Key points are not available for this paper at this time.
Glycoprotein VI (GPVI) is a platelet-specific glycoprotein that has been indicated to react with collagen and activate platelets. Its structure was recently identified by cDNA cloning (Clemetson, J. M., Polgar, J., Magnenat, E., Wells, T. N., and Clemetson, K. J. (1999) J. Biol. Chem. 274, 29019–29024). However, the mechanism of the interaction between collagen and GPVI has not been analyzed in detail because both collagen and GPVI are insoluble molecules. In this study, we expressed the extracellular domain of GPVI as soluble forms as follows: the monomeric form (GPVIex) and the dimeric form of GPVI fused with the human immunoglobulin Fc domain (GPVI-Fc2). Purified GPVIex strongly inhibited convulxin (Cvx)-induced platelet aggregation but only weakly inhibited that induced by collagen-related peptide. However, only GPVI-Fc2, and not GPVIex, inhibited collagen-induced platelet aggregation. The dimeric form of GPVI exhibits high affinity for collagen, as concluded from measurements of GPVI binding to immobilized collagen by both the enzyme-linked immunosorbent assay and surface plasmon resonance methods. GPVI-Fc2 bound to the surface of immobilized collagen with a dissociation constant (K D) of 5.76 × 10−7m, but the binding of GPVIex was too weak to allow estimation of this parameter. Cvx did not inhibit the binding of dimeric GPVI to collagen, indicating that the binding site of GPVI to collagen was different from that to Cvx. Taken together, our data indicate that the high affinity binding site for collagen is composed from two chains of GPVI. Furthermore, they suggest that the binding sites for Cvx are different from the collagen-binding sites and do not need to be formed by two GPVI molecules. Because dimeric GPVI is the only form that shows high affinity to fibrous collagen, our results indicate that GPVI would be present as a dimeric form on the platelet. Moreover, surface plasmon resonance indicated that there is no detectable interaction between soluble collagen and GPVI, supporting our previous observation that GPVI only reacts with fibrous collagen. Glycoprotein VI (GPVI) is a platelet-specific glycoprotein that has been indicated to react with collagen and activate platelets. Its structure was recently identified by cDNA cloning (Clemetson, J. M., Polgar, J., Magnenat, E., Wells, T. N., and Clemetson, K. J. (1999) J. Biol. Chem. 274, 29019–29024). However, the mechanism of the interaction between collagen and GPVI has not been analyzed in detail because both collagen and GPVI are insoluble molecules. In this study, we expressed the extracellular domain of GPVI as soluble forms as follows: the monomeric form (GPVIex) and the dimeric form of GPVI fused with the human immunoglobulin Fc domain (GPVI-Fc2). Purified GPVIex strongly inhibited convulxin (Cvx)-induced platelet aggregation but only weakly inhibited that induced by collagen-related peptide. However, only GPVI-Fc2, and not GPVIex, inhibited collagen-induced platelet aggregation. The dimeric form of GPVI exhibits high affinity for collagen, as concluded from measurements of GPVI binding to immobilized collagen by both the enzyme-linked immunosorbent assay and surface plasmon resonance methods. GPVI-Fc2 bound to the surface of immobilized collagen with a dissociation constant (K D) of 5.76 × 10−7m, but the binding of GPVIex was too weak to allow estimation of this parameter. Cvx did not inhibit the binding of dimeric GPVI to collagen, indicating that the binding site of GPVI to collagen was different from that to Cvx. Taken together, our data indicate that the high affinity binding site for collagen is composed from two chains of GPVI. Furthermore, they suggest that the binding sites for Cvx are different from the collagen-binding sites and do not need to be formed by two GPVI molecules. Because dimeric GPVI is the only form that shows high affinity to fibrous collagen, our results indicate that GPVI would be present as a dimeric form on the platelet. Moreover, surface plasmon resonance indicated that there is no detectable interaction between soluble collagen and GPVI, supporting our previous observation that GPVI only reacts with fibrous collagen. Collagen is one of the major components of the vessel wall. When the vessel wall becomes damaged, platelets adhere to and are activated on the exposed collagen surface, leading to thrombus formation. Many proteins on the platelet surface were reported to be putative collagen receptors, but among them, only two glycoproteins have properties consistent with them being relevant collagen receptors under normal physiological conditions: one is glycoprotein (GP) 1The abbreviations used for: GP, glycoprotein; PBS, phosphate-buffered saline; CRP, collagen-related peptide; ELISA, enzyme-linked immunosorbent assay; SPR, surface plasmon resonance; Cvx, convulxin; HEK, human embryonic kidney; PRP, platelet-rich plasma. VI and the other is integrin α2β1 (GPIa/IIa). Platelets deficient in either integrin α2β1 (1Handa M. Watanabe K. Kawai Y. Kamata T. Koyama T. Nagai H. Ikeda Y. Thromb. Haemostasis. 1995; 73: 521-528Crossref PubMed Scopus (89) Google Scholar, 2Nieuwenhuis H.K. Akkerman J.W. Houdijk W.P. Sixma J.J. Nature. 1985; 318: 470-472Crossref PubMed Scopus (399) Google Scholar) or GPVI (3Sugiyama T. Okuma M. Ushikubi F. Sensaki S. Kanaji K. Uchino H. Blood. 1987; 69: 1712-1720Crossref PubMed Google Scholar, 4Moroi M. Jung S.M. Okuma M. Shinmyozu K. J. Clin. Invest. 1989; 84: 1440-1445Crossref PubMed Scopus (371) Google Scholar) show loss of reactivity toward collagen, and antibodies against integrin α2β1, such as 6F1 (5Coller B.S. Beer J.H. Scudder L.E. Steinberg M.H. Blood. 1989; 74: 182-192Crossref PubMed Google Scholar) and P1E6 (6Keely P.J. Parise L.V. J. Biol. Chem. 1996; 271: 26668-26676Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar), and the Fab fragment of an anti-GPVI antibody (7Sugiyama T. Ishibashi T. Okuma M. Int. J. Hematol. 1993; 58: 99-104PubMed Google Scholar) inhibited collagen-induced platelet aggregation. Snake venom convulxin (Cvx) (8Francischetti I.M. Saliou B. Leduc M. Carlini C.R. Hatmi M. Randon J. Faili A. Bon C. Toxicon. 1997; 35: 1217-1228Crossref PubMed Scopus (96) Google Scholar) and collagen-related peptide (CRP), which mimic the collagen triple helix (9Morton L.F. Hargreaves P.G. Farndale R.W. Young R.D. Barnes M.J. Biochem. J. 1995; 306: 337-344Crossref PubMed Scopus (285) Google Scholar), can each activate platelets by binding specifically to GPVI, so both are useful tools for analyzing the function of GPVI. A number of studies showed that the binding of collagen, CRP, and Cvx to GPVI induced platelet activation through tyrosine phosphorylation of the Fc receptor γ-chain, Syk, phospholipase C, and many other proteins (10Watson S. Berlanga O. Best D. Frampton J. Platelets. 2000; 11: 252-258Crossref PubMed Scopus (70) Google Scholar), thus indicating that GPVI is a key receptor for collagen-induced platelet activation. The cDNA cloning of GPVI revealed that GPVI belongs to the immunoglobulin superfamily that contains two C2 immunoglobulin-like domains and an Arg residue in the transmembrane region that makes a salt bridge with the Asp residue of the Fc receptor γ-chain (11Clemetson J.M. Polgar J. Magnenat E. Wells T.N. Clemetson K.J. J. Biol. Chem. 1999; 274: 29019-29024Abstract Full Text Full Text PDF PubMed Scopus (366) Google Scholar, 12Miura Y. Ohnuma M. Jung S.M. Moroi M. Thromb. Res. 2000; 98: 301-309Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 13Jandrot-Perrus M. Busfield S. Lagrue A.H. Xiong X. Debili N. Chickering T. Le Couedic J.P. Goodearl A. Dussault B. Fraser C. Vainchenker W. Villeval J.L. Blood. 2000; 96: 1798-1807Crossref PubMed Google Scholar). Although its structure was identified, the mechanism for the reaction of GPVI with collagen remains unclear. Our previous results obtained by analyzing platelet binding to fibrous collagen suggested that GPVI is reactive only with fibrous collagen and not reactive with soluble collagen (4Moroi M. Jung S.M. Okuma M. Shinmyozu K. J. Clin. Invest. 1989; 84: 1440-1445Crossref PubMed Scopus (371) Google Scholar, 18Moroi M. Jung S.M. Shinmyozu K. Tomiyama Y. Ordinas A. Diaz-Ricart M. Blood. 1996; 88: 2081-2092Crossref PubMed Google Scholar). In the present study, to facilitate the analysis of these complex reactions, we used a simplified model system in which we assessed the collagen binding ability of two forms of the extracellular domain of GPVI. We expressed the extracellular domain of GPVI as the soluble monomeric protein (GPVIex) and the soluble dimer consisting of two molecules of the fusion protein GPVI-Fc domain (from human immunoglobulin) (GPVI-Fc2). The dimeric form, and not the monomeric form, shows high affinity to collagen, and only GPVI-Fc2 inhibited collagen-induced platelet aggregation. These results indicated that the specific with high affinity for fibrous collagen is from two GPVI molecules and thus suggested that GPVI would be present as a dimeric form on the platelet surface, with two GPVI molecules being by the Fc receptor The cDNA of GPVI the extracellular two domains of human GPVI the Y. Ohnuma M. Jung S.M. Moroi M. Thromb. Res. 2000; 98: 301-309Abstract Full Text Full Text PDF PubMed Scopus (46) Google was obtained by the GPVI cDNA as the and the site and the site as the and was The was by a with and to the The contains and the and the obtained fusion protein contains these its The was The obtained was by and The between the and GPVI was by the to the The of this was by of the a dimeric GPVI, of the human immunoglobulin Fc domain was from T. T. S. 1993; Full Text PDF PubMed Scopus Google Scholar) and as and to the site of was as embryonic were with the or the C. H. Biol. 1987; PubMed Scopus Google Scholar). protein monomeric GPVI (GPVIex) and dimeric GPVI-Fc were in the of GPVI-Fc the was and the obtained was to a of protein with phosphate-buffered GPVI-Fc2 protein was by The were by an and to a of with GPVIex was a and a The of the GPVIex protein was to be by an the assay to against platelet were against and were with of collagen in was immobilized to a each with of in The were with and with and for the ELISA, of GPVI-Fc2 in were to the and for with of Fc antibody was to the and for The were with and was an for to the were in and with different GPVI to was as the and each of the binding was by this the of of monomeric GPVIex on binding to collagen, GPVIex of was with of antibody for and with immobilized collagen. The binding of GPVIex to the collagen surface was by the of Cvx bound to the with the immobilized collagen for and the with the were with of Cvx for and the bound Cvx was with a The was as indicated the GPVIex binding the of bound GPVIex was the antibody by the with the the of GPVIex bound to immobilized collagen or were by each with of antibody for with of for and as the of and Cvx on GPVI-Fc2 binding to collagen, GPVI-Fc2 was with of or Cvx, and the binding to collagen was by the were expressed as of the and as the from were with the system collagen in was to a an to the GPVI-Fc2 in was to a by the of the collagen surface was by of through the two A surface was with the in the of and with GPVIex and GPVI-Fc2 of were the immobilized collagen a of and the resonance were The of the surface was by that of the surface, and the data thus obtained were analyzed by of the binding with The binding of soluble collagen was by soluble collagen in the surface of immobilized a of The of soluble collagen was in detail S.M. Moroi M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). was from the of of The platelet-rich was with the of and for to the platelets. The obtained platelets were with and the platelets were with of × aggregation was by a with or platelets in Platelets were activated with collagen or Cvx and the of was was by the of (9Morton L.F. Hargreaves P.G. Farndale R.W. Young R.D. Barnes M.J. Biochem. J. 1995; 306: 337-344Crossref PubMed Scopus (285) Google Scholar) as M. Jung S.M. Shinmyozu K. Tomiyama Y. Ordinas A. Diaz-Ricart M. Blood. 1996; 88: 2081-2092Crossref PubMed Google Scholar). has a platelet of or Cvx was from to the (8Francischetti I.M. Saliou B. Leduc M. Carlini C.R. Hatmi M. Randon J. Faili A. Bon C. Toxicon. 1997; 35: 1217-1228Crossref PubMed Scopus (96) Google Scholar). Cvx was by as Y. Ohnuma M. Jung S.M. Moroi M. Thromb. Res. 2000; 98: 301-309Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). and were obtained from The antibody was from or The extracellular domain of GPVI was expressed as the monomeric fusion protein and as the dimeric human immunoglobulin Fc domain fusion These were expressed as soluble proteins a which and of the expressed as E. of the were obtained from the for both forms of GPVI. The of GPVIex is and under and in and GPVI-Fc2 has a of and under the and in and The of the GPVI-Fc2 was to be from not that the GPVI-Fc2 is present as a Cvx Y. Ohnuma M. Jung S.M. Moroi M. Thromb. Res. 2000; 98: 301-309Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar) and GPVI antibody J. S. M. Y. T. K. and S. M. for GPVI and GPVIex and GPVI-Fc2 only under the that these have to that of GPVI not we the of the monomeric and the dimeric forms of GPVI against platelet aggregation. in GPVIex did not inhibit collagen-induced platelet aggregation a high but did inhibit platelet weakly In GPVI-Fc2 strongly inhibited platelet aggregation. platelet aggregation was inhibited by both GPVIex and GPVI-Fc2 of and not of GPVIex and GPVI-Fc2 on platelet of GPVIex and were analyzed as in the of of was used as the GPVIex did not inhibit collagen-induced platelet aggregation a of but GPVI-Fc2 collagen-induced platelet aggregation These results indicate that the two forms of GPVI show different binding to collagen and the The binding of monomeric and dimeric to immobilized was analyzed by the proteins showed and binding to the surface did not the binding of either of the to and collagen not Although is to the of of GPVIex and GPVI-Fc2 because we used different antibodies to these we not binding of GPVIex to and the was by a only a in was a high of GPVIex, not In to GPVIex, the of GPVI-Fc2 to and were each These results indicated that collagen to dimeric The results suggested that the of GPVI be to a specific with a high affinity for collagen. this we the monomeric GPVIex with the antibody which to dimeric GPVIex because each of the antibody to one GPVIex through the its the of on the affinity of GPVIex for collagen. GPVIex with binding to collagen, with the binding on the of to the of the binding was with a of binding that of GPVIex When the binding of GPVIex was with that of GPVI-Fc2, from the the the binding of GPVIex to the of binding obtained indicating that the binding of the GPVIex was of that of GPVI-Fc2 not These indicate that an of the binding of GPVIex to collagen, which would be by a in the of GPVIex the of the antibody to GPVIex, there is a for antibody with only one GPVIex and thus not a These results the that the of GPVI would be for the high affinity binding of GPVI-Fc2 to collagen and the that the high affinity is to GPVI-Fc2 a different from that of Because Cvx and were reported to be specific for GPVI, we ability to with collagen for the binding to GPVI-Fc2 by the inhibited the binding of GPVI-Fc2 to immobilized collagen in a The for of binding was to be However, Cvx did not inhibit the binding of GPVI-Fc2 to collagen but the binding to collagen A for the of Cvx can be on the of its structure J. Clemetson J.M. M. Magnenat Wells T.N. Clemetson K.J. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). Cvx has binding sites for GPVI, and GPVI with Cvx sites different from the collagen-binding a Cvx would be to molecules of GPVI and to GPVI the collagen Furthermore, Cvx did not inhibit GPVI-Fc2 binding to collagen, that the site of GPVI is different from its collagen-binding Because the binding to collagen was inhibited by CRP, the collagen-binding site of GPVI be the or with that for analysis of the binding of collagen and the was by the Collagen and were immobilized on under and the between GPVI-Fc2 or GPVIex and immobilized collagen or were under physiological The different were obtained and by the from the collagen or The data obtained from are in In this dissociation were obtained by two on and and from the binding resonance the indicated that our data to a model a that there be a of the complex the of GPVI-Fc2 with collagen However, in the the are not different from of the and the is a of the of the complex to GPVI of GPVI-Fc2 indicated that dimeric GPVI has affinity to collagen The binding of GPVIex to the collagen surface was so weak that not be from the obtained data In to collagen, the of GPVIex for was being × in the × × × × × × × × in a in the × × × × × × × × × × in a of the interaction of GPVIex with immobilized collagen and The of GPVIex to was with immobilized collagen and immobilized The data were as indicated in resonance In the the interaction of GPVI and immobilized collagen was These immobilized would fibrous collagen because the was under physiological the collagen was immobilized to the under the interaction of GPVI with only the soluble form of collagen, we soluble collagen as under the and we analyzed the interaction by the in the that there is no interaction of the immobilized GPVI-Fc2 with soluble collagen, the of soluble collagen not be because only a of soluble collagen be that GPVI not have affinity for soluble collagen, and the interaction between immobilized collagen and GPVI is to the interaction with fibrous collagen GPVI is a platelet-specific protein structure was recently identified from cDNA cloning (11Clemetson J.M. Polgar J. Magnenat E. Wells T.N. Clemetson K.J. J. Biol. Chem. 1999; 274: 29019-29024Abstract Full Text Full Text PDF PubMed Scopus (366) Google Scholar, 12Miura Y. Ohnuma M. Jung S.M. Moroi M. Thromb. Res. 2000; 98: 301-309Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 13Jandrot-Perrus M. Busfield S. Lagrue A.H. Xiong X. Debili N. Chickering T. Le Couedic J.P. Goodearl A. Dussault B. Fraser C. Vainchenker W. Villeval J.L. Blood. 2000; 96: 1798-1807Crossref PubMed Google Scholar). as a physiological collagen receptor on its function is to to collagen and activate as concluded from studies on platelets from (3Sugiyama T. Okuma M. Ushikubi F. Sensaki S. Kanaji K. Uchino H. Blood. 1987; 69: 1712-1720Crossref PubMed Google Scholar, 4Moroi M. Jung S.M. Okuma M. Shinmyozu K. J. Clin. Invest. 1989; 84: 1440-1445Crossref PubMed Scopus (371) Google Scholar, 18Moroi M. Jung S.M. Shinmyozu K. Tomiyama Y. Ordinas A. Diaz-Ricart M. Blood. 1996; 88: 2081-2092Crossref PubMed Google Scholar). has been that the high affinity interaction of platelets with collagen through integrin α2β1, collagen in platelet and the affinity interaction between collagen and GPVI to activation in platelets (10Watson S. Berlanga O. Best D. Frampton J. Platelets. 2000; 11: 252-258Crossref PubMed Scopus (70) Google Scholar, M. Jung S.M. Thromb. Haemostasis. 1997; PubMed Scopus Google Scholar). Although studies on the interaction between integrin α2β1 and collagen have been S.M. Moroi M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google S.M. Moroi M. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, S.M. Moroi M. J. Biochem. PubMed Scopus (46) Google Scholar), no of the interaction between GPVI and collagen have been In this study, we soluble forms of GPVI, and we analyzed interaction with collagen. a of the we the of the in the The the of the which be to the in the of GPVI not We expressed the extracellular domain of GPVI with and the (GPVIex) and the fused form of this extracellular domain with the Fc domain (GPVI-Fc2). GPVIex is a monomeric form, and GPVI-Fc2 is a dimeric form, in which two GPVI-Fc molecules are by formed from the in the Fc domain of each GPVI has been indicated to form a complex with Fc receptor γ-chain through the between Arg of GPVI and Asp of the γ-chain in the transmembrane domains Y. Ohnuma M. Jung S.M. Moroi M. Thromb. Res. 2000; 98: 301-309Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, C. H. D. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). Because γ-chain is present as a dimer by a GPVI be present as a dimer form on the platelet M. Busfield S. Lagrue A.H. Xiong X. Debili N. Chickering T. Le Couedic J.P. Goodearl A. Dussault B. Fraser C. Vainchenker W. Villeval J.L. Blood. 2000; 96: 1798-1807Crossref PubMed Google Scholar) reported that the GPVI extracellular domain fusion protein inhibited collagen-induced platelet aggregation collagen was with Our data indicating that only GPVI-Fc2, and not GPVIex, inhibited collagen-induced platelet aggregation is consistent with the of M. Busfield S. Lagrue A.H. Xiong X. Debili N. Chickering T. Le Couedic J.P. Goodearl A. Dussault B. Fraser C. Vainchenker W. Villeval J.L. Blood. 2000; 96: 1798-1807Crossref PubMed Google with collagen was for the of our dimeric In both the and the dimer inhibited platelet aggregation a The of the GPVI proteins on aggregation was between its on and platelet The GPVIex weakly the platelet aggregation. These results suggested that GPVI-Fc2 has a binding affinity to collagen, GPVIex, the monomeric form, is that a in the different reactivity of GPVIex and but of our indicate that this is In we GPVIex by the antibody with the of GPVIex, and we showed that the GPVIex the ability to to immobilized collagen, its binding was not the as that of The binding of the GPVIex with that of GPVI-Fc2 can be from the we that the of binding becomes antibody to GPVIex the of by the These results the that the of the form of GPVI is to the ability to collagen, thus the that the collagen binding of GPVI-Fc2 has a different from that of and were used to the indicated that GPVI-Fc2 to collagen, but GPVIex has no affinity for collagen. indicated that GPVI-Fc2 has a of 5.76 × is that we obtained for the soluble α2β1 × S.M. Moroi M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, S.M. Moroi M. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). that the activated integrin α2β1 is to the binding of platelets to collagen. GPVIex did not show interaction with collagen by and methods. high of GPVIex only of platelet aggregation or collagen binding the other GPVIex in a and inhibit platelet aggregation. the and Cvx binding of GPVIex are to of the dimeric GPVI and is only the affinity toward collagen that In of monomeric GPVIex with antibody its binding to immobilized collagen. Taken together, our results indicate that the high affinity to collagen is to the dimeric structure of GPVI. of affinity induced by receptor has been reported for superfamily receptors M. Y. P.J. S. A. 1995; PubMed Scopus Google Scholar). In one of our previous S.M. Moroi M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), we showed that platelet binding to soluble collagen is different from platelet binding to fibrous collagen. binding to soluble collagen is strongly on and strongly inhibited by α2β1 platelet binding to fibrous collagen is of the and only by the The binding of platelets to fibrous collagen is in the of (4Moroi M. Jung S.M. Okuma M. Shinmyozu K. J. Clin. Invest. 1989; 84: 1440-1445Crossref PubMed Scopus (371) Google Scholar), which that GPVI not to soluble collagen and specifically to fibrous collagen. this we the binding of soluble collagen to the immobilized GPVI-Fc2 by the and we that there is no interaction between GPVI-Fc2 and soluble collagen suggested that the GPVI binding to immobilized collagen by the or is not to monomeric collagen but is to the binding to fibrous collagen that is formed the or are with physiological the to the GPVI-Fc2 has affinity to inhibited the platelet aggregation and bound to immobilized The of the interaction between GPVI-Fc2 and was × The with the for the interaction with collagen would be to the dissociation which suggested that of the collagen structure other the would be in the dissociation of the bound collagen from GPVI. Although both the and showed the binding of dimeric and monomeric GPVI to A and GPVIex was against platelet aggregation GPVI-Fc2 would from the between the immobilized and GPVI-Fc2 is suggested to strongly to GPVIex and GPVI-Fc2 strongly inhibited platelet aggregation not Cvx did not inhibit GPVI-Fc2 binding to immobilized collagen, but These results indicated that the sites of GPVI are different from the collagen-binding sites of GPVI. is by the results of B. W. K. J.P. C. H. J. PubMed Scopus Google Scholar) that the anti-GPVI antibody the collagen-induced aggregation but not the C. H. D. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) reported that did not react with collagen, the react with Cvx with a to that of platelets. These show weak reactivity to The of these to collagen receptor are to the of our monomeric GPVIex to these GPVIex not to collagen but reacts with Cvx a to that reactive with GPVIex reacts weakly with These results suggest that the GPVI expressed on the would be present as a monomeric form with the the studies and showed that the GPVI on the surface a to that of platelets can react with collagen and H. D. Y. C. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). the of GPVI in platelets showed reactivity to collagen. we that would indicate of GPVI a would suggest that the of the γ-chain would be in a with monomeric GPVI. These results our that the dimeric form of GPVI would be the form of GPVI, and thus GPVI be expressed a high to an GPVI in the However, platelets from the of a reported (4Moroi M. Jung S.M. Okuma M. Shinmyozu K. J. Clin. Invest. 1989; 84: 1440-1445Crossref PubMed Scopus (371) Google Scholar) and platelets from H. D. Y. C. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) expressed only the normal of GPVI but normal reactivity to collagen. Although studies are to the of the dimeric form of GPVI on the platelet surface, our present results would suggest the of a for the γ-chain complex that specifically a dimeric complex in platelets and In this study, we two forms of GPVI, the monomeric form GPVIex and the dimeric form GPVI-Fc2, and our data indicate that the dimeric GPVI-Fc2 has high affinity to fibrous collagen. These with other data from our and by other suggested that GPVI would be present as a dimeric form in platelets. Our studies show that the dimeric form of GPVI, GPVI-Fc2, be useful for analyzing the interaction of GPVI with insoluble collagen. We for the Fc fusion
Miura et al. (Fri,) studied this question.