The role of pleckstrin & pleckstrin-2 in platelet biology.

描述（由申请人提供）：本提案的总体假设是，pleckstrin和pleckstrin-2在动脉止血和血栓形成中发挥着关键而独特的作用。Pleckstrin约占血小板和白细胞中总细胞蛋白的1%，是一种以含有两个原型Pleckstrin同源（PH）结构域而闻名的蛋白质。血小板激活后，PKC迅速磷酸化pleckstrin，诱导其结合膜结合磷脂，如磷脂酰肌醇4,5二磷酸（PIP2）。血小板还含有一种广泛表达的pleckstrin类似物，称为pleckstrin- 2。虽然pleckstrin的活性是通过蛋白磷酸化调节的，但pleckstrin-2不是磷酸化蛋白，而是通过结合PI3K产生的特异性磷脂磷脂酰肌醇3,4二磷酸（pi3,4p2）而激活的。为了了解这两种蛋白在体内的真正作用，我们在小鼠pleckstrin和pleckstrin-2基因中引入了破坏性突变，并分析了缺乏pleckstrin同种异构体的转基因小鼠的血小板表型。缺乏pleckstrin或pleckstirin-2的血小板会损害PKC-或pi3k介导的聚集、肌动蛋白组装和分泌的缺陷。缺乏这两种pleckstrin亚型的血小板在肌动蛋白成核和胞外分泌方面几乎完全缺陷。总之，这些数据证明了pleckstrin和pleckstrin-2在血小板生物学的几个方面发挥的关键作用。基于我们的初步小鼠实验，我们假设pleckstrin同种异构体通过促进磷脂向肌动蛋白组装和分泌所需的蛋白质的呈现，有助于血小板生物学。本提案的总体目标是对血小板中pleckstrin和pleckstrin-2的功能进行全面和系统的研究，并了解两种不同亚型之间的差异。在Aim 1中，我们将确定两种pleckstrin亚型与血小板肌动蛋白动力学之间的分子联系。在Aim 2中，我们将正式确定pleckstrin和pleckstrin-2如何通过生物化学和小鼠遗传学的结合来驱动血小板分泌。在目标3中，我们将分析结构机制，通过这两种普莱克斯琴同种异构体介导其作用

DESCRIPTION (provided by applicant): The overall hypothesis of this proposal is that pleckstrin and pleckstrin-2 play critical, and distinct roles in arterial hemostasis and thrombosis. Pleckstrin, which makes up approximately one percent of the total cellular protein within platelets and leukocytes, is a protein best known for containing the two prototypic Pleckstrin Homology (PH) domains. Following platelet activation, PKC rapidly phosphorylates pleckstrin, inducing it to bind membrane bound phospholipids such as phosphatidylinositol 4,5 bisphosphate (PIP2). Platelets also contain a widely expressed paralog of pleckstrin, called pleckstrin- 2. Although the activity of pleckstrin is regulated through protein phosphorylation, pleckstrin-2 is not a phosphoprotein, but is instead activated by binding a specific PI3K generated phospholipid, phosphatidylinositol 3,4 bisphosphate (PI3,4P2). To understand the true in vivo role of these two proteins, we have introduced disruptive mutations within the murine pleckstrin and pleckstrin-2 genes, and analyzed the platelet phenotype in genetically modified mice lacking either pleckstrin isoform. Platelets lacking either pleckstrin or pleckstirin-2 have impaired defects of PKC- or PI3K-mediated aggregation, actin assembly, and secretion. Platelets lacking both pleckstrin isoforms have an almost total defect in actin nucleation and exocytosis. Together, these data demonstrate the critical roles that pleckstrin and pleckstrin-2 play in several aspects of platelet biology. Based on our preliminary murine experiments, we hypothesize that pleckstrin isoforms contribute to platelet biology by facilitating the presentation of phospholipids to proteins that are required for actin assembly and for secretion. The overall goal of this proposal is to perform a comprehensive and systematic study of the function of pleckstrin and pleckstrin-2 within platelets, and to understand the differences between the two distinct isoforms. In Aim 1, we will determine the molecular link between the two pleckstrin isoforms and platelet actin dynamics. In Aim 2, we will formally identify how pleckstrin and pleckstrin-2 drive platelet secretion by using a combination of biochemistry and murine genetics. In Aim 3, we will analyze the structural mechanisms by which both pleckstrin isoforms mediate their effects PUBLIC HEALTH RELEVANCE: Heart disease and strokes are among the leading causes of morbidity and mortality in this country. Blood clots that form at sites of atherosclerosis are often the precipitating event for both of these diseases. Our work demonstrates that two platelet proteins, pleckstrin and pleckstrin-2 are important for the activation of platelets. We hope that a better understanding of the events that regulate platelet activation, including pleckstrin-mediated signaling, will lead to new therapeutic approaches to prevent vascular occlusion, as well as lead to a better understanding of platelet biology.