INVOLVEMENT OF THE TETHERED LIGAND-RECEPTOR IN THROMBIN-INDUCED ENDOTHELIUM-MEDIATED RELAXATIONS

The mechanisms by which the serine protease, alpha-thrombin, mediates relaxations were examined in isolated dog and pig coronary arteries an d dog saphenous veins. In rings of coronary arteries and saphenous vei ns contracted submaximally with prostaglandin F2alpha or U46619, alpha -thrombin (0.1-10 nM) caused relaxations that were abolished by treatm ent with N(omega)-nitro-L-arginine (L-NNA) or removal of the endotheli um, indicating that the relaxations were mediated by endothelium-deriv ed nitric oxide. These relaxations were blocked by the thrombin active site inhibitor, MD-805, indicating the requirement of thrombin's cata lytic site to induce the relaxations. The thrombin exosite inhibitor, BMS-180742, decreased the sensitivity to alpha-thrombin without alteri ng maximal relaxations. Indomethacin, a cyclooxygenase inhibitor, had no inhibitory effect on the relaxations caused by alpha-thrombin, indi cating that the relaxations were not mediated by cyclooxygenase produc ts. Similar to alpha-thrombin, the thrombin receptor activating peptid e (human sequence: SFLLRNP, 1-100 muM) caused relaxations in pig coron ary artery and dog saphenous vein but not in dog coronary artery. Thes e relaxations were blocked by L-NNA but not by indomethacin. The resul ts indicate that alpha-thrombin induces endothelium-dependent relaxati ons by a novel signaling mechanism that involves proteolytic cleavage of the thrombin receptor to expose a new amino terminus that functions as a ''tethered peptide ligand'' to activate thrombin receptors on th e endothelial cells and release nitric oxide.