Activated platelets secrete a number of proteins, many of which are specifically localized within the platelet Alpha-granule. While not all of these secreted proteins are platelet specific, the platelet serves as the primary cellular source for their biological circulation. Upon release, proteins such as platelet factor 4 (PF-4), beta-thromboglobulin (Beta-TG) and platelet thrombospondin (TS) share the common property of heparin binding. Bovine platelet secreted proteins can be resolved from one another heprin affinity matricies, eluting respectively with 0.55M (TS), 0.65M (Beta-TG) and l (PF-4) buffered sodium chloride. Platelet TS, a protein of reduced molecular size 180,000 daltons has the amino terminal sequence: Asp-Arg-Ile-Glu-Glu-Ser-Gly-Gly-Asp-Asp-Ser-Val and bears virtual identity to human platelets TS. When hydrolyzed by thrombin in the presence of 2mM calcium, the 180,000 chain yields a 150,000 fragment and a 30,000 heparin binding peptide. This 30,000 peptide has been isolated in quantities sufficient to complete its primary structure. TS has additional affinities for fibrinogen, fibronectin, arginine and thiols. TS proteolyzed separately by either thrombin, plasmin, trypsin and chymotrypsin, in the presence of calcium, generates a large fragment which does not bind to heparin but does bind to fibrinogen and other ligands. This large peptide has the amino terminal sequence: Ser-Ser-Asn-Pro-Gln-Phe. Hydrolysis of TS by the above mentioned enzymes in the presence of EDTA yields fragments of lower molecular weight; these will be characterized with respect to amino terminal sequence and ligand affinity interactions. Electron microscopic imaging will be carried out on the large TS fragments in the presence and absence of calcium. Similar studies will be carried out when TS or its binding fragments are complexed with fibrinogen. Polyclonal and monoclonal anitbodies will be raised against TS and its principal fragments and these will be utilized to determine ligand binding changes following proteolysis. PF-4 will be isolated to homogeneity and crystallized by a hanging drop technique from polyethylene glycol solutions. Heavy atom derivatives and heparin conjugates of these crystals will be searched for, in order to achieve atomic resolution to 3.0 angstroms and its correlation with the known PF-4 pprimary sequence. The amino acid sequence of bovine Beta-TG related protein will be completed and its structure compared with the other heparin binding proteins secreted by the platelet.
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