Murine Models of Platelet Integrin Function

稳定的血小板粘附、聚集和血栓形成取决于激活和调节粘附配体与整联蛋白α IIb-beta3结合的由内而外的信号，以及来自α IIb-beta3的由外而外的信号，调节血小板中的细胞骨架事件。alphaIIb-beta3信号传导缺陷可导致出血素质，但这些缺陷的分子基础 目前还不清楚，α Ⅱ b-β 3信号在巨核细胞中的作用也不清楚，巨核细胞是血小板的来源。该项目的目标是通过关注最近已被证明与血小板和巨核细胞中的β 3相互作用的两种主要类型的蛋白激酶Src和蛋白激酶C（PKC）来阐明血小板和巨核细胞中β 3信号传导的分子基础。这项工作将解决三个特定的施舍，利用一个独特的实验系统，小鼠巨核细胞和血小板来源于胚胎干细胞或骨髓，结合基因表达，敲低和敲除策略。首先，我们将检验c-Src被调节和由外向内信号传导在细胞内启动的假设。 通过c-Src与β 3胞质尾区的直接相互作用，这些实验将评估c-Src调控的结构基础，酪氨酸磷酸酶在这种相互作用中的作用，以及Src激活可以被选择性地破坏而不损害其他alphaIIb-beta3信号功能的想法。二是 将研究PKC与β 1 Ib-β 3相互作用的机制和结果。特别地，将表征特定PKC同种型与α 11b-β 3的相互作用，并建立它们在由内向外或由外向内的α 11b-β 3信号传导中的功能。将评估一种或多种PKC调节talin（另一种与β 3活化有关的整合素结合蛋白）的可能性。第三，将研究α 11-β 3、Src和PKC在巨核细胞成熟和前血小板形成中的可能作用。因此，通过在相关的原代小鼠细胞中建立alphaIIb-beta3/Src/PKC相互作用的机制和结果，这些研究将为正常巨核细胞的研究提供新的见解。 和血小板功能，在止血缺陷期间的血小板功能障碍，以及在动脉血栓形成中药理学阻断β 1 Ib-β 3信号传导的新治疗靶点的鉴定。

Stable platelet adhesion, aggregation and thrombus formation are dependent on inside-out signals that activate and regulate adhesive ligand binding to integrin alphaIIb-beta3, and on outside-in signals from alphalI-beta3 that regulate cytoskeletal events in platelets. Defects in alphaIIb-beta3 signaling can cause a bleeding diathesis, but the molecular basis of these defects remains unclear, as does the role of alphaIIb-beta3 signaling in megakaryocytes, cells from which platelets are derived. The goal of this project is clarify the molecular basis of alphalIb-beta3 signaling in platelets and megakaryocytes by focusing on two major classes of protein kinases, Src and protein kinase C (PKC), which have been shown recently to interact with alphalIb-beta3. This work will address three specific alms by utilizing a unique experimental system, mouse megakaryocytes and platelets derived from embryonic stem cells or bone marrow, in conjunction with gene expression, knock-down and knock-out strategies. First, we will test the hypothesis that c-Src is regulated and outside-in signaling is initiated in megakaryocytes and platelets by direct interaction of c-Src with the beta3 cytoplasmic tail. These experiments will evaluate the structural basis of c-Src regulation by alphallb-beta3, the role of tyrosine phosphatases in this interplay, and the idea that Src activation can be disrupted selectively without impairing other alphaIIb-beta3 signaling functions. Second, the mechanisms and consequences of PKC interaction with alphalIb-beta3 will be investigated. In particular, the interaction of specific PKC isoforms with alphalIb-beta3 will be characterized, and their functions in inside-out or outside-in alphaIlb-beta3 signaling established. The possibility that one or more PKCs regulate talin, another integrin-binding protein implicated in alphalIb-beta3 activation, will be assessed. Third, possible roles for alphaIl-beta3, Src and PKC in megakaryocyte maturation and proplatelet formation will be investigated. Thus, by establishing the mechanisms and consequences of alphaIIb-beta3/Src/PKC interactions in the relevant primary murine cells, these studies should provide new insights into normal megakaryocyte and platelet function, into platelet dysfunction during defective hemostasis, and into identification of new therapeutic targets for pharmacological blockade of alphalIb-beta3 signaling in arterial thrombosis.