Anti-inflammatory potential of FVIIa-released EEVs. (A) An equal number of EEVs (2 × 10⁸) were isolated from HUVECs labeled with fluorescent PKH67 dye and then treated for 24 hours with a control vehicle (Con EVs) or FVIIa (100 nM). FVIIa-EVs were incubated with THP-1 cells for 4 hours. EV uptake by THP-1 cells was analyzed by monitoring PKH67 fluorescence (green) in the cells. DAPI was used to stain the nuclei, and anti-CD14 antibodies were used to stain the monocytic cell surface marker CD14. Images were taken by a confocal microscope with a 63× objective lens (LSM 510 Meta; Zeiss). (B-D) THP-1 cells were incubated with a control vehicle (Control) or EVs derived from HUVECs treated with a control vehicle (Con EVs) or FVIIa (FVIIa EVs) for 4 hours. After the cells were washed to remove free EVs, they were challenged with LPS (200 ng/mL). After 12 hours, the levels of proinflammatory cytokines, TNF-α (B), IL-6 (C), or IL-1 β (D) in the supernatant medium was determined by ELISA. (E-G) PBMCs isolated from human blood were incubated with a control vehicle or EEVs and challenged with LPS, as described for panels B, C, and D. TNF-α (E), IL-6 (F), and IL-1β (G) in the supernatant medium were measured by ELISA. (H) Murine endothelial cells (bEND.3) were treated with 100 nM of hFVIIa, mFVIIa, or mFVIIa_L4F for 24 hours. EEVs released into the supernatant medium were quantified by NTA. (I-K) An equal number of EVs (2 × 10⁸) isolated from bEND.3 cells treated with a control vehicle (Con EVs), hFVIIa (hFVIIa EVs), mFVIIa (mFVIIa EVs), or mFVIIa_L4F (mFVIIa_L4F EVs) were incubated with murine peritoneal macrophages for 4 hours and then challenged with LPS for 12 hours. The levels of TNF-α (I), IL-6 (J), and IL-1β (K) in the supernatant medium were determined by ELISA. *P < .05; **P < .01; ***P < .001; ****P < .0001; ns, not significant.