MURINE MODELS OF PLATELET INTEGRIN FUNCTION

对人类血小板和模型细胞系统的研究表明，通过整合素alphaIIbbeta3进行的双向信号传导调节了细胞与可溶性粘附配体的相互作用（内向外信号传导），并触发了血小板与血管基质稳定粘附所必需的细胞骨架变化（外向内信号传导）。然而，整合素信号通路可以随着细胞环境的变化而变化，并且在了解人类血小板中的基因表达方面取得了进展。因此，本项目将利用重组技术研究小鼠巨核细胞和血小板中的alphaIIbbeta3信号，包括离体和体内。具体目标将解决关于alphaIIbbeta3在止血、动脉血栓形成和巨核细胞发育中的功能的基本未解决的问题。首先，将通过逆转录病毒介导的基因转移、利用生长因子扩大巨核细胞池以及使用一组敏感试验评估alphaIIb β 3功能，在体外巨核细胞中检测alphaIIb和β 3细胞质尾部在信号传导中的作用。其次，将在这些细胞中评估β -内啡肽、H-Ras在调节alphaIIbbeta3亲和力/亲和度中的潜在作用，因为这些蛋白质在模型细胞系统中与整合素调节剂有关。此外，将确定β -内啡肽基因缺失在体内的影响。第三，表征小鼠巨核细胞粘附于alphaIIbbeta3配体所触发的outside-in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用于靶向修饰的alphaIIb基因在小鼠血小板中的体内表达，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小鼠模型。具体来说，alphaIIb将融合到串联FKBP“二聚化”重复序列中，当与β 3在体内表达时，这将使我们能够聚集alphaIIbbeta3，并通过给药FKBP二聚体刺激纤维蛋白原与血小板的结合。综上所述，这些研究将提供关于allphaIIbbeta3信号在止血、血栓形成和巨核细胞生成中的分子基础的新信息，并有助于更清楚地了解由内向外和由外向内信号在这些生物学过程中的相对作用。它们也可能导致细胞内靶标的鉴定，从而开发出下一代干扰整合素信号传导的抗血栓药物。

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.