Megakaryocytes have a unique differentiation process in which they become polyploid through endomitosis and then undergo extensive 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. We have developed a procedure to purify guinea pig megakaryocytes, and perhaps more importantly, their immediate platelet-antigen expressing precursors, based on their ability to be specifically aggregated by bovine von Willebrand factor. In addition, we found that recognizable megakaryocytes can be fractionated into immature and mature populations based on differences in their buoyant densities. these methodologies provide the opportunity to examine differentiation events within the recognizable megakaryocyte compartment as well as in immediate megakaryocyte precursors. A major aim of this proposal is to define the repertoire of proteins turned on or off at specific points in megakaryocyte differentiation by studying synthesis and post-translational modifications of proteins in selected megakaryocyte and megakaryocyte precursor populations using two-dimensional gel electrophoretic analyses after specific labeling procedures. Our working hypothesis is that during megakaryocyte differentiation, changes occur at the macromolecular level which can be detected by available techniques. Guinea pig megakaryocytes will be used as a model in these studies. Another aspect of this proposal will be investigation of mechanisms involved in determination of platelet size during platelet formation through study of a rat strain in which we have discovered hereditary macrothrombocytopenia. This characteristic shows an autosomal recessive mode of inheritance which suggests that these rats have a qualitative or quantitative defect in a component necessary for proper subdivision of megakaryocyte cytoplasm into platelets. Studies are proposed to define this defect in platelet formation, physiologically and at the molecular level. These studies should yield basic insights into megakaryocyte differentiation and platelet formation as well as provide some understanding of megakaryocyte abnormalities in hereditary and acquired diseases.
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