Megakaryocytes have a unique differentiation process in which they become polyploid through endomitosis and then undergo extensive cytoplasmic growth and maturation leading to the formation of several thousand platelets per cell. Although the cell kinetics of this process have been studied in detail, little is known about differentiation at the macromolecular level. Recent advances in methodology such as isolation and purification of megakaryocytes in large quantities, analysis of their cell contents by flow cytometry and assays of megakaryocyte precursors in vitro now allow the study of megakaryocyte differentiation in more detail. By modifying a technique (density fractionation) used in megakaryocyte purification, we have been able to fractionate megakaryocytes into immature and mature populations. This separation will permit us to examine differentiation within the recongnizable megakaryocyte compartment. A major aim of this proposal is to compare the labeling patterns of proteins of these two megakaryocyte populations by SDS slab gel electrophoresis and flow cytometry after specific protein labeling in normal and altered states of megakaryocytopoiesis. Our working hypothesis is that during megakaryocyte differentiation, changes occur at the macromolecular level which can be detected by available techniques. Rat megakaryocytes will be used as a model for these studies. Similar studies will be done on human megakaryocytes if sufficient numbers can be obtained by marrow aspiration. Another aspect of this proposal concerns the precursors that form single megakaryocytes in vitro. We hypothesize that these cells are the immediate precursors of megakaryocytes and are more responsive to changes in platelet demand than are colony-forming cells. To test this hypothesis we will determine whether the number of these precursors is altered early after induction of acute thrombocytopenia in rats as well as examine their biological and physical characteristics. Finally, we propose to use the irradiated rat as a model for determining the relationship between megakaryocyte colony-stimulating activity produced in vivo and platelet and megakaryocyte concentration. These studies should provide basic insights into differentiation and regulation of megakaryocytopoiesis as well as provide some understanding of megakaryocyte abnormalities in diseases such as idiopathic thrombocytopenic purpura and chronic myelocytic leukemia.
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