Plasmin improves oedematous blood-gas barrier by cleaving epithelial sodium channels
Background and PurposeLung oedema in association with suppressed fibrinolysis is a hallmark of lung injury. We aimed to test whether plasmin cleaves epithelial sodium channels (ENaC) to resolve lung oedema fluid. Experimental ApproachesHuman lungs and airway acid-instilled mice were used for analysing fluid resolution. In silico prediction, mutagenesis, Xenopus oocytes, immunoblotting, voltage clamp, mass spectrometry, protein docking, and alveolar fluid clearance were combined for identifying plasmin specific cleavage sites and benefits. Key ResultsPlasmin led to a marked increment in lung fluid resolution in both human lungs ex vivo and injured mice. Plasmin specifically activated {beta}{gamma}ENaC channels in oocytes in a time-dependent manner. Deletion of four consensus proteolysis tracts ({Delta}432-444, {gamma}{Delta}131-138, {gamma}{Delta}178-193, and {gamma}{Delta}410-422) eliminated plasmin-induced activation significantly. Further, immunoblotting assays identified 7 cleavage sites (K126, R135, K136, R153, K168, R178, K179) for plasmin to trim both furin-cleaved C-terminal fragments and full-length human {gamma}ENaC proteins. In addition to confirming the 7 cleavage sites, 9 new sites (R122, R137, R138, K150, K170, R172, R180, K181, K189) in synthesized peptides were found to be cleaved by plasmin with mass spectrometry. These cleavage sites were located in the finger and the thumb, particularly the GRIP domain of human ENaC 3D model composed of two proteolytic centres for plasmin. Novel uncleaved sites beyond the GRIP domain in both and {gamma} subunits were identified to interrupt the plasmin cleavage-induced conformational change in ENaC channel complexes. Additionally, plasmin could regulate ENaC activity via the G protein signal. Conclusion and ImplicationsWe demonstrate that plasmin could cleave ENaC to benefit the blood-gas exchange by resolving oedema fluid as a potent fibrinolytic therapy for oedematous pulmonary diseases. Bullet point summaryO_ST_ABSWhat is already knowC_ST_ABSO_LISerine proteases proteolytically cleave epithelial sodium channels, including plasmin and uPA acutely. C_LIO_LIActivity of epithelial sodium channels is increased post proteolysis. C_LI What this study addsO_LIPlasmin cleaves up to 16 sites composed of two proteolytic centres in both full-length and furin-cleaved human {gamma} subunit of epithelial sodium channels in hours. C_LIO_LINon-proteolytic sites in both and {gamma} subunits interrupt the plasmin cleavage-induced channel gating. C_LIO_LIIntratracheally instilled plasmin facilitates alveolar fluid clearance in normal human and injured mouse lungs. C_LI Clinical significanceO_LIActivation of human lung epithelial sodium channels by plasmin may benefit lung oedema resolution as a novel therapy for ARDS. C_LI