, Viles AF, Kestin AS.
Downregulation of the platelet surface glycoprotein Ib-IX complex in whole blood stimulated by thrombin, adenosine diphosphate, or an in vivo wound.
Blood.
1991;77:770-779[Abstract/Free Full Text].
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Abstract
Introduction