Thrombin receptor-activating peptide sensitizes the human endothelial thrombin receptor

Abstract

Receptor-operated effects of alpha-thrombin and of the thrombin receptor-activating peptide TRAP14 on cytoplasmic Ca2+ concentration ([Ca2+]i) were examined in fura 2-loaded endothelial cells. Experiments with hirudin showed that alpha-thrombin-induced Ca2+ influx requires the continuous presence of active alpha-thrombin. YFLLRNP, known to antagonize alpha-thrombin- and TRAP7-induced [Ca2+]i transients in platelets, did not antagonize [Ca2+]i transients in response to alpha-thrombin and TRAP14 in human umbilical vein endothelial cells (HUVEC). Repetitive short-term stimulations with alpha-thrombin desensitized [Ca2+]i transients to subsequent stimulations with either alpha-thrombin or TRAP14. In contrast, repeated short-term stimulations with TRAP14 sensitized [Ca2+]i transients to subsequent stimulations with either agonist. Blockade of Ca2+ influx by SKF-96365 abolished the sensitizing effect of TRAP14. The results indicate distinct characteristics of platelet and endothelial thrombin receptors and suggest that alpha-thrombin and TRAP14 activate the receptor differently. It appears that receptor desensitization occurs independently of TRAP14 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 TRAP14/tethered ligand binding site of the receptor. TRAP14 primes the receptor by a mechanism mediated by Ca2+ influx.