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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 92 No. 6 (September 15), 1998:
pp. 1852-1858
RAPID COMMUNICATION
Collagen Induces Tyrosine Phosphorylation of Wiskott-Aldrich Syndrome
Protein in Human Platelets
By
Atsushi Oda,
Hans D. Ochs,
Brian J. Druker,
Katsutoshi Ozaki,
Chiaki Watanabe,
Makoto Handa,
Yoshitaka Miyakawa, and
Yasuo Ikeda
From the Division of Hematology, Department of Internal Medicine, and
the Blood Center, Keio University, Tokyo, Japan; the Department of
Pediatrics, University of Washington, School of Medicine, Seattle, WA;
and the Division of Hematology and Medical Oncology, Oregon Health
Sciences University, Portland, OR.
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ABSTRACT |
Wiskott-Aldrich syndrome (WAS) and X-linked thrombocytopenia (XLT)
are caused by mutations of the WAS protein (WASP) gene. All
hematopoietic stem cell-derived lineages, including platelets, express
WASP. Platelets from WAS patients are smaller than their normal
counterparts and defects in platelet aggregation and actin polymerization have been reported. To determine if WASP is important for normal platelet function, we examined its role in signal
transduction. We found that collagen but not thrombopoietin or thrombin
induces a rapid and robust increase in tyrosine phosphorylation of
platelet-associated WASP. Collagen-induced tyrosine phosphorylation of
WASP was inhibited by cytochalasin D and wortmannin, respectively,
suggesting that actin polymerization and phosphatidylinositol 3-kinase
(PI3-kinase) play a role in the induction of tyrosine phosphorylation
of WASP. Binding of glutathion S-transferase (GST)-Grb2 to WASP was
seen in the lysate of resting platelets. The binding was reduced when lysates from collagen-stimulated platelets were incubated with GST-Grb2, suggesting that tyrosine phosphorylation of WASP may directly
or indirectly modulate the adapter function of WASP. Although thrombin-
and thrombopoietin-induced increase in tyrosine phosphorylation of WASP
is negligible or marginal, WASP from thrombin-activated platelets
became incorporated into the Triton X-100-insoluble 10,000g
sedimentable residue in an aggregation-dependent manner, suggesting
that it may have a regulatory role in platelet cytoskeletal processes
during aggregation. Lastly, we found that WASP is cleaved in response
to activation of calpain, a protease that may have a role in
postaggregation signaling processes. Our data suggest that collagen
specifically induces an increase in tyrosine phosphorylation of WASP
and that WASP is involved in signaling during thrombin-induced aggregation by its redistribution to the cytoskeleton and its cleavage
during aggregation.
© 1998 by The American Society of Hematology.
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INTRODUCTION |
MUTATIONS IN THE classic Wiskott-Aldrich
syndrome (WAS) protein (WASP) gene may result in WAS or in a
milder phenotype, X-linked thrombocytopenia (XLT).1-4
Whereas the immune defect in XLT is either mild or absent,
thrombocytopenia and platelets of small size are common features for
both phenotypes, suggesting that WASP may have a unique and essential
role in platelet production and the regulation of platelet function in
the circulation.5-8 WASP is a 64-kD protein expressed
exclusively in hematopoietic cells.9,10 Because WASP does
not have a known catalytic domain, it has been postulated that it may
serve as an adapter protein for other signaling and cytoskeletal
molecules. This possibility has been further supported by the reports
that WASP binds in vitro to several tyrosine kinases (c-Src, Fyn, Tec
family kinases, and cERG), Grb2, p47nck, and phospholipase
C 1. Fyn and p47nck have also been shown to associate
with WASP in vivo. The interaction of WASP with these molecules is
thought to be mediated through the binding of the PXXY motif of WASP to
Src homology (SH) 3 domains of the signaling
molecules.10-16 Many adapter molecules, such as Shc, Grb2,
p47nck, and the 85-kD subunit for phosphatidylinositol
3-kinase (PI3-kinase), are known to become tyrosine phosphorylated and
thus create the binding sites for SH2 or protein tyrosine binding (PTB)
domains of other signaling molecules, contributing to the efficient
assembly of a large signaling complex.17-19 Although WASP
has five tyrosine residues, no inducible tyrosine phosphorylation of
WASP has been documented so far. For example, it was reported that, in
HL60 cells, various types of stimulation, including treatment with interleukin-4 (IL-4) or cross-linking of Fc receptors, failed to
induce tyrosine phosphorylation of WASP.16 Similarly, the cross-linking of B-cell receptors (BCRs) did not seem to induce a
significant increase in tyrosine phosphorylation of WASP,12 although a role for WASP in BCR signaling has been suggested and WASP
has been reported to be associated with BTK, a B-cell-specific tyrosine kinase expressed in B cells. Interestingly, recent studies have suggested that collagen may activate platelets through signal transduction pathways, analogous to BCR signaling in B
cells.20-23 Thus, glycoprotein VI (gpVI), one of the
receptors for collagen on platelets, has been found to be
associated with the subunit of Fc receptor.20,21 The
cross-linking of the subunit of the Fc receptor may trigger
activation of src family kinases and the syk tyrosine kinase, leading
to activation of phospholipase C2, which may, in turn, mediate
hydrolysis of polyphosphoinositides. The essential role of the syk
kinase and the subunit of Fc receptor in collagen-induced
platelet activation were confirmed by studies of platelets from syk
tyrosine kinase-null or Fc receptor-null mice generated by gene
targeting. Platelets, which are deficient in both of the
two molecules, failed to respond to
collagen.23
Given the similarity between BCR signaling in B cells and
collagen-induced signalings in platelets, we postulated that normal platelets, which express WASP and BTK (unpublished
observation), represent a model system to investigate the
role of WASP in signal transduction.
To accomplish this, we examined whether WASP becomes uniquely tyrosine
phosphorylated after treatment with collagen. We found that
collagen-treated but not thrombin- or thrombopoietin-treated platelets
substantially increase tyrosine phosphorylation of WASP. In contrast,
thrombin induces redistribution of WASP into the Triton
X-100-insoluble residue in an aggregation-dependent fashion. Finally,
similar to many proteins incorporated into the Triton X-100-insoluble
residue after platelet aggregation, such as integrin  3, c-Src,
talin, and actin-binding protein, we found that WASP was an endogenous
substrate for calpain, suggesting a role for WASP in postaggregation
events.
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MATERIALS AND METHODS |
Materials.
Prostaglandin E1 (PGE1), cytochalasin D,
wortmannin, calcium ionophore A23187, Arg-Gly-Asp-Ser (RGDS) peptide,
dibucaine, dimethyl sulfoxide (DMSO), aspirin, apyrase (type VIII),
N-2-hydroxyethylpiperazine-N -2-ethanesulfonic acid (HEPES),
sodium dodecyl sulfate (SDS), 2-mercaptoethanol, sodium orthovanadate,
chicken egg albumin, protein A-Sepharose, Triton X-100, and Tris
(hydroxymethyl) aminomethane (Tris) were purchased from Sigma (St
Louis, MO). Polyvinylidene difluoride (PVDF) membranes (pore size, 0.45 mm) were obtained from Millipore Corp (Bedford, MA). SDS-polyacrylamide
gel electrophoresis (SDS-PAGE) molecular standards and enhanced
chemiluminesence (ECL) reagents, including secondary antibodies, were
purchased from Amersham (Arlington Heights, IL). Calpeptin was obtained
from Calbiochem (La Jolla, CA). The antiphosphotyrosine murine
monoclonal antibody (4G10) and the anti-WASP rabbit polyclonal antibody
(503) were described previously.10,24-26 Purified human
thrombin was generously provided by Green Cross Co Ltd (Osaka, Japan).
Agarose conjugated-glutathion S-transferase (GST), -GST-Grb2,
and -GST-Grb2-SH2 domain were purchased from Santa Cruz (Santa Cruz,
CA). Recombinant thrombopoietin was a generous gift from Kirin Brewery
Co Ltd (Maebashi, Japan). Horse tendon collagen (type I) was purchased
from Hormon Chemie (Munich, Germany).
Platelet preparation.
Human blood from healthy volunteers was drawn by venipuncture into 1/10
vol of 3.8% (wt/vol) trisodium citrate and gently mixed. Platelet-rich
plasma (PRP) was prepared by centrifugation of whole blood at
200g for 20 minutes. PRP was aspirated and incubated with
aspirin (2 mmol/L) for 30 minutes at room temperature. After the
addition of PGE1 (1 µmol/L) from a stock solution in
absolute ethanol (1 mmol/L), the PRP was spun at 800g to form a
soft platelet pellet. The pellet was resuspended in 1 mL of a modified
HEPES-Tyrode buffer (129 mmol/L NaCl, 8.9 mmol/L NaHCO3,
0.8 mmol/L KH2PO4, 0.8 mmol/L
MgCl2, 5.6 mmol/L dextrose, and 10 mmol/L HEPES, pH 7.4)
also containing apyrase (2 U/mL) and washed twice. Platelets were
resuspended at a concentration of 3 × 108 cells/mL in
the same buffer containing apyrase (2 U/mL) at 37°C. RGDS peptide
(200 µmol/L) was added whenever it was necessary to inhibit platelet
aggregation.
Gel electrophoresis and Western blotting to detect tyrosine
phosphorylated proteins or WASP.
Platelet stimulation was terminated by the addition of an equal volume
of 2× concentrated Laemmli's sample buffer (10% glycerol, 1%
SDS, 5% 2-mercaptoethanol, 50 mmol/L Tris-HCl [pH 6.8], and 0.002%
bromophenol blue), 10 mmol/L EGTA, and 1 mmol/L sodium orthovanadate. After boiling at 95°C for 5 minutes, one-dimensional SDS-electrophoresis was performed on 10% or 7.5% to 15%
polyacrylamide gels.24-26 Separated proteins were
electrophoretically transferred from the gel onto PVDF membranes in a
buffer containing Tris (25 mmol/L), glycine (192 mmol/L), and 20%
methanol at 0.2 amps for 12 hours at room temperature. To block
residual protein binding sites, membranes were incubated in TBST
(Tris-buffered saline [TBS]: 10 mmol/L Tris, 150 mmol/L NaCl, pH 7.6 with 0.1% Tween 20) with 10% chicken egg albumin. The blots were
washed with TBST and incubated overnight with primary antibodies at a
final concentration of 1.0 µg/mL for antibody 4G10 and 1:1,000
dilution for antibody 503 in TBST. The primary antibody was removed and
the blots were washed four times in TBST and incubated with horse
radish peroxidase-conjugated second antibody diluted 1:3,000 in TBST.
Blots were then washed four times in TBST. Antibody reactions were
detected with chemiluminescence according to the manufacturer's
instructions.
Immunoprecipitation.
Platelet stimulation was terminated by the addition of an equal amount
of lysis buffer (15 mmol/L HEPES, 150 mmol/L NaCl, 1 mmol/L
phenylmethyl sulfonyl fluoride [PMSF], 10 mmol/L EGTA, 1 mmol/L sodium orthovanadate, 0.8 µg/mL leupeptin, 2% Triton X-100
[vol/wt], pH 7.4). After 20 minutes on ice, the lysates were
centrifuged at 10,000g (at 4°C) for 20 minutes. The
supernatant was removed, incubated with protein A-Sepharose (40 µL of
50% slurry) for 1 hour, and centrifuged to obtain the precleared
supernatant. The anti-WASP polyclonal antibody 503 was then added and
the mixtures were incubated for 2 to 3 hours on ice (concentration, 2 µL/mL supernatant). Protein A-Sepharose (40 µL of 50% slurry/mL
supernatant) was added and incubated for several hours. The immune
complexes were washed with 1 mL of cold washing buffer (the same as the lysis buffer except the concentration of Triton X-100 [vol/wt] was
1%) three times and then resuspended in Laemmli's sample buffer.
Isolation of platelet cytoskeleton.
The Triton X-100-insoluble cytoskeleton was isolated as
described,25,26 with the following
modification. An equal amount of lysis buffer was added to
platelet suspensions to solubilize platelets. After 5 minutes on ice,
the lysates were centrifuged at 10,000g. The resulting pellet
was washed twice in washing buffer. For one-dimensional SDS
electrophoresis, the Triton X-100-insoluble residue was solubilized in
SDS sample buffer. The supernatant was diluted with an equal volume of
2× concentrated SDS sample buffer.
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RESULTS |
Collagen-induced tyrosine phophorylation of WASP.
Platelets suspended in the presence of 200 µmol/L RGDS peptide were
treated with either collagen (50 µg/mL), thrombin (1 U/mL), or
thrombopoietin (100 ng/mL). After incubation for various times, the
platelets were lysed and the lysates were incubated with anti-WASP antiserum 503 and protein A Sepharose. The same amount of
a 64-kD protein recognized by the anti-WASP antiserum was
immunoprecipitated from all cellular lysates in each set of experimemts
(Fig 1A through D, lower panels). After
treatment with collagen, WASP was increasingly tyrosine phosphorylated
over the low basal level (Fig 1A, upper panel). Tyrosine
phosphorylation reached a plateau within 2 minutes. In contrast,
thrombin and thrombopoietin induced a marginal increase in tyrosine
phosphorylation of WASP in platelets (Fig 1B and C), but the degree of
phosphorylation by either of the reagents was much smaller than that
induced by collagen. When platelet suspensions were stirred in the
absence of RGDS peptide, thrombin-induced increase in tyrosine
phosphorylation of WASP was still minimal (Fig 1D).
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| Fig 1.
(A through D, upper panels) Tyrosine phosphorylation of
WASP in platelets stimulated by collagen (50 µg/mL), thrombopoietin
(TPO; 100 ng/mL), or thrombin (1 U/mL) at the time intervals indicated.
Platelets were lysed by the addition of an equal amount of a buffer
containing 2% Triton X-100 before and after exposure to collagen, TPO,
or thrombin. To prevent platelet aggregation, platelets were treated
with RGDS (200 µmol/L), except in lanes 1 through 3 in (D). For
experiments shown in (D), we used lysis buffer containing 0.2% SDS and
sonicated the lysates. WASP was immunoprecipitated with the polyclonal
anti-WASP antiserum (503). Immune complexes were resuspended in SDS
sample buffer and divided into two. Proteins were separated by 10%
SDS-PAGE and transferred onto PVDF membranes. One immunoblot was probed
with antiphosphotyrosine antibodies and bands were visualized by
chemiluminescence (upper panels, A through D). The other blot was
probed for WASP (lower panels, A through D).
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Collagen-induced tyrosine phosphorylation was inhibited by
cytochaslsin D and wortmannin.
Because of recent reports suggesting a role of WASP in the
initiation of actin polymerization in platelets27 as well
as in T cells28 and because of evidence that PI3-kinase is
also involved in the regulation of cytoskeletal reorganization of
platelets,29,30 we examined the effects of cytochalasin D
(an inhibitor of actin polymerization) and wortmannin (a PI3-kinase
inhibitor) on WASP tyrosine phosphorylation. Platelets were treated
with 0.1% DMSO (the vehicle for cytochalasin D and wortmannin) for 30 minutes, cytochalasin D (10 µmol/L) for 10 minutes, or wortmannin (50 nmol/L) for 30 minutes. Cytochalasin D or wortmannin but not DMSO
inhibited the collagen-induced increase in tyrosine phosphorylation of
WASP (Fig 2).
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| Fig 2.
Collagen-induced tyrosine phosphorylation of WASP is
inhibited by cytochalasin D or wortmannin. Platelet suspensions were
incubated in the presence of RGDS peptide (200 µmol/L) for 5 minutes.
After treatment with DMSO (0.1%) for 30 minutes, cytochalasin D (10 µmol/L) for 10 minutes, or wortmannin (50 nmol/L) for 30 minutes,
collagen (50 µg/mL) was added to the suspension for 5 minutes as
indicated (lanes 2 through 4). Platelets were lysed by the addition of
an equal amount of a buffer containing 2% Triton X-100 before
(resting, lane 1) and after the addition of collagen (lanes 2 through
4). Tyrosine phosphorylation of WASP was detected as described in Fig
1.
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WASP associates with the cytoskeleton after platelet aggregation.
We next examined the possibility that WASP plays a role in signaling
after platelet aggregation. Because it has been shown that many
signaling molecules, including c-Src, become incorporated into the
integrin-rich cytoskeleton after platelet
aggregation,25,26,31-33 we tested the possibility that WASP
may be incorporated into the cytoskeleton. In resting platelets, WASP
was found predominantly in Triton X-100-soluble fraction
(Fig 3). However, after stimulation of
platelets by adding thrombin and stirring to induce platelet aggregation, WASP became associated with the Triton X-100-insoluble residue. This association of WASP with the cytoskeleton was inhibited by omitting stirring to minimize platelet aggregation (lane 6, Fig 3),
as has been reported for c-Src, Vav, Crkl, Grb2, c-Cbl, and
 IIb3.25,26,31-33 Because activation of calpain is
known to accompany platelet aggregation,33-37 we examined
whether WASP is a substrate for calpain and, if so, is cleaved during
platelet aggregation. Activation of µ-calpain is known to be
accompanied by cleavage of the enzyme, although it is unclear whether
cleavage is necessary for activation.33,34 When platelets
were treated with calcium ionophore A23187 or dibucaine, both known to
induce calpain activation in platelets in the presence of
extracellular calcium, we observed a significant loss of immunoreactive
WASP and the generation of bands (Fig 4A,
indicated by the arrows) of lower molecular weights reactive with
anti-WASP (lane 2 and 5, Fig 4A). Inhibition of calpain activation by
adding EGTA or calpeptin resulted in an inhibition of cleavage of WASP
(lanes 3, 4, and 7, Fig 4A). Activation of calpain by dibucaine does not require extracellular calcium, whereas A23187 does. As
expected, WASP cleavage induced by dibucaine was not inhibited by EGTA
(lane 6, Fig 4A), but A23187-induced cleavage was (lane 3, Fig 4A). Thus, only under conditions in which calpain is activated was WASP
cleaved. These data suggest that WASP may be a substrate for calpain,
although we cannot rule out the possibility that activation of calpain
may result in activation of other proteases that are responsible for
the cleavage of WASP. When platelets were activated with thrombin and
simultaneously stirred to induce platelet aggregation, a significant
loss of WASP immunoreactivity was also observed (Fig 4B), resulting in
the appearance of the same set of cleaved bands as observed in Fig 4A
(compare lanes 1 and 2, Fig 4B). Omitting stirring to minimize
aggregation resulted in an inhibition of the loss of immunoreactive
WASP (lane 3, Fig 4B). Because activation of calpain could occur during
lysis of platelets, these experiments were performed by the addition of EGTA and EDTA immediately before lysis of platelets to prevent artificial cleavage.38
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| Fig 3.
Association of WASP with the Triton X-100-insoluble
residue. Platelets were lysed with Triton X-100-EGTA buffer before or
after stimulation with thrombin (1 U/mL), with or without stirring.
Lysates were separated by high speed centrifugation into soluble and
insoluble residues. Proteins from each fraction were separated by 10%
SDS-PAGE and immunoblotted with anti-WASP antiserum 503. Lane 1, Triton
X-100-soluble residue of resting cells (7.5 × 106
cells). Lane 2, Triton X-100-insoluble residue of resting cells (3.0 × 107 cells). Lanes 3 through 5, Triton X-100-insoluble
residue from 3.0 × 107 cells, prepared 15 seconds, 1 minute, and 5 minutes after exposure to thrombin (1 U/mL) with
stirring, respectively. Lane 6, Triton X-100-insoluble residue of
cells (3.0 × 107 cells) stimulated for 5 minutes with
thrombin but without stirring.
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| Fig 4.
Cleavage of WASP. (A) Platelets were incubated with
either calpeptin (20 µmol/L) or DMSO (vehicle of calpeptin; final
concentration, 0.1%) for 5 minutes. Platelets were then
treated with calcium ionophore A23187 (1 µmol/L for 5 minutes) or
dibucaine (1 mmol/L for 15 minutes) in the presence of 1 mmol/L
CaCl2 or 5 mmol/L EGTA. Whole platelet lysates (1.5 × 107 cells/lane) were analyzed by 10% SDS-PAGE. Separated
proteins were electrophoretically transferred from the gel onto
nitrocellulose membranes. WASP was detected by immunoblotting with
polyclonal antibody 503. The top arrow indicates the relative position
of the intact 64-kD subunit of WASP. The lower arrows indicate the
positions of apparently cleaved products of WASP. Lane 1, control, DMSO + CaCl2; lane 2, A23187 + CaCl2; lane 3, A23187 + EGTA; lane 4, A23187 + CaCl2 + calpeptin ; lane 5, dibucaine + CaCl2; lane 6, dibucaine + EGTA; lane 7, dibucaine + CaCl2 + calpeptin. (B)
Cleavage of WASP during platelet aggregation. Platelets were stimulated
with thrombin (1 U/mL) for 30 minutes with or without stirring. After
the addition of EGTA (5 mmol/L) and EDTA (5 mmol/L), platelets were
lysed by boiling in SDS sample buffer. WASP was detected by
immunoblotting as described in Fig 1. Lane 1, resting platelets; lane
2, thrombin stimulation of platelets for 30 minutes with stirring; lane
3, thrombin stimulation for 30 minutes without stirring. The arrows
indicate the same cleaved fragments of WASP as in (A).
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Binding of GST-Grb2 but not GST to WASP.
Because WASP is thought to function as an adapter protein in
lymphocytes, we next asked whether WASP in platelets may also bind to
other proteins and, if so, whether collagen-induced stimulation may
affect the adapter function of WASP. Because it is impossible to
examine all SH2/SH3 proteins expressed in platelets for binding to
WASP,25,26,31-33 we focused our attention on Grb2, which is known to be constitutively associated with WASP in
lymphocytes.10,12 Platelet lysates obtained before and 5 minutes after collagen stimulation were equally divided for
immunoprecipitation with anti-WASP antiserum or for GST-fusion
protein-binding studies. WASP was immunoprecipitated by WASP antiserum
503 but not by the preimmune serum from both lysates equally well (data
not shown), confirming that the lysates have the same amount of WASP.
As expected, tyrosine phosphorylation of the 64-kD protein in platelets
was dramatically increased after collagen stimulation (data not shown). The 64-kD tyrosine phosphorylated protein was not precipitated by
preimmune serum. GST-Grb2 but not GST on agarose precipitated WASP from
the same lysates (Fig 5). The amount of
WASP precipitated by GST-Grb2 on agarose was significantly reduced when
lysates from collagen-stimulated platelets were used (Fig 5, lane 2).
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| Fig 5.
Binding of GST-Grb2 but not GST to WASP. Platelets were
stimulated in nonaggregating conditions and lysed as described for Fig
1. Lysates before ( ) and 5 minutes after (+) collagen stimulation
were equally divided into four aliquots. The two samples from each of
the lysates were incubated either with agarose-bound GST-Grb2 (10 µg,
lanes 1 and 2) or GST (10 µg, lanes 3 and 4) on ice for 3 hours.
After three washes in phosphate-buffered saline (150 mmol/L, pH 7.4)
containing orthovanadate (200 µmol/L), the precipitates were
incubated in SDS sample buffer. The SDS samples were subjected to
Western blotting to detect WASP by polyclonal anti-WASP antiserum
503.
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DISCUSSION |
Although platelet abnormalities are a hallmark of WAS, the role of WASP
in platelet release and function is not
understood.5-8,27,28 Because of reports that, in other
cells, WASP may serve as an adapter protein for other molecules
involved in signaling or cytoskeletal reorganization, we explored
signal transduction in human platelets and found that collagen, a
natural agonist for platelets, is a strong inducer of tyrosine
phosphorylation of WASP. Because WASP is believed to be an adapter-like
molecule and protein tyrosine phosphorylation is essential for
collagen-induced platelet activation,20-23 it is tempting
to speculate that the induction of tyrosine phosphorylation of WASP is
generating the potential binding site(s) for proteins containing SH2
and/or PTB domains, thus contributing to collagen-induced signaling in platelets. The robust induction of tyrosine
phosphorylation of WASP seems to be a unique response to
collagen-induced stimulation, because exposure to thrombin or
thrombopoietin resulted in only a small increase in tyrosine
phosphorylation of WASP. The observation that tyrosine phosphorylation
of WASP during thrombin-induced platelet aggregation was also marginal
may be misleading, being caused by the artificial dephosphorylation
during the lysis of platelet aggregates, as has been reported by Law et
al39 for integrin 3. Thus, it is possible that the
increase in tyrosine phosphorylation of WASP has been underestimated by
the current study. The observation that both cytochalasin D and
wortmannin inhibited collagen-induced tyrosine phosphorylation of WASP
suggests that the kinase(s) responsible for WASP phosphorylation may
require both actin polymerization and activation of PI-3 kinase.
Candidates for such kinases in platelets include Fak, RAFTK, and Tec.
However, these kinases are not exclusively expressed in
platelets.31,32,40,41 Thus, platelets may have unique
mechanisms that enable these kinases to phosphorylate WASP or they may
express unique and unknown platelet-specific WASP kinase(s).
WASP may also play a role during platelet aggregation, as evidenced by
the redistribution of WASP to the cytoskeleton after thrombin-stimulated platelet aggregation. This is similar to what has
been described for Cdc42, c-Src, Fyn, c-Cbl, Vav, Crkl, and IIb3
integrin.25,26,31-33 The carboxyl terminal portion of WASP
contains regions that show homology to several actin-binding proteins,
such as verprolin and cofilin, which may allow binding of WASP to
filamentous actin.1,2 This is also consistent with the
proposed role for WASP in the regulation of actin polymerization through interaction with Cdc42 and/or WIP.42-44 On
the other hand, we prefer to interpret our data to suggest that
tyrosine phosphorylation of WASP acts downstream of actin
polymerization. Thus, WASP may not simply regulate actin polymerization
in platelets, but the function of WASP, in turn, could be modulated by
polymerization of actin. WASP was shown to be associated with Grb2 in
studies of other hematopoietic cells.9-16 Because the
experiments reported here have demonstrated that platelet WASP is
associated in vitro with Grb2, WASP and Grb2 may recruit more signaling
molecules into the cytoskeleton. This hypothesis is also supported by
our previous finding that Grb2 redistributes to the platelet
cytoskeleton in an aggregation-dependent fashion.45 Such
redistribution could be essential for platelet aggregation, because WAS
platelets show defective aggregation in response not just to collagen,
but also to other agonists such as thrombin or ADP.8,46
Binding of Grb2 to WASP appreared to be reduced after stimulation of
platelets by collagen.
In other cellular elements studied, WASP was not significantly tyrosine
phosphorylated after stimulation of the cells, although WASP was found
to be constitutively associated with Grb2.10-16 Because
induction of tyrosine phosphorylation seems to be unique to
collagen-stimulated platelets, it is attractive to suggest that
tyrosine phosphorylation of WASP may directly or indirectly reduce
avidity or accessibility of Grb2 to bind WASP. However, because we have
not determined the stoichiometry of tyrosine phosphorylation of WASP in
collagen-stimulated platelets, we cannot rule our the possibility that
Grb2 binding to WASP is reduced by a mechanism that is unrelated to
WASP tyrosine phosphorylation.
The finding that WASP is an endogenous substrate for calpain and that
it is cleaved during aggregation is intriguing in view of the recent
report that the integrin 3 subunit is also cleaved during platelet
aggregation.36 It appears possible that WASP, 3, and
calpain colocalize during platelet aggregation and that WASP and 3
are cleaved by the same protease. The hypothesis is in aggreement with
the findings by Remold-O'Donnell et al47 suggesting that
the protein defective in WAS is related to the regulation of calpain
and that inappropriate activation of calpain in platelets and its
release may be responsible for the consistent loss of CD43 and other
glycoproteins on the surface of T lymphocytes. They further argue that,
as a consequence, lymphocytes, especially T lymphocytes, lose
functionally important cell surface molecules and surface-associated
receptors, contributing to the immunodeficiency observed in WAS
patients.47 Although the degree of cleavage of WASP in
aggregated platelets seems to be small, this is generally true for most
substrates for calpain in platelets.33 For example, most of
the talin or actin binding protein that is cleaved in calcium
ionophore- or dibucaine-treated platelets in the presence of
extracellular ionic calcium shows cleavage that is less than 10% of
total. It has been suggested that this small degree of cleavage may
play a significant role in the shedding of procoagulant microparticles
from platelets or in the relaxation of fibrin-platelet clots.33,48
Although we have shown that tyrosine phosphorylation of WASP may
modulate its binding to Grb2 in vitro, it is imperative to search for
the physiological ligands for WASP associated phosphotyrosines. Furthermore, it is also essential to examine the effects of tyrosine phosphorylation of WASP on the binding of other SH3-containing proteins, including PSTPIP, a recently characterized ligand for WASP.49 The unique and robust tyrosine phosphorylation of
WASP observed in collagen-stimulated platelets makes them an attractive system to further analyze the physiologic functions of WASP. This becomes even more attractive in view of the fact that platelets contain
many SH2/SH3 proteins that are potential candidates for WASP
ligands.25,26,31-33,45 Finally, our data suggest that WASP may also play a role in aggregation events, based on our observation that WASP redistributes to the cytoskeleton during platelet aggregation and that it is an endogenous substrate for calpain.
| |
FOOTNOTES |
Submitted April 3, 1998;
accepted June 26, 1998.
Supported in part by grants from The Ministry of Education, Science,
Technology and Sport of Japan (Y.I. and A.O.), The Ryoichi Naito
Foundation for Medical Research (A.O.), Research Grants for Life
Sciences and Medicine, Keio University Medical Science Fund (A.O.), the
National Institutes of Health (HD17427; H.D.O.), and the DeJoria
Wiskott-Aldrich Research Fund (H.D.O.).
Address reprint requests to Hans D. Ochs, MD, Professor, Department of
Pediatrics, RD-20, 1959 NE Pacific St, Box 356320, University of
Washington Medical School, Seattle, WA 98195.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
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Abstract
Introduction
Abstract