The objective of this research is to enhance understanding of reversible ethanol induced thrombocytopenia, a common and serious complication of alcoholism. I have developed a tissue culture system for the assay of human megakaryocyte progenitors (CFU-M) in vitro, to study the effects of ethanol on thrombocytopoiesis. Ethanol suppresses human platelet production independently of nutritional deficiency and inhibits proliferation of myeloid and erythroid progenitors in vitro. In have done preliminary experiments showing that both a cell closely related to human CFU-M and human megakaryocytes are sensitive to ethanol at or near concentrations occurring in vivo. To render this system more useful for studying the effects of ethanol, I propose to characterize it further as to the influence of cell-cell interactions on CFU-M proliferation. I then propose to delineate more fully the degree of ethanol sensitivity of human CFU-M and related cells. Bone marrow cells will be fractionated before exposure to ethanol to reveal the contribution of accessory cells (adherent leukocytes and T-lymphocytes) to ethanol sensitivity. Similar experiments will also be performed with acetaldehyde, the principal metabolite of ethanol. To investigate a possible basis for the effects of ethanol and acetaldehyde. I will attempt to reverse their inhibitory effect on CFU-M by supplementation of cultures with micronutrients (folinic acid, pyridoxine) or metabolites (a-ketoglutarate, L-asparate) implicated in the effects of ethanol on other hematopoietic cells or other tissues. Since platelet production may be regulated at two levels, I also propose to isolate human megakaryocytes from bone marrow by established techniques to determine the effect of ethanol and acetaldehyde on DNA and protein synthesis and maturation of these cells and its potential reversibility by micronutrients or metabolites. To study the effect of chronic alcoholism on CFU-M in vivo, I shall collect bone marrow and serum from consenting patients hospitalized with ethanol thrombocytopenia. Bone marrow will be assayed for CFU-M number and proportion in S-phase in comparison to assays for BFU-E and CFU-GM and with results from normal volunteers. Serum will be assayed for the possible presence of factors inhibitory to CFU-M proliferation in culture in an autologous system. These studies will increase knowledge of mechanism by which ethanol produces significant end-organ damage and will lead to increased understanding of normal thrombocytopoiesis by its contrast with the diseased state.
