The overall objective of the proposed project is to understand the role of thrombospondin (TSP) and its cellular receptor (GPIV) in vascular biology. TSP is a major component of platelets and the vascular subendothelium and is involved in mediating platelet- platelet, platelet-monocyte and perhaps other cell-cell and cell- matrix interactions. An 88Kd integral membrane protein has recently been identified as a TSP receptor on platelets and 2 tumor ell lines. This protein is identical to platelet glycoprotein IV (GPIV) and probably to a monocyte protein of unknown function now known as CD36. TSP also modulates arterial smooth muscle cell growth and plays a role in the regulation of plasminogen activation and heparin function. Thus, study of TSP and its receptor is important in understanding both physiological and pathological events at the vessel wall (e.g. thrombosis and atherosclerosis). The specific objectives are to characterize structure-function relationships of the TSP-GPIV interaction by isolating, characterizing and cloning the cDNA for the TSP receptor (GPIV) from human endothelial cell and U937 cell cDNA libraries. The latter is a human cell line of monocyte lineage. Structural domains of GPIV, in particular those involved in mediating TSP binding, membrane insertion and intracytoplasmic functions (e.g. cytoskeletal interactions) will be characterized by analyzing function of mutated (truncated) receptor molecules in transfected cells. In a complementary manner the TSP domain involved in mediating GPIV binding will also be identified using monoclonal antibodies and partial protease digestion of the intact molecule. The gene for GPIV will be identified from a human genomic lambda phage library and partially characterized. The cellular biology of the TSP-GPIV interaction will be studied with particular reference to known TSP functions. The role of GPIV in mediating TSP-dependent cytoadhesive interactions, such as that among platelets, monocytes, and endothelia, between malaria infected erythrocytes and endothelia, and between tumor cells and extracellular matrix will be studied as will the expression and regulation of both TSP and GPIV in vascular cells (endothelium and smooth muscle) and mononuclear phagocytes using specific immunological (monoclonal and polyclonal) and molecular probes.
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