The role of hematopoietic growth factors in the promotion of megakaryocytic differentiation in experimental animals and man will be analyzed. Interleukin-3 (IL-3), a murine hematopoietic growth factor with broad target cell specificity, promotes both the proliferation of the megakaryocytic progenitor cell and the differentiation of megakaryocytes in vitro. To determine if IL-3 has a role in megakaryocytopoiesis in vivo, expression of the IL-3 gene will be measured following thrombocytopenia induced by 1) administration of anti-platelet serum to mice; and 2) exchange transfusion of rats with platelet-poor blood. Gene expression will be assessed directly at the transcriptional level by measuring steady-state splenic IL-3 messenger RNA by dot and Northern blotting, and/or solution hybridization with RNA probes for IL-3. Production of IL-3 activity in response to thrombocytopenia will be assessed indirectly by testing the effects of freeze-thawed extracts of spleen cells on the proliferation of an IL-3-dependent cell line and on the synthesis of acetylcholinesterase in liquid marrow cultures. In man, megakaryocytic differentiation will be studied in vitro by measuring the synthesis of specific proteins in short-term serumless liquid marrow cultures in response to the recombinant hematopoietic growth factors erythropoietin (rEpo) and granulocytemacrophage colony-stimulating factor (rGM-CSF). Glycoprotein (GP) IIb/IIIa, GP Ib and von Willebrand factor will be quantitated by two-site immunoradiometric or amplified enzyme immunoassays employing monoclonal antibodies to these proteins. To determine if growth factors promote megakaryocytic differentiation directly or act via accessory cells, human megakaryocytes labelled with a monoclonal antibody to GP IIb/IIIa will be purified to homogeneity by fluorescence-activated cell sorting and cultured in the presence of rEpo or rGM-CSF. DNA and specific protein synthesis potentially induced by these factors will be quantitated in short-term cultures by tritiated thymidine incorporation and two-site immunoassays. To determine if the action of growth factors is stage-specific, human megakaryocytes sorted according to size (determined by forward angle light scatter) and ploidy (with the supravital DNA dye Hoechst 33324) will be incubated with varying concentrations of rEpo or rGM- CSF, and size, DNA and specific protein synthesis will measured following culture.
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