Analysis of the interaction of platelet collagen receptor glycoprotein VI (GPVI)with collagen - A dimeric form of GPVI, but not the monomeric form, shows affinity to fibrous collagen
Analysis of the interaction of platelet collagen receptor glycoprotein VI (GPVI)with collagen - A dimeric form of GPVI, but not the monomeric form, shows affinity to fibrous collagen
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DOI：
10.1074/jbc.m204029200
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发表时间：
2002-11-29
影响因子：
4.8
通讯作者：
Moroi, M
中科院分区：
文献类型：
--
作者：
Miura, Y;Takahashi, T;Moroi, M
糖蛋白VI（GPVI）是血小板特异性糖蛋白，其已被指示与胶原反应并活化血小板。最近通过cDNA克隆鉴定了其结构（Clemetson，J.M.，Polgar，J.，Magnenat，E.，威尔斯，T. N.，Clemetson，K. J.（1999）J.Biol.Chem.274，29019 - 29024）。然而，胶原蛋白和GPVI之间的相互作用的机制还没有详细分析，因为胶原蛋白和GPVI都是不溶性分子。在这项研究中，我们表达了可溶形式的GPVI的胞外结构域，如下所示：单体形式（GPVIex）和与人免疫球蛋白Fc结构域融合的GPVI的二聚体形式（GPVI-Fc（2））。纯化的GPVIex强烈抑制惊厥素（Cvx）诱导的血小板聚集，但只有弱抑制胶原相关肽诱导的。然而，只有GPVI-Fc（2），而不是GPVIex，抑制胶原诱导的血小板聚集。GPVI的二聚体形式表现出对胶原蛋白的高亲和力，如从通过酶联免疫吸附测定和表面等离子体共振方法测量GPVI结合到固定化胶原蛋白得出的结论。GPVI-Fc（2）与固定化胶原表面结合，解离常数（K-D）为5.76 × 10（-7）m，但GPVIex的结合太弱，无法估计该参数。Cvx不抑制二聚体GPVI与胶原的结合，表明GPVI与胶原的结合位点与Cvx不同。综上所述，我们的数据表明，胶原蛋白的高亲和力结合位点由两条GPVI链组成。此外，他们认为Cvx的结合位点与胶原结合位点不同，不需要由两个GPVI分子形成。因为二聚体GPVI是唯一对纤维胶原显示高亲和力的形式，我们的结果表明GPVI将作为二聚体形式存在于血小板上。此外，表面等离子体共振表明，可溶性胶原蛋白和GPVI之间没有可检测到的相互作用，支持我们以前的观察，即GPVI只与纤维胶原蛋白反应。
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-Fc(2)). Purified GPVIex strongly inhibited convulxin (Cvx)-induced platelet aggregation but only weakly inhibited that induced by collagen-related peptide. However, only GPVI-Fc(2), and not GPVIex, inhibited collagen-induced platelet aggregation. The dimeric form of GPVI exhibits high affinity for collagen, as concluded from measurements of GPVI bindling to immobilized collagen by both the enzyme-linked immunosorbent assay and surface plasmon resonance methods. GPVI-Fc(2) bound to the surface of immobilized collagen with a dissociation constant (K-D) of 5.76 x 10(-7) m,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.