The agonist of the protease-activated receptor-1 (PAR1) but not PAR3 mimics thrombin-induced vascular endothelial growth factor release in human vascular smooth muscle cells

Thrombin, a serine protease generated by the activation of the blood coagulation cascade following vessel injury, induces vascular endothelial growth factor (VEGF) release. However, the molecular mechanism of thrombin-induced VEGF release is largely unknown. An agonist of protease-activated receptor-1 (PAR1), SFLLRNPNDKYEPF, mimicked thrombin-induced VEGF release in human vascular smooth muscle (HVSM) cells, as determined by enzyme-linked immunosorbent assay, reverse transcriptase-polymerase chain reaction, and Northern blotting. In contrast, the agonist of PAR3, TFRGAP, did not affect VEGF release or expression. SFLLRNPNDKYEPF, but not TFRGAP, up-regulated [Ca2+]i. Moreover, the calcium ionophone A23187 was found to trigger VEGF release in HVSM cells. Thrombin-induced VEGF release was blocked by anti-thrombin, heparin, a synthetic thrombin receptor inhibitor E5510, the calcium chelator BAPTA, the protein kinase C inhibitor calphostin C, and the MEK1/2 inhibitor U0126. Thus, our data show that thrombin caused VEGF release via PAR1 activation in a manner dependent on [Ca2+]i and p44/42 downstream from the receptor activation.