The objectives of this project are to define and characterize molecular evens of megakaryocytopoiesis and platelet production by analyzing the in vivo expression of platelet glycoprotein (GP) receptors. Our studies target the GP Ib-IX-V complex, a membrane multi subunit receptor providing platelets with two properties essential for normal function. First, GP Ib-IX-V supports platelet adhesion on thrombogenic surfaces, a process exemplified by bleeding disorders which result from a congenital absence of the receptor or its ligand, von Willerbrand factor. Second, is an assumed role for GP Ib-IX-V in normal platelet morphogenesis, suggested by the release of abnormal giant platelets coinciding with the congenital absence of the receptor, the Bernard- Soulier syndrome. However, the mechanisms responsible for platelet release from the megakaryocyte and the maintenance of a normal structure of recirculating platelets are poorly understood primarily due to the lack of in vitro models to examine and manipulate either process. A distinctive feature of this proposal is the utilization of in vivo models to examine platelet receptor expression in the unique intracellular environment of the megakaryocyte and platelet. Proposed studies will provide fundamental information on the mechanisms of in vivo regulation of GP Ib supporting a long-term objective of defining molecular events and/or factors responsible for the transcription of megakaryocytic-specific genes (Aims 1 and 2). Other experiments will evaluate the expression of septin genes during megakaryocytopoiesis. These have been made possible by the identification of human septin genes expressed by megakaryocytes, which are directly linked to the expression of the beta-subunit of GP Ib (Aim 3). We will also test the structural hypothesis linking platelet GP Ib-IX-V expression to normal platelet structure by proposing experiments to examine the in vivo relevance of interactions between the cytoplasmic domain of the alpha- subunit of GP Ib and the platelet membrane cytoskeleton in a murine model of the Bernard-Soulier syndrome (Aim 4).
These aims will be achieved through the combined use of megakaryocytic-specific gene promoters, the expression of variant GP Ibalpha molecules on the surface of murine blood platelets, and an examination of in vivo and in vitro megakaryocytic gene expression. These studies will generate basic information that can be applied to therapeutic strategies for pathological conditions affecting megakaryocytopoiesis and platelet production.
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