Plasmin cleaves human epithelial sodium channels to resolve lung edema

Lung edema is a very common life-threatening dysfunction among infectious and non-infectious cardiopulmonary diseases, including systemic sepsis, pneumonia, hypoxia, high altitude induced mountain sickness, smoke inhalation, occupational exposure of gaseous irritants, and air pollutants. Anti-fibrinolytic compounds, including plasminogen activator inhibitor, antitrypsin, a2-antiplasmin, and a2 macroglobulin, are increased dramatically in the edematous injured lungs. Fibrinolytics administration may therefore be a pharmaceutical approach to restore normal fibrinolysis by neutralizing these elevated anti-proteases. A recent meta-analysis of preclinical studies demonstrates that fibrinolytic therapy benefits acute lung injury in animal models. We thus set out to study the potential therapeutic effects of plasmin on alveolar fluid clearance and underlying mechanisms. Instilled plasmin intratracheally expedited the rate to clear fluid from human lungs ex vivo significantly. Moreover, plasmin restores alveolar fluid clearance in a mouse model of gastric acid aspiration induced lung injury. For the subsequent mechanistic study, we heterologously expressed HA- and V5-tagged human epithelial sodium channels (ENaC), a major pathway for maintaining alveolar fluid homeostasis, in Xenopus oocytes. A combination of in silico prediction, mutagenesis, immunoblotting, and functional validation was applied to find potential cleavage sites in ENaC proteins for plasmin to modify ENaC activity proteolytically. Our results showed that plasmin specifically activated aßY ENaC channels in a time-dependent manner. Deletion of four consensus proteolysis tracts (aΔ432-444, YΔ131-138, YΔ178-193, and YΔ410-422) eliminated plasmin-induced activation significantly. Furthermore, we newly identified K126, R153, K168, R178, and K179 amino acid residues as new catalytic domains for plasmin; while R135 and K136 amino acid residues could be responsible for catalysis of furin-trimmed C-terminal fragments. In addition, plasmin may activate the G protein signals to regulate ENaC activity. Conclusively, our study uncovers multiple novel cleavage domains in human Y ENaC proteins for plasmin. ENaC could be a pharmaceutic target for plasmin to alleviate lung edema.