The roles of pleckstrin and pleckstrin-2 in platelet biology

描述(由申请人提供)：本提案的目标是了解pleckstrin和pleckstrin-2在血小板黏附和血栓形成中的作用。Pleckstrin是一种重要的PKC底物，约占血小板细胞总蛋白的1%。它由氨基和羧基末端的Pleckstrin同源(PH)结构域和一个中间的DEP结构域组成。虽然pleckstrin最初是在血小板中被描述的，但它在血小板激活中的作用仍然不完全清楚。我们在过表达研究中发现，pleckstrin一旦被PKC磷酸化，就能调节由磷脂酶C和磷脂酰肌醇3-激酶产生的磷脂第二信使，并促进整合素介导的细胞骨架变化和黏附。我们还克隆了pleckstrin-2的cdna，pleckstrin-2是一种广泛表达的类似蛋白，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来调节。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，对缺乏这两种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基因中引入了零突变，并产生了嵌合鼠，目前正在培育这些嵌合鼠来生产pleckstrin基因敲除动物。我们还针对pleckstrin-2基因，最近产生了pleckstrin-2零突变纯合的小鼠。Pleckstrin-2基因敲除的血小板对凝血酶和胶原反应的聚集功能受损，致密颗粒分泌缺陷，并阻碍了固定化纤维蛋白原的扩散。我推测，在血小板中，Pleckstrin和Pleckstrin-2是中等磷脂第二信使，调节血小板的胞吐，并与整合素协同作用，诱导肌动蛋白重组和稳定的血小板黏附。基于这一假设，我提出了三个具体目标。在目标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。在目标2中，通过使用几种体外和体内模型，我们将确定pleckstrin和pleckstrin-2对稳定血小板黏附的贡献，并验证这两种异构体都是血栓形成所必需的假设。在目标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亚型与肌醇磷脂的结合。

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