The procoagulant effects of extracellular vesicles derived from hypoxic endothelial cells can be selectively inhibited by inorganic nitrite

Extracellular vesicles (EVs) derived from endothelial cells are elevated in cardiovascular disease and promote inflammation and coagulation. Hypoxia is often a key feature and is itself a potent stimulator of increased EV production. Inorganic nitrite (NO2−) has beneficial and protective effects that are enhanced in hypoxia. Investigate the impact of hypoxia on the functional capacity of EV derived from endothelial cells under hypoxia, and assess whether pre-treatment of endothelial cells with NO2− can alter EV function. Differential ultracentrifugation was used to isolate EV from the cultured endothelial cell line HECV (CEV), and from primary human umbilical cord derived endothelial cells (PEV), with time-resolved fluorescence used to assess EV protein composition. Clot formation was induced by thrombin and calcium in two assays; using an Alexa Fluor 594 human fibrinogen conjugate assay and standard turbidometry. Platelet aggregation was determined using multiple electrode aggregometry. Scanning electron microscopy was used to visualise fibrin clots. Hypoxia exposure (1% O2) significantly increased CEV production in comparison to normoxia (21% O2) (1825 ± 72 EVs/cell vs 117 ± 9 EVs/cell, p 0.05). Hypoxia-derived PEVs contained significantly more tissue factor than normoxia-derived EVs (Relative Fluorescence Units (RFU) = 7666 ± 1698 vs 5958 ± 1644, p