Endophilin A2参与调控血小板活化及血栓形成的机制研究

Our previous work has revealed that upregulation of endophilin A2 can prolong tail bleeding time, inhibit platelets adhesion and aggregation, and endophilin A2 can interact with platelet surface collagen receptors, integrin β1 and GPVI, suggesting endophilin A2 may be the critical contributor to platelets activation and thrombosis by regulating integrin β1 and GPVI, however, the underlying mechanisms are still unclear. In this study, we will evaluate the effects of endophilin A2 on platelets activation and thrombosis by using endophilin A2 gene knockout and transgenic mice. Furthermore, we will identify the critical binding domain of endophilin A2 which is required for interaction with integrin β1 and GPVI, and its outside-in signaling pathways. Our study will gain a more sophisticated insight into the mechanism of platelets activation and thrombosis by which endophilin A2 regulates integrin β1 and GPVI, and provide the evidence that modulation of endophilin A2 expression may be a novel therapeutic stratage for platelets activation and thrombosis.

我们预实验发现endophilin A2转基因小鼠鼠尾出血时间延长，血小板粘附聚集能力降低，endophilin A2与血小板表面胶原受体integrin β1和GPVI有直接结合，提示endophilin A2可能是调控integrin β1和GPVI活化以及血小板功能和血栓形成的关键分子，但机理不清。本研究拟采用基因敲除和转基因小鼠，首先明确endophilin A2在调控血小板活化及血栓形成中的作用，进而阐明endophilin A2与integrin β1及GPVI结合的关键结构域和调控机理，揭示endophilin A2调控integrin β1和GPVI活化及其下游信号通路。该研究将从endophilin A2调控integrin β1和GPVI活性的角度揭示血小板活化及血栓形成的新机制，为评估endophilin A2是否可作为改善血小板功能及血栓形成的新靶点提供实验室依据。

本项目主要从endophilin A2参与调控血小板功能及血栓形成的分子机制展开系统研究，主要发现有：1.通过构建endophilin A2基因敲除小鼠，观察endophilin A2基因敲除小鼠的鼠尾出血时间明显缩短，但是endophilin A2转基因小鼠及基因敲除小鼠的血小板数量并无显著性差异，而血小板聚集率明显增加；同时发现基因敲除后肺血栓形成更加明显，小鼠生存率降低。2.Endophilin A2和Talin及 Kindlins竞争性结合于Integrin β1相同的序列，抑制Integrin β1的活化，进而抑制血小板活化及血栓形成3.Endophilin A2通过SH3结构域与血小板胶原受体GPVI的PRD结构域相互作用，进一步参与调控GPVI蛋白活化及其下游信号通路。本研究采用基因修饰小鼠，在整体动物实验明确endophilin A2在血小板功能和血栓形成中的作用，阐明endophilin A2是调控血小板表面受体integrin β1及GPVI蛋白活化及其下游信号通路的关键分子。为评估endophilin A2蛋白作为改善血小板功能和血栓形成的新靶点提供实验室依据。