FVIIa-induced EVs exhibit higher procoagulant activity in vitro, and this is dependent on anionic phospholipids and independent of TF. (A) EVs released from HUVECs upon control vehicle or FVIIa treatment (100 nM for 24 hours) were isolated and suspended into equal amounts of a calcium-containing buffer. EVs were incubated with either annexin V (400 nM), TF neutralizing antibodies (10 µg/mL), or control IgG (10 µg/mL) for 1 h. After that, FVIIa (10 nM) was added to the EV suspension, and FX (175 nM) was added 5 minutes later. The rate of FX activation was measured in a chromogenic assay. (B) EVs were prepared and treated with annexin V as described in panel A. The ability of EVs to support prothrombin activation was measured by adding FVa (10 nM) and FXa (1.0 nM), followed by the substrate prothrombin (1.4 µM). Thrombin generated in the reaction mixture was measured in a chromogenic assay. (C-D) EVs, isolated from control- or FVIIa-treated HUVECs, were quantified, and an equal number of EVs were used to compare their ability to activate FX (C) and prothrombin (D). The assay conditions for measuring the rate of FX and prothrombin activation were the same as those used in panels A and B, respectively. (E-F) HUVECs were treated with FVIIa (100 nM) for 5 hours. Then, cells were washed twice to remove FVIIa and incubated with annexin V (400 nM), TF neutralizing antibody (10 µg/mL), or control IgG (10 µg/mL) for 1 hour. Intact cells were used to assess the cell surface associated-procoagulant activity in FX (E) or prothrombin (F) activation assays, as described for panels A and B. (G) HUVECs, cultured in glass coverslips, were treated with a control vehicle or FVIIa (100 nM) for 5 hours. Cells were washed and incubated with fluorescein isothiocyanate–conjugated annexin V (dilution 1:20) for 1 hour. Cells were then washed, fixed with 4% paraformaldehyde, and subjected to fluorescence microscopy (original magnification, ×40). **P < .01; ****P < .0001.