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

Abstract

Background: 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.

Objectives: 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.

Methods: 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.

Results: Hypoxia exposure (1% O₂) significantly increased CEV production in comparison to normoxia (21% O₂) (1825 ± 72 EVs/cell vs 117 ± 9 EVs/cell, p < 0.001, respectively) but had no effect on CEV mean size (221 ± 6 nm vs 203 ± 4 nm, 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 < 0.001, respectively) and less tissue factor pathway inhibitor (RFU = 9799 ± 2353 vs 19723 ± 2698, p < 0.05). Hypoxia significantly increased CEV induced fibrin polymer formation compared to normoxia (% area = 46.98 ± 0.97 vs 36.36 ± 0.72, p < 0.05). Pre-treatment of endothelial cells with NO₂^- in hypoxia abrogated this effect (% area = 15.70 ± 1.99, p < 0.001). Hypoxia derived CEV non-significantly increased the maximum clot formed, shortened time to max clot, and increased time to clot lysis by turbidometry. ADP-mediated platelet aggregation was significantly elevated with PEV derived from hypoxia compared to normoxia (888.0 ± 32.2 AU*min vs 671.5.2 ± 28.3 AU*min, p < 0.01). This was abrogated by pre-treatment of hypoxic endothelial cells with NO₂^- (716.5 ± 744.3 AU*min, p < 0.001).

Conclusions: Hypoxia-derived PEVs and CEVs exhibit increased procoagulant activity compared to normoxia-derived EVs, which we confirm to be mediated by an imbalance of TF/TFPI. Pre-treatment of endothelial cells with NO₂^- reduces the pro-coagulant activity of EVs via a mechanism that is Hypoxia-inducible factor 1 (HIF-1) dependent, but independent of TF/TFPI.

Keywords: Coagulation; Extracellular vesicles; Haemostasis; Nitrite; Thrombosis.