ROLE OF PLECKSTRIN HOMOLOGY DOMAINS IN PLATELET ACTIVATION

当血小板在血管损伤部位被凝血酶、胶原蛋白、ADP和凝血素A2等激动剂激活时，它们会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，我们已经了解了很多；然而，巨大的差距仍然存在。基于与其他磷脂信号蛋白的同源性，我们最初提出PKC中突出的底物pleckstrin可能是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。我们发现过表达的pleckstrin调节磷脂酶C和磷脂酰肌醇3-激酶介导的信号。并通过依赖于整合素和Rho家族的小gtp结合蛋白的信号通路诱导细胞骨架重组。我们的假设是，pleckstrin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Specific Aim #1中，我们将扩展我们的观察，即pleckstrin通过确定由pleckstrin调节的下游信号蛋白来帮助调节血小板细胞骨架。特异性目标#2将识别在体内与pleckstrin相互作用的分子。特异性目标#3将研究靶向破坏pleckstrin对小鼠巨核细胞和血小板信号传导的影响。

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.