Abstract-Thrombospondin-1 (TSP-1) is a matricellular protein that is expressed in negligible amounts in normal blood vessels but is markedly upregulated in vascular injury. Although TSP-1 can act as a pleiotropic regulator for human vascular smooth muscle cells (HVSMCs), the intracellular signaling pathways stimulated by this protein remain obscure. In cultured HVSMCs derived from saphenous vein, TSP-1 induces tyrosine phosphorylation of a number of cellular proteins, with a complex temporal pattern of activation. Immunoprecipitation techniques have identified the early tyrosine-phosphorylated signals as being the p85 regulatory subunit of phosphatidylinositol 3-kinase (PI 3-K) and focal adhesion kinase (FAK). Tyrosine phosphorylation of the p85 subunit of PI 3-K showed a biphasic response to TSP-1 stimulation, which corresponded to a biphasic activation of the lipid kinase. Treatment with both wortmannin and LY294002 inhibited PI 3-K activity of HVSMCs but did not affect tyrosine phosphorylation of the p85 regulatory subunit. TSP-1-stimulated FAK phosphorylation, however, was substantially reduced by these inhibitors, as was the TSP-1-induced chemotaxis of these cells. These results suggest that activation of PI 3-K is an early signal induced by TSP-1 and is critical for chemotaxis. Activation of this kinase precedes and may occur upstream from FAK phosphorylation, although the nature of the interaction between these 2 enzymes remains obscure. Key Words: thrombospondin-1 Ⅲ focal adhesion kinase Ⅲ phosphatidylinositol 3-kinase Ⅲ human vascular smooth muscle T hrombospondin-1 (TSP-1), a large, homotrimeric glycoprotein, is a member of the TSP family of matricellular proteins. This family currently consists of 5 isoforms: TSP-1 through TSP-4 and TSP-5/cartilage oligomeric matrix protein, 1 which, although implicated in a wide variety of biological processes, 2 show differential tissue expression both temporally and spatially. 3,4 TSP-1 was originally described as a product of platelet ␣-granules 5 but has since been shown to be synthesized by a number of different cell types, including vascular endothelial and smooth muscle cells. 6,7 TSP-1 is present in negligible amounts in normal human blood vessels, but the expression of this protein is markedly upregulated in injured and diseased vasculature, 8 -10 suggesting a possible role for TSP-1 in the abnormal cellular response to vascular injury.Literature evidence would suggest that TSP-1 levels are regulated by growth factors. Both platelet-derived growth factor (PDGF) and transforming growth factor- (TGF-) have been shown to induce TSP-1 secretion from vascular smooth muscle cells (VSMCs). 11,12 TSP-1 mRNA levels have been shown to be similarly regulated. Basic fibroblast growth factor upregulates TSP-1 mRNA in 3T3 cells, 13 and PDGF induces upregulation in a manner similar to that of c-myc and c-fos, with TSP-1 being classified as an immediate early response gene. 14 These data, in conjunction with the role of TSP-1 in potentiating the chemotactic response of VS...