凝血酶诱导糖蛋白Ibα酶切的分子机制研究

Molecular mechanism of thrombin-induced GPIbα shedding

目的探讨凝血酶诱导糖蛋白(GP)Ibα酶切的分子机制。方法取健康志愿者静脉血,分离得到洗涤血小板。洗涤血小板分别与钙离子依赖蛋白酶(calpain)抑制剂、活性氧(ROS)抑制剂、还原型烟酰胺腺嘌呤二核苷酸磷酸(NAD-PH)氧化酶抑制剂、线粒体靶向的ROS抑制剂、含半胱氨酸的天冬氨酸蛋白水解酶(caspase)抑制剂、前列腺素E1(PGE1)、解离素-金属蛋白酶17(ADAM17)抑制剂或溶剂对照等预孵育,再与凝血酶(在搅拌或非搅拌条件下)孵育。流式细胞仪检测ROS浓度,Western blot检测GPIbα的酶切和calpain底物talin的酶切。结果 ROS抑制剂二硫苏糖醇(DTT)和calpain抑制剂MDL单独使用时,均部分抑制凝血酶(搅拌条件下)诱导的GPIbα酶切;联合使用时,则完全抑制凝血酶(搅拌条件下)诱导的GPIbα酶切。DTT完全抑制凝血酶(非搅拌条件下)诱导的GPIbα酶切,而MDL没有抑制作用。另外,caspase抑制剂和血小板活化抑制剂(PGE1)不抑制凝血酶诱导的GPIbα酶切。凝血酶(非搅拌条件下)剂量依赖地引起血小板ROS浓度升高;NAD(P)H氧化酶抑制剂抑制凝血酶引起的ROS浓度升高,线粒体靶向的ROS抑制剂则没有抑制作用。结论在搅拌条件下,ROS和cal-pain两条信号通路共同调控凝血酶诱导的GPIbα酶切;在非搅拌条件下,通过NAD(P)H氧化酶产生的ROS调控凝血酶诱导的GPIbα酶切。

Objective To investigate the molecular mechanism of thrombin-induced GPIbct shedding. Methods Washed platelets were obtained from anti-coagulated healthy volunteers, whole blood by centrifugation and washing. Washed platelets were pre-incubated with calpain inhibitors, reactive oxygen species（ROS） antagonist,NAD（P） H oxidase inhibitor, mitochondrial ROS antagonist, caspase inhibitor, PGE1, ADAM17 inhibitor, or DMSO, and then incubated with thrombin under stirring/non-stirred conditions. ROS levels were measured with flow cytome- try. Cleavage of GPIbα and talin was detected by Western blot. Results Thrombin-induced GPIbα shedding understirring conditions was partially inhibited by calpain inhibitor MDL or ROS antagonist DTT respectively, however, when used in tandem, MDL and DTT completely reduced GPIbα shedding as effectively as ADAM17 inhibitor GM6001. On the other hand, DTY completely inhibited thrombin-induced GPIbα shedding under non-stirred condi- tion, but MDL did not. In addition, caspase inhibitor and PGE1 did not inhibit thrombin-induced GPIbα shedding under stirring/non-stirred conditions. Thrombin dose-dependently elevated ROS levels. Thrombin-induced ROS production was inhibited by NAD（P） H oxidase inhibitor, but not by mitochondrial ROS antagonist. Conclusion Under non-stirred condition, ROS regulated thrombin-induced GPIboL shedding. However, ROS and calpain togeth- er regulated thrombin-induced GPIbα shedding under stirring condition.