The objective of this research proposal is to determine the significance of polyploidy in megakaryocytopoiesis, because mature megakaryocytes (MK) are the immediate precursors of circulating platelets. The basic premise is that polyploidization represents a pivotal point in MK differentiation, since changes in size, ploidy and maturation are landmark features of MK accompanying perturbations in platelet production. Specific objectives and hypotheses are: 1. To produce an objective, quantitative, computerized data base MK maturation. hypothesis - cytoplasmic maturation of recognizable MK is directly related to nuclear differentiation. hypothesis - this relationship is similar across the predominant ploidy classes observed in mature MK. 2. To compare specific cytoplasmic (platelet markers) features across ploidy classes. hypothesis - ploidy class determines the ultimate functional maturity of individual MK. 3. To determine the effects of cytokines or hematopoietic growth factors on MK maturation. hypothesis - cytoplasmic maturation is differentially regulated by various cytokines. hypothesis - cells from different ploidy classes are distinctively responsive to specific external influences. 4. To characterize the interrelationship between proliferation and polyploidization. hypothesis - the mitotic history of committed MK progenitors influences the degree of cell polyploidy, and as a result, the functional maturity of recognizable MK. Three different in vitro assays will help delineate the interrelationships between cell proliferation of committed progenitors (BFU-MK and CFU-MK), polyploidization, and the functional maturity of recognizable MK. Specific markers will be used to quantitate biochemical events in individual cells undergoing terminal cytoplasmic maturation, and the same MK will be analyzed for the degree of nuclear differentiation and polyploidy. Specific membrane glycoproteins, alpha-granule constituents (immunocytochemistry) and nuclear chromatin patterns (Feulgen) will be analyzed by computer-assisted microscopy and image cytometry. Cellular development in the presence of purified and recombinant cytokines will help distinguish which level(s) of megakaryocytopoiesis can be altered by environmental conditions. And of great importance, what effect a cytokine(s) has on functional maturity, which is an indication of the hemostatic potential of mature MK, the immediate precursors of circulating platelets. This research will provide a greater understanding of the biological implications of polyploidy in megakaryocytopoiesis, and generate a reference data base applicable to studying the effects of cytokines on thrombopoiesis in vivo, as well as experimentally induced thrombocytopenia and thrombocytosis. Similar work with human MK will have potentially beneficial applications in the diagnosis and management of patients with various thrombopathic problems.
