FVIIa cleaves PAR1 on endothelial cells. Confluent monolayers of HUVECs were incubated at 37°C with (A) control buffer (○) or FVIIa (10nM; ●) for varying time periods, (B) varying concentrations of FVIIa for 1 hour, or (C) FVIIa (10nM), FVIIai (10nM), APC (10nM), or thrombin (1nM) for 1 hour. At the end of treatment, the supernatants were removed, the cell monolayer was fixed, and the residual intact (uncleaved) PAR1 at the cell surface was detected by cell surface ELISA using a conformation-specific PAR1 mAb (ATAP2). The spectrophotometric reading obtained for ATAP2 binding to untreated cells was taken as 100% (n = 5-7; mean ± SEM).