Bone marrow megakaryocytes give rise to blood platelets by fragmentation of their cytoplasm and release of the fragments into the blood stream. There is considerable evidence that megakaryocyte maturation is controlled by changes in the circulating platelet population via a humoral mechanism. One source of a thrombopoietic stimulation is the plasma of thrombocytopenic animals. A certain fraction of this plasma stimulates platelet production in vivo. Therefore, the effect of fractionated thrombocytopenic plasma on isolated megakaryocytes will be studied. Two in vitro stimulators of megakaryocytic colonies, interleukin-3 and megakaryocyte colony potentiator, will also be studied for their effects on isolated megakaryocytes. These studies will be performed with the intention of determining whether or not megakaryocyte maturation can be controlled at the level of the recognizable megakaryocyte. The effect of these agents on megakaryocytes will be investigated in three areas: metabolism, morphology and maturation. Using radiolabeled precursors, protein synthesis and phosphorylation and lipid synthesis will be studied. Analysis of material will be performed using electrophoretic and chromatographic procedures. Glycolysis will be studied by measuring glucose uptake and lactic acid production. Morphology will be investigated using phase and both scanning and transmission electron microscopy. Cytoskeletal structures will be studied using immunofluorescence and immunoelectron microscopy. Maturation will be studied using pulse chase experiments with 3H-thymidine and following the movement of label through successive levels of maturation as determined by Wright-Giemsa stained specimens. Also the effect on megakaryocyte ploidy distribution will be studied. In addition fluorescent activated cell sorting will be used to separate megakaryocytes by ploidy. The separated cells, will be used to investigate the possible metabolic differences between megakaryocytes of different ploidys. These studies provide the basis for a long term program on the study of biochemical and metabolic changes that occur during megakaryocyte maturation, as well as explaining the mechanism of action of thrombopoietic stimulators at the cellular level. In addition these studies may lead to a useful in vitro assay for the purification of thrombopoietin. Overall, a better understanding of the control of megakaryocytopoiesis will be the outcome of this project.
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