Platelets, the end product of megakaryocytopoiesis, are critical for maintaining chemostatic integrity. Platelets transfusions provide life- sustaining support for many thrombocytopenic patients, especially those undergoing chemotherapy and bone marrow transplantation. Currently predictions suggest a serious supply-demand problem for blood products beginning in the year 2000, highlighting an urgent need for novel methods to treat and prevent thrombocytopenia, therapy ideally aimed at reducing platelet need in patients and increasing platelet yields during donation. A long-term goal of my laboratory is to understand the molecular basis of megakaryocytopoiesis, the developmental process whereby hematopoietic pleuripotential progenitor cells generate cells ultimately produce circulating platelets, and understanding megakaryocytopoiesis should prove useful for developing such novel therapies. Thrombopoietin (TPO) the central effector of megakaryocytopoiesis, binds to MAP on the surface of megakaryocyte precursors, initiating a cascade of intracellular signals that orchestrate the molecular changes required for megakaryocytopoiesis. While the forced expression of MPL in a variety of cell lines has helped identify a number of signaling pathways activated by TPO engagement of MPL, the mechanism by which TPO stimulates megakaryocytopoiesis remains unknown. We recently developed transgenic mice with avian leukosis virus receptor (TVA) expression restricted to cells of the megakaryocyte lineage. These mice provide the first system for efficient genetic manipulation of primary murine megakaryocyte-lineage These mice provide the first system for efficient genetic manipulation of primary murine megakaryocyte-lineage cells, and they afford us the unique opportunity to isolate, expand, and study large numbers of cells from early in the megakaryocyte developmental pathway. We propose to use these mice to better understand the role of Ras and JAK/STAT signaling affect megakaryocytopoiesis. We will use avian retroviral vectors to express conditionally active and dominant-negative mutants of specific effectors of Ras and JAK/STAT signaling. In combination with the use of chemical inhibitors of these pathways, transgenic mice that uniquely perturb these pathways in megakaryocyte-lineage cells, and murine expression array experiments, the proposed experiments help us how these two signal pathways affect megakaryocytopoiesis.
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