FVIIa-induced p44/42 MAPK activation and barrier protection is mediated via Rac1. (A) HUVECs were treated with specific inhibitors of Rac1 (NSC 23766, 100μM) or RhoA (Y-27632, 10μM) for 2 hours before being treated with FVIIa (10nM) for 20 minutes. Activation of p44/42 MAPK was evaluated by immunoblot analysis. (B) Same treatment as in panel A, but p44/42 MAPK activation was quantified by densitometry (n = 3). (C) HUVECs cultured in Transwells were treated with FVIIa (10nM) for 2 hours in the absence or presence of Rac1 inhibitor (NSC 23766, 100μM) or RhoA inhibitor (Y-27632, 10μM), and then thrombin (5nM) for 10 minutes to induce permeability. (D) HUVECs cultured in Transwells were infected with adenovirus (20 MOI/cell) encoding the dominant-negative variant of Rac1 (Rac DN) or RhoA (Rho DN). After culturing cells for 48 hours, confluent monolayers were treated with FVIIa (10nM) for 2 hours, and then thrombin (5nM) for 10 minutes. Permeability was measured as described in Figure 4. *Statistically significant (P < .05) compared with thrombin treatment alone.