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

We studied dynamics of cell surface expression ofproteolytically activated thrombin receptor (PAR-1) in human pulmonaryartery endothelial cells (HPAEC). PAR-1 activation was measured bychanges in cytosolic calcium concentration(Ca²⁺]_i)and HPAEC retraction response (determined by real-time transendothelial monolayer electrical resistance).Ca²⁺]_iincrease in response to thrombin was abolished by preexposure to 25 nMthrombin for >60 min, indicating PAR-1 desensitization, butpreexposure to 25 nM thrombin for only 30 min or to 10 nM thrombin forup to 2 h did not desensitize PAR-1. Exposure to 10 or 25 nM thrombindecreased monolayer electrical resistance 40-60%. Cellspreexposed to 10 nM thrombin, but not those preexposed to 25 nMthrombin, remained responsive to thrombin 3 h later. Loss of cellretractility was coupled to decreased cell surface PAR-1 expression asdetermined by immunofluorescence. Cell surface PAR-1 disappeared uponshort-term (30 min) thrombin exposure but reappeared within 90 minafter incubation in thrombin-free medium. Exposure to 25 nM thrombinfor >60 min prevented rapid cycloheximide-insensitive PAR-1reappearance. Cycloheximide-sensitive recovery of cell surface PAR-1expression required 18 h. Therefore, both duration and concentration ofthrombin exposure regulate the time course of recovery of HPAEC surfacePAR-1 expression. The results support the hypothesis that initialrecovery of PAR-1 surface expression in endothelial cells results froma rapidly mobilizable PAR-1 pool, whereas delayed recovery results fromde novo PAR-1 synthesis. We conclude that thrombin itself regulatesendothelial cell surface PAR-1 expression and that decreased surfaceexpression interferes with thrombin-induced endothelial cell activation responses.