FVIIa pretreatment reduces thrombin-induced barrier disruption. (A) HUVECs (5 × 104 cells) were plated on 12-well Transwell chambers and cultured for 4 days. Confluent monolayers were treated with varying concentrations of FVIIa or APC (20nM) for 2 hours, and then FVIIa (or APC) was removed before the monolayers were treated with thrombin (5nM) for 10 minutes. At the end of 10 minutes, thrombin was removed, the cells were washed with PBS, Evans Blue-BSA was added to the upper chamber, and the amount of dye leaked to the bottom chamber at 10 minutes was measured by measuring optical density at 650 nm. The optical density reading obtained with thrombin treatment of cells not exposed to any prior treatment was taken as 100%. *P < .05 compared with thrombin control (n = 6-10). (B) HUVECs (2 × 105 cells) were plated onto each well of an 8-well ECIS chamber slide. After 48 hours, the cells were equilibrated with serum-free medium for 1 hour and left alone (top line) or exposed to FVIIa (10nM, 3rd line from the top), APC (20nM, 2nd line from the top), or control vehicle (bottom line) for 2 hours, followed by thrombin (5nM). Uninterrupted continuous resistance was collected with the ECIS measuring apparatus from the beginning. Because there was no change in the resistance before the addition of thrombin, this portion of the readings was not included in the figure. The arrow indicates the time of thrombin addition. (C) HUVECs cultured to confluence in Transwells as described in panel A were treated with control vehicle (CV), FVIIa (10nM) ± EPCR mAb (25 μg/mL), or FVIIai (10nM) for 2 hours, and then with thrombin (5nM) for 10 minutes. Cell permeability after thrombin treatment was determined by the migration of Evans Blue-BSA from the apical to the bottom chamber (n = 3-6). (D) HUVECs transfected with scrambled siRNA or EPCR-specific siRNA were treated with FVIIa (10nM) for 2 hours, followed by thrombin (5nM) for 10 minutes. Permeability was measured as described in panel A. *P < .05 compared with thrombin treatment alone. (E) HUVECs transfected with scrambled siRNA or PAR2 specific siRNA were treated with FVIIa (10nM) for 2 hours, followed by thrombin (5nM) for 10 minutes. Permeability was measured as described in panel A. *Statistically significant (P < .05) compared with thrombin treatment alone.
