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Mechanism of platelet activation mediated by GPIa/IIa, a receptor for collagen.

由胶原蛋白受体 GPIa/IIa 介导的血小板活化机制。

基本信息

批准号：
11670989
负责人：
OZAKI Yukio
金额：
$ 2.24万
依托单位：
Yamanashi Medical University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2000
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11670989/
关键词：
Platelet activation collagen receptor GPIa IIa rhodocytin tyrosine kinase Src Cas GPla lla

项目摘要

Although glycoprotein Ia/IIa (GpIa/IIa, integrin α_2β_1) has established its role as a collagen receptor, it remains unclear whether GPIa/IIa mediates activation signals. In this study, we show that rhodocytin, purified from the Calloselasma rhodostoma venom, induces platelet aggregation, which can be blocked by anti-GPIa mAbs. Studies with rhodocytin- coupled beads and liposomes loaded with recombinant GPIa/IIa demonstrated that rhodocytin directly binds to GPIa/IIa independently of divalent cations. In vitro kinase assays and Western blotting of GPIa immunoprecipitates revealed that Src and Lyn constitutively associate with GPIa/IIa and that Src activity increases transiently after rhodocytin stimulation. Src specifically associates with p130 Crk-associated substrate (Cas) in a manner dependent upon Cas phosphorylation, suggesting that Src is responsible for Cas tyrosine phosphorylation. While all these phenomena occur early after rhodocytin stimulation in a cAMP-resistant manner-tyrosine phosphorylation of Syk and phospholipase C γ2 (PLC γ2), intracellular Ca^<2+> mobilization, and platelet aggregation occur later in a cAMP-sensitive manner. Cytochalasin D, which interferes with actin polymerization and blocks receptor clustering, inhibits all the rhodocytin-mediated signals we examined in this study. We suggest that rhodocytin, by clustering GPIa/IIa, activates GPIa/IIa-associated Src, which then mediates downstream activation signals.

尽管糖蛋白Ia/IIa（GPIa/IIa，integrin α_2β_1）作为胶原受体的作用已被证实，但GPIa/IIa是否介导激活信号仍不清楚。在这项研究中，我们表明，从Calloselasma rhodostoma毒液中纯化的rhodocytin诱导血小板聚集，这可以被抗GPIa单克隆抗体阻断。用红细胞素偶联的珠粒和装载有重组GPIa/IIa的脂质体进行的研究表明，红细胞素不依赖于二价阳离子而直接结合GPIa/IIa。在体外激酶测定和蛋白质印迹的GPIa免疫沉淀物显示，Src和林恩组成型与GPIa/IIa和Src活性增加后，rhodocytin刺激短暂。Src以依赖于Cas磷酸化的方式与p130 Crk相关底物（Cas）特异性结合，表明Src负责Cas酪氨酸磷酸化。虽然所有这些现象都发生在红细胞素刺激后的早期，但都是以cAMP抵抗的方式发生的--Syk和磷脂酶C γ2（PLC γ2）的酪氨酸磷酸化、细胞内Ca^2+动员和血小板聚集以cAMP敏感的方式发生。细胞松弛素D，干扰肌动蛋白聚合和阻断受体聚集，抑制所有的红细胞素介导的信号，我们在这项研究中检查。我们认为，红细胞素，通过聚集GPIa/IIa，激活GPIa/IIa相关的Src，然后介导下游激活信号。