Thrombospondin" Synthesis and Secretion by Cells in Culture

Thrombospondin, a high molecular weight glycoprotein secreted by platelets in response to activation by thrombin, has been identified by immunofluorescence in bovine aortic endothelial cells, human foreskin fibroblasts, and human aortic smooth muscle cells. Immunofluorescence patterns were found to be similar using antisera raised to thrombospondins purified either from bovine aortic endothelial cells or from human platelets. Radioimmune precipitation of pulse-labeled cellular proteins confirmed the presence of thrombospondin in positively stained cells. A sensitive quantitative enzyme-linked immunosorbent assay (ELISA) was developed and used to determine that the accumulation of secreted thrombospondin was similar for endothelial cells and fibroblasts but was higher for smooth muscle cells. The presence of thrombospondin in a variety of cells suggests that its function may not be limited to an involvement in platelet interactions. Thrombospondin (TS), a high molecular weight glycoprotein, is released from a-granules after activation of platelets by thrombin (1, 2). After release, the protein binds to the activated platelet surface in a calcium-dependent fashion (3) and may participate in platelet-platelet interactions (4). Earlier work in this laboratory (5) identified a high molecular weight glycoprotein secreted by endothelial cells in culture that represented a substantial portion of the noncollagenous protein synthesized and secreted by these cells. This glycoprotein was subsequently shown to be indistinguishable from TS by a variety of criteria, including co-purification, molecular weight, amino acid composition, immunological cross-reactivity, and peptide maps (6). Although TS has been shown to have lectinlike activity in platelet-platelet interactions (4), its function in endothelial cells is not known. This led us to examine other cells in culture for the synthesis and secretion of TS. We have now identified TS in a variety of mesenchymal cells. Since TS is secreted and deposited in the cell layer of these cells in culture, we postulate that it may function as a matrix protein in vivo. MATERIALS AND METHODS