Effect of agonists for PARs and S1P receptors on endothelial barrier function. (A) Confluent HUVECs were incubated for 10 minutes in a dual-chamber system with the indicated agonists (5 nM thrombin, 10 μM PAR1 agonist TFLLRN-PNDK, 100 μM PAR2 agonist SLIGRL) in the absence (▪) or presence (□) of cleavage-blocking anti-PAR1 (combination of ATAP2 and WEDE15), and permeability was determined using Evans blue–labeled albumin in the upper chamber and measuring the optical density at 650 nm (OD650) in the lower chamber (mean ± SD, n = 3). (B) Confluent HUVECs were incubated for 10 minutes with the indicated concentrations of S1P (○) or SEW2871 (•) followed by a 10-minute incubation with 10 μM PAR1 agonist (means ± SD, n = 4, *P < .005 compared to no agonist).