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目的 检测凝血酶受体活化过程中血小板膜糖蛋白 (GP)Ⅰbα的动态分布及其细胞骨架蛋白的改变,揭示GPⅠbα的异位分布机制与凝血酶受体在血小板信号传递中的作用。方法 以凝血酶受体活化肽 (thrombinreceptoractivatingpeptide,TRAP)诱导血小板活化,应用流式细胞术比较刺激前后血小板膜表面GPⅠbα及P 选择素的表达,并通过转移电泳及免疫沉淀技术,分析细胞骨架中GPⅠbα、肌动蛋白、肌球蛋白的变化。观测细胞松弛素 (Cytochalasin) D与腺苷三磷酸双磷酸酶(Apyrase)Ⅶ对血小板活化过程的影响。结果 TRAP作用后促使血小板活化,流式细胞术检测结果显示其P 选择素水平显著升高,而GPⅠbα先呈现进行性减少,而后逐渐回升的可逆性变化。抑制剂作用后这一过程受到不同程度影响,其中细胞松弛素D能阻滞GPⅠbα的内移, ApyraseⅦ对活化过程中GPⅠbα的逆转没有影响,只对它的恢复起作用,促使GPⅠbα迅速返回细胞表面。细胞骨架蛋白分析结果也表明肌动蛋白、肌球蛋白与GPⅠbα受刺激后呈先增加后减少的动态变化趋势。细胞松弛素D能阻滞GPⅠbα、肌动蛋白与肌球蛋白向骨架中心的转位, ApyraseⅦ则能加快骨架蛋白在骨架中心的消散。免疫印迹证实GPⅠbα的这种可逆性变化与血小板膜骨架紧密相关。结论 凝血酶受体活化
Objective To detect the dynamic distribution of platelet glycoprotein (Ⅰbα) and its cytoskeletal protein during the activation of thrombin receptor (GPR), and to reveal the ectopic distribution of GPⅠbα and the role of thrombin receptor in platelet signal transduction. Methods Thrombin receptor activating peptide (TRAP) was used to induce platelet activation. Flow cytometry was used to compare the expression of GP Ⅰ bα and P-selectin on platelet membrane before and after stimulation. The expression of GP Ⅰ bα in the cytoskeleton was analyzed by transfer electrophoresis and immunoprecipitation , Actin, myosin changes. To observe the effects of Cytochalasin D and Apyrase Ⅶ on platelet activation. Results TRAP induced platelet activation, flow cytometry results showed that the P-selectin levels were significantly increased, while GP Ⅰ bα presented a progressive decrease, and then gradually rose reversible changes. Inhibitory effect of this process is affected to varying degrees, of which cytochalasin D can block GP Ⅰ bα internal migration, Apyrase Ⅶ on the activation of GP Ⅰ bα reversal has no effect, only its recovery, prompting GP Ⅰ b α quickly returned to the cell surface . Cytoskeleton protein analysis also showed that actin, myosin and GP Ⅰ bα stimulated the first increase and then decrease the dynamic trend. Cytochalasin D blocks the translocation of GPIbα, actin and myosin to the center of the framework, and Apyrase VII accelerates the dissipation of the backbone protein in the center of the matrix. Immunoblotting demonstrated that this reversible change in GPIbα is closely associated with the platelet membrane matrix. Conclusion Thrombin receptor activation