Human platelet thrombospondin (TSP) is a high molecular weight glycoprotein (Mr 450,000) composed of three identical disulphide bonded peptide chains. TSP is a constituent of platelet alpha- granules and an essential component of the secondary phase of platelet aggregation. TSP is also synthesized by a variety of cells including smooth muscle cells, endothelial cells and fibroblasts. In these cells the synthesized TSP is secreted and incorporated into the extracellular matrix (ECM). However there is some recent work which suggests that TSP may serve to regulate aortic smooth muscle cell (SMC) growth. TSP may also serve as a focus for protease generation in the ECM by its ability to bind both plasminogen and tissue plasminogen activator (t-PA). Since TSP is constituent of both the platelet and the vessel wall and involved in platelet-platelet and platelet vessel interactions its study should contribute to the better understanding of its role in both physiologic and pathologic states (thrombosis, atheroma). In order to gain greater knowledge about the structure and regulation of TSP synthesis in the vessel wall the following studies are proposed: 1) Investigation of the role of the heparin binding domain (HBD) of TSP with respect to aortic smooth muscle cell growth. These studies will use a bacterial expression system that synthesizes the HBD. The isolated HBD will be used to study its role in aortic smooth muscle cell proliferation. 2) Study of the gene structure of TSP to better understand the organization and evolution of the gene. Questions addressed will include, a) Do the exons code for seperate functional domains? b) Did the TSP gene evolve to its present form by mechanisms such as gene duplication and exon shuffling? 3) Investigate the mechanism by which platelet derived growth factor (PDGF) increases TSP synthesis in aortic SMC's. Does PDGF increase TSP mRNA by increasing the transcriptional rate or by stabilizing the mRNA for TSP? 4) Define the PDGF responsive elements within the TSP gene by use of gene constructions that consist of the putative regulatory region fused to a reporter gene (bacterial chloramphenicol acetyl transferase (CAT)). 5) Study the interaction of TSP with plasminogen using purified proteins and isolated aortic SMC matrices. 6) Exploit the ability of carcinoma cells which use TSP as an attachment factor in trying to localize the cell binding domain on TSP.
