The hemostatic potential of FVIIa-derived EVs from murine brain endothelial cells isolated from various genotypes. Brain endothelial cells were isolated from WT, EPCR-KO, EPCR-OX, PAR1-R41Q, and PAR1-R46Q mice and cultured ex vivo. Confluent monolayers of endothelial cells were treated with a control vehicle or FVIIa (100 nM) for 24 hours in serum-free medium. (A-B) Equal volumes of EVs isolated from the conditioned medium were evaluated for their ability to activate FX (A) or prothrombin (B) in the presence of either control vehicle or annexin V (400 nM). (C-D) WT murine endothelial cells were treated with a control vehicle or FVIIa (100 nM) for 5 hours. The cells were washed to remove FVIIa and then treated with annexin V (400 nM) for 30 minutes. (C-D) Cell surface–associated procoagulant activity was evaluated in FX activation assay by adding FVIIa (10 nM) and FX (175 nM) (C) or prothrombin activation assay by adding FVa (10 nM), FXa (1.0 nM), and prothrombin (1.4 µM) (D). *P < .05; ***P < .001; ****P < .0001.