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

尽管糖蛋白Ia/IIa(GpIa/IIa，整合素α_2β_1)作为胶原受体的作用已经确立，但目前尚不清楚GPIa/IIa是否介导了激活信号。在这项研究中，我们发现，从红腹蛇毒液中提纯的柔红菌素可以诱导血小板聚集，这可以被抗GPIa单抗所阻断。对红霉素偶联微球和重组GPIa/IIa脂质体的研究表明，红霉素直接与GPIa/IIa结合，而不依赖于二价阳离子。体外蛋白激酶分析和免疫印迹分析表明，Src和Lyn与GPIa/IIa结合，并且在刺激红霉素后，Src的活性短暂增加。SRC以依赖于Cas磷酸化的方式与p130Crk相关底物(Cas)特异性结合，提示Src参与了Cas酪氨酸的磷酸化。而所有这些现象在罗多克刺激后早期以cAMP抵抗的方式发生-Syk和磷脂酶Cγ2(PLCγ2)的酪氨酸磷酸化，细胞内钙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，阻断受体聚集，抑制我们在本研究中检测到的所有罗多克介导的信号。我们认为，红霉素通过聚集GPIa/IIa，激活与GPIa/IIa相关的Src，然后介导下游激活信号。

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.