A Novel Association of Fc Receptor (cid:103) -Chain with Glycoprotein VI and Their Co-expression as a Collagen Receptor in Human Platelets*

The mechanism by which occupancy of collagen re- ceptors is coupled to platelet activation has been uncer-tain. Our group previously demonstrated that glycopro- tein (GP) VI, an uncharacterized platelet membrane protein, is specifically required for collagen-platelet in- teraction leading to activation of protein-tyrosine kinase Syk. Since collagen stimulation of platelets has recently been found to induce tyrosine phosphorylation of Fc receptor (FcR) (cid:103) -chain, a signal-generating subunit of FcR, we further investigated the relationships between FcR (cid:103) -chain and GPVI in human platelets. Our present study revealed the following. FcR (cid:103) -chain was physically and stably associated with GPVI in human platelets; both FcR (cid:103) -chain and GPVI were proportion-ally absent in GPVI-deficient platelets; GPVI cross-link- ing or collagen stimulation of platelets resulted in tyrosine phosphorylation of GPVI-associated FcR (cid:103) -chain accompanied by Syk association and activation. These findings strongly suggest that the associated complex of GPVI and FcR (cid:103) -chain is a collagen receptor featuring the signaling through immune receptors. an aggregometer with continuous stirring at 800 rpm. Platelet Lysis and Immunoprecipitation— Unstimulated or stimu- lated platelets were lysed in an ice-cold lysis buffer (16), and immunoprecipitation of each specified protein was performed as described previously (8, 10). Fusion Protein Precipitation Studies— Precipitation of platelet pro- tein with GST-Syk-SH2 was performed as described previously (16). Immunoblotting Studies— Immunoblotting analysis of whole platelet lysates, immunoprecipitated proteins, or precipitated proteins with GST-Syk-SH2 followed by enhanced chemiluminescence (ECL) detec- tion was performed as described previously (8, 10). When immunoblotting was performed with biotinylated anti-GPVI IgG, the blots were then incubated with avidin-horseradish peroxidase and developed with ECL. In some experiments, immunoblotted protein bands were meas- ured by densitometry (Densitorol DMU-33C, Toyo Kagaku Sangyo Ltd., Osaka, Japan). Absorption of proteins in normal control samples was set to 100%. buffer.

Despite the widely accepted consensus that the interactions between the extracellular matrix protein collagen and platelets are vital for the maintenance of hemostasis, the exact nature of collagen receptor on platelets has been a great enigma to date except for well characterized integrin heterodimer ␣ 2 ␤ 1 (1,2), also called glycoprotein (GP) 1 Ia-IIa, as a principal adhesion receptor for collagen. Among several candidates that have been proposed to be platelet collagen receptors (3)(4)(5)(6)(7), we have recently provided biochemical evidence that GPVI, as yet an unidentified 62-kDa platelet membrane protein (6,7), is specifically required for collagen-induced platelet activation other than GPIa-IIa (integrin ␣ 2 ␤ 1 ). GPVI cross-linking with the F(abЈ) 2 fragments of anti-GPVI IgG (F(abЈ) 2 ␣GPVI) induces cAMP-insensitive activation of protein-tyrosine kinase Syk accompanied by tyrosine phosphorylation of phospholipase C␥2 (PLC␥2) in a manner similar to collagen stimulation (8); GPVIdeficient platelets (6,7,9) expressing a normal amount of GPIa-IIa exhibit lack of collagen-stimulated Syk activation and tyrosine phosphorylation of PLC␥2 (10). However, the question of how GPVI is involved in collagen receptor and transduces signals leading to Syk activation accompanied by tyrosine phosphorylation of PLC␥2 still remains unsolved.
One of the mechanisms by which Syk is activated is achieved via interaction between its tandem Src homology 2 (SH2) domains and a tyrosine-phosphorylated activation motif, termed the immunoreceptor tyrosine-based activation motif, found in receptors of the immune system or their associated chains (11). In platelets, this mechanism of Syk activation is a prerequisite for the activation through a low affinity Fc receptor for IgG (Fc␥R) (12,13). Among known Fc receptors belonging to the immunoglobulin superfamily (14), human platelets express only a single Fc␥R encoded by the Fc␥RIIA gene (15). Recently Gibbins et al. (16) demonstrated the presence of the immunoreceptor tyrosine-based activation motif-containing Fc receptor (FcR) ␥-chain, a 20-kDa disulfide-linked, homodimeric, signalgenerating subunit in human platelets, and showed its tyrosine phosphorylation, Syk association, and subsequent phosphorylation of PLC␥2 upon collagen stimulation but not upon Fc␥RIIA cross-linking. FcR ␥-chain has been shown to be associated with all three classes of Fc␥R, the high affinity IgE FcR (Fc⑀RI) and the IgA FcR (Fc␣R), and the T-cell receptor-CD3 complex (14,(17)(18)(19)(20)(21). Since platelets express Fc␥RIIA but lack these other known FcR ␥-chain-associated membrane proteins, FcR ␥-chain may be associated with a novel counterpart in platelets.
Therefore, we hypothesized that FcR ␥-chain might associate with GPVI composing a collagen receptor and play a pivotal role in collagen-induced Syk activation through GPVI in human platelets. In testing this hypothesis, we studied the following points: whether a complex of FcR ␥-chain and GPVI can be demonstrated in human platelets; how FcR ␥-chain is present in GPVI-deficient platelets; whether GPVI cross-linking of platelets with F(abЈ) 2 ␣GPVI induces tyrosine phosphorylation of FcR ␥-chain and its association with Syk; and how GPVI is involved in the binding of the tandem SH2 domains of Syk to FcR ␥-chain in collagen-stimulated platelets. Here we report that FcR ␥-chain is physically and stably associated with platelet membrane GPVI but not expressed in GPVI-deficient platelets and also that GPVI cross-linking or collagen stimulation of platelets induces tyrosine phosphorylation of GPVI-associated FcR ␥-chain accompanied by Syk association and activation. * This work was supported by grants-in-aid for scientific research from the Ministry of Education, Science, and Culture of Japan, by the Ryoichi Naito Foundation Grant for Medical Research, and by the Yamanouchi Foundation for Research on Metabolic Disorders. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Materials-Glutathione S-transferase
Preparation of anti-GPVI IgG and F(abЈ) 2 ␣GPVI was performed as described previously (8), using the serum of a patient with GPVI deficiency (6) who was followed up as an outpatient in our department. For immunoblotting, anti-GPVI IgG was labeled with biotin using a standard method as described previously (26).
Preparation and Stimulation of Platelets-Blood samplings were performed by venipuncture from healthy donors or from patients with GPVI-deficient platelets with informed consent. Anticoagulation of blood and preparation of washed platelets were performed as described previously (8). Washed platelets (1.0 ϫ 10 9 cells/ml) were stimulated with 150 g/ml F(abЈ) 2 ␣GPVI or 100 g/ml collagen for the indicated times at 37°C in an aggregometer with continuous stirring at 800 rpm.
Platelet Lysis and Immunoprecipitation-Unstimulated or stimulated platelets were lysed in an ice-cold lysis buffer (16), and immunoprecipitation of each specified protein was performed as described previously (8,10).
Fusion Protein Precipitation Studies-Precipitation of platelet protein with GST-Syk-SH2 was performed as described previously (16).
Immunoblotting Studies-Immunoblotting analysis of whole platelet lysates, immunoprecipitated proteins, or precipitated proteins with GST-Syk-SH2 followed by enhanced chemiluminescence (ECL) detection was performed as described previously (8,10). When immunoblotting was performed with biotinylated anti-GPVI IgG, the blots were then incubated with avidin-horseradish peroxidase and developed with ECL. In some experiments, immunoblotted protein bands were measured by densitometry (Densitorol DMU-33C, Toyo Kagaku Sangyo Ltd., Osaka, Japan). Absorption of proteins in normal control samples was set to 100%.
Preparation of Erythrocytes, Granulocytes, and Lymphocytes-Washed erythrocytes were prepared and erythrocyte membranes were obtained by hemolysing cells as described previously (27). Erythrocyte hemoglobin-free membrane was lysed in the lysis buffer.
Washed granulocytes and lymphocytes were prepared with a modification of a previously described method (28). Briefly, after removing of platelet-rich plasma, erythrocytes were eliminated from the remaining blood by dextran sedimentation. The upper leukocyte layer was centrifuged over Ficoll-Paque (Pharmacia Biotech Inc.) at 400 ϫ g for 30 min. Each granulocyte-or lymphocyte-containing fraction was taken and washed once with phosphate-buffered saline. Contaminating erythrocytes in the granulocyte pellets were lysed in hypotonic saline. The granulocytes or lymphocytes were further washed and lysed in the lysis buffer.
Protein concentration in cell lysates was measured with BCA protein assay reagent (Pierce). After addition of SDS sample buffer and boiling, the samples containing an equal amount of protein (20 g/lane) were subjected to SDS-PAGE and subsequent immunoblotting as described above.

RESULTS AND DISCUSSION
FcR ␥-Chain Is Physically Associated with Platelet Membrane GPVI-We first examined whether there is physical association between FcR ␥-chain and GPVI. Cell lysates of unstimulated washed platelets were prepared and tested for the interaction between FcR ␥-chain and GPVI by immunoprecipitation, followed by immunoblotting with anti-FcR ␥-chain serum and anti-GPVI IgG. When FcR ␥-chain and associated proteins were immunoprecipitated from platelet cell lysates with anti-FcR ␥-chain serum, GPVI was co-precipitated with FcR ␥-chain (Fig. 1A). Conversely, when GPVI and associated proteins were immunoprecipitated from platelet cell lysates with anti-GPVI IgG, FcR ␥-chain was co-precipitated with GPVI (Fig. 1B). Immunoblotting with anti-FcR ␥-chain serum revealed the presence of an apparent doublet of FcR ␥-chain, which has been observed previously (16,29). We further studied the possible association of FcR ␥-chain with integrin ␣ 2 ␤ 1 , Fc␥RII, or GPIV (CD36), which is another candidate for collagen receptor (3). Unstimulated washed human platelets were lysed and subjected to immunoprecipitation with antibodies specific to integrin ␣ 2 ␤ 1 , Fc␥RII, or GPIV. Immunoblotting with anti-FcR ␥-chain serum revealed the absence of FcR ␥-chain in those immunoprecipitates (Fig. 1B). These findings demonstrate that FcR ␥-chain is physically associated with GPVI but not with integrin ␣ 2 ␤ 1 , Fc␥RII, or GPIV in human platelets.
FcR ␥-Chain Is Absent Specifically in GPVI-deficient Platelets-We then questioned how FcR ␥-chain is present in GPVIdeficient platelets. Whole cell lysates of platelets obtained from a patient with complete GPVI deficiency (6) were tested for the presence of FcR ␥-chain by immunoblotting with anti-FcR ␥-chain serum. Surprisingly, FcR ␥-chain was not immunodetected with anti-FcR ␥-chain serum in the GPVI-deficient platelets, indicating the complete deficiency of FcR ␥-chain in these platelets ( Fig. 2A). We next examined the presence of FcR ␥-chain in the other peripheral blood cells such as granulocytes, lymphocytes, and erythrocytes obtained from the same patient (Fig. 2B). The results showed that FcR ␥-chain was normally present in those blood cells compared with normal control. Therefore, these data indicate that the expression of FcR ␥-chain is altered specifically in GPVI-deficient platelets. This suggested to us that FcR ␥-chain might associate only with GPVI and require GPVI for its expression in human platelets. If so, the amount of FcR ␥-chain should be proportional to that of GPVI in human platelets. The same study as described above was then performed using another patient, who showed incomplete deficiency of GPVI in platelets (9). Densitometric analysis of the levels of FcR ␥-chain and GPVI revealed that both FcR ␥-chain and GPVI were approximately 10% normal in platelets ( Fig. 2A), while FcR ␥-chain was intact in the other peripheral blood cells (data not shown), demonstrating the proportional co-deficiency of FcR ␥-chain with GPVI in GPVI-deficient platelets. Therefore, we concluded that both GPVI and FcR ␥-chain were co-expressed in human platelets and that the lack of GPVI might cause the expression deficiency of FcR ␥-chain. Although it has been reported that the expression of some of FcR requires the expression of FcR ␥-chain (30,31), this may suggest another possibility that the expression of FcR ␥-chain also requires its counterpart.
F(abЈ) 2 ␣GPVI Induces Tyrosine Phosphorylation of GPVI-associated FcR ␥-Chain Followed by Syk Association-We reported previously that the cross-linking of platelet GPVI with F(abЈ) 2 ␣GPVI-induced activation of Syk in a similar manner to collagen stimulation (8). Therefore we questioned how GPVIassociated FcR ␥-chain is involved in GPVI cross-linking or collagen-induced activation of Syk. Washed human platelets were stimulated with F(abЈ) 2 ␣GPVI for various times up to 10 min and lysed for immunoprecipitation with anti-FcR ␥-chain serum. The immunoprecipitates were subjected to immunoblotting analysis with anti-FcR ␥-chain serum, anti-Syk antibody, FIG. 2. Proportional co-deficiency of FcR ␥-chain with GPVI in GPVI-deficient platelets. A, washed platelets were obtained from one patient (Pt1) with complete deficiency of platelet GPVI, another patient (Pt2) with incomplete deficiency of platelet GPVI, and a normal subject (N) and lysed in the lysis buffer. Whole cell lysates were resolved on SDS-PAGE and immunoblotted (WB) with anti-FcR ␥-chain serum (␣FcR ␥-chain) or biotinylated anti-GPVI IgG (␣GPVI IgG) as described in Fig. 1. B, washed granulocytes (Gr), lymphocytes (Lym), and erythrocytes (Ery) were obtained from normal control (N) and the patient (Pt1). Immunoblotting (WB) with anti-FcR ␥-chain serum or biotinylated anti-GPVI IgG was performed as described in Fig. 1. The data of platelets (Plt) obtained from normal control are shown for comparison. The same results were also confirmed by the other patient (data not shown).

FIG. 4. Tyrosine phosphorylation of GPVI-associated FcR
␥-chain and its association with the SH2 domains of Syk upon collagen stimulation. Washed platelets were stimulated with collagen (100 g/ml) for various times up to 10 min and lysed in the lysis buffer. Both GST fusion protein containing the tandem SH2 domains of Syk (GST-Syk-SH2) and GST alone as a control on glutathione-agarose were used to analyze their binding proteins in the whole cell lysates. Precipitated proteins with GST-Syk-SH2 or GST were resolved on SDS-PAGE and immunoblotted (WB) with biotinylated anti-GPVI IgG (␣GPVI IgG), anti-FcR ␥-chain serum (␣FcR␥-chain), or anti-phosphotyrosine antibody (␣PY). The blots were then incubated with avidin-or anti-IgG-horseradish peroxidase and developed with ECL.
anti-GPVI IgG, or anti-phosphotyrosine antibody. As shown in Fig. 3, FcR ␥-chain was tyrosine-phosphorylated in a time-dependent manner. Immunoblot analysis with anti-FcR ␥-chain serum confirmed similar and specific efficiencies of immunoprecipitaion of FcR ␥-chain. Protein-tyrosine kinase Syk became associated with FcR ␥-chain in a time-dependent manner upon GPVI cross-linking, and the associated Syk was tyrosinephosphorylated. During these events, FcR ␥-chain was stably associated with GPVI. Similar findings were also obtained with collagen-stimulated platelets: collagen induced time-dependent tyrosine phosphorylation of FcR ␥-chain, which was stably associated with GPVI and recruited Syk (data not shown). Since parallel increases in the activity of Syk with its tyrosinephosphorylated state are well known and also confirmed in our previous reports (8,10), these data strongly suggest that GPVI engagement including collagen stimulation causes tyrosine phosphorylation of FcR ␥-chain, which is involved in recruitment and activation of Syk.
Binding of the Tandem SH2 Domains of Syk to Tyrosinephosphorylated FcR ␥-Chain Associated with GPVI in Collagen-stimulated Platelets-It has been demonstrated that Syk binds tyrosine-phosphorylated FcR ␥-chain through its tandem SH2 domains (11). To show the relationships between GPVI, FcR ␥-chain, and the tandem SH2 domains of Syk in collagenstimulated platelets, we used GST-Syk-SH2. Following collagen stimulation, platelets were lysed and the lysates were incubated with GST-Syk-SH2. Precipitated proteins with GST-Syk-SH2 were resolved on SDS-PAGE and subjected to immunoblotting analysis with anti-FcR ␥-chain serum, anti-GPVI IgG, or anti-phosphotyrosine antibody. As shown in Fig. 4, the co-precipitation of FcR ␥-chain with the fusion protein reached a maximum after 60 s of collagen stimulation and declined thereafter. Both GPVI and FcR ␥-chain showed similar timedependent appearances in the fusion protein precipitates, where FcR ␥-chain was tyrosine-phosphorylated but GPVI was not (data not shown). Therefore, taken together with our previous findings that GPVI is specifically required for collagenplatelet interaction leading to Syk activation (8,10), these data indicate that collagen signals through GPVI, and induces tyrosine phosphorylation of its associated FcR ␥-chain, followed by the recruitment of Syk through the tandem SH2 domains.
FcR ␥-chain has been widely accepted to be involved in immune responses in association with Fc receptors and T-cell receptor. Our present work demonstrates that FcR ␥-chain is associated and co-expressed with a so far uncharacterized membrane GPVI in human platelets and that this novel association plays essential roles in tyrosine phosphorylation of FcR ␥-chain followed by the association of Syk, which is triggered by signaling through GPVI upon collagen stimulation. This could emphasize that FcR ␥-chain is involved not only in immune responses but also in a nonimmune response with a novel counterpart GPVI as shown here.
Although we do not deny that GPVI may associate with more than one protein, our findings are relevant to the concept that the associated complex between GPVI and FcR ␥-chain is a collagen receptor featuring the signaling through immune receptors.