ROLE OF PLECKSTRIN HOMOLOGY DOMAINS IN PLATELET ACTIVATION

当血小板在血管损伤部位被激动剂如凝血酶、胶原、ADP和血栓烷A2激活时，它们经历肌动蛋白细胞骨架的重组。这一事件是由这些激动剂激活其细胞表面受体引发的，导致脂质第二信使的形成和蛋白激酶的激活。关于磷脂第二信使在血小板活化中的关键作用，人们已经了解了很多;然而，仍然存在很大的差距。基于与其他磷脂信号传导蛋白的同源性，我们最初提出，主要的PKC底物，pleckstrin，可以被证明是G蛋白偶联受体，磷脂信号和肌动蛋白细胞骨架之间的联系。我们已经发现，过表达的pleckstrin调节由磷脂酶C和磷脂酰肌醇3-激酶介导的信号，并通过依赖于Rho家族的整联蛋白和小GTP结合蛋白的信号传导途径诱导细胞骨架重组。我们的假设是普列克底物蛋白调节血小板中磷脂第二信使的形成，并与整合素一起诱导肌动蛋白重组，从而促进血小板活化。基于我们的假设，我们建议做以下工作：在具体目标#1中，我们将扩展我们的观察，即普列克底物蛋白通过确定受普列克底物蛋白调节的下游信号蛋白来帮助调节血小板细胞骨架。具体目标#2将鉴定与普列克底物蛋白在体内相互作用的分子。具体目标#3将检查靶向破坏普列克底物蛋白对鼠巨核细胞和血小板上的信号传导的后果。

When platelets become activated at sites of vascular injury by agonists such as thrombin, collagen, ADP and thromboxane A2, they undergo a reorganization of their actin cytoskeleton. This event is initiated by these agonists activating their cell surface receptors, leading to the formation of lipid second messengers and activation of protein kinases. A great deal has been learned about the critical role of phospholipid second messengers in platelet activation; however, large gaps remain. Based on homology with other phospholipid signaling proteins, we originally proposed that the prominent PKC substrate, pleckstrin, could prove to be a link between G-protein coupled receptors, phospholipid signals and the actin cytoskeleton We have found that over-expressed pleckstrin moderates signals mediated by phospholipase C and phosphatidylinositol 3-kinase, and induces cytoskeletal reorganization through a signaling pathway dependent on integrins and small GTP-binding proteins of the Rho family. It is our hypothesis that pleckstrin moderates phospholipid second messengers formation in platelets, and in concert with integrins, induces actin reorganization contributing to platelet activation. Based on our hypothesis, we propose to do the following: In Specific Aim #1, we will extend our observation that pleckstrin helps regulate the platelet cytoskeleton by determining downstream signaling proteins that are regulated by pleckstrin. Specific Aim #2 will identify the molecules with which pleckstrin interacts in vivo. Specific Aim #3 will examine the consequences of targeted disruption of pleckstrin on signaling on murine megakaryocytes and platelets.