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

描述（由申请方提供）：该提案的总体假设是普列克底物蛋白和普列克底物蛋白-2在动脉止血和血栓形成中发挥关键且不同的作用。普列克底物蛋白约占血小板和白细胞内总细胞蛋白的1%，是一种因含有两个原型普列克底物蛋白同源（PH）结构域而最为人所知的蛋白质。血小板活化后，PKC迅速磷酸化普列克底物蛋白，诱导其结合膜结合磷脂，如磷脂酰肌醇4，5二磷酸（PIP 2）。血小板还含有广泛表达的普列克底物蛋白，称为普列克底物蛋白-2。尽管普列克底物蛋白的活性通过蛋白磷酸化调节，但普列克底物蛋白-2不是磷蛋白，而是通过结合特异性PI 3 K产生的磷脂，磷脂酰肌醇3，4二磷酸（PI 3，4P 2）来活化。为了了解这两种蛋白质在体内的真实作用，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同种型的转基因小鼠的血小板表型。缺乏pleckstrin或pleckstrin-2的血小板具有PKC或PI 3 K介导的聚集、肌动蛋白组装和分泌缺陷。缺乏两种普列克底物蛋白亚型的血小板在肌动蛋白成核和胞吐方面几乎完全缺陷。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。基于我们初步的小鼠实验，我们假设pleckstrin亚型有助于血小板生物学通过促进磷脂的蛋白质，所需的肌动蛋白组装和分泌的介绍。本提案的总体目标是对血小板内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。在目的1中，我们将确定两个pleckstrin亚型和血小板肌动蛋白动力学之间的分子联系。在目标2中，我们将正式确定如何pleckstrin和pleckstrin-2驱动血小板分泌通过使用生物化学和小鼠遗传学的组合。在目标3中，我们将分析两种普列克底物异构体介导其效应的结构机制

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