MURINE MODELS OF PLATELET INTEGRIN FUNCTION

通过对人类血小板和模型细胞系统的研究已经确定，通过整合素αIIbbeta3的双向信号调节细胞与可溶性黏附配体的相互作用(由内向外信号)，并触发细胞骨架的变化，从而使血小板与血管基质稳定黏附(由外向内信号)。然而，整合素信号通路可以随着细胞环境的不同而变化，并朝着了解人类血小板基因表达的方向发展。因此，本项目将使用重组技术来研究体内和体外小鼠巨核细胞和血小板中的AlphaIIbbeta3信号。具体目标将解决有关AlphaIIbbeta3在止血、动脉血栓形成和巨核细胞发育中的功能的基本悬而未决的问题。首先，将通过逆转录病毒介导的基因转移、用生长因子扩大巨核细胞库以及使用一组敏感的检测方法评估AlphaIIb Beta3的功能，在体外研究AlphaIIb和Beta3细胞质尾巴在巨核细胞信号转导中的作用。其次，将评估这些细胞中Beta3-endonexin，H-RAS在调节alphaIIbbeta3亲和力/亲和力方面的潜在作用，因为这些蛋白在模型细胞系统中被认为是整合素调节因子。此外，还将确定Beta3-endonexin基因在体内缺失的影响。第三，将描述由小鼠巨核细胞与AlphaIIbbeta3配体黏附触发的由外向内的信号通路，并研究这些通路在巨核细胞发育和功能中的潜在作用。第四，基于逆转录病毒的技术将在体内靶向表达修改的AlphaIIb基因在小鼠血小板中的表达，目标是创建一种有条件的初级血小板高反应性和动脉血栓形成的小鼠模型。具体地说，AlphaIIb将与串联的FKBP“二聚化”重复序列融合，当在体内与Beta3一起表达时，这将使我们能够聚集AlphaIIbbeta3，并通过注射FKBP二聚体来刺激纤维蛋白原与血小板的结合。综上所述，这些研究将为AlphaIIbbeta3信号在止血、血栓形成和巨核细胞生成中的分子基础提供新的信息，并将使人们更清楚地理解内向外和外向内信号在这些生物过程中的相对作用。它们还可能导致识别细胞内靶点，以开发干扰整合素信号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.