DISTINCT RECEPTORS AND SIGNALING PATHWAYS IN ALPHA-THROMBIN-RECEPTOR AND THROMBIN-RECEPTOR PEPTIDE-INDUCED VASCULAR CONTRACTIONS

The vasoactive mechanisms of the serine protease alpha-thrombin were e xamined in isolated coronary arteries from dogs. In resting coronary a rteries with endothelium, alpha-thrombin caused concentration-dependen t contractions that were characterized by an initial transient relaxat ion followed by slowly developing sustained contractions. The vascular actions of alpha-thrombin were mimicked by the thrombin receptor-acti vating peptide (TRAP) SFLLRNP, a synthetic peptide based on the cleave d amino terminus of the thrombin receptor domain. Treatment of the art eries with N omega-nitro-L-arginine or removal of endothelium abolishe d the transient relaxations and enhanced the contractions, indicating that the transient relaxations were mediated by the concurrent release of endothelium-derived nitric oxide. alpha-Thrombin that had been cat alytically inactivated with the irreversible inhibitor by use of D-Phe -Pro-Arg-chloromethyl ketone did not cause contractions, indicating th e requirement of proteolytic cleavage by alpha-thrombin to induce cont ractions. In contrast to TRAP, alpha-thrombin-induced contractions wer e blocked by hirudin (a specific thrombin inhibitor), nifedipine and d iltiazem (Ca2+ channel blockers), or staurosporine and calphostin C (p rotein kinase C inhibitors). Unlike alpha-thrombin, which undergoes ho mologous desensitization, TRAP failed to cause desensitization to subs equent stimulation by alpha-thrombin or TRAP. These observations suppo rt the hypothesis that vasoactive actions of alpha-thrombin are mediat ed by a mechanism that involves cleavage at the active site to expose a new NH2 terminus that activates the thrombin receptor. Further, the dissociation between alpha-thrombin and the synthetic receptor peptide in signal transduction and dissimilar desensitizing properties sugges t the existence of distinct thrombin receptor subtypes and/or signalin g events in vascular smooth muscle.