Plasmin improves oedematous blood-gas barrier by cleaving epithelial sodium channels

Abstract

Background and Purpose: Lung 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 Approaches: Human 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 Results: Plasmin led to a marked increment in lung fluid resolution in both human lungs ex vivo and injured mice. Plasmin specifically activated abgENaC channels in oocytes in a time-dependent manner. Deletion of four consensus proteolysis tracts (aD432-444, gD131-138, gD178-193, and gD410-422) 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 gENaC 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 a and g 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 Implications:We 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.

PAI-1 regulates AT2-mediated re-alveolarization and ion permeability

“…Serpine1 increased transepithelial permeability. The proteolytic link between the fibrinolytic niche and ENaC proteins has been provided by other groups and us (39)(40)(41)(42). uPA cleaves human gENaC subunits to alter ion transport across the airway epithelium (39).…”

Why COVID-19 Transmission Is More Efficient and Aggressive Than Viral Transmission in Previous Coronavirus Epidemics?

“…Such plasmin-induced cleavage of the ENaC subunits promotes the flow of Na + ions into the epithelial cells, leading to the dehydration of the air-facing surface of the lungs and alveoli, which is normally lined by a thin film of liquid, and hypertension [ 64 ]. Plasmin is a potent protease that cleaves the human γ ENaC subunit at 16 sites, including the cleavage sites of trypsin, chymotrypsin, prostasin, and elastases [ 66 ]. Significant harm is induced by the uncontrolled proteolysis of these proteins, which are highly expressed on epithelial cells, considered as the major pathways for Na + entry, and play important roles in maintaining the proper depth of airway and alveolar lining fluids, the reabsorption of edema fluid in injured lungs, and the regulation of salt retention in the collecting tubules [ 64 , 67 , 68 , 69 , 70 , 71 , 72 , 73 , 74 ].…”

Functional prediction and comparative population analysis of variants in genes for proteases and innate immunity related to SARS-CoV-2 infection

“…The SARS-CoV-2 relies on the host cell proteases to cut its S protein in two parts thus forming N-terminal part which recognizes ACE2 receptor and C-terminal part involved in the viral entry which must be further cleaved by furin and/or other furin-like enzymes ( Coutard et al, 2020 ). It has been shown that plasmin is also capable to cleave furin sites ( Zhao et al, 2020 ) and that individuals with elevated plasmin demonstrated higher susceptibility to COVID-19 ( Ji et al, 2020 ). In addition to plasmin, S protein of coronaviruses may be cleaved by other airway proteases such as trypsin-1 and TMPRSS11a ( Ji et al, 2020 ).…”