The goals of this work are to obtain a complete understanding of the structure, metabolism and function of the sulfated macromolecules, i.e. the proteoglycans and sulfated proteins, of megakaryocytes and platelets. Our previous work has demonstrated the existence of three proteoglycans and several sulfated proteins in guinea pig megakaryocytes and platelets, and the existence of proteoglycans of the same Mr range in human platelets. We will extend these studies as follows. (1) The core proteins of each proteoglycan will be characterized by N-terminal amino acid sequence analysis and study of total amino acid composition. Antibodies prepared against the intact proteoglycans, peptides synthesized on the basis of selected sequences in the N-terminal peptides of the core proteins, and oligonucleotide probes based on the peptide sequences will be used to screen a cDNA library prepared from HEL cells in order to obtain cDNA for the core proteins; we can then determine total amino acid sequence. (2) The sulfated and non-sulfated oligosaccharides of platelet proteoglycans will be completely characterized. (3) (The subcellular localization of the three proteoglycans will be probed ultrastructurally at the light and electron microscopy level to determine their specific localization. Antibodies generated against the intact molecules and the N-terminal sequences will bc used as probes. The cDNA will bc used as a probe for in situ hybridization studies to determine whether the different core proteins are expressed at different stages of megakaryocyte maturation. (4) The role of poteoglycan synthesis in alpha granule formation will be assessed using an in vitro model in which megakaryocytes are incubated in the presence of specific inhibitors of proteoglycan synthesis, and by studying patients with the Gray Platelet Syndrome to see if they lack proteoglycans. (5) The role of surface proteoglycans in platelet function will be assessed by digesting the surface of the cells with chondroitinase or incubating the cells with anti-membrane proteoglycan antibody, and monitoring the effect on platelet function. (6) Sulfated proteins of human platelets will be identified and the nature of the sulfate-containing moieties characterized. Their functional role will be assessed by surface digestion of platelets with sulfatases. These studies will provide an understanding of the roles of the sulfated macromolecules in platelet function, and a basis for determining whether and how they are altered in abnormalities which affect platelet function.
