The roles of pleckstrin and pleckstrin-2 in platelet biology

描述（由申请人提供）：本提案的目的是了解pleckstrin和pleckstrin-2在血小板粘附和血栓形成中的作用。Pleckstrin是一种突出的PKC底物，约占血小板总细胞蛋白的1%。它由氨基端和羧基端Pleckstrin同源结构域（PH）和中间的DEP结构域组成。尽管pleckstrin首先在血小板中被描述，但其在血小板活化中的作用仍未完全了解。我们在过表达研究中发现，pleckstrin一旦被PKC磷酸化，就可以调节磷脂酶C和磷脂酰肌醇3-激酶产生的磷脂第二信使，并增强整合素介导的细胞骨架变化和粘附。我们还克隆了pleckstrin-2的cDNA，这是一个广泛表达的类似物，也存在于血小板中。Pleckstrin-2不被PKC磷酸化，而是通过与磷脂酰肌醇3-激酶（PI3K）的特定磷脂产物结合而受到调节。虽然过表达研究有助于深入了解这两种蛋白的潜在作用，但我认为，对缺乏这些酶的血小板的研究对于全面了解它们对血小板生物学的贡献至关重要。因此，我们在小鼠pleckstrin基因中引入了一个零突变，并产生了嵌合小鼠，目前正在培育生产pleckstrin敲除动物。我们也瞄准了pleckstrin-2基因，并且最近产生了pleckstrin-2零突变的纯合子小鼠。Pleckstrin-2敲除的血小板对凝血酶和胶原蛋白的聚集受损，致密颗粒分泌缺陷，并损害固定纤维蛋白原的扩散。我假设在血小板中，pleckstrin和pleckstrin-2调节磷脂第二信使，调节血小板胞吐，并与整合素协同作用，诱导肌动蛋白重组和稳定血小板粘附。基于这一假设，我提出三个具体目标。在Aim 1中，我们将确定血小板肌动蛋白动力学和pleckstrin亚型之间的分子联系。在Aim 2中，通过使用几个离体和体内模型，我们将确定pleckstrin和pleckstrin-2对稳定血小板粘附的贡献，并验证这两种异构体都是血栓形成所必需的假设。在Aim 3中，我们将使用体外方法，包括结构研究，进一步研究两种pleckstrin亚型的磷酸肌苷结合

DESCRIPTION (provided by applicant): The goal of this proposal is to understand the roles of pleckstrin and pleckstrin-2 in platelet adhesion and thrombosis. Pleckstrin is a prominent PKC substrate that makes up about 1 percent of total cellular protein in platelets. It consists of amino- and carboxy-terminal Pleckstrin Homology (PH) domains and an intervening DEP domain. Although pleckstrin was first described in platelets, its role in platelet activation is still not completely understood. We have found in overexpression studies that pleckstrin, once it was phosphorylated by PKC, regulates phospholipid second messengers generated by both phospholipase C and phosphatidylinositol 3- kinase, and enhances integrin mediated cytoskeletal changes and adhesion. We have also cloned a cDNA for pleckstrin-2, which is a widely expressed paralog that is also present in platelets. Pleckstrin-2 is not phosphorylated by PKC, but instead is regulated by binding to specific phospholipid products of phosphatidylinositol 3-kinase (PI3K). Although overexpression studies have been useful for gaining insight into the potential role of these two proteins, I believe that studies of platelets lacking these enzymes are critical for a complete understanding of their contributions to platelet biology. Consequently, we have introduced a null mutation into the murine pleckstrin gene, and have generated chimeric mice that are currently being bred to produce pleckstrin knockout animals. We have also targeted the pleckstrin-2 gene, and have recently generated mice that are homozygous for a pleckstrin-2 null mutation. Pleckstrin-2 knockout platelets have impaired aggregation in response to thrombin and collagen, have defective dense granule secretion, and have impaired the spreading of immobilized fibrinogen. I hypothesize that in platelets, pleckstrin, and pleckstrin-2 moderate phospholipid second messengers, regulate platelet exocytosis, and function in concert with integrins to induce actin reorganization and stable platelet adhesion. Based upon this hypothesis, I propose three specific aims. In Aim 1, we will determine the molecular link between platelet actin dynamics and pleckstrin isoforms. In Aim 2, by using several ex vivo and in vivo models we will determine the contribution of pleckstrin and pleckstrin-2 to stable platelet adhesion, and test the hypothesis that both of these isoforms are required for thrombus formation. In Aim 3, we will use in vitro approaches, including structural studies, to further investigate phosphoinositide binding by the two pleckstrin isoforms