Transgenic Analysis of Platelet Receptor Expression

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The objectives of this proposal are to examine the molecular events controlling maturation of the megakaryocyte, platelet biogenesis and normal platelet function. The experimental plan utilizes transgenic animals and the expression of variant membrane receptor. Our goals are to characterize the unique biological events occurring during megakaryocytopoiesis and the consequences of these events on normal platelet function. A murine model of the human Bernard-Soulier syndrome (BSS) has been established via a targeted deletion of the a-subunit of GP Ib (GP Iba), a subunit of the platelet receptor, GP Ib-IX. The mouse model displays a severe bleeding phenotype with macrothrombocytopenia mirroring the human Bernard-Soulier syndrome. The murine phenotype is rescued by expression of a human GP Iba subunit. New data is presented establishing the cytoplasmic tail of GP Iba controls platelet morphology and platelet release from the megakaryocyte. Moreover, a signal transduction pathway via the tail of GP Iba is described with functional consequences for platelet release and platelet function. Experiments are proposed to test the hypotheses: i) The GP Ib-IX receptor controls aspects of megakaryocyte maturation and platelet release via structural elements within the GP Iba subunit and ii) the GP Ib-IX complex contributes to megakaryocyte development via controlled signaling pathways that also become relevant for normal platelet function. Aim 1 determines the GP Iba structural requirements necessary for facilitating normal platelet release and defines the molecular defect responsible for the macrothrombocytopenia in BSS. Aim 2 characterizes the megakaryocytopoiesis defect in the GP Iba nu_ mouse. Aim 3 characterizes signaling through GP Iba and its role in thrombopoiesis. Aim 4 determines the hemostastic relevance of GP Iba cytoplasmic interactions in genetically engineered platelets. Although clearly important, the role of the GP Ib-IX complex in thrombopoiesis is poorly understood reflecting the lack of appropriate in vitro models. However, models, such as the murine BSS, present an opportunity to study these unique events and will provide new information on the mechanisms controlling megakaryocytopoiesis, the release of blood platelets and role of platelets in thrombus formation.