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.
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