The role of cAMP in PAR2-mediated release of VWF and P-selectin. (A) HUVECs were stimulated with forskolin (20 μM), isoproterenol (10 μM), or vehicle for 15 minutes and then assayed for cAMP. Figure is representative of an experiment conducted 3 times. (B) Dose response of forskolin and PAR2-AP in HUVECs. Forskolin data are representative of an experiment done 3 times, whereas PAR2-AP data are the average of an experiment done 3 times. Dose-response curves were generated using the area under the curve function in SOFTmax PRO. (A-B) Cells were stimulated for 15 minutes with agonist and IBMX (100 μM) and then were assayed for cAMP. (C) Protein kinase A inhibition with H-89 decreases PAR2-AP and forskolin stimulation of VWF. HUVECs were pretreated with H-89 (10 μM) for 10 minutes and then stimulated with PAR1-AP (100 μM), PAR2-AP (200 μM), or forskolin (20 μM with 100 μM IBMX) for 20 minutes, and medium was collected and assayed for VWF. Figure represents the mean ± SEM of 3 experiments conducted in triplicate. *Statistically significant difference between control and H-89 (P < .05 as determined by 2-tailed t test). (D) Forskolin and H-89 do not significantly alter PAR1-AP–mediated release of P-selectin. HUVECs were pretreated with H-89 (10 μm) for 10 minutes or stimulated with forskolin (20 μM) along with PAR1-AP (100 μM) or PAR2-AP (200 μM) or vehicle for 15 minutes, and P-selectin was measured. Values represent the fold induction and are corrected for the effect of forskolin on basal P-selectin release. Figure represents the mean ± SEM of 3 experiments conducted in triplicate.