This project is aimed at understanding the events that occur during differentiation along the megakaryocyte-platelet axis, a process in which multipotent cells give rise to progenitors restricted to the megakaryocyte linkage. We will approach this general objective with parallel investigations of murine and human megakaryocytopoiesis. Three principal approaches will be used to analyze the process. The first of these involves characterization of the expression of lineage-associated proteins during differentiation. In this approach, we will make use of specific immune reagents directed against membrane-specific, granule-specific and cytoplasmic platelet-precursor proteins to identify precursor cells. We will attempt, utilizing immune reagents, to probe cell surface antigens on early progenitors and to isolate, or enrich, progenitors. The antibodies will also be used in studies of the biosynthesis of platelet-specific proteins in megakaryocytes in vitro. In our second specific aim, we will attempt to isolate human and murine megakaryocyte-colony-stimulating activities using a composite of conventional techniques for protein isolation and recently developed monoclonal antibody techniques which have been successfully applied to other proteins by this laboratory. The isolated proteins will be characterized with respect to structure and function, and radioimmunoassays to the active proteins will be developed. In the third specific aim we will apply the information regarding lineagerelated proteins and quantitation of colony-stimulating activities to explore disturbances of megakaryocytopoiesis in human subjects and in mice.
