Murine Models of Platelet Integrin Function

血小板整合素功能的小鼠模型

• 批准号: 6968160
• 负责人: SANFORD J SHATTIL
• 金额: $ 39.19万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-12-01 至 2009-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6968160
• 关键词: actin binding protein; biological signal transduction; clinical research; genetically modified animals; hematopoiesis; integrins; laboratory mouse; megakaryocytes; platelets; protein kinase C; protein protein interaction; protein tyrosine kinase; protein tyrosine phosphatase

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.

稳定的血小板粘附，聚集和血栓形成取决于激活和调节与整联蛋白alphaiib-beta3结合的内而外信号，以及来自α-beta3的外部信号，这些信号调节了调节血小板中细胞骨架事件的α-beta3。 Alphaiib-Beta3信号传导中的缺陷可能会引起出血的素质，但是这些缺陷的分子基础 尚不清楚，α-beta3信号在巨核细胞中的作用，来自其血小板的细胞。该项目的目的是阐明血小板和巨核细胞中α-beta3信号传导的分子基础，通过关注两种主要类别的蛋白激酶，SRC和蛋白激酶C（PKC），这些蛋白激酶C（PKC）最近已证明与Alphalib-Beta3相互作用。这项工作将通过利用独特的实验系统：源自胚胎干细胞或骨髓衍生的小鼠巨核细胞和血小板来解决三个特定的施舍，并与基因表达，敲除和敲除策略结合使用。首先，我们将检验以下假设：C-SRC受到调节，并在外部信号传导中启动 通过C-SRC与Beta3细胞质尾部直接相互作用的巨核细胞和血小板。这些实验将通过alphallb-beta3，酪氨酸磷酸酶在此相互作用中的作用来评估C-SRC调节的结构基础，并且可以选择性地破坏SRC激活而不会损害其他Alphaiib-Beta3信号功能的想法。第二， PKC与α-Beta3相互作用的机理和后果将研究。特别是，将表征特定的PKC同工型与α-Beta3的相互作用，并确定其在内而外或外部或外部的功能。一个或多个PKC调节塔林（另一种与α-Beta3激活有关的结合蛋白结合蛋白）的可能性将被评估。第三，将研究Alphail-Beta3，SRC和PKC在巨核细胞成熟和预言形成中的可能作用。因此，通过建立相关原代鼠细胞中Alphaiib-Beta3/src/pkc相互作用的机制和后果，这些研究应为正常的巨核细胞提供新的见解 和血小板功能，在止血缺陷期间进入血小板功能障碍，并鉴定出动脉血栓形成中α3信号的药理阻断的新治疗靶标。