From the Department of Hematology and Oncology, Clinical Sciences for Pathological Organs,
Graduate School of Medicine, Kyoto University, Kyoto 606-8507, Japan
We have previously shown that uncharacterized glycoprotein VI (GPVI), which is constitutively associated and coexpressed with Fc receptor
chain (FcR
) in human platelets, is essential for collagen-stimulated tyrosine phosphorylation of FcR
, Syk, and phospholipase C
2
(PLC
2), leading to platelet activation. Here we investigated involvement of the Src family in
the proximal signals through the GPVI-FcR
complex, using the snake venom convulxin from Crotalus durissus terrificus, which specifically recognizes GPVI and activates platelets
through cross-linking GPVI. Convulxin-coupled beads precipitated the GPVI-FcR
complex
from platelet lysates. Collagen and convulxin induced tyrosine phosphorylation of FcR
, Syk,
and PLC
2 and recruited tyrosine-phosphorylated Syk to the GPVI-FcR
complex. Using
coprecipitation methods with convulxin-coupled beads and antibodies against FcR
and the
Src family, we showed that Fyn and Lyn, but not Yes, Src, Fgr, Hck, and Lck, were physically associated with the GPVI-FcR
complex irrespective of stimulation. Furthermore, Fyn was
rapidly activated by collagen or cross-linking GPVI. The Src family-specific inhibitor PP1
dose-dependently inhibited collagen- or convulxin-induced tyrosine phosphorylation of proteins including FcR
, Syk, and PLC
2, accompanied by a loss of aggregation and ATP release
reaction. These results indicate that the Src family plays a critical role in platelet activation via
the collagen receptor GPVI-FcR
complex.
Key words:
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Introduction |
Platelets adhere to collagen fibers of extracellular matrix
and become activated through specific membrane receptors, resulting in shape change, granule release, and aggregation. These processes are vital for the formation of a
hemostatic plug under a physiologic or pathologic condition. Many platelet surface glycoproteins (GPs)1 have been
proposed as receptors for collagen, including the integrin
2
1 (GPIaIIa; reference 1), GPIV (GPIIIb, CD36; reference 2), an uncharacterized protein GPVI (p62; references
3, 4), the type I collagen receptor (p65; reference 5), and 85-90-kD GPs (6). It has been well established that
2
1
plays a major role in platelet adhesion to collagen (1), although it remains unclear whether
2
1 directly transduces
signals leading to platelet activation. Our laboratory and
others have reported patients with mild bleeding disorders
whose platelets lack GPVI but not
2
1 and show selective
deficiency in collagen-induced platelet aggregation and release reaction (3, 4, 7). Several lines of evidence have demonstrated that GPVI is implicated in collagen-induced
tyrosine phosphorylation of proteins (8), including Fc
receptor
chain (FcR
), Syk tyrosine kinase, and phospholipase C
2 (PLC
2), which are essential for platelet activation upon collagen stimulation (12). GPVI is constitutively associated with FcR
(10, 11), while both of them
are proportionally absent in GPVI-deficient platelets (10),
suggesting their coexpression. Cross-linking GPVI by the
F(ab')2 of anti-GPVI IgG (anti-GPVI F(ab')2) stimulates tyrosine phosphorylation of FcR
, Syk, and PLC
2, and recruits Syk to FcR
, leading to Syk activation (8, 10, 11),
whereas GPVI-deficient platelets specifically lack activation
of Syk in response to collagen (9). These findings have revealed the GPVI-FcR
complex as a collagen receptor responsible for platelet activation.
To further investigate the mechanisms involved in the
proximal signals through the GPVI-FcR
complex, we used
the snake venom convulxin from Crotalus durissus terrificus,
which has been known to be a powerful platelet activator
(15, 16), and the Src family-specific inhibitor PP1 (4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine; reference 17). Recent reports have shown that convulxin is a heterodimeric C-type lectin and binds to platelet surface
with a high affinity on a small number of binding sites (18,
19), that 125I-convulxin specifically recognizes a glycosylated 62-kD membrane protein immunoprecipitated by the
anti-GPVI IgG (18, 20), and that cross-linking GPVI with
convulxin induces tyrosine phosphorylation of proteins including FcR
, Syk, and PLC
2 (18). In this report, we
demonstrated that the Src family tyrosine kinases Fyn and
Lyn were physically associated with the GPVI-FcR
complex and that Fyn was activated by collagen stimulation or
cross-linking GPVI. PP1 dose-dependently inhibited collagen- or convulxin-induced tyrosine phosphorylation of
proteins including FcR
, Syk, and PLC
2, accompanied
by a loss of aggregation and ATP release reaction. Analogy
to the antigen and Fc receptor signals, the Src family kinases
were involved in platelet activation through the collagen receptor GPVI coupled with FcR
as a signal transducing
subunit containing an immunoreceptor tyrosine-based activation motif (ITAM; references 21, 22).
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Materials and Methods |
Reagents.
Convulxin was purified with gel filtration chromatography as described by Polgár et al. (18) from lyophilized Crotalus durissus terrificus venom obtained from Sigma Chemical Co.
(St. Louis, MO). CNBr-activated Sepharose 4B beads, Sepharose
4B beads, and protein A-Sepharose beads were purchased from
Pharmacia Biotechnology Inc. (Uppsala, Sweden). As previously
described, human anti-GPVI IgG and its F(ab')2 fragments were
prepared from serum of a patient with GPVI deficiency (3, 8),
who has been followed up as an outpatient in our department.
Anti-FcR
IgG was produced by immunizing rabbits with a synthetic peptide (residues 80-86) from FcR
as described previously (23). mAbs against Syk (clone Syk101), Lyn (clone Lyn9),
and Yes (clone 3H9) were purchased from Wako Pure Chemical
Industries, Ltd. (Osaka, Japan). Anti-phosphotyrosine (clone 4G10)
and anti-Src (clone 327) mAbs were obtained from Upstate Biotechnology, Inc. (Lake Placid, NY) and Oncogene Science, Inc.
(Uniondale, NY), respectively. Anti-low affinity Fc receptor for
IgG (Fc
RII) mAb (clone IV.3) was from Medarex Inc. (Annandale, NJ). Affinity-purified rabbit IgG specific to Fyn (FYN3),
Hck (N-30), and PLC
2 (C19) and affinity-purified goat IgG to
Lck (2102) were purchased from Santa Cruz Biotechnology Inc.
(Santa Cruz, CA). Affinity-purified rabbit anti-Fgr Ab was obtained from Transduction Laboratories (Lexington, KY). Mouse
mAb (clone 19-1; reference 24) against the
chain of human
high affinity Fc receptor for IgE (Fc
RI) was a kind gift from Dr.
M.-H. Jouvin and Dr. J.-P. Kinet (Beth Israel Hospital, Boston,
MA). Acid soluble fibrillar collagen prepared from equine tendon
was purchased from Horm-Chemie (Munich, Germany). PP1
(17) and PGE1 were kindly provided by Pfizer Central Research
(Groton, CT) and Ono Pharmaceutical Co. (Osaka, Japan), respectively. Arg-Gly-Asp-Ser (RGDS) and enolase were obtained
from Sigma Chemical Co.
-[32P]ATP (3,000 Ci/mmol, 10 mCi/ml) was supplied by DuPont NEN (Boston, MA). All other
reagents were obtained as previously reported (25).
Preparation and Stimulation of Platelets.
After informed consent was obtained, venous blood was collected from healthy adult
donors. All experiments involving human subjects were conducted
according to the principles expressed in the Declaration of Helsinki. Anticoagulation of blood and preparation of platelet-rich
plasma were performed as described previously (26). Platelet-rich
plasma was incubated with 1 mM aspirin for 30 min at 37°C.
Gel-filtered platelets were then prepared as described previously
(27) at a final concentration of 1 × 109 cells/ml in Hepes buffer
(137 mM NaCl, 2.7 mM KCl, 1 mM MgCl2, 1 mM CaCl2, 5.6 mM
glucose, 1 mg/ml BSA, 3.3 mM NaH2PO4, and 20 mM Hepes,
pH 7.4), containing 1 U/ml apyrase and 500 µM RGDS to avoid
secondary effects via released ADP and aggregation. Platelets were stimulated by 50 µg/ml of collagen, 50 ng/ml of convulxin, or 150 µg/ml of anti-GPVI F(ab')2 with gentle agitation for appropriate periods at 37°C. In some experiments, gel-filtered
platelets were incubated for 3 min at 37°C with indicated concentrations of PP1 or DMSO (0.25%) as a control before stimulation. Resting or stimulated platelets were lysed with an equal
volume of ice-cold 2× Triton X-100 lysis buffer (150 mM NaCl,
10 mM EGTA, 2% Triton X-100, 2 mM PMSF, 2 mM Na3VO4,
40 µg/ml leupeptin, 40 µg/ml aprotinin, and 20 mM Hepes, pH
7.4) or 2× radioimmunoprecipitation assay (RIPA) buffer (150 mM NaCl, 10 mM EGTA, 2% Triton X-100, 2% sodium deoxycholate, 0.2% SDS, 2 mM PMSF, 2 mM Na3VO4, 40 µg/ml leupeptin, 40 µg/ml aprotinin, and 20 mM Hepes, pH 7.4). For
preparation of whole platelet lysates, gel-filtered platelets were
suspended in Hepes buffer without BSA and directly lysed in an
equal volume of 2× SDS sample buffer (×1; 2% SDS, 5% glycerol, 5%
-ME, and 62.5 mM Tris-HCl, pH 6.8), and boiled for
5 min.
Affinity Precipitation of Convulxin-binding Proteins.
100 µg of
purified convulxin was covalently coupled to 1 ml of CNBr-activated Sepharose 4B beads according to the manufacturer's instructions. 1.2 ml of platelet lysates (from 6 × 108 cells/sample)
was clarified by centrifugation at 16,000 g for 30 min, incubated
for 1 h with 40 µl of Sepharose 4B (50% slurry), and then precleared by sedimentation at 16,000 g for 30 min. These steps and
all subsequent steps were carried out at 4°C. The supernatants were incubated for 1 h with 40 µl of convulxin-coupled
Sepharose 4B (50% slurry) or Sepharose 4B as a control. The
beads were sedimented by brief centrifugation and washed four
times in lysis buffer containing 0.5 M NaCl. Proteins were eluted
from the beads in 90 µl of SDS sample buffer and boiled for 5 min.
Immunoprecipitation.
Immunoprecipitation of each protein
and its associated proteins was performed as described previously
(25), followed by two-step elution with different buffers. 1.2 ml
of lysates prepared from 6 × 108 platelets in Triton X-100 lysis
buffer was clarified by centrifugation at 16,000 g for 30 min, precleared with protein A-Sepharose beads, and incubated for 2 h
with 5 µg of anti-FcR
IgG, preimmune rabbit IgG, anti-Fc
RII
mAb, anti-Fyn Ab, anti-Lyn mAb, anti-Yes mAb, anti-Src mAb,
and anti-PLC
2 Ab. This step and all subsequent steps were performed at 4°C. For immunoprecipitation of Yes and Src, lysates
were further incubated for 1 h with 25 µg of rabbit anti-mouse
IgG. Immune complexes were then precipitated with protein A-Sepharose beads for 1 h, sedimented by brief centrifugation, and washed four times in Triton X-100 lysis buffer. Coprecipitated proteins were first eluted in 60 µl of RIPA buffer. Eluates
were diluted with 30 µl of 3× SDS sample buffer and boiled for
5 min. The left beads were further washed four times in RIPA
buffer. Immunoprecipitates were then eluted in 60 µl of SDS
sample buffer and boiled for 5 min. These samples eluted by SDS
sample buffer were checked with immunoblotting whether each
Ab precipitated its recognizing protein.
Immunoblotting.
Immunoblot analysis for each protein was
done as described previously (25). In brief, samples (the whole
platelet lysates from 5 × 106 cells/lane or affinity precipitates and
immunoprecipitates from 1 × 108 cells/lane) were subjected to
10% SDS-PAGE and transferred electrophoretically to nitrocellulose membranes (Bio-Rad Laboratories, Hercules, CA) with a
semidry blotter. For detection of FcR
, samples were resolved on 12.5% SDS-PAGE with Tris-Tricine buffer system. The
membranes were treated with Ab against phosphotyrosine (1 µg/
ml), GPVI (2.5 µg/ml), FcR
(1 µg/ml),
chain of Fc
RI (0.5 µg/
ml), Syk (2 µg/ml), Fyn (1 µg/ml), Lyn (5 µg/ml), Yes (1 µg/ml),
Src (0.2 µg/ml), Fgr (1 µg/ml), Hck (2 µg/ml), Lck (1 µg/ml), or
PLC
2 (1 µg/ml), followed by the ECL chemiluminescence reaction (Amersham International plc., Little Chalfont, UK). For
detection of GPVI, anti-GPVI IgG was biotinylated to enhance
sensitivity (10). The densities of some protein bands on scanned
images were analyzed using the public domain NIH Image program (written by Wayne Rasband at the National Institutes of
Health).
Immune Complex Kinase Assay.
To examine in vitro kinase
activity of Fyn and Lyn, the kinases were isolated by immunoprecipitation with 1 µg of specific Abs as described above from
platelet lysates (from 3 × 108 cells/sample) prepared in RIPA
buffer. Immunoprecipitates were washed four times with RIPA
buffer and twice with kinase buffer (20 mM Hepes, pH 7.5, and
10 mM MnCl2), and then incubated for 5 min at room temperature with 10 µg of acid-treated enolase in 30 µl of kinase buffer
including 5 µCi of
-[32P]ATP and 1 µM of ATP. Reactions
were stopped by addition of 30 µl of 2× SDS sample buffer. The
samples were boiled for 3 min and subjected to 10% SDS-PAGE.
Radioactivity of each protein was visualized and quantified with
the Bio-Imaging Analyzer BAS-2000II (Fuji Photo Film Co.,
Tokyo, Japan).
Platelet Aggregation and ATP Release Reaction.
Gel-filtered platelets were prepared from platelet-rich plasma as described above
but without aspirin pretreatment, suspended at a final concentration of 3 × 108 cells/ml in Hepes buffer without RGDS, and incubated with PP1 or DMSO for 3 min at 37°C before stimulation
by collagen, convulxin, or thrombin. Standard aggregometry of
platelets was performed according to the Born method (28) in an
aggregometer (HEMA TRACER 1; Nikko Bioscience, Tokyo,
Japan). Release reaction from dense granules of platelets was
monitored as ATP release using luciferin-luciferase reaction in a
lumi-aggregometer (Chrono-Log, Harverton, PA) as described
previously (8).
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Results |
Precipitation of the GPVI-FcR
Complex with Convulxin-coupled Beads from Platelet Lysates.
Convulxin specifically
and strongly binds GPVI expressed on the platelet surface
(19). 125I-convulxin also specifically recognizes GPVI blotted on polyvinylidene difluoride or nitrocellulose membranes (18, 20). Here we used Sepharose 4B beads that
were covalently coupled with convulxin to isolate GPVI
and associated proteins from platelet lysates. Immunoblot
analysis showed that convulxin-coupled Sepharose 4B but
not Sepharose 4B alone effectively precipitated both GPVI and FcR
, which are constitutively associated with each
other (10, 11), when we used Triton X-100 lysis buffer
(Fig. 1, A and B). If we used more stringent RIPA buffer to
solubilize platelets, only GPVI but not FcR
was precipitated with convulxin-coupled Sepharose 4B from platelet
lysates (data not shown). These findings indicate that GPVI
and FcR
were dissociated in RIPA buffer and that convulxin directly and strongly binds to GPVI. Thus, we used Triton X-100 lysis buffer to analyze molecules associated
with the GPVI-FcR
complex in the rest of studies.

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Fig. 1.
Affinity precipitation of the GPVI-FcR complex with convulxin-coupled Sepharose 4B from platelet lysates. Unstimulated gel-filtered
platelets were lysed directly in SDS sample buffer or in Triton X-100 lysis
buffer. Whole lysates (lane 1) and proteins precipitated by affinity with
convulxin-coupled Sepharose 4B (lane 2) or Sepharose 4B alone (lane 3)
were resolved by 10% (A) or 12.5% SDS-PAGE (B), transferred to nitrocellulose membranes, and immunoblotted with anti-GPVI IgG (A) or
anti-FcR IgG (B). Molecular mass markers are indicated in kD on the
right of panels.
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Collagen- or Convulxin-induced Tyrosine Phosphorylation of Proteins Associated with the GPVI-FcR
Complex.
Collagen, con-vulxin, and anti-GPVI F(ab')2 stimulate tyrosine phosphorylation of signaling molecules including FcR
, Syk, and
PLC
2, which are essential for platelet activation by these agonists (8, 18). Recent studies have shown that
convulxin activates platelets via cross-linking GPVI (18,
20). We examined tyrosine phosphorylation of proteins coprecipitated with the GPVI-FcR
complex purified by
convulxin-coupled Sepharose 4B from intact platelets and
platelets stimulated by collagen or convulxin. Upon collagen and convulxin stimulation, we observed an increase
in tyrosine phosphorylation of proteins with molecular
masses of 140, 95, 85, 72, 53/60, and 10/12 kD (Fig. 2 A).
We confirmed that the 72-kD band corresponded to accumulation of tyrosine-phosphorylated Syk (Fig. 2 B) and
that the 10/12-kD doublets were FcR
, which showed
mobility retardation on SDS-PAGE due to phosphorylation (Fig. 2 D). GPVI, whose molecular mass is 62 kD
under the reduced condition (3, 4, 8), was equally precipitated (Fig. 2 C) but not tyrosine phosphorylated irrespective
of stimulation as previously reported (8, 10). Whereas collagen and convulxin stimulated tyrosine phosphorylation of
PLC
2 as previously reported (8, 14, 18, 29, 30), we could
not detect PLC
2 in the precipitated proteins with convulxin-coupled Sepharose 4B regardless of stimulation (data
not shown).

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Fig. 2.
Tyrosine phosphorylation of proteins coprecipitated with the
GPVI-FcR complex in collagen- and convulxin-stimulated platelets.
Gel-filtered platelets were unstimulated (lane 1) or stimulated for 1 min at
37°C with 50 µg/ml of collagen (lane 2) or 50 ng/ml of convulxin (lanes
3 and 4) and lysed in Triton X-100 lysis buffer. Precipitated proteins with
convulxin-coupled Sepharose 4B (lane 1-3) or Sepharose 4B (lane 4)
were resolved by 10% (A, top, B, and C) or 12.5% SDS-PAGE (A, bottom
and D), transferred to nitrocellulose membranes, and immunoblotted
with Abs against phosphotyrosine (A), Syk (B), GPVI (C), and FcR (D).
Molecular mass markers are indicated in kD on the right of panels. In A
(bottom) and D open and closed arrows mark unphosphorylated and phosphorylated FcR , respectively.
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Physical Association of Fyn and Lyn with the GPVI-FcR
Complex.
We next focused on the heavily tyrosine-phosphorylated proteins with molecular masses of 53-60 kD.
The Src family kinases are cytosolic protein tyrosine kinases
with molecular masses ~60 kD and usually tyrosine phosphorylated themselves. Previous reports have shown that
platelets abundantly express the Src family kinases including
Src, Fyn, Yes, Lyn, Hck, Fgr, and Lck (31). We then examined whether the Src family kinases existed in the precipitated proteins with convulxin-coupled Sepharose 4B by
immunoblotting with Ab specific to each kinase. Fig. 3
shows that convulxin-coupled Sepharose 4B precipitated
Fyn (59 kD) and Lyn (53-56 kD), but not Yes (62 kD), Src
(60 kD), Fgr (59 kD), and Lck (56 kD, data not shown),
from lysates of resting platelets and collagen- or convulxin-stimulated platelets. Upon stimulation, protein levels of
precipitated Fyn and Lyn did not change (Fig. 3, A and B). Sepharose 4B alone as a control precipitated none of these
kinases (Fig. 3). We could not detect Hck with immunoblotting in whole platelet lysates or in the precipitated proteins with convulxin-coupled Sepharose 4B (data not
shown). To confirm whether Fyn and Lyn precipitated
with convulxin-coupled Sepharose 4B were associated with the GPVI-FcR
complex, we next analyzed proteins
coimmunoprecipitated with anti-FcR
IgG and Abs
against the Src family kinases. There was a problem that H
chain of IgG used for immunoprecipitation overlapped the
Src family kinases or GPVI on immunoblots due to their
similar molecular masses. Hence, RIPA buffer was used to elute only coprecipitated proteins from immunoprecipitates
as described in Materials and Methods, since RIPA buffer
could not elute IgG and its recognizing proteins from immune complexes bound to protein A-Sepharose beads
(data not shown). Fyn and Lyn but not Src were coimmunoprecipitated with anti-FcR
IgG from platelet lysates
(Fig. 4, A-C). Yes, Fgr, Hck, and Lck were not detected in
coimmunoprecipitates with anti-FcR
IgG (data not
shown). Conversely, anti-Fyn or anti-Lyn Ab, but not
anti-Yes or anti-Src mAb coimmunoprecipitated both
GPVI and FcR
(Fig. 4, D and E). These data clearly demonstrate that Fyn and Lyn were constitutively and specifically associated with the GPVI-FcR
complex. Because
very recent studies indicated that platelets heterogeneously
express Fc
RI (37, 38), which is associated with FcR
and
Lyn in other cells (39, 40), we examined whether Fc
RI
existed in the precipitated proteins with convulxin-coupled
Sepharose 4B. We did not detect the
chain of Fc
RI
among them with immunoblotting (data not shown). Although collagen stimulates tyrosine phosphorylation of
Fc
RIIA in platelets (14), convulxin did not induce it (data
not shown). Convulxin-coupled Sepharose 4B did not coprecipitate Fc
RIIA (data not shown), confirming our previous findings that it is not associated with the GPVI-FcR
complex (10).

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Fig. 3.
Coprecipitation of Fyn and Lyn, but not Yes, Src, and Fgr,
with the GPVI-FcR complex from platelet lysates. Gel-filtered platelets
were unstimulated (lanes 1 and 2) or stimulated for 1 min at 37°C with 50 µg/ml of collagen (lane 3) or 50 ng/ml of convulxin (lanes 4 and 5) and
lysed in Triton X-100 lysis buffer (lanes 2-5) or directly in SDS sample
buffer (lane 1). Whole lysates (lane 1) and precipitated proteins with convulxin-coupled Sepharose 4B (lanes 2-4) or Sepharose 4B (lane 5) were
resolved by 10% SDS-PAGE, transferred to nitrocellulose membranes,
and immunoblotted with Abs against Fyn (A), Lyn (B), Yes (C), Src (D),
and Fgr (E).
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Fig. 4.
Coimmunoprecipitation of Fyn and Lyn with anti-FcR IgG and coimmunoprecipitation of GPVI and FcR
with anti-Fyn and anti-Lyn Abs.
Unstimulated gel-filtered platelets were lysed directly in SDS
sample buffer or in Triton X-100
lysis buffer. (A-C) Whole lysates
(lane 1) and immunoprecipitates
with preimmune rabbit IgG
(lane 2) and anti-FcR IgG (lane
3) were resolved by 10% SDS-PAGE, transferred to nitrocellulose membranes, and immunoblotted with Abs against Fyn (A),
Lyn (B), and Src (C). (D and E)
Whole lysates (lane 1) and immuno-precipitates with Abs
against Fyn (lane 2), Lyn (lane 3),
Yes (lane 4), and Src (lane 5)
were resolved by 10% (D) or
12.5% (E) SDS-PAGE, transferred to nitrocellulose membranes, and immunoblotted with
anti-GPVI IgG (D) and anti-FcR IgG (E).
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Rapid Activation of Fyn upon Collagen Stimulation or GPVI
Cross-linking.
Next we examined whether collagen stimulation or cross-linking GPVI activated Fyn and Lyn. Fyn
and Lyn were immunoprecipitated and subjected to an in
vitro kinase assay with enolase as an exogenous substrate.
We confirmed with immunoblotting that an equal amount of each kinase was immunoprecipitated (data not shown).
Judging from both autophosphorylating activity and kinase
activity toward enolase, Fyn was very rapidly activated by
collagen stimulation or by cross-linking GPVI with convulxin or anti-GPVI F(ab')2 (Fig. 5, A-C). Activation of
Fyn reached the maximum level at 10 s and returned to the
basal level within 60 s upon stimulation of convulxin or
anti-GPVI F(ab')2, whereas collagen-stimulated activation
of Fyn was observed through 2 min of stimulation. We did
not detect an increase in kinase activity of Lyn by collagen
or cross-linking GPVI (data not shown). In some but not
all experiments, we observed a slight decrease in kinase activity of Lyn upon convulxin (Fig. 5 D) or collagen stimulation (data not shown).

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Fig. 5.
Kinase activity of Fyn and Lyn in platelets stimulated by collagen, convulxin, or anti-GPVI F(ab')2. Gel-filtered platelets were stimulated for the indicated periods at 37°C with collagen (A, 50 µg/ml), convulxin (B and D, 50 ng/ml), or anti-GPVI F(ab')2 (C, 150 µg/ml) and
lysed in RIPA buffer. Fyn (A-C) and Lyn (D) were immunoprecipitated
from lysates and subjected to in vitro kinase assay with enolase as an exogenous substrate, followed by 10% SDS-PAGE. Radioactivity of each protein was visualized (A-D) and quantified with the Bio-Imaging Analyzer
BAS-2000II. Kinase activity toward enolase was calculated (H). Positions
of Fyn, Lyn, and enolase are indicated on the right.
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Inhibition of the Collagen- or Convulxin-induced Protein Tyro-sine Phosphorylation by the Src Family-specific Inhibitor PP1.
We used the Src family-specific inhibitor PP1 (17) to examine whether activation of Src family kinases was involved in the collagen- or convulxin-induced tyrosine
phosphorylation of molecules including FcR
, Syk, and
PLC
2. Gel-filtered platelets were preincubated with or
without various concentrations of PP1 for 3 min and then stimulated with 50 µg/ml of collagen or 50 ng/ml of convulxin for appropriate times. Whole lysates were prepared
from those platelets and subjected to the analysis of immunoblotting with anti-phosphotyrosine mAb (Fig. 6). Collagen stimulation induced tyrosine phosphorylation of proteins with molecular masses of 140, 120, 100, 85, 72/75,
65, 38/40, and 28/30 kD. Preincubation of platelets with
PP1 inhibited the collagen-induced tyrosine phosphorylation of these proteins in a dose-dependent manner (Fig. 6
A). Convulxin stimulation induced tyrosine phosphorylation of proteins with the same molecular masses as described above, but the intensity of those protein bands was
much stronger than upon collagen stimulation (Fig. 6 B).
Preincubation of platelets with PP1 also inhibited the convulxin-induced tyrosine phosphorylation in a dose-dependent manner as was observed with collagen stimulation (Fig. 6 B). The heavily tyrosine-phosphorylated 60-kD
protein band in both resting and activated platelets corresponded to Src with immunoblotting analysis (data not
shown). Next we examined the effects of PP1 on the collagen- or convulxin-induced tyrosine phosphorylation of
FcR
, Syk, and PLC
2 in platelets. PP1 (1-20 µM) dose-dependently inhibited the collagen-stimulated tyrosine phosphorylation of GPVI-associated FcR
(Fig. 7 C) as well as the recruitment of tyrosine-phosphorylated Syk to the
GPVI-FcR
complex (Fig. 7, A and B). We confirmed
that convulxin-coupled Sepharose 4B equally precipitated
both GPVI and FcR
, irrespective of PP1 pretreatment
(Fig. 7, D and E). PP1 also reduced collagen-stimulated tyrosine phosphorylation of PLC
2 in a dose-dependent
fashion (Fig. 7 F). Essentially similar results on the PP1 effects were obtained with convulxin-stimulated platelets
(data not shown).

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Fig. 6.
The Src family-specific inhibitor
PP1 dose-dependently inhibits collagen- or
convulxin-induced tyrosine phosphorylation of
whole lysates. Gel-filtered platelets were preincubated for 3 min at 37°C with 0.25% DMSO
(lanes 1 and 2) or the indicated concentrations
of PP1 (lanes 3-7). Platelets were unstimulated
(lane 1) or stimulated with 50 µg/ml of collagen
for 30 s (A, lanes 2-7) or 50 ng/ml of convulxin
for 15 s (B, lanes 2-7), directly lysed in SDS
sample buffer, resolved by 10% SDS-PAGE,
transferred to nitrocellulose membranes, and immunoblotted with anti-phosphotyrosine mAb.
Molecular mass markers are indicated in kD on
the right of panels. Arrows mark the position
of Src.
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Fig. 7.
PP1 dose-dependently inhibits collagen-induced Syk recruitment and tyrosine phosphorylation of FcR and PLC 2. Gel-filtered
platelets were preincubated for 3 min at 37°C with 0.25% DMSO (lanes 1 and 2) or the indicated concentrations of PP1 (lanes 3-6). Platelets were
unstimulated (lane 1) or stimulated with 50 µg/ml of collagen for 30 s
(lanes 2-6) and lysed in Triton X-100 lysis buffer. Precipitated proteins
with convulxin-coupled Sepharose 4B (A-E) or immunoprecipitates with
anti-PLC 2 Ab (F and G) were resolved by 10% (A, B, and E-G) or
12.5% (C and D) SDS-PAGE, transferred to nitrocellulose membranes,
and immunoblotted with Abs against phosphotyrosine (A, C, and F), Syk
(B), FcR (D), GPVI (E), and PLC 2 (G). In H the band densities from
tyrosine-phosphorylated Syk (A), FcR (C), and PLC 2 (F) were quantified and graphed as a percentage of the band density in lane 2 of each
panel. The position of each protein is indicated on the right of panels.
|
|
Inhibition of the Collagen- or Convulxin-induced Aggregation
and ATP Release Reaction by PP1.
To investigate functional
involvement of the Src family kinases in platelet activation
through the GPVI-FcR
complex, we examined the effects of PP1 on aggregation and ATP release reaction upon collagen or convulxin stimulation. PP1 alone did not cause
any shape change, aggregation, or ATP release up to 20 µM
of concentrations (data not shown). PP1 ranging in concentrations from 1 to 10 µM dose-dependently inhibited
aggregation and ATP release caused by 2 µg/ml of collagen
(Fig. 8, A and a), whereas PP1 little affected the extent of
shape change but markedly delayed it (Fig. 8 A). Similar
findings were also obtained when platelets were pretreated with 1-10 µM of PP1 and stimulated with 10 ng/ml of
convulxin (Fig. 8, B and b). In contrast, 20 µM of PP1 did
not affect aggregation and ATP release induced by 0.1 U/ml
of thrombin (Fig. 8, C and c).

View larger version (20K):
[in this window]
[in a new window]
|
Fig. 8.
PP1 dose-dependently inhibits collagen- or convulxin-
induced aggregation and ATP release reaction. Gel-filtered platelets were
preincubated for 3 min at 37°C with 0.25% DMSO or various concentrations of PP1 (µM) as indicated. Platelets were stimulated with 2 µg/ml of
collagen (A and a), 10 ng/ml of convulxin (B and b), or 0.1 U/ml of
thrombin (C and c) under constant stirring conditions. Aggregation curves
(A-C) and ATP release (a-c) were monitored. This experiment is representative of four similar experiments from different donors.
|
|
 |
Discussion |
Because the snake venom convulxin specifically binds to
GPVI (18, 20), we used convulxin-coupled Sepharose 4B
beads for affinity precipitation of the GPVI-FcR
complex
and associated proteins from platelet lysates. Convulxin-coupled Sepharose 4B efficiently precipitated GPVI, FcR
,
and their associated proteins such as Syk. We also found
that collagen or convulxin stimulation induced tyrosine
phosphorylation of FcR
associated with GPVI and recruitment of Syk to the GPVI-FcR
complex, confirming
the complex as a collagen receptor. These experiments
showed that convulxin-coupled Sepharose 4B was a powerful tool to isolate and study the GPVI-FcR
complex.
We noticed that convulxin-coupled Sepharose 4B coprecipitated tyrosine-phosphorylated proteins with molecular
masses of 53-60 kD with the GPVI-FcR
complex. It has been established that the Src family tyrosine kinases are essential for generating initial signals through the immune receptors, which consist of ITAM-containing subunits, leading
to tyrosine phosphorylation of ITAMs and Syk activation
(22, 39, 40). Platelets abundantly express the Src family kinases, whose molecular masses are 53-62 kD and usually
tyrosine phosphorylated (31). Using coprecipitation methods with convulxin-coupled Sepharose 4B, anti-FcR
IgG, and Abs to the Src family kinases, we clearly showed
that Fyn and Lyn, but not Yes, Src, Fgr, Hck, and Lck,
were constitutively associated with the GPVI-FcR
complex, irrespective of stimulation. Although GPIV (CD36) is
also stably associated with Fyn, Lyn, and Yes in platelets
(36), GPIV is not associated with either GPVI or FcR
(10). Recent reports showed that platelets heterogeneously express Fc
RI (37, 38), which consists of the
,
, and
chains, and is associated with Lyn in other cells such as mast cells (39, 40). However, we denied association of the
chain of Fc
RI with the GPVI-FcR
complex. Fc
RIIA,
which is another ITAM-containing receptor in platelets, is
not associated with the GPVI-FcR
complex (10). These
data indicate that the GPVI-FcR
complex is a newly
identified receptor that is physically associated with the Src
family kinases in platelets.
Rapid activation of Src (41) and Lyn (42) but not of Fyn
has been previously reported in thrombin-stimulated platelets under specific experimental conditions, such as weak
stimulation or specified lysis buffers. We have reported that
Src is activated upon collagen stimulation or cross-linking
GPVI (8, 9). In addition, here we showed for the first time
that Fyn became rapidly activated by collagen or cross-linking GPVI. We could detect the Src activation only if
we used 1% Triton X-100 containing lysis buffer to lyse
platelets as previously reported (8, 9). In contrast to Src, we
could observe the Fyn activation only if we used RIPA
buffer as described in Materials and Methods. However, we
could not detect Lyn activation by collagen stimulation or cross-linking GPVI with either RIPA buffer or Triton X-100
lysis buffer. It has been well documented that members
of the Src family kinases are differently distributed in resting platelets (35) and relocate to the membrane skeleton or
the cytoskeleton (41), which is insoluble in 1% Triton
X-100 containing lysis buffer, upon stimulation. These different natures of the kinases may cause the different requirements for lysis buffers to detect their activation. We
observed a slight decrease in kinase activity of Lyn upon
convulxin or collagen stimulation in some but not all experiments. Such a decrease in Lyn activity seems to be similar to Src activity as reported previously (9, 41). Although
Src is activated by either collagen or cross-linking GPVI in
normal platelets, Src is still activated by collagen in GPVI-deficient platelets lacking collagen-induced Syk activation
and PLC
2 phosphorylation (9). Taken together with our
new findings that Src was not physically associated with the
GPVI-FcR
complex, Src does not seem to be directly involved at least in Syk activation and subsequent tyrosine
phosphorylation of PLC
2 upon collagen stimulation.
It has been demonstrated that PP1 selectively inhibits the
Src family kinases, including Lck, Fyn, Src, Hck, and Lyn,
but not the Syk family kinases, including ZAP-70 and Syk,
even at concentrations over 100 µM (17, 45). We observed
that PP1 (1-20 µM) dose-dependently inhibited collagen-
or convulxin-induced tyrosine phosphorylation of whole
lysates. PP1 also abolished tyrosine phosphorylation of
GPVI-associated FcR
, Syk, and PLC
2 as well as recruitment of Syk to the GPVI-FcR
complex. The concentrations of PP1 used here were similar ones that have been reported to inhibit induction of tyrosine phosphorylation or
cell functions upon stimulation in T cells, RBL-2H3 cells,
fibroblasts, or Mo7e cells (17, 45). As for immune receptors, it has been reported that FcR
is phosphorylated
on tyrosine by the Src family kinases that are associated
with the receptors, such as Lyn for Fc
RI (22, 39, 40).
Therefore, Fyn and Lyn that were associated with the
GPVI-FcR
complex would be most probable candidate
kinases responsible for phosphorylating FcR
and subsequent signals in GPVI-mediated platelet activation by collagen. Finally, we showed that PP1 inhibited collagen- or
convulxin-stimulated aggregation and ATP release reaction
but not extent of shape change in a dose-dependent manner. The inhibition of aggregation and release reaction by PP1 was observed parallel to that of the tyrosine phosphorylation discussed above, suggesting that PP1 blocks platelet
functions through inhibiting activation of the Src family
and its downstream signals. However, when we used higher
concentrations of collagen (20-50 µg/ml), aggregation and
ATP release reactions became somewhat insensitive to
even 20 µM of PP1, although PP1 still notably delayed beginning of aggregation and ATP release by these concentrations of the agonists (data not shown). It is probably because PP1 could not completely inhibit activation of the
Src family upon strong stimulation of collagen. If it is not,
there may be another possibility that PP1-insensitive bypass
signals might function in platelets stimulated with higher
concentrations of collagen. Although previous studies have
shown that platelets are enriched in the Src family kinases
(31), this is the first report demonstrating that the Src
family is functionally related to platelet activation.
The collagen-stimulated tyrosine phosphorylation including Syk and PLC
2 is compromised by blocking of
2
1 with anti-
2
1 mAbs (14, 48) or proteolytic cleavage
of the
1 subunit with the snake venom metalloproteinase
Jararhagin (49), suggesting that
2
1 is also essential for the
collagen signals. However, direct cross-linking of
2
1
alone with mAbs does not cause platelet activation and
protein tyrosine phosphorylation (14). In contrast to our
present findings on the GPVI-FcR
complex,
2
1 has not been shown to be physically associated with protein tyrosine kinases in platelets (48, 49). On the other hand, cross-linking the GPVI-FcR
complex with anti-GPVI F(ab')2,
convulxin, or collagen-related peptides, which also stimulate platelets via GPVI (50), causes platelet activation and
tyrosine phosphorylation of proteins including FcR
, Syk,
and PLC
2 independently of
2
1 (8, 10, 18, 51, 52).
In GPVI-deficient platelets, collagen stimulates
2
1-dependent activation of Src and tyrosine phosphorylation of multiple proteins but not that of Syk, PLC
2, and Vav (9). These data seem to support the two-site, two-step model of
platelet activation by collagen (53, 54). At the first step of
the platelet-collagen interaction,
2
1 plays a critical role in
adhesion and might modulate the GPVI-FcR
complex-mediated responses including protein tyrosine phosphorylation as a coreceptor, leading to aggregation and release reaction. Roles of the other receptors (2, 5, 6) remain to be less
clear than
2
1 and the GPVI-FcR
complex in the collagen signals. Platelets from patients lacking GPIV (the
Naka-negative phenotype) aggregate (55) and show tyrosine
phosphorylation (56) normally to collagen. However, GPIV
may play some roles in the collagen-induced tyrosine phosphorylation, since GPIV is physically associated with Fyn,
Yes, and Lyn (36). Further studies are necessary to elucidate
the complete nature of collagen signals through the GPVI-
FcR
complex,
2
1, and possibly others.
Address correspondence to Hiroshi Takayama, The Department of Hematology and Oncology, Clinical Sciences for Pathological Organs, Graduate School of Medicine, Kyoto University, 54 Shogoin-Kawaracho, Sakyo-ku, Kyoto 606-8507, Japan. Phone: 81-75-751-3151; Fax: 81-75-751-3201; E-mail: hiro{at}kuhp.kyoto-u.ac.jp
We thank Dr. M.-H. Jouvin and Dr. J.-P. Kinet for mAb to the
chain of Fc
RI, Pfizer Central Research
for PP1, and Ono Pharmaceutical Co. for PGE1. We also thank Dr. K. Hirai for critical reading of the
manuscript and I. Nakamura for secretarial assistance.
This work was supported by grants-in-aid for scientific research from the Ministry of Education, Science and
Culture of Japan, funds for comprehensive research on aging and health from the Ministry of Health and
Welfare of Japan, and the Ryoichi Naito Foundation Grant for Medical Research. Y. Ezumi is a Research Fellow of the Japan Society for the Promotion of Science.
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