The goal of this project is to understand the role of proteoglycans in normal megakaryocyte and platelet development and in disorders such as hypercholesterolemia. We have observed, along with changes in lipid metabolism, changes in (S35) sulfate incorporation into platelets of hypercholesterolemic animals which suggest substantial changes in synthesis of proteoglycans and other sulfated proteins in the megakaryocytes of the hypercholesterolemic animals. The current proposal will extend these studies. (1) Proteoglycan synthesis and metabolism will be studied in vitro in megakaryocytes isolated from normal and hypercholesterolemic guinea pigs. The cells will be incubated with (S35) sulfate. (H3)glucosamine and (H3)leucine to label the proteoglycans and hyaluronic acid. The proteoglycans will be characterized, the time course of synthesis and the turnover rates of the various molecules will be determined, and the subcellular locations of the sulfated molecules will be assessed by treating the cells with agents which may release labeled molecules selectively from either the granules or surface of the cells. The regulation of proteoglycan synthesis by hypercholesterolemic serum will be studied both in freshly-isolated megakaryocytes and in long-term marrow cultures. (2) Proteoglycan synthesis in megakaryocytes and the proteoglycan content of platelets will be studied in vivo, using megakaryocytes and platelets isolated from normal and hypercholesterolemic guinea pigs which have been injected with (S35)sulfate. The time course of incorporation of radiolabel into specific proteoglycans and other sulfated macromolecules will be studied in both megakaryocytes and platelets over a period of five days in order to provide information concerning whether megakaryocytes at different stages of maturation synthesize different types of sulfated molecules, and whether the pattern is changed in hypercholesterolemia. The subcellular localization of the various sulfated molecules will be probed in megakaryocytes and platelets labeled in vivo. The functional role of proteoglycans in guinea pig platelets will be assessed. (3) The mass distribution of proteoglycans of guinea pig blatelets and megakaryocytes will be determined for both normal and hypercholesterolemic animals. (4) The studies will be extended to analysis of proteoglycans of platelets from normal and hypercholesterolemic humans. This research is expected to define the poorly-understood role of proteoglycans in megakaryocyte and platelet biology and development, and their importance in the development of platelet abnormalities in patients with hypercholesterolemia.
