MURINE MODELS OF PLATELET INTEGRIN FUNCTION

对人血小板和模型细胞系统的研究已经确定，通过整合素α IIb β 3的双向信号传导调节细胞与可溶性粘附配体的相互作用（由内向外信号传导），并触发血小板稳定粘附于血管基质所必需的细胞骨架变化（由外向内信号传导）。然而，整合素信号通路可以随着细胞环境而变化，并朝着理解人类血小板中的基因表达的方向发展。因此，本项目将使用重组技术研究小鼠巨核细胞和血小板中的α IIb β 3信号，包括体外和体内。具体的目标将解决根本的未解决的问题，在止血，动脉血栓形成和巨核细胞发育的α IIb β 3的功能。首先，将通过逆转录病毒介导的基因转移、用生长因子扩增巨核细胞库和使用一组敏感测定评估α IIb β 3功能的组合，在巨核细胞中离体检查α IIb和β 3胞质尾区在信号传导中的作用。其次，β 3-内切蛋白，H-Ras在α IIb β 3亲和力/亲合力调节中的潜在作用将在这些细胞中进行评价，因为这些蛋白质在模型细胞系统中被认为是整联蛋白调节剂。此外，还将确定β 3-内切蛋白基因缺失在体内的影响。第三，由外向内的信号转导途径，由小鼠巨核细胞的粘附α IIb β 3配体的特点，这些途径在巨核细胞的发育和功能的潜在作用将进行研究。第四，基于逆转录病毒的技术将用于靶向小鼠血小板中修饰的alphaIIb基因的体内表达，目的是建立原发性血小板高反应性和动脉血栓形成的条件性小鼠模型。具体地说，α IIb将融合到串联FKBP“二聚化”重复序列，并且当与β 3在体内表达时，这将使我们能够通过施用FKBP二聚化剂使α IIb β 3聚集并刺激纤维蛋白原与血小板结合。 综上所述，这些研究将提供新的信息allphaIIbbeta 3信号在止血，血栓形成和巨核细胞生成的分子基础，他们应该导致更清楚地了解在这些生物过程中的相对作用的由内而外和由外而外的信号。它们还可能导致识别细胞内靶点，用于开发干扰整合素信号传导的下一代抗血栓药物。

Work with human platelets and model cell systems has established that bi-directional signaling through integrin alphaIIbbeta3 regulates the interaction of cells with soluble adhesive ligands (inside-out signaling) and triggers cytoskeletal changes necessary for stable platelet adhesion to vascular matrices (outside-in signaling). However, integrin signaling pathways can vary with the cell context, and progress toward understanding the gene expression in human platelets. Therefore, this project will use recombinant techniques to study alphaIIbbeta3 signaling in murine megakaryocytes and platelets, both ex vivo and in vivo. The specific aims will address fundamental unresolved issues regarding the function of alphaIIbbeta3 in hemostasis, arterial thrombosis and megakaryocyte development. First, the roles of the alphaIIb and beta3 cytoplasmic tails in signaling will be examined in megakary0ocytes ex vivo through a combination of retroviral-mediated gene transfer, expansion of the megakaryocyte pool with growth factors, and assessment of alphaIIb beta3 function using a panel of sensitive assays. Second, the potential roles of beta3-endonexin, H-Ras in the regulation of alphaIIbbeta3 affinity/avidity will be evaluated in these cells, since these proteins have been implicated as integrin regulators in model cell systems. In addition, the effects of deletion of the beta3-endonexin gene in vivo will be determined. Third, the outside-in signaling pathways that are triggered by adhesion of mouse megakaryocytes to alphaIIbbeta3 ligands will be characterized, and the potential role of these pathways in megakaryocyte development and function will be studied. Fourth, retroviral-based techniques will be used to target expression of a modified alphaIIb gene in mouse platelets in vivo, with the goal of creating a conditional mouse model of primary platelet hyperreactivity and arterial thrombosis. Specifically, alphaIIb will be fused to tandem FKBP "dimerization" repeats, and when expressed with beta3 in vivo, this will enable us to cluster alphaIIbbeta3 and stimulate fibrinogen binding to platelets by administration of an FKBP dimerizer. Taken together, these studies will provide new information about the molecular basis of allphaIIbbeta3 signaling in hemostasis, thrombosis and megakaryocytopoiesis, and they should lead to clearer understanding of the relative roles of inside-out and outside-in signaling in these biological processes. They may also lead to the identification of intracellular targets for the development of a future generation of anti-thrombotic drugs that interfere with integrin signaling.