The roles of pleckstrin and pleckstrin-2 in platelet biology

这项建议的目标是了解pleckstrin和pleckstrin-2在血小板黏附和 血栓形成。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组成 域。虽然pleckstrin最先在血小板中被描述，但它在血小板激活中的作用仍然不完全。 明白了。我们在过度表达研究中发现，pleckstrin一旦被PKC磷酸化， 调节磷脂酶C和磷脂酰肌醇3产生的磷脂第二信使- 并增强整合素介导的细胞骨架改变和黏附。我们还克隆了一个cdna 对于pleckstrin-2，它是一种广泛表达的类似物，也存在于血小板中。Pleckstrin-2不是 被PKC磷酸化，但通过与特定的磷脂产物结合来调节 磷脂酰肌醇3-激酶(PI3K)。尽管过度表达研究对获得洞察力很有用 对于这两种蛋白质的潜在作用，我相信对缺乏这些酶的血小板的研究是 对于全面了解它们对血小板生物学的贡献至关重要。因此，我们有 将零突变引入小鼠pleckstrin基因，并产生了嵌合小鼠 目前正在培育产生Pleckstrin基因敲除动物。我们还针对pleckstrin-2基因， 最近产生的小鼠是pleckstrin-2零突变的纯合子。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，密度有缺陷 颗粒分泌，阻碍了固定化纤维蛋白原的扩散。我假设在血小板中， Pleckstrin和Pleckstrin-2中等磷脂第二信使，调节血小板胞吐，以及 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。在此基础上 假设，我提出了三个具体目标。在目标1中，我们将确定血小板之间的分子联系 肌动蛋白动力学和Pleckstrin亚型。在目标2中，通过使用几个体外和体内模型，我们将 确定Pleckstrin和Pleckstrin-2对稳定血小板黏附的作用，并检验假设 这两种异构体都是血栓形成所必需的。在目标3中，我们将使用体外方法， 包括结构研究，以进一步研究这两种pleckstrin亚型与肌醇磷脂的结合。

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 ~1% 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. InAim 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.