The roles of pleckstrin and pleckstrin-2 in platelet biology

pleckstrin 和 pleckstrin-2 在血小板生物学中的作用

基本信息

批准号：
7028484
负责人：
CHARLES S. ABRAMS
金额：
$ 39.25万
依托单位：
UNIVERSITY OF PENNSYLVANIA
依托单位国家：
美国
项目类别：
财政年份：
2006
资助国家：
美国
起止时间：
2006-02-01 至 2010-01-31
项目状态：
已结题

项目摘要

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

描述（由申请人提供）：该提案的目的是了解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％。它由氨基和羧基末端Pleckstrin同源（pH）域以及中间的DEP域组成。尽管Pleckstrin首先在血小板中描述，但其在血小板激活中的作用仍未完全了解。我们在过表达的研究中发现，一旦将其磷酸化，PLECKSTRIN被PKC磷酸化，可以调节由磷脂酶C和磷脂酰肌醇3-激酶产生的磷脂第二信使，并增强整联蛋白介导的细胞骨架变化和粘附。我们还为Pleckstrin-2克隆了一个cDNA，这是一个广泛表达的旁系同源物，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来调节。尽管过表达的研究对于了解这两种蛋白质的潜在作用很有用，但我认为缺乏这些酶的血小板的研究对于完全理解它们对血小板生物学的贡献至关重要。因此，我们已经在鼠Pleckstrin基因中引入了无效的突变，并产生了目前正在繁殖以生产Pleckstrin敲除动物的嵌合小鼠。我们还瞄准了pleckstrin-2基因，并且最近产生的小鼠是纯合子pleckstrin-2 null突变。 PLECKSTRIN-2基因敲除血小板因响应凝血酶和胶原蛋白而造成了损害的聚集，具有有缺陷的密集颗粒分泌，并且损害了固定的纤维蛋白原的扩散。我假设在血小板，pleckstrin和pleckstrin-2中度磷脂第二使者中，调节血小板外胞毒性，并与整联蛋白一起起作用，以诱导肌动蛋白重组和稳定的血小板粘附。基于这个假设，我提出了三个具体目标。在AIM 1中，我们将确定血小板肌动蛋白动力学和Pleckstrin同工型之间的分子联系。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，并检验以下假设：这两种同工型都是血栓形成所必需的。在AIM 3中，我们将使用包括结构研究在内的体外方法来进一步研究由两种Pleckstrin同工型的磷酸肌醇结合