α_vβ₃-Integrin antagonists inhibit thrombin-induced proliferation and focal adhesion formation in smooth muscle cells

α_vβ₃-Integrin antagonists reduced neointimal formation following vascular injury in eight different animal models. Because α-thrombin contributes to neointimal formation, we examined the hypothesis that α_vβ₃-integrins influence α-thrombin-induced signaling. Cultured rat aortic smooth muscle cells (RASMC) expressed α_vβ₃-integrins as demonstrated by immunofluorescence microscopy and fluorescence-activated cell sorting analysis. Proliferative responses to α-thrombin were partially inhibited by anti-β₃-integrin monoclonal antibody F11 and by cyclic RGD peptides. Immunofluorescence microscopy showed that α-thrombin stimulated a rapid increase in the formation of focal adhesions as identified by vinculin staining and that this effect was partially inhibited by α_vβ₃ antagonists. β₃-Integrin staining was diffuse in quiescent RASMC and did not concentrate at sites of focal adhesions following thrombin treatment. α-Throm bin elicited a time-dependent increase in activation of c-Jun NH₂-terminal kinase-1 (JNK1) and in tyrosine phosphorylation of focal adhesion kinase (FAK). α_vβ₃-Integrin antagonists partially inhibited increases in JNK1 activity but had no effect on FAK phosphorylation. In SMC isolated from β₃-integrin-deficient mice, focal adhesion formation was impaired in response to thrombin but not sphingosine-1-phosphate, a potent activator of Rho. In summary, α_vβ ₃-integrins play an important role in α-thrombin-induced proliferation and focal adhesion formation in RASMC.