THROMBIN RECEPTOR-ACTIVATING PEPTIDE SENSITIZES THE HUMAN ENDOTHELIAL THROMBIN RECEPTOR

Receptor-operated effects of alpha-thrombin and of the thrombin receptor-activating peptide TRAP(14) on cytoplasmic Ca2+ concentration ([Ca2+](i)) were examined in fura 2-loaded endothelial cells. Experiments with hirudin showed that ol-thrombin-induced Ca2+ influx requires the continuous presence of active a-thrombin. YFLLRNP, known to antagonize alpha-thrombin- and TRAP(7)-induced [Ca2+](i) transients in platelets, did not antagonize [Ca2+](i) transients in response to alpha-thrombin and TRAP(14) in human umbilical vein endothelial cells (HUVEC). Repetitive short-term stimulations with a-thrombin desensitized [Ca2+](i) transients to subsequent stimulations with either alpha-tkrombin or TRAP(14). In contrast, repeated short-term stimulations with TRAP(14) sensitized [Ca2+](i) transients to subsequent stimulations with either agonist. Blockade of Ca2+ influx by SKF-96365 abolished the sensitizing effect of TRAP(14). The results indicate distinct characteristics of platelet and endothelial thrombin receptors and suggest that alpha-thrombin and TRAP(14) activate the receptor differently. It appears that receptor desensitization occurs independently of TRAP(14) binding and, hence, tethered ligand binding to and activation of the receptor. Persistent receptor desensitization after alpha-thrombin seems to depend on both alpha-thrombin binding to the hirudin-like receptor domain and the irreversible proteolytic cleavage of the receptor. It does not involve the TRAP(14)/ tethered ligand binding site of the receptor. TRAP(14) primes the receptor by a mechanism mediated by Ca2+ influx.