FVIIa induces the release of EVs from endothelial cells. (A) HUVECs were incubated with PKH67 dye (20 nM) for 30 minutes at 37°C in a serum-free medium. The cells were washed 3 times with Hanks balanced salt solution, fixed with 4% paraformaldehyde, and stained with 4′,6-diamidino-2-phenylindole (DAPI) for 30 minutes. The labeled cells were subjected to fluorescence (Fl.) microscopy. (B) PKH67-labeled cells were serum starved for 1 hour, followed by treatment with FVIIa (100 nM) or a control (Con) vehicle for varying times. EVs isolated from the supernatant at the indicated times were quantified by measuring the fluorescence intensity of PKH67 dye. (C) Cells grown onto 6-well culture dishes were treated with a control vehicle or FVIIa (100 nM) for 24 hours. EVs isolated from culture supernatants were quantified by NTA NanoSight. (D) A spectrum showing the diameter range of EVs as analyzed by NTA NanoSight. (E) HUVECs were treated with a control vehicle or FVIIa (100 nM) for 24 hours. EVs isolated from cell supernatants and cell lysates were subjected to immunoblotting to probe endothelial cell surface markers, EPCR, and VE-cadherin (loading, EVs from 1 × 10⁶ cells; cell lysates from 0.5 × 10⁵ cells). (F-G) Band intensities were quantified by densitometric analysis. (H) PKH67-labeled cells were treated with a control vehicle, FVIIa (100 nM) or FVIIai (100 nM) for 24 hours. EVs were quantified by fluorescence measurement. (I) HUVECs were incubated with hirudin (H; 4U/mL) for 1 hours, followed by FVIIa (100 nM) for 24 hours. EV generation was quantified by NTA NanoSight. *P < .05; **P < .01; ***P < .001; ****P < .0001. ns, not statistically significantly different.