THROMBIN RECEPTOR-ACTIVATING PEPTIDE SENSITIZES THE HUMAN ENDOTHELIALTHROMBIN RECEPTOR

Receptor-operated effects of alpha-thrombin and of the thrombin recept or-activating peptide TRAP(14) on cytoplasmic Ca2+ concentration ([Ca2 +](i)) were examined in fura 2-loaded endothelial cells. Experiments w ith hirudin showed that ol-thrombin-induced Ca2+ influx requires the c ontinuous 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). Repet itive short-term stimulations with a-thrombin desensitized [Ca2+](i) t ransients to subsequent stimulations with either alpha-tkrombin or TRA P(14). In contrast, repeated short-term stimulations with TRAP(14) sen sitized [Ca2+](i) transients to subsequent stimulations with either ag onist. Blockade of Ca2+ influx by SKF-96365 abolished the sensitizing effect of TRAP(14). The results indicate distinct characteristics of p latelet and endothelial thrombin receptors and suggest that alpha-thro mbin and TRAP(14) activate the receptor differently. It appears that r eceptor desensitization occurs independently of TRAP(14) binding and, hence, tethered ligand binding to and activation of the receptor. Pers istent receptor desensitization after alpha-thrombin seems to depend o n both alpha-thrombin binding to the hirudin-like receptor domain and the irreversible proteolytic cleavage of the receptor. It does not inv olve the TRAP(14)/ tethered ligand binding site of the receptor. TRAP( 14) primes the receptor by a mechanism mediated by Ca2+ influx.