凝血酶受体活化与血小板膜糖蛋白Ⅰbα的异位分布机制

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GPIⅠbα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(thrombin receptor activating peplide，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GPIbd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对活化过程巾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，Apyrase Ⅶ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在血小板信号传递过程中发挥了重要作用，导致GPⅠbα由血小板膜表面向内转移而后又逐渐返回至胞膜外，这种动态变化与细胞骨架重组及二磷酸腺苷(ADP)有关。

Functions of thrombin receptors in the reversible distribution of platelet surface glycoprotein Ⅰ bα in activated platelets

OBJECTIVE: To detect the redistribution of platelet surface glycoprotein (GP)Ibalpha and cytoskeleton reorganization in the course of thrombin receptor activation, and investigate the mechanism of GPIbalpha retranslocation and the role of thrombin receptors in platelet signal transduction. METHODS: The thrombin receptor activating peptide (PAR1-AP, TRAP) was used for stimulating platelet at different time points (0 - 60 min), then the platelet surface GPIbalpha and P-selectin were examined with flowcytometry, and the alterations of GPIbalpha, actin and myosin were analyzed in cytoskeleton by Western blot and GPIbalpha immunoprecipitation. Cytochalasin D and/or Apyrase VII were used for investigating their inhibitory effect on platelet activation. RESULTS: An increase of P-selectin and reversible internalization of GPIbalpha were observed within platelets upon TRAP activation, and transient changes of actin, myosin and GPIbalpha/myosin, GPIbalpha/actin association were also found in this course. These changes were apparently blocked by cytochalasin D, which inhibited the incorporation of GPIbalpha, actin and myosin into cytoskeleton. Apyrase VII had a weak effect on GPIbalpha internalization, although it accelerated the return of GPIbalpha to platelet surface. In addition, Apyrase VII also quickened the GPIbalpha disappearance in cytoskeleton and the dissociation of GPIb/myosin or GPIb/actin during activation. CONCLUSION: Thrombin receptor activation takes part in platelet signal transduction, inducing a reversible redistribution of GPIbalpha. This process is related to cytoskeleton reorganisation and ADP.