Collagen-stimulated Activation of Syk but Not c-Src Is Severely Compromised in Human Platelets Lacking Membrane Glycoprotein VI*

(Received for publication, July 2, 1996)

Tatsuo Ichinohe Dagger , Hiroshi Takayama Dagger §, Yasuharu Ezumi Dagger , Morio Arai , Naomasa Yamamoto par , Hoyu Takahashi ** and Minoru Okuma Dagger

From the Dagger  Department of Hematology and Oncology, Clinical Sciences for Pathological Organs, Graduate School of Medicine, Kyoto University, 54 Shogoin-Kawaramachi, Sakyo-ku, Kyoto 606-01,  Department of Clinical Pathology, Tokyo Medical College, Tokyo 160, par  Department of Cardiovascular Research, Tokyo Metropolitan Institute of Medical Science, Tokyo 113, and ** Blood Transfusion Division, Niigata University Medical Hospital, Niigata 951, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
Acknowledgments
REFERENCES


ABSTRACT

Activation of circulating platelets by subendothelial collagen is an essential event in vascular hemostasis. In human platelets, two membrane glycoprotein (GP) abnormalities, integrin alpha 2beta 1 deficiency and GPVI deficiency, have been reported to result in severe hyporesponsiveness to fibrillar collagen. Although it has been well established that integrin alpha 2beta 1, also known as the GPIa-IIa complex, functions as a primary platelet adhesion receptor for collagen, the mechanism by which GPVI contributes to collagen-platelet interaction has been ill defined to date. However, our recent observation that GPVI cross-linking couples to cyclic AMP-insensitive activation of c-Src and Syk tyrosine kinases suggested a potential role for GPVI in regulating protein-tyrosine phosphorylation by collagen (Ichinohe, T., Takayama, H., Ezumi, Y., Yanagi, S., Yamamura, H., and Okuma, M. (1995) J. Biol. Chem. 270, 28029-28036). To further investigate this hypothesis, here we examined the collagen-induced protein-tyrosine phosphorylation in GPVI-deficient platelets expressing normal amounts of alpha 2beta 1. In response to collagen, these platelets exhibited alpha 2beta 1-dependent c-Src activation accompanied by tyrosine phosphorylation of several substrates including cortactin. In contrast, severe defects were observed in collagen-stimulated Syk activation and tyrosine phosphorylation of phospholipase C-gamma 2, Vav, and focal adhesion kinase, implicating a specific requirement of GPVI for recruiting these molecules to signaling cascades evoked by collagen-platelet interaction.


INTRODUCTION

Upon interaction with subendothelial collagen, platelets undergo a series of biochemical events that eventually lead to cell spreading, secretion of granular contents, and activation of major fibrinogen receptor, integrin alpha IIbbeta 3, to form platelet aggregates (1). To accomplish this essential role in primary hemostasis, platelets are believed to express specific surface receptors for fibrillar collagen. Although a number of candidates have been proposed as a platelet collagen receptor to date (2), true participation of such molecules in physiological collagen-platelet interaction remains largely unclarified, and the molecular mechanism by which collagen induces platelet activation has yet to be elucidated. Several lines of evidence have indicated that the integrin heterodimer alpha 2beta 1 (3, 4), also called glycoprotein (GP)1 Ia-IIa, is a principal platelet adhesion receptor for collagen. Clinical deficiency of platelet alpha 2beta 1 results in a mild to severe bleeding tendency, accompanied by defective platelet aggregation in response only to collagen (5, 6, 7). Specific monoclonal antibodies against alpha 2beta 1 inhibits platelet adhesion to fibrillar collagen (8, 9), and liposomes containing alpha 2beta 1 can adhere to collagen types I, II, III, and IV (10). In addition, the molecular analysis of this complex revealed the presence of a putative collagen-binding domain within the alpha 2 subunit (11, 12). Although alpha 2beta 1-mediated adhesion thus appears to be an essential primary step in collagen-platelet interaction, it is still unknown whether collagen-alpha 2beta 1 binding is sufficient for stimulating a full picture of collagen-induced platelet activation.

Protein-tyrosine phosphorylation is now considered a key signaling event in the activation of platelets (13). Collagen is also known to stimulate activation of protein-tyrosine kinases (PTKs), c-Src (14), Syk (15), and focal adhesion kinase (Fak) (16) and promotes rapid tyrosine phosphorylation of a number of proteins in platelets including cortactin (13), phospholipase C (PLC)-gamma 2 (17, 18) and Vav (19). Unlike the case with other platelet agonists, collagen-stimulated protein-tyrosine phosphorylation is unique in that it is insensitive to the inhibition by cAMP-increasing reagents (20), which are known to potently arrest platelet activation by most agonists. Although integrin alpha 2beta 1-mediated cell adhesion is believed to be essential for the induction of such tyrosine phosphorylation (21, 22), another strong candidate responsible for the collagen-stimulated protein-tyrosine phosphorylation is a so-far uncharacterized 62-kDa membrane protein, GPVI (23, 24).

Previous clinical reports have suggested an important role of GPVI for the induction of collagen-stimulated signaling events in platelets. Despite the normal expression of alpha 2beta 1 complex, platelets lacking GPVI do not become aggregated by collagen (23, 24, 25, 26, 27) and exhibit profound defects in collagen-induced platelet responses such as calcium mobilization (25) and thromboxane synthesis (23). We demonstrated recently that signaling through GPVI is considerably dependent on the activation of PTKs in platelets and that GPVI cross-linking leads to cAMP-insensitive activation of c-Src and Syk tyrosine kinases (28). In addition, GPVI cross-linking also stimulates cAMP-insensitive tyrosine phosphorylation of PLC-gamma 2 in a manner similar to collagen stimulation (28). These observations support a concept that collagen-stimulated protein-tyrosine phosphorylation in platelets may be partly regulated through a GPVI-dependent mechanism.

To further test this hypothesis, we chose to examine whether collagen could induce protein-tyrosine phosphorylation in platelets lacking GPVI but expressing normal amounts of alpha 2beta 1 complex. In these platelets, collagen stimulated prompt activation of c-Src accompanied by tyrosine phosphorylation of a number of proteins in a manner dependent on collagen-alpha 2beta 1 interaction. However, GPVI-deficient platelets exhibited severe defects in the activation of Syk and tyrosine phosphorylation of several substrates by collagen. Our results indicated that collagen-stimulated protein-tyrosine phosphorylation in platelets are regulated through both alpha 2beta 1-dependent and GPVI-dependent mechanisms and that GPVI-mediated signaling is required for collagen-stimulated activation of Syk.


EXPERIMENTAL PROCEDURES

Reagents

Acid-soluble fibrillar collagen prepared from equine tendon was purchased from Horm-Chemie (Munich, Germany). Prostacyclin (PGI2) was kindly provided by Ono Pharmaceutical Co. (Osaka, Japan). Arg-Gly-Asp-Ser (RGDS) tetrapeptide and histone from calf thymus (subgroup f2b) were obtained from Sigma. Phenylmethylsulfonyl fluoride, aprotinin, and sodium orthovanadate was from Nacalai Tesque, Inc. (Kyoto, Japan). All other reagents were obtained as reported previously (28, 29).

Antibodies

Monoclonal antibody (mAb) against c-Src (327) was obtained from Oncogene Science, Inc. (Uniondale, NY). Anti-Syk mAb (101) was from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Anti-phosphotyrosine mAb (4G10), anti-Fak mAb (2A7), and mAb against p80/85 cortactin (4F11) were obtained from Upstate Biotechnology, Inc. (Lake Placid, NY). mAb against human alpha 2beta 1 (Gi9) was from Immunotech S.A. (Marseille, France), and anti-Fcgamma RII mAb (IV.3) was from Medarex, Inc. (Annandale, NJ). Platelet-nonbinding mouse monoclonal IgG1 (R118) was a generous gift from Dr. Kina (Chest-Disease Institute, Kyoto University). Rabbit polyclonal IgG against PLC-gamma 2 or p95Vav was purchased from Santa Cruz Biotechnology, Inc.(Santa Cruz, CA). Rabbit anti-mouse IgG and peroxidase-conjugated goat anti-mouse or anti-rabbit IgG were from Cappel Organon Teknika Co. (Durham, NC).

Preparation and Activation of Platelets

After informed consent was obtained, venous blood was collected from healthy adult donors or three donors with platelet GPVI deficiency reported previously (23, 26, 27). Washed platelets were prepared as described previously (28), suspended at 0.5 or 1.0 × 109/ml, and stimulated with 20 µg/ml of collagen while being stirred at 800 rpm in a glass cuvette. In the experiments to evaluate the effects of inhibition of collagen-alpha 2beta 1 interaction, platelet suspensions from GPVI-deficient donors were preincubated for 15 min with 20 µg/ml anti-alpha 2beta 1 mAb Gi9 to inhibit collagen-alpha 2beta 1 interaction, anti-Fcgamma RII mAb IV.3 as platelet-binding control, or mouse monoclonal IgG1 R118 as platelet-nonbinding control. In some other experiments, GPVI-deficient platelet suspensions were also pretreated with several inhibitors for platelet activation including 3 µM PGI2 (3 min), 20 µM cytochalasin D (10 min), and 1 mM RGDS (5 min).

Platelet Lysis and Immunoprecipitation

To prepare whole platelet lysates, each reaction was terminated at the indicated time points by the addition of an equal volume of 2 × SDS sample buffer (28). The samples were boiled for 5 min and subjected to immunoblotting analysis. Immunoprecipitation of each specified protein was performed essentially as described previously (28). Whereas c-Src was immunoprecipitated from lysates prepared in 1% Triton X-100-containing buffer reported by Clark and Brugge (30), the other proteins were precipitated in radioimmunoprecipitation assay (RIPA) buffer (1% Triton X-100, 0.1% SDS, 1% deoxycholate sodium, 20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 5 mM EDTA, 5 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, 20 µg/ml leupeptin, 20 µg/ml aprotinin, and 1 mM sodium orthovanadate). In some experiments, platelet lysates were incubated for 40 min with protein G-agarose (Santa Cruz Biotechnology) prior to the immunoprecipitation to remove preincubating mAbs.

Immunoblotting

Immunoblotting analysis of whole platelet lysates or immunoprecipitated proteins was performed as described previously (28). Membranes were probed as indicated with anti-phosphotyrosine 4G10 (1 µg/ml), anti-Src 327 (1 µg/ml), anti-Syk 101 (3 µg/ml), anti-Fak 2A7 (5 µg/ml), anti-cortactin 4F11 (5 µg/ml), rabbit anti-PLC-gamma 2 (1 µg/ml), or rabbit anti-Vav (1 µg/ml) followed by enhanced chemiluminescence detection of peroxidase-conjugated secondary antibody binding.

Immune Complex Kinase Assay

In vitro kinase activity of c-Src or Syk was measured as described previously (28). Histone phosphorylating activity was determined by the ratio of incorporation of 32P quantitated by a BAS 2000 imaging analyzer (Fuji) to the relative amount of immunoprecipitated c-Src or Syk estimated by densitometric analysis of quantitative immunoblots corresponding to each sample.


RESULTS

Collagen Stimulates alpha 2beta 1-dependent Tyrosine Phosphorylation of Multiple Proteins in GPVI-deficient Platelets

GPVI-deficient platelets were obtained from three subjects reported previously (23, 26, 27). Immunoblotting analyses confirmed that GPVI was undetectable in one of these patients (donor 1) (23) and less than 10% normal in the other two (donors 2 and 3) (26, 27). These platelets subnormally adhered to fibrillar collagen (types I and III) but specifically lacked collagen-induced aggregation despite expressing normal amounts of cell surface alpha 2beta 1 complex (23, 26, 27). Because protein-tyrosine phosphorylation is suggested to be one of the earliest platelet responses to collagen (22), we first examined whether the interaction with collagen promotes tyrosine phosphorylation in GPVI-deficient platelets. Although previous reports have demonstrated that GPVI-deficient platelets were almost totally defective in collagen-stimulated signaling events (23, 25), a substantial degree of rapid protein-tyrosine phosphorylation was induced in response to collagen in the platelets from all three donors we examined (Fig. 1A). Although no apparent difference was found in a profile of major tyrosine-phosphorylated protein bands (150, 130, a bundle of 68-72, and 64 kDa) between normal and GPVI-deficient platelets, time-dependent dephosphorylation of these bands was not observed in the GPVI-deficient platelets (Fig. 1B), which could be explained by the lack of alpha IIbbeta 3-mediated signaling required for the regulation of proteintyrosine phosphatases as we have demonstrated previously (31, 32).


Fig. 1. Collagen-stimulated protein-tyrosine phosphorylation in GPVI-deficient platelets. A, washed platelets obtained from a normal donor or donors with platelet GPVI deficiency (Donors 1, 2, and 3) were unstimulated (Collagen -) or stimulated with collagen for 1 min (Collagen +) under stirring conditions, lysed in SDS sample buffer, resolved by 8% SDS-polyacrylamide gel electrophoresis, transferred onto nitrocellulose membrane, and probed with anti-phosphotyrosine mAb, 4G10. In each lane, lysate from 5 × 106 platelets was loaded. The positions of molecular weight standards (in kilodaltons) are shown on the left. B, washed platelets from a normal or a donor with GPVI deficiency (donor 1) were stirred and stimulated with collagen for the times indicated below each lane, lysed, and subjected to anti-phosphotyrosine immunoblotting as described above. Similar results were obtained when platelets from donors 2 and 3 were examined. Shown in the middle are the positions of molecular weight standards.
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Integrin alpha 2beta 1 is believed to mediate primary platelet adhesion to collagen (4) and is also implicated in the induction of protein-tyrosine phosphorylation by collagen (21, 22). Confirming this, pretreatment of GPVI-deficient platelets with anti-alpha 2beta 1 mAb completely abolished collagen-induced tyrosine phosphorylation, whereas the preincubation with either platelet-binding control antibody (anti-Fc receptor mAb) or platelet-nonbinding mouse IgG1 (Fig. 2A) did not. In addition, cytochalasin D, an inhibitor of F-actin formation, also significantly abrogated the induction of such protein-tyrosine phosphorylation, whereas the presence of PGI2, which most potently inhibits platelet activation by elevating intracellular cAMP, or RGDS tetrapeptide, which blocks ligand-alpha IIbbeta 3 binding, was without effect (Fig. 2B). These findings indicated that collagen stimulates protein-tyrosine phosphorylation even in the absence of GPVI in a manner dependent on both collagen-alpha 2beta 1 interaction and subsequent cytoskeletal rearrangement.


Fig. 2. Effects of anti-alpha 2beta 1 mAb, PGI2, cytochalasin D, and RGDS on collagen-stimulated protein-tyrosine phosphorylation in GPVI-deficient platelets. A, platelet suspensions from a donor with GPVI deficiency were pretreated with anti-Fcgamma RII mAb (+ Anti-FcR), control mouse IgG1 (+ mG1), or anti-alpha 2beta 1 mAb (+ Anti-alpha 2beta 1) for 15 min, stimulated with 20 µg/ml of collagen for the times indicated below each lane, lysed, and subjected to anti-phosphotyrosine immunoblotting. B, whole lysates from unstimulated (UN, lane 1) or collagen-activated GPVI-deficient platelets (lanes 2-5) in the absence (COL, lane 2) or presence of PGI2 (PGI2/COL, lane 3), cytochalasin D (CytD/COL, lane 4), or RGDS (RGDS/COL, lane 5) were resolved on SDS-PAGE and subjected to anti-phosphotyrosine immunoblotting. In A and B, representative data of platelets obtained from a GPVI-deficient donor 1 were shown because similar results were obtained from two other GPVI-deficient donors we examined. The positions of molecular weight standards are indicated on the left.
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Collagen Does Not Stimulate Integrin-mediated Tyrosine Phosphorylation of Fak in GPVI-deficient Platelets

In normal platelets, collagen has been reported to promote activation of c-Src (14), Syk (15), and Fak (16) tyrosine kinases. Although the mechanism of how collagen-platelet interaction leads to activation of these PTKs is not fully clarified, previous analysis suggested the involvement of integrin alpha 2beta 1 in collagen-induced activation of Fak (21). However, under our experimental conditions, it appeared unlikely that Fak becomes initially activated solely by collagen-alpha 2beta 1 interaction because tyrosine phosphorylation of Fak occurred later than peak activation of c-Src and Syk in normal platelets (Fig. 3A and Fig. 4A). Furthermore, collagen did not induce tyrosine phosphorylation of Fak in GPVI-deficient platelets (Fig. 4A), suggesting that Fak is not involved in the alpha 2beta 1-mediated tyrosine phosphorylation observed in the GPVI-deficient platelets. Because collagen does not stimulate significant calcium mobilization in GPVI-deficient platelets (25), these findings may be consistent with the hypothesis suggested by Shattil et al. (33) that tyrosine phosphorylation of Fak requires both ligand-integrin interaction and signaling from other receptors leading to either calcium mobilization or protein kinase C activation.


Fig. 3. Collagen-stimulated activation of c-Src but not of Syk in GPVI-deficient platelets. A and B, washed platelets obtained from normal or GPVI-deficient donors were stirred and stimulated with 20 µg/ml of collagen for the indicated times, lysed, and precipitated with mAb against c-Src or Syk as described under "Experimental Procedures." The anti-Src immunoprecipitate was divided into two aliquots; one was analyzed on a quantitative immunoblotting with anti-Src (alpha Src), and the other was subjected to an in vitro kinase assay using histone as an exogenous substrate. Similarly, one-half of the anti-Syk immunoprecipitate was analyzed on an anti-phosphotyrosine (alpha PY) immunoblotting followed by reprobing with anti-Syk (alpha Syk), and the other half was used for an immune complex kinase assay as described above. The representative results of in vitro kinase assay are shown in A as autoradiographies exhibiting c-Src autophosphorylation (c-Src) and phosphorylation of exogenous histone by immunoprecipitated c-Src (H2b(Src)) or Syk (H2b(Syk)). Values shown in B represent the mean results of two different experiments. Anti-Src immunoprecipitates from donor 3 was not available because of the limited amount of kindly donated blood. C, GPVI-deficient platelets obtained from donor 1 were preincubated for 15 min with anti-alpha 2beta 1 mAb Gi9 or control mouse IgG1, stimulated with collagen for the indicated times under stirring conditions, lysed, and precipitated with mAb against c-Src. Prior to the immunoprecipitation, preexisting mAbs were removed from platelet lysates by protein G-agarose beads. Anti-Src immunoprecipitates were subjected to in vitro kinase assay as described above. Values represent the mean results of two different experiments.
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Fig. 4. Tyrosine phosphorylation of cortactin but not of Fak, PLC-gamma 2, and Vav in collagen-stimulated, GPVI-deficient platelets. A and B, washed platelets from a normal donor or a donor with GPVI deficiency (donor 1) were stirred and stimulated with 20 µg/ml of collagen for the times indicated below each lane, lysed in RIPA buffer, and precipitated with anti-Fak mAb 2A7 (A), anti-p80/85 mAb 4F11, rabbit anti-PLC-gamma 2, or rabbit anti-Vav (B). Immunoprecipitates (IP) with each specified antibody as shown on the right were resolved by SDS-PAGE, subjected to immunoblotting with anti-phosphotyrosine mAb 4G10 (alpha PY), and then reprobed as shown on the left with anti-cortactin (alpha p80/85), anti-PLC-gamma 2 (alpha PLCgamma 2), and anti-Vav (alpha Vav), respectively. C, anti-cortactin immunoprecipitates were prepared as described above from unstimulated (lane 1) or collagen-stimulated (lanes 2-4) GPVI-deficient platelets in the absence (lane 2) or presence of RGDS (lane 3) or anti-alpha 2beta 1 mAb Gi9 (lane 4), resolved by SDS-PAGE, subjected to anti-phosphotyrosine (alpha PY) immunoblots, then reprobed with anti-cortactin mAb (alpha p80/85). In all panels, representative data obtained from a GPVI-deficient donor 1 were shown because similar results were obtained when platelets from two other GPVI-deficient donors were likewise examined.
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Collagen Rapidly Stimulates Activation of c-Src but not of Syk in GPVI-deficient Platelets

To investigate responsible PTKs for the collagen-induced tyrosine phosphorylation in GPVI-deficient platelets, collagen-stimulated activities of c-Src and Syk were next evaluated by their in vitro histone phosphorylating activity (Fig. 3, A and B). In response to collagen, the activity of c-Src from GPVI-deficient platelets exhibited a 1.5-fold increase that was comparable to normal platelets. Such activation of c-Src was found to be also alpha 2beta 1-dependent because it was significantly inhibited in the presence of anti-alpha 2beta 1 mAb (Fig. 3C).

By sharp contrast with such rapid activation of c-Src, collagen-stimulated activation of Syk was invariably impaired in GPVI-deficient platelets. In response to collagen, Syk in normal platelets became rapidly phosphorylated on tyrosine and exhibited a 5-fold maximal increase in its activity. However, in the GPVI-deficient platelets, an increase in collagen-stimulated Syk activity was markedly blunted, and tyrosine phosphorylation of Syk was undetectable (Fig. 3, A and B). This defect in Syk activation in GPVI deficiency was specific to stimulation with collagen, because other platelet agonists such as thrombin and STA2 (an agonistic analogue of thromboxane A2) induced prompt activation and subsequent deactivation of Syk in GPVI-deficient platelets in a profile indistinguishable from that of normal platelets (data not shown).

Collagen-stimulated Tyrosine Phosphorylation of PLC-gamma 2 and Vav Is Not Observed in GPVI-deficient Platelets

Because a number of platelet agonists usually stimulate parallel activation of c-Src and Syk in normal platelets, it was highly implicative to us that c-Src was preferentially activated in collagen-treated GPVI-deficient platelets. To gain some insight into the subsequent cellular events, we examined the difference in substrates that become tyrosine-phosphorylated in response to collagen between normal and GPVI-deficient platelets (Fig. 4B). In collagen-stimulated normal platelets, we could readily detect tyrosine phosphorylation of cortactin, an F-actin binding protein originally identified in v-Src-transformed cells (34, 35), PLC-gamma 2 that may be involved in collagen-stimulated calcium mobilization (17), and Vav, an adapter oncoprotein that may participate in Syk-dependent signaling (36, 37). Tyrosine phosphorylation of these substrates by collagen was independent of platelet aggregation, because it was still observed in the presence of inhibitors for platelet aggregation such as RGDS peptide or PGI2 (data not shown). In GPVI-deficient platelets, collagen also induced tyrosine phosphorylation of cortactin in an alpha 2beta 1-dependent manner (Fig. 4, B and C) but failed to stimulate that of PLC-gamma 2 and Vav (Fig. 4B). On the other hand, in normal platelets, antibody-mediated GPVI cross-linking is sufficient to stimulate cAMP-insensitive tyrosine phosphorylation of Syk, PLC-gamma 2 (28), and Vav (data not shown). Very recently, Cichowski et al. (19) reported that collagen-stimulated tyrosine phosphorylation of Vav is an alpha 2beta 1-dependent event, but our above findings suggested an additional requirement of GPVI-mediated signaling for Vav to become phosphorylated on tyrosine.


DISCUSSION

Thus far, several clinical reports have suggested the involvement of GPVI in the events responsible for collagen-induced platelet aggregation, but its precise role in collagen-platelet interaction has been poorly defined. Although the lack of collagen-stimulated responses in alpha 2beta 1-deficient platelets is considered to be a consequence of their defective adhesion to collagen (2, 4), previous studies could not explain the mechanism of how GPVI-deficient platelets with normal amounts of alpha 2beta 1 became severely hyporesponsive to collagen. In this study, we demonstrated for the first time that collagen was capable of stimulating protein-tyrosine phosphorylation in GPVI-deficient platelets that lack most other signaling events evoked by collagen. In addition, we showed that such tyrosine phosphorylation was almost totally diminished in the presence of anti-alpha 2beta 1 mAb, indicating that collagen-alpha 2beta 1 interaction is substantially retained to stimulate protein-tyrosine phosphorylation in the absence of GPVI.

Among three PTKs that have been reported to become activated by collagen in normal platelets (14, 15, 16), we could detect increased activity of only c-Src in collagen-stimulated GPVI-deficient platelets. Such activation of c-Src that accompanied tyrosine phosphorylation of its putative substrate, cortactin (35), was also inhibited when GPVI-deficient platelets were pretreated with anti-alpha 2beta 1 mAb. These results indicated that integrin alpha 2beta 1-mediated events were responsible for c-Src-dependent protein-tyrosine phosphorylation when GPVI-deficient platelets reacted with collagen. By contrast, GPVI-deficient platelets did not exhibit detectable tyrosine phosphorylation of Syk, Fak, PLC-gamma 2, and Vav in response to collagen. Therefore, although it has been believed that protein-tyrosine phosphorylation in collagen-activated platelets is mostly dependent on collagen-alpha 2beta 1 interaction (21), our present observations indicated an additional requirement of GPVI as a signaling counterpart to complement alpha 2beta 1-mediated signalings for the collagen-stimulated tyrosine phosphorylation of certain specific substrates. Furthermore, the significant abolishment of collagen-induced c-Src activation in anti-alpha 2beta 1 mAb-treated, GPVI-deficient platelets strongly suggests that collagen-stimulated activation of c-Src and Syk in platelets are, for the most part, regulated through alpha 2beta 1 and GPVI.

Collagen-platelet interaction has been considered to be a complex process involving alpha 2beta 1-dependent and alpha 2beta 1-independent mechanisms (2, 38, 39). Although whether GPVI can mediate alpha 2beta 1-independent platelet interaction with collagen is still unclarified, a recent clinical report (7) describing that alpha 2beta 1-deficient platelets expressing normal amounts of GPVI did not respond to collagen raised a question as to the role of GPVI as an independent signaling receptor for collagen. When we tested the effects of anti-alpha 2beta 1 mAb on collagen-stimulated activation of c-Src and Syk in normal platelets, this treatment almost totally abolished such events under conditions of stasis.2 However, as partly shown in a previous report (40), we observed delayed but significant activation of both kinases by collagen when anti-alpha 2beta 1 mAb-treated normal platelets were stirred,2 suggesting that anti-alpha 2beta 1-mediated inhibition of collagen-alpha 2beta 1 interaction is insufficient for totally preventing collagen-induced activation of normal platelets, especially under dynamic conditions. Although the involvement of GPVI-mediated signaling under such conditions has yet to be elucidated, GPVI may cooperate with alpha 2beta 1-mediated signaling in a manner dependent on the extent of flow conditions where platelets react with collagen.

Another candidate regulator for collagen-stimulated protein-tyrosine phosphorylation in platelets is GPIV (CD36) (41), which is shown to associate with Src family tyrosine kinases Fyn, Lyn, and Yes (42). However, in contrast to the alpha 2beta 1-deficient or GPVI-deficient platelets, platelets lacking GPIV became normally aggregated (43) and exhibited essentially normal protein-tyrosine phosphorylation in response to collagen (44). Therefore, although GPIV has been suggested to be an important mediator of platelet adhesion to collagen (39, 45), its role in collagen-induced signaling events seems to be relatively minor when compared with those of alpha 2beta 1 or GPVI.

According to our present study, alpha 2beta 1-mediated activation of c-Src and tyrosine phosphorylation of cortactin did not link with either activation of Syk or tyrosine phosphorylation of PLC-gamma 2 and Vav in the absence of GPVI. Given the observations that the earliest known event following GPVI engagement is activation of c-Src and Syk tyrosine kinases (28) and that collagen is capable of activating c-Src also via an alpha 2beta 1-dependent mechanism as shown in this study, GPVI appears to possess a unique function unshared with alpha 2beta 1 in regulating tyrosine phosphorylation and activation of Syk. If this is the case, the lack of Syk-dependent signalings in response to collagen should be the most reasonable explanation for a distinctive molecular pathology leading to defective collagen-induced aggregation in GPVI-deficient platelets. Of note here, the essential requirement of Syk-dependent signalings for PLC-mediated calcium mobilization and subsequent cell activation was demonstrated in other cells pharmacologically or genetically engineered to compromise Syk tyrosine kinase activity (46, 47). In this sense, GPVI deficiency could be regarded as the first clinical example for an impaired cellular function due to uncoupling of ligand-cell interaction with the activation of Syk family PTKs.

In summary, we have described altered collagen-stimulated protein-tyrosine phosphorylation in GPVI-deficient platelets where alpha 2beta 1-dependent signalings were substantially retained. Although GPVI engagement leads to activation of both c-Src and Syk in platelets, collagen-stimulated activation of c-Src is also independently shared by alpha 2beta 1-mediated events in contrast to the Syk activation, which seems to exclusively require the presence of GPVI. Fig. 5 summarizes our working hypothesis for the differential roles of alpha 2beta 1 and GPVI in collagen-stimulated protein-tyrosine phosphorylation in platelets.


Fig. 5. A working hypothesis for the roles of alpha 2beta 1 and GPVI in collagen-stimulated protein-tyrosine phosphorylation in platelets. A, GPVI-deficient platelets bind to fibrillar collagen via integrin alpha 2beta 1, which leads to activation of c-Src and tyrosine phosphorylation of a number of cellular substrates including cortactin. However, when GPVI is absent, collagen-alpha 2beta 1 interaction does not promote either activation of Syk or tyrosine phosphorylation of Fak, PLC-gamma 2, and Vav. Such defects in collagen-stimulated tyrosine phosphorylation may be a responsible mechanism for defective activation of GPVI-deficient platelets in response to collagen. B, when both alpha 2beta 1 and GPVI are present, collagen-platelet interaction leads to the activation of Syk as well as c-Src followed by tyrosine phosphorylation of PLC-gamma 2 and Vav. In addition, Fak becomes also phosphorylated on tyrosine, probably through coordinated signalings from alpha 2beta 1 and GPVI. Because c-Src can be also activated by GPVI cross-linking alone in normal platelets (28), collagen-stimulated activation of c-Src appears to be regulated through either an alpha 2beta 1-dependent or GPVI-dependent mechanism. On the other hand, GPVI-mediated signalings are specifically required for the activation of Syk and tyrosine phosphorylation of several substrates such as PLC-gamma 2, Vav, and Fak following collagen-platelet interaction.
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FOOTNOTES

*   This work was supported in part by grants-in-aid from the Ministry of Education, Science, and Culture of Japan and by The Ryoichi Naito Foundation Grant for Medical Research. The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§   To whom correspondence should be addressed. Tel.: 81-75-751-3151; Fax: 81-75-751-3201.
1    The abbreviations used are: GP, glycoprotein; Fak, focal adhesion kinase; PTK, protein-tyrosine kinase; PLC, phospholipase C; PGI2, prostacyclin; mAb, monoclonal antibody.
2    T. Ichinohe, H. Takayama, and M. Okuma, unpublished observations.

Acknowledgments

We thank Dr. Tatsuo Kina for generously providing us platelet nonbinding mouse monoclonal IgG1 R118; Dr. Kenjiro Tanoue for valuable discussions; Drs. Kenjiro Tomo and Kenzo Hirai for encouragement and suggestions; and Hiroko Nakagawa and Ikuko Nakamura for excellent technical and secretarial assistance.


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