This project is aimed at understanding the events and regulatory mechanisms involved in the control of human megakaryocyte growth and maturation and platelet production, with special emphasis on the events that occur from endoreduplication through platelet shedding. Two principal approaches will be used in our analyses. The first of these is to rigorously characterize one step of the regulatory process. In this approach, we will isolate growth stimulatory factor(s) from aplastic human plasma and cell conditioned medium using standard biochemical isolation techniques. This approach will be facilitated by a radioimmunoassay for in vitro megakaryocyte growth developed in our laboratory. Monoclonal and polyclonal antibodies will be produced against the isolated factor(s), and immunoabsorbant columns prepared. These columns will allow production of factor-deficient plasms and sera that may be used to analyze the physiologic role of the isolated factor, and to determine the presence of other regulatory molecules. We will use these immunoabsorbant columns to isolate the factor(s) in sufficient quantities for structual analysis and determination of biological mechanisms. We will also develop antisera to growth factors previously found to influence stem cell growth in order to deplete megakaryocyte growth factor containing samples of these factors. The antifactor antibodies will also be used to develop radioimmunoassays for use in patient studies. The second approach will involve the study of the interaction of platelet products and of coagulation proteins with megakaryocytes and cells that produce megakaryocyte regulatory molecules. Platelet products, such as type beta-transforming growth factor, will be analyzed for their ability to act as regulators along the platelet production process. Coagulation proteins will be tested for a role in feedback regulation of megakaryocytopoiesis. Rigorous characterization of discrete regulatory factor(s) in megakaryocytopoiesis, and the ability to quantitate them in patients, will expand our understanding of clinical disorders in which specific controls of megakaryocytopoiesis are either inadequate or ineffective and lead to thrombocytopenia or thrombocytosis despite normal numbers of red blood cells and white blood cells.
