III型PI3K-VPS34在血小板活化和血栓形成中的机制研究

Class III PI3K, also known as vacuolar protein sorting 34 (VPS34), is a member of PI3K family, which play important roles in diverse cellular functions. The role of class III PI3K in hemostasis and thrombosis is ill defined. With platelet specific VPS34 deficient mice, we have demonstrated that class III PI3K is vital in thrombosis, while dispensable in hemostasis. VPS34-/- platelets exhibited impaired activation in response to multiple agonists, which was phenocopied by using 3-methyladenine, a specific class III PI3K inhibitor in human platelets. These data indicate that class III PI3K might be a safe antiplatelet target. The present project aims to elucidate the signal transduction mechanism of platelet class III PI3K, with special emphasis on NADPH Oxidase activation pathway, autophagy pathway, and mTOR-regulated cytoskeleton reorgnization pathway. By unveiling the mechanism of class III PI3K VPS34, this project will strengthen the understanding of platelet activation process and thrombosis, and provide evidences for this molecule to be developed as a safe target in the better management of thrombotic diseases.

磷酯酰肌醇-3激酶（PI3K）激酶是重要的细胞功能调控蛋白家族。该家族成员中的III型PI3K（VPS34）在血小板中的功能和机制未知。我们首先报道了III型PI3K在血小板中的表达，并发现体外抑制III型PI3K可以减弱血小板活化。为探索III型PI3K的体内作用，我们构建了血小板特异敲除III型PI3K的小鼠，发现该小鼠的病理性血栓形成受到抑制，但正常止血功能并未受影响，提示该分子可能是一个安全的新的抗血栓靶点。本课题在前期功能研究的基础上提出III型PI3K下游通过NOX活化、自噬、以及mTOR调控的细胞骨架重组的通路假说，拟通过基因敲除模型和分子生物学手段进行体内体外研究验证，最终明确III型PI3K在血小板中的信号通路机制。本课题将填补血小板领域内对III型PI3K研究的空白，加深对血小板精细调控机制的理解，发现并验证针对血栓性疾病的新的安全干预靶点。

通过本课题的实施，完成了预定的研究目标。首先，我们获得了III型PI3K催化亚基VPS34敲除小鼠，以此为研究工具，检测了体内血栓，体外刺激剂作用下的血小板聚集和分泌等功能，发现VPS34血小板特异性敲除不影响小鼠止血，但导致血小板血栓形成受损。VPS34缺失血小板在低浓度胶原和凝血酶刺激下，血小板聚集和致密颗粒释放均受损；VPS34缺失抑制NOX的组装，导致由NOX介导的血小板ROS释放减少；VPS34缺失还会抑制mTOR信号，进而影响血小板活化。此外，我们还发现VPS34缺失还会改变血小板本底自噬流变化。因此，该研究深入阐释了VPS34在血小板中的功能和发挥作用的分子机制，加深了对血栓性疾病的分子病理机制的理解，为寻找新的有效干预靶点提供了理论依据和支持。