Time course of recovery of endothelial cell surface thrombin receptor (PAR-1) expression

We studied dynamics of cell surface expression of proteolytically activated thrombin receptor (PAR-1) in human pulmonary artery endothelial cells (HPAEC). PAR-1 activation was measured by changes in cytosolic calcium concentration ([Ca2+](i)) and HPAEC retraction response (determined by real-time transendothelial monolayer electrical resistance). [Ca2+](i) increase in response to thrombin was abolished by preexposure to 25 nM thrombin for >60 min, indicating PAR-1 desensitization, but pre-exposure to 25 nM thrombin for only 30 min or to 10 nM thrombin for up to 2 h did not desensitize PAR-I. Exposure to 10 or 25 nM thrombin decreased monolayer electrical resistance 40-60%. Cells preexposed to 10 nM thrombin, but not those preexposed to 25 nM thrombin, remained responsive to thrombin 9 h later. Loss of cell retractility was coupled to decreased cell surface PAR-I expression as determined by immunofluorescence. Cell surface PAR-I disappeared upon short-term (30 min) thrombin exposure but reappeared within 90 min after incubation in thrombin-free medium. Exposure to 25 nM thrombin for >60 min prevented rapid cycloheximide-sensitive PAR-1 reappearance. Cycloheximide-sensitive recovery of cell surface PAR-1 expression required 18 h. Therefore, both duration and concentration of thrombin exposure regulate the time course of recovery of HPAEC surface PAR-1 expression. The results support the hypothesis that initial recovery of PAR-1 surface expression in endothelial cells results from a rapidly mobilizable PAR-1 pool, whereas delayed recovery results from de novo PAR-1 synthesis. We conclude that thrombin itself regulates endothelial cell surface PAR-1 expression and that decreased surface expression interferes with thrombin-induced endothelial cell activation responses.