(Received for publication, August 7, 1995)
From the
Platelet glycoprotein (GP) VI is a so-far uncharacterized 62-kDa
membrane protein, whose deficiency results in selective impairment in
collagen-induced platelet aggregation. Our group previously reported a
human polyclonal antibody (anti-p62 IgG) that induces activation of
normal, but not of GPVI-deficient, platelets in an Fc-independent
manner. The F(ab`) fragments of this antibody
(F(ab`)
-anti-p62) stimulated tyrosine phosphorylation of
numerous proteins, which was not prevented even in the presence of
cAMP-increasing agents such as prostacyclin. Pretreatment of platelets
with the protein-tyrosine kinase (PTK) inhibitor tyrphostin A47
completely abolished F(ab`)
-anti-p62-induced platelet
aggregation in parallel with dose-dependent inhibition of
protein-tyrosine phosphorylation, indicating an essential requirement
of PTK activity for generating GPVI-mediated signaling. We found that
two cytosolic PTKs, c-Src and Syk, became rapidly activated in response
to F(ab`)
-anti-p62 in a way insensitive to elevation of
cAMP. In contrast, in the presence of prostacyclin,
F(ab`)
-anti-p62 did not stimulate tyrosine phosphorylation
of the focal adhesion kinase. cAMP-insensitive activation of c-Src and
Syk was also observed in collagen- but not thrombin-stimulated
platelets. Moreover, either F(ab`)
-anti-p62 or collagen
stimulated cAMP-insensitive tyrosine phosphorylation of phospholipase
C-
2. These results indicate that the receptor-mediated activation
of several PTKs in platelets is regulated through a cAMP-sensitive or
-insensitive mechanism depending on the nature of each stimulus, and
also suggest that GPVI engagement is coupled to cAMP-insensitive
activation of c-Src and Syk accompanied by tyrosine phosphorylation of
numerous substrates including phospholipase C-
2 in a manner
similar to collagen stimulation.
Investigations of platelet membrane glycoproteins have unveiled
a great deal about how the interaction between cell surface protein
molecules and their specific ligands harmoniously regulates diverse
platelet functions, such as adhesion and aggregation. Although the
structures and functions of several major glycoproteins on platelets
have been steadily clarified, the molecular characterization of
platelet glycoprotein (GP) ()VI, a 62-kDa membrane protein
that was originally described in the pioneer studies by Phillips and
Agin(1, 2) , remains a great enigma. A breakthrough in
the elucidation of this molecule was a clinical report on a platelet
activating antibody (anti-p62 IgG) found in a patient with autoimmune
thrombocytopenia whose platelets showed selective deficiency in
collagen-induced platelet aggregation(3) . This antibody
immunoprecipitated a 62-kDa/57-kDa (reducing/nonreducing) membrane
protein from normal platelets but did not significantly react with
membrane proteins of the patient's platelets. The 62-kDa protein
recognized by this antibody was later identified as GPVI through the
analysis of platelets from a patient with familial
GPVI-deficiency(4) , and there have since been two other
reports on patients with GPVI-deficiency(5, 6) .
Platelets from these patients were not reactive with anti-p62 IgG and
also showed defective responses only to collagen despite the normal
expression of GPIa-IIa (integrin
), a
primary platelet adhesion receptor for
collagen(7, 8) . Although these clinical findings have
suggested a potential role of GPVI in collagen-induced platelet
activation(9, 10) , very little is known about the
mechanism for signaling through GPVI.
Like other platelet agonists,
collagen stimulates an increase in intracellular Ca,
phosphoinositide metabolism, activation of protein kinase C, and
accumulation of phosphatidic acid (11, 12, 13) . However, recent studies
pointed out that collagen-induced signal transduction is unique
compared to other stimuli; while elevation of cyclic AMP is thought to
prevent many aspects of platelet activating signal transduction, it
does not inhibit several features of collagen-induced signaling events.
This cAMP-insensitive signaling stimulated by collagen includes
intracellular Ca
mobilization, phosphatidic acid
formation, and protein-tyrosine
phosphorylation(14, 15) . Among these, a key signaling
event insensitive to cAMP appears to be protein-tyrosine
phosphorylation(16) , because the recent reports revealed that
collagen stimulates tyrosine phosphorylation of phospholipase C
(PLC)-
2 which could lead to cytosolic Ca
mobilization and phosphatidic acid
formation(17, 18) .
Platelets have been known to
possess a number of nonreceptor protein-tyrosine kinases (PTKs)
including five Src family kinases (c-Src, Fyn, Yes, two variants of
Lyn, and Hck)(19, 20) , focal adhesion kinase
(FAK)(21) , and Syk(22, 23) . Thrombin
stimulation affects four of five Src family kinases. c-Src is
enzymatically activated(24, 25, 26) ; c-Src
and Fyn form a signaling complex with phosphatidylinositol
3-kinase(27) ; Fyn, Lyn, and Yes become associated with the
GTPase-activating protein of p21(28) .
Although all of these events occur independently of platelet
aggregation mediated by fibrinogen binding to GPIIb-IIIa (integrin
), redistribution of c-Src, Fyn,
Lyn, and Yes into the Triton-insoluble ``cytoskeleton''
fraction takes place in an aggregation-dependent
manner(26, 29) . In addition, activation of FAK which
does not belong to the Src family is believed to be brought about by
fibrinogen binding or adherence to GPIIb-IIIa and to be involved in the
relatively late phase of protein-tyrosine phosphorylation in activation
of platelets(21, 30) .
Another cytosolic PTK that
is also implicated in platelet activation by a variety of platelet
agonists is Syk, a member of the Syk/ZAP-70
family(23, 31) . Unlike the Src family PTKs, Syk
possesses two tandemly arranged SH2 (src homology 2) domains, which are
considered essential for the association of Syk with its target
molecules that contain a cytoplasmic consensus motif called a
tyrosine-based activation motif (32) . It has been reported
that cross-linking of multiple glycoproteins by wheat germ
agglutinin(21) , ligation of GPIIb-IIIa with disintegrin or
fibrinogen(33) , and clustering of the low affinity Fc
receptors (FcRIIA) (34) lead to tyrosine phosphorylation
and activation of Syk. In addition, antibody-mediated CD9 clustering
has been shown to induce activation of Syk which becomes associated
with c-Src(35) .
With these backgrounds in mind, it was of
great interest to us to clarify the nature of GPVI-mediated signaling
with special reference to protein-tyrosine phosphorylation and
activation of PTKs, since specific stimulation of GPVI can be achieved
by use of the F(ab`) fragments of anti-p62 IgG
(F(ab`)
-anti-p62). Surprisingly, stimulation of GPVI with
F(ab`)
-anti-p62 was found to induce cAMP-insensitive
tyrosine phosphorylation of numerous substrates including PLC-
2,
as is the case with collagen stimulation. In this paper, we report the
unique nature of F(ab`)
-anti-p62-induced activation of PTKs
through GPVI and its comparison with collagen stimulation.
Figure 1:
Radioimmunoprecipitation of platelet
GPVI, and the induction of platelet aggregation and protein-tyrosine
phosphorylation by anti-p62 IgG in normal and GPVI-deficient platelets. Panel A,I-labeled platelets from a normal donor (lanes 1 and 2) or a donor with GPVI-deficiency (lanes 3 and 4) were lysed in 1% Triton X-100,
precipitated with 10 µg of anti-p62-IgG (lanes 1 and 3) or control human IgG (lanes 2 and 4),
resolved by 8% SDS-polyacrylamide gel electrophoresis under reducing (left) or nonreducing (right) conditions, and
subjected to autoradiography. The arrows on the left indicate the positions of GPVI. The positions of molecular weight
standards (in kDa) are shown on the right. Panel B, washed platelets from a normal donor (left) or a donor
with GPVI-deficiency (right) were stimulated with 150
µg/ml of F(ab`)
-anti-p62 (
p62) under
stirring conditions. Platelet aggregations were concurrently monitored
and the representative tracings were shown (upper panels).
Platelets were lysed in SDS sample buffer at the times indicated below each lane, resolved by 8% SDS-polyacrylamide gel
electrophoresis, transferred onto nitrocellulose filter, and
immunoblotted with an anti-phosphotyrosine mAb, 4G10 (lower
panels). In each lane, lysate from 5
10
platelets was loaded. The positions of molecular weight standards are
shown on the right.
We first evaluated the profile of
time-dependent protein-tyrosine phosphorylation in
F(ab`)-anti-p62-stimulated platelets. Whole lysates from
resting and F(ab`)
-anti-p62-stimulated platelets (5.0
10
platelets/each lane) were subjected to analysis
on anti-phosphotyrosine immunoblots (Fig. 1B, lower
panels). In normal platelets, F(ab`)
-anti-p62 induced
tyrosine phosphorylation of multiple proteins, which were subdivided
into the following three groups. The first group with the earliest
appearance peaked at 1 min and gradually diminished (bands at 150-,
130-, 72-, and 42-kDa). The second group also appeared early but
declined more slowly (102- and 68-kDa). Coincident with platelet
aggregation, the last group with sustained tyrosine phosphorylation
emerged (125-115-kDa bundle and 105-kDa). In contrast,
F(ab`)
-anti-p62 did not stimulate protein-tyrosine
phosphorylation in the GPVI-deficient platelets.
Since GPIIb-IIIa is
a crucial regulator of protein-tyrosine phosphorylation in
platelets(16) , we analyzed the profile of
F(ab`)-anti-p62-stimulated protein-tyrosine phosphorylation
in platelets treated with RGDS tetrapeptide, which blocks fibrinogen
binding to GPIIb-IIIa, and in Glanzmann's thrombasthenic
platelets. F(ab`)
-anti-p62 induced tyrosine phosphorylation
of numerous proteins as well as ATP secretion in RGDS-treated platelets (Fig. 2, A and C) or in thrombasthenic
platelets (data not shown). In these platelets, however,
dephosphorylation of the tyrosine-phosphorylated proteins was blunted
as was consistent with our previous report on the profiles of thrombin-
or STA
-induced protein-tyrosine phosphorylation (38, 40) . These results indicated that
F(ab`)
-anti-p62 did induce protein-tyrosine phosphorylation
through GPVI engagement independently of fibrinogen binding to
GPIIb-IIIa on platelets.
Figure 2:
Effects of RGDS and PGI on
platelet ATP release, aggregation, and protein-tyrosine phosphorylation
induced by F(ab`)
-anti-p62. Panels A and B, platelet-rich plasma (3.0
10
platelets/ml) was
incubated with 1 mM RGDS tetrapeptide (A) or 3
µM PGI
(B) and stimulated with 150
µg/ml of F(ab`)
-anti-p62 under stirring conditions.
Representative tracings of platelet aggregation and ATP release were
shown. Panels C and D, washed platelets (5.0
10
/ml) were incubated with 1 mM RGDS (C)
or 3 µM PGI
(D) and stimulated with
150 µg/ml of F(ab`)
-anti-p62 under stirring conditions
for the times indicated below each lane. Platelets were lysed
in SDS sample buffer and subjected to anti-phosphotyrosine
immunoblotting as described in the legend to Fig. 1. The
positions of molecular weight standards are shown on the right.
Figure 3:
Effects of tyrphostin A47 and
chelerythrine on F(ab`)-anti-p62-induced platelet
aggregation and protein-tyrosine phosphorylation. Panel A, washed platelets were preincubated with 0.5% (v/v) dimethyl
sulfoxide (DMSO) (a), chelerythrine (b, 20
µM), or tyrphostin A47 (c, 50 µM; d, 200 µM), and stimulated with 150 µg/ml of
F(ab`)
-anti-p62 (
p62) under stirring
conditions. Platelet aggregations were monitored and their
representative tracings were shown. Panel B, washed platelets
were preincubated with 0.5% Me
SO (lanes 1 and 2), tyrphostin A47 (A47) (lanes 3-5, 50, 100, and 200 µM, respectively), or chelerythrine (CLR) (lane 6, 10 µM; lane 7, 20 µM), and unstimulated (lane 1) or
stimulated for 1 min with 150 µg/ml of F(ab`)
-anti-p62
under stirring conditions (lanes 2-7). Platelets were
lysed in SDS sample buffer and subjected to anti-phosphotyrosine
immunoblotting as described in the legend to Fig. 1. The
positions of molecular weight standards are indicated on the right.
Figure 4:
Time course of
F(ab`)-anti-p62-stimulated tyrosine phosphorylation and
activation of Syk in normal, PGI
-treated, and
Glanzmann's thrombasthenic platelets. Panel A, washed
platelets from a normal donor were stirred and stimulated with 150
µg/ml of F(ab`)
-anti-p62 for the times indicated below each lane in the absence (PGI
,
-) or presence (PGI
, +) of 3
µM PGI
. Platelets were lysed in RIPA buffer,
precipitated with a rabbit anti-Syk serum. The immunoprecipitates were
divided into two; one-half was analyzed on anti-phosphotyrosine
immunoblotting (
PY) followed by reprobing with an
anti-Syk mAb (
Syk), and the other half was subjected to
an in vitro kinase assay using histone as an exogenous
substrate (Histone). Panel B, washed platelets from a
donor with Glanzmann's thrombasthenia (GT) were stirred
and stimulated with 150 µg/ml of F(ab`)
-anti-p62 for
the times indicated below each lane. Anti-Syk
immunoprecipitates were prepared and analyzed as described above. In
both A and B, the arrows on the right indicate the positions of Syk.
Previous reports have indicated that
activated Syk became incorporated into the 1% Triton X-100-insoluble
cytoskeleton fraction in thrombin-stimulated
platelets(31, 39) . Since F(ab`)-anti-p62
elicited cAMP-insensitive activation of Syk, we examined if the
elevation of cAMP would affect subcellular redistribution of Syk in
F(ab`)
-anti-p62-stimulated platelets. The Triton
X-100-soluble and -insoluble fractions were prepared from resting or
F(ab`)
-anti-p62-activated platelets, and subcellular
distribution of Syk in these two fractions was analyzed on anti-Syk
immunoblots (Fig. 5A). In the absence of
PGI
, increasing amounts of Syk became relocalized to the
Triton-insoluble cytoskeleton fraction in a time-dependent manner with
a concomitant decrease in Syk recovered in the Triton-soluble fraction.
In contrast, F(ab`)
-anti-p62-induced association of Syk
with the cytoskeleton was not observed in either PGI
- or
RGDS-pretreated platelets. We further studied the tyrosine
phosphorylation state of Syk in the cytoskeleton fraction (Fig. 5B). Tyrosine-phosphorylated Syk was found in the
cytoskeleton at 45 s following F(ab`)
-anti-p62 stimulation
but significantly decreased at 90 s, although a comparable amount of
Syk was recovered at both time points. This observation was consistent
with the tyrosine phosphorylation state of Syk in the whole cell
lysates prepared in RIPA buffer (Fig. 4A). These
results indicated that although F(ab`)
-anti-p62-induced
activation of Syk was cAMP-insensitive, subsequent cytoskeletal
association of Syk and its dephosphorylation on tyrosine were
cAMP-sensitive or dependent on GPIIb-IIIa-mediated aggregation.
Figure 5:
Effects of PGI and RGDS on
F(ab`)
-anti-p62-stimulated subcellular redistribution and
phosphorylation state of Syk in the 1% Triton X-100-insoluble fraction. Panel A, washed platelets were stirred and stimulated with 150
µg/ml of F(ab`)
-anti-p62 for the times indicated below each lane in the absence (
p62) or presence
of 3 µM PGI
(PGI
/
p62) or in the presence of 1
mM RGDS (RGDS/
p62). Platelets were lysed and
fractionated into 1% Triton X-100-soluble (SOL) and -insoluble
cytoskeleton (CSK) fractions. The Triton-soluble and insoluble
fractions from 1
10
and 4
10
cells, respectively, were resolved by 10% SDS-polyacrylamide gel
electrophoresis and immunoblotted with an anti-Syk mAb. Panel B, the cytoskeleton fractions prepared as described above were
resolubilized in 0.5 M NaCl, precipitated with anti-Syk serum,
resolved by 8% SDS-polyacrylamide gel electrophoresis, and analyzed on
anti-phosphotyrosine immunoblotting (
PY) followed by
reprobing with an anti-Syk mAb (
Syk). In both A and B, the arrows on the right indicate
the positions of Syk.
Figure 6:
Effects of PGI on
F(ab`)
-anti-p62-stimulated kinase activity of c-Src and
tyrosine phosphorylation of FAK. Panel A, washed platelets
were stirred and stimulated with 150 µg/ml of
F(ab`)
-anti-p62 for the times indicated below each
lane in the absence (PGI
, -) or presence (PGI
, +) of 3 µM
PGI
. Platelets were lysed in 1% Triton X-100-containing
buffer and precipitated with an anti-Src mAb. The anti-Src
immunoprecipitates were divided into two aliquots; one was analyzed on
a quantitative immunoblotting with anti-Src (
Src), and
the other was subjected to an in vitro kinase assay using
histone as an exogenous substrate. Autoradiographies of c-Src
autophosphorylation (c-Src) and histone phosphorylation (Histone) are shown. The arrows on the right indicate the positions of c-Src. Panel B, washed
platelets were unstimulated (UN, lane 1) or stimulated for 5
min with F(ab`)
-anti-p62 (
p62, lanes 2 and 3) or collagen (COL, lanes 4 and 5) under
stirring conditions in the absence (lanes 1, 2, and 4) or presence (lanes 3 and 5) of
PGI
, lysed, precipitated with an anti-FAK mAb, and
subjected to anti-phosphotyrosine immunoblotting (
PY).
The arrow on the right indicates the position of FAK.
The comparable amount of FAK in each lane was verified on the reprobing
of the same membrane with anti-FAK mAb (data not
shown).
Figure 7:
Effects of PGI on collagen- or
thrombin-induced tyrosine phosphorylation of Syk and activation of
c-Src. Washed platelets were unstimulated (UN, lane 1) or
stimulated with 20 µg/ml of collagen for 30 s (COL, lanes 2 and 3), or with 0.1 unit/ml of thrombin for 10 s (THR, lanes 4 and 5) under stirring conditions in the
absence (lanes 1, 2, and 4) or presence (lanes 3 and 5) of 3 µM PGI
. Tyrosine
phosphorylation of Syk and activation of c-Src in each condition were
examined as described in the legends to Fig. 4and Fig. 6, respectively. Longer incubation with thrombin for up to
5 min did not lead to tyrosine phosphorylation of Syk or activation of
c-Src in the presence of PGI
(data not shown). The arrows and arrowheads on the right indicate
the positions of Syk and c-Src,
respectively.
Figure 8:
Tyrosine phosphorylation of PLC-1 and
PLC-
2 stimulated by F(ab`)
-anti-p62 in the absence or
presence of PGI
. Washed platelets were unstimulated (UN, lane 1) or stimulated for 30 s with 150 µg/ml of
F(ab`)
-anti-p62 under stirring conditions (
p62,
lanes 2 and 3) in the absence (lanes 1 and 2) or presence of 3 µM PGI
(lanes
3), lysed in RIPA buffer, precipitated with rabbit polyclonal
anti-PLC-
1 or anti-PLC-
2 IgG, and subjected to
anti-phosphotyrosine immunoblotting (
PY) followed by
reprobing with anti-PLC-
1 (
PLC
1) or
anti-PLC-
2 (
PLC
2). The arrows and arrowheads on the right indicate the positions of
PLC-
1 and PLC-
2, respectively.
In this study, we demonstrated that stimulation of platelet
GPVI with F(ab`)-anti-p62 induced tyrosine phosphorylation
of multiple proteins, which was not inhibited by agents that increase
cAMP. We further showed that at least two cytosolic PTKs of different
families, c-Src and Syk, were involved in the cAMP-insensitive
protein-tyrosine phosphorylation in GPVI-mediated or
collagen-stimulated platelet activation. In addition, the inhibition of
PTKs activity by tyrphostin dramatically abrogated
F(ab`)
-anti-p62-induced platelet aggregation in a
dose-dependent fashion. These results indicate that signaling through
GPVI considerably depends on the activation of PTKs, which are free of
suppressive regulation by cAMP.
The validity of
F(ab`)-anti-p62 as a selective agonist for platelet GPVI
was supported by the following points. First, anti-p62 IgG is strongly
reactive with platelet
GPVI(3, 4, 5, 6) , as was confirmed
by the immunoprecipitation from membrane-labeled normal platelets that
yielded a single major band corresponding to GPVI. Second, anti-p62 IgG
did not stimulate protein-tyrosine phosphorylation as well as platelet
aggregation in the GPVI-deficient platelets. Third, the involvement of
platelet Fc receptors was negligible because anti-p62 IgG-induced
platelet activation was unaffected by the Fc-blocking antibody and we
used only the F(ab`)
fragments depleted of the Fc portions.
Anti-p62 IgG requires divalent interaction to exert its effect because
its Fab fragments cannot activate platelets(3) , suggesting
that the mechanism for the F(ab`)
-anti-p62-triggered
signaling is Fc-independent cross-linking of GPVI. Although most of
platelet activating antibodies have been shown to induce platelet
aggregation via an interaction with Fc receptors, a recent report
described the Fc-independent platelet activation induced by a specific
monoclonal antibody that recognizes 45- and 36-kDa membrane
proteins(41) .
cAMP has been thought to inhibit nealy all
aspects of signaling events induced by various platelet
agonists(42) . Inhibition of protein-tyrosine phosphorylation
is also implicated in the negative regulation of platelet function by
cAMP. Pumiglia et al.(43) first reported that
thrombin-induced protein-tyrosine phosphorylation was inhibited by
cAMP-increasing agents such as PGI or dibutyryl cAMP.
Subsequently, Smith and his colleagues (15) reported that
elevation of cAMP with iloprost inhibited thrombin- but not
collagen-stimulated protein-tyrosine phosphorylation. Under the
experimental conditions where platelets were not allowed to aggregate,
fibrinogen binding to GPIIb-IIIa has been reported to evoke
cAMP-sensitive or -insensitive protein-tyrosine phosphorylation
depending on whether fibrinogen is immobilized or
soluble(21, 30) . Recently, anti-CD9 antibody has been
reported to induce cAMP-sensitive protein-tyrosine phosphorylation with
help of its Fc portion(35) . In confirmation of these previous
reports, we observed that cAMP increasing agents abrogated
protein-tyrosine phosphorylation induced by thrombin or
STA
, but did not with those induced by collagen. Although
the mechanisms for cAMP-sensitive or -insensitive protein-tyrosine
phosphorylation are to be investigated further, the unique nature that
F(ab`)
-anti-p62-stimulated platelet activation is dependent
on cAMP-insensitive induction of protein-tyrosine phosphorylation may
support the concept that activation of PTKs is one of the earliest
signaling responses coupled to GPVI engagement.
In search for
responsible PTKs for cAMP-insensitive protein-tyrosine phosphorylation,
we found that the effects of PGI on the activation of
c-Src, Syk, and FAK varied between agonists employed in this study.
While F(ab`)
-anti-p62 and collagen stimulated activation of
c-Src and Syk in a cAMP-insensitive fashion, activation of these
kinases by thrombin was inhibited in the presence of PGI
.
Furthermore, collagen did stimulate tyrosine phosphorylation of FAK in
PGI
-treated platelets, but F(ab`)
-anti-p62 did
not. These results indicate that both cAMP-sensitive and -insensitive
activation mechanisms exist for such PTKs as c-Src, Syk, and FAK, and
that either of them is operating depending on the nature of each
different receptor-coupled signal.
Although c-Src and Syk have been
reported to become activated by a variety of platelet agonists, the
present work is the first report demonstrating activation of c-Src
through clustering of platelet surface glycoproteins. Furthermore, GPVI
is newly identified as a member of platelet membrane glycoproteins
whose engagement leads to tyrosine phosphorylation and activation of
Syk. In other cells, it has been reported that the Src family and
Syk/ZAP-70 family kinases are recruited to the phosphorylated
tyrosine-based activation motif-containing receptors and
synergistically involved in generating cellular
responses(44, 45, 46) . Although we tried
several immunoprecipitation analyses under various lysis conditions, we
could not reproducibly detect association of GPVI with c-Src or Syk,
and did not observe tyrosine phosphorylation of GPVI through the time
course of F(ab`)-anti-p62-stimulated platelet activation. (
)However, it appears that the possibility of interaction of
PTK with GPVI cannot be ruled out, because activated GPVI may be poorly
recovered by intact anti-p62 IgG in the presence of an excess amount of
its F(ab`)
fragments. In fact, we could observe
coprecipitation of Syk with GPVI when we stimulated platelets with
anti-p62 IgG instead of F(ab`)
-anti-p62 and directly
collected GPVI-anti-p62 IgG complex.
Very recently, Ozaki et al.(35) have reported that both persistent
activation of Syk and association of c-Src with Syk are evoked by
antibody- induced clustering of CD9 but are not functional per se with respect to tyrosine phosphorylation of substrate proteins and
the full picture of platelet aggregation, although the recruitment of
these PTKs to CD9 remains to be clarified(35) . Clearly, their
work is in sharp contrast to our novel observations that clustering of
GPVI induces functional activation of Syk and c-Src leading to platelet
activation as well as cAMP-insensitive tyrosine phosphorylation of many
cellular substrates.
Clark et al.(31) reported that the agonist-induced activation of Syk was enhanced by GPIIb-IIIa-dependent mechanisms, implying the possible role of Syk not only in the ``early'' phase but in the aggregation-dependent protein-tyrosine phosphorylation in platelets. On the contrary, in agreement with another recent report(35) , our results showed that activity of Syk was rather negatively regulated also in an aggregation-dependent fashion, because tyrosine-phosphorylated Syk was rapidly dephosphorylated on tyrosine and deactivated as platelets aggregated. This was not explained solely by the translocation of activated enzyme to the cytoskeleton fraction which was true for c-Src(26) , because such deactivation of Syk was also observed in RIPA lysates containing the solubilized cytoskeleton fraction. We further observed that the cytoskeletal association of Syk following GPVI cross-linking was GPIIb-IIIa-dependent and demonstrated for the first time that relocated Syk subsequently became dephosphorylated on tyrosine in the cytoskeleton fraction. These findings indicate that cytoskeletal reorganization is involved in dephosphorylation of Syk through the GPIIb-IIIa-dependent mechanisms. The dual regulation, that is, GPIIb-IIIa-mediated activation and deactivation, may differently modulate the activity of Syk in such a way as to depend on each platelet agonist or ligand. We have recently provided evidence that dephosphorylation of tyrosine-phosphorylated proteins in platelets occurs mostly on the cytoskeleton by the action of protein-tyrosine phophatases regulated by GPIIb-IIIa-mediated aggregation(38) . The same mechanisms could be applied to the deactivation of Syk by the interaction with protein-tyrosine phosphatase in the cytoskeleton.
Although some clinical reports from our laboratory and others have
implicated the possible involvement of GPVI in collagen-induced
platelet activation, no biochemical evidence has been provided to date
for this concept. It is noteworthy that signaling through GPVI
resembles collagen stimulation in the sense that both induce
cAMP-insensitive activation of c-Src and Syk accompanied by tyrosine
phosphorylation of PLC-2. In order to obtain more conclusive
evidence for the involvement of GPVI in collagen-platelet interaction,
it should be of great value to study collagen-induced signaling events
in the GPVI-deficient platelets. Interestingly enough, our preliminary
results showed that the GPVI-deficient platelets from the few patients
so far reported specifically lacked activation of Syk but possessed
normal activation of c-Src when stimulated by collagen, although both
of these kinases became normally activated when these platelets were
stimulated by other agonists including thrombin. (
)Given the
fact that F(ab`)
-anti-p62-induced clustering of GPVI evoked
activation of c-Src and Syk as was shown in this study, a model is
emerging that GPVI-mediated signaling may be essential for
collagen-stimulated activation of Syk while c-Src could be activated
not only through GPVI but also presumably via GPIa-IIa in
collagen-platelet interaction. Based on the present work, our efforts
are currently directed to more complete understanding of the mechanisms
for collagen-induced platelet activation which possibly involve
coordinated signalings from different membrane receptors including
GPVI.