Thrombospondin is a high molecular weight glycoprotein which has been identified in platelets, endothelial cells, smooth muscle cells, fibroblasts and, most recently, in an epithelial cell line from rat alveolar. The objective of the proposed study is to evaluate the hypothesis that thrombospondin is a multifunctional protein which mediates the macromolecular associations involved in cell-to-cell and cell-to-extracellular matrix interactions. Specific focus will be directed to the following areas: (1) The interaction of thrombospondin with calcium. The sedimentation coefficient, intrinsic viscosity, circular dichrosim, electron microscopic appearance and resistance to proteolysis of thrombospondin are profoundly affected by the removal of divalent cations with EDTA. Titration of both the circular dichroism and the peptide pattern produced by limited tryptic digestion imply that the interaction of calcium with thrombospondin involves cooperative interactions between multiple sites. In the proposed study, binding experiments will be performed with calcium and lanthanide ions to directly measure the number of binding sites, to confirm the presence of cooperative interactions and to determine the dissociation constants. (2) Structural domains of thrombospondin.
A specific aim of the proposed study is to establish a detailed map of the domain structure of thrombospondin. Methods for the purification of these domains and monoclonal antibodies directed against these domains will be developed and used in the functional studies described below. Electron microscopy, peptide mapping by reverse phase high pressure liquid chromatography and N-terminal amino acid sequencing will be used to orient the structural domains within the thrombospondin molecule. (3) Functional domains of thrombospondin. Thrombospondin has the ability to bind to calcium, heparin, fibrin(ogen), fibronectin, histidine-rich glycoprotein, type V collagen, laminin and cell surfaces.
A specific aim of the proposed study is to identify the structural domain of thrombospondin which contains each of these functional activities. In addition, the thrombospondin-binding portion of some of the above mentioned compounds will be identified and the effect of divalent cations on these interactions will be investigated. (4) Cell surface receptors for thrombospondin. Several different solid-phase binding assays will be developed and employed to identify proteins on the surface of platelets, endothelial cells, smooth muscle cells, fibroblasts, red cells and macrophages which bind to thrombospondin. The functional significance of potential receptors will be evaluated by determination of their subcellular location and the ability of physiologically relevant inhibitors to inhibit their binding to thrombospondin.
