The overall research objective of this program is an understanding of megakaryocytopoiesis and regulation of the production of platelets. Our program consists of two major components: Studies of Thrombopoietin, the presumed humoral regulator of thrombopoiesis; and Investigations of Megakaryocyte Colony-Forming Cells, precursors of megakaryocytes which can be identified in culture systems, in vitro. Thrombopoietin. Purification of thrombopoietin (TPO) will continue, using as starting material a fraction of rabbit plasma proteins precipitated by a (NH4)2SO4 saturation of 60-80% and then further purified by affinity chromatography will various lectins. Additional purification will be based on ion exchange and gel sieving chromatographic techniques. Characterization of TPO will be undertaken. The biological sites of action of TPO and their relative importance in regulating thrombopoiesis will be determined with a variety of assays. Megakaryocyte Colony-Forming Cells (Meg-CFC). We will use Meg-CFC obtained from both the bone marrow and spleen of mice to study the regulation of megakaryocytopoiesis. The effects of TPO on the frequency, total numbers, and some characteristics of Meg-CFC, in vivo and in vitro, will be measured. The ploidy distribution in megakaryocyte colonies (i.e., DNA content of the individual megakaryocytes in colonies) will be analyzed in order to determine if DNA levels are altered by TPO or by various agents which perturb bone marrow function (e.g., 5-fluorouracil, cyclophosphamide, irradiation). We will attempt to identify different mechanisms by which megakaryocyte precursors respond to TPO, thrombocytopenia, and certain classes of drugs. Relationships between numbers and characteristics of human Meg-CFC, detectable megakaryocytes in the bone marrow, and circulating platelets will be determined in patients with abnormal thrombopoiesis. The results will provide information about important aspects of the regulation of megakaryocytopoiesis and on the potentially unique role of DNA in the response of polyploid megakaryocytes to stimulation and suppression and in the production of platelets. An additional goal is to design therapeutic regimens for myelotoxic agents, used for cancer chemotherapy, that minimize the production of thrombocytopenia or long-term marrow toxicity.
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