FVIIa-released EEVs induce barrier protection in target endothelial cells. (A) An equal number of EVs (2 × 10⁸), released from HUVECs prelabeled with PKH67 dye and treated with a control vehicle (Con EVs) or FVIIa (FVIIa EVs), were incubated with naive HUVECs for 4 hours. The uptake of EEVs by target naive endothelial cells was determined by analyzing PKH67 fluorescence (green). Endothelial cells were immunostained for VE-cadherin to identify the cell periphery. Nuclei were stained with DAPI. (B) HUVECs grown to confluence in transwells were incubated with a control vehicle (Control) or EVs (2 × 10⁸) released from HUVECs treated with control vehicle (Con EVs) or FVIIa (FVIIa EVs) for 4 hours. After 4 hours, the monolayer was washed twice and challenged with LPS (200 ng/mL). Barrier permeability was measured 12 hours after the addition of LPS, as described in “Materials and methods.” The barrier permeability (OD readings) observed in cells treated with LPS that were not exposed to EVs were taken as 100%. (C) bEND.3 cells grown to confluence in transwells were exposed to control vehicle (Control) or EVs (2 × 10⁸) released from bEND.3 cells treated with a control vehicle (Con EVs), hFVIIa (hFVIIa EVs), mFVIIa (mFVIIa EVs), or mFVIIa_L4F (mFVIIa_L4F EVs). LPS-induced barrier permeability was evaluated as described in panel B. ***P < .001; ****P < .0001; ns, not significant.