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. 2020 Jul;177(13):3091-3106.
doi: 10.1111/bph.15038. Epub 2020 May 11.

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

Runzhen Zhao  1 , Gibran Ali  1 , Hong-Guang Nie  1   2 , Yongchang Chang  3 , Deepa Bhattarai  1 , Xuefeng Su  1 , Xiaoli Zhao  4 , Michael A Matthay  5 , Hong-Long Ji  1   6
Affiliations

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

Runzhen Zhao et al. Br J Pharmacol. 2020 Jul.

Abstract

Background and purpose: Lung oedema in association with suppressed fibrinolysis is a hallmark of lung injury. Here, we have tested whether plasmin cleaves epithelial sodium channels (ENaC) to resolve lung oedema fluid.

Experimental approach: Human lungs and airway acid-instilled mice were used for analysing fluid resolution. In silico prediction, mutagenesis, Xenopus oocytes, immunoblotting, voltage clamp, mass spectrometry, and protein docking were combined for identifying plasmin cleavage sites.

Key results: Plasmin improved lung fluid resolution in both human lungs ex vivo and injured mice. Plasmin activated αβγENaC channels in oocytes in a time-dependent manner. Deletion of four consensus proteolysis tracts (αΔ432-444, γΔ131-138, γΔ178-193, and γΔ410-422) 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 γENaC proteins. In addition, 9 new sites (R122, R137, R138, K150, K170, R172, R180, K181, K189) in synthesized peptides were found to be cleaved by plasmin. 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 α and γ 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: Plasmin can cleave ENaC to improve blood-gas exchange by resolving oedema fluid and could be a potent therapy for oedematous lungs.

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

FIGURE 1
FIGURE 1
Intratracheally administered plasmin up‐regulates alveolar fluid clearance in normal human lungs ex vivo. (a) Total 60‐min alveolar fluid clearance (AFC60) in the presence and absence (control) of plasmin (Pl, 60 μg·ml−1, 0.7 μM) and/or amiloride (Amil, 1 mM). Adjacent human lung lobes within one resected sample were selected, and AFC values were measured in parallel for both control and treated groups. Data are presented as medians, means, 25% and 75% percentiles, and SEM; n = 6 per group. * P < .05, significantly different as indicated; NS, not significantly different from both control and Amil. (b) Amiloride sensitive (AS) fraction of AFC was computed as ENaC contribution in human lung lobes instilled with PBS only (control) and plasmin. * P < .05, significantly different from control; n = 6 per group
FIGURE 2
FIGURE 2
Plasmin stimulates alveolar fluid clearance in mouse lungs with acid aspiration injury. (a) Lung water content (wet/dry ratio). Mice were intratracheally instilled with 1‐N HCl (50 μl) as a model of gastric acid aspiration induced lung injury. Control animals were intratracheally instilled with 50‐μl PBS. Plasmin (60 and 100 μg·ml−1) or a mixture of plasmin and equal amount of α2‐antiplasmin (Pl + AP) were intratracheally instilled, 4 hr post HCl instillation. Lungs were dissected and weighed before and after drying. * P < .05, significantly different as indicated. Dashed line indicates the difference from more than one group. (b) Plasmin restores in vivo 30 min (AFC30) in mice injured by HCl. n = 20. * P < .05, significantly different from control. # P < .05, significantly different from HCl group. & P < .05, significantly different from plasmin only groups
FIGURE 3
FIGURE 3
Plasmin restores deficient ENaC activity in monolayers of alveolar type 2 (AT2) epithelial cells from uPA deficient mice. (a) Transepithelial resistance in WT and plau −/− monolayers up to 10 days post seeding. * P < .05, significantly different from WT cells. n = 6. (b) Equivalent short‐circuit (IEQ) in polarized AT2 monolayers cultured at the air‐liquid mode. IEQ = ET/RT. ET is the transepithelial potential difference, and RT is the resistance across the monolayer read by an EVOM meter. Data are presented as means ±SEM; n = 6.. * P < .05, significantly different from WT group; NS, not significant. (c) Effects of plasmin on IEQ in polarized AT2 monolayers. Data were presented as medians, means, 25% and 75% percentiles, and SEM for wild type (WT; n = 5), plau knockout (plau −/− ; n = 7), and addition of plasmin (20 μg·ml−1; n = 6 ). * P < .05, significantly different as indicated
FIGURE 4
FIGURE 4
Plasmin specifically activates human αβγ‐ENaCs expressed in Xenopus oocytes. (a) Amiloride‐sensitive sodium currents (ENaC) were digitally sampled at the membrane potential of −100 mV in cells heterologously expressing human αβγ‐ENaCs, incubated with two‐chain uPA (10 μg·ml−1, 30 min at room temperature), plasmin (Pl, 10 μg·ml−1), and a mixture of plasmin (Pl, 10 μg·ml−1) and α2‐antiplasmin (α2‐AP, 10 μg·ml−1). n = 20 per group, * P < .05, significantly different from controls in the absence of fibrinolytic molecules; NS, not significant. (b) Time course of ENaC activation by plasmin. ENaC activity in the set of oocytes was recorded at designated time points after exposure to plasmin. n = 6 per group. * P < .05, significantly different from the corresponding time point of controls. (c) Effects of plasmin on the current levels of α, α + β (αβ), and α + γ (αγ) channels; n = 12 for α alone, n = 10 for αβ, and n = 12 for αγ group. * P < .05, significantly different from the corresponding controls in the absence of plasmin; NS, not significant. (d) Normalized incremental fold of ENaC activity. Ratio of current amplitudes recorded after over before addition of plasmin or perfusate was computed as fold of increase in channel activity; . * P < .05, significantly different from controls; NS, not significant.. (e,f) Detection of full‐length (F) and cleavage (C) of α ENaC by plasmin. HA (attached to the N‐terminal tail) and V5 (attached to the C‐terminal tail) tagged α subunit (αV5HA) was co‐expressed with βγ subunits complementarily. Biotinylated plasma membrane proteins were run on a 7.5% SDS‐PAGE gel and probed with anti‐V5 (e) and anti‐HA (f) monoclonal antibodies. Lanes from left to right were loaded with plasma membrane proteins of cells without plasmin treatment (Ctl), pretreated with plasmin (Pl, 10 μg·ml−1 for 30 min at room temperature), and chymotrypsin (Chy, 10 μg·ml−1) as a positive control. (g,h) Cleavage of HA and V5 tagged γ ENaC (γV5HA) by plasmin as recognized by anti‐V5 (g) and anti‐HA (h) antibodies. Cleaved bands are labelled with white lines at the same level. These experiments were repeated at least four times with similar observations
FIGURE 5
FIGURE 5
Effects of plasmin on α and γ‐ENaC mutants missing consensus cleavage domains. (a) Basal ENaC currents were recorded in oocytes expressing αβγ‐ENaC and three deletion mutants lack of putative cleavage domains, namely, αΔ173–178βγ (n = 24), αΔ201–204βγ (n = 26) and αΔ432–444βγ (n = 26). The activated currents were collected again 1 hr post incubation with plasmin (10 μg·ml−1, 30 min at room temperature) at the room temperature. The activated current levels were normalized to that basal currents. * P < .05, significantly different from αβγ‐ENaC; NS, not significant. n = 19–26. (b) Activation of three γ deletion mutants of αβγΔ131–138, αβγΔ178–193, and αβγΔ410–422 by plasmin. * P < .05, significantly different from αβγ‐ENaC; NS, not significant. (c–f) Immunoblotting detection of cleavage in full‐length and three deletion γ mutants. The most left lane is protein marker. Untreated cells were controls (−). Endogenous furin cleaved band in the absence of plasmin and plasmin‐cleaved band in the presence of plasmin (+) were labelled with white lines. These blots represent four experiments with similar observations. (g) Plasmin activates the activity of mouse mutant (αβγK189A) for plasmin cleavage site. Whole‐cell currents were digitized and then repeated 1 hr post incubation with plasmin (10 μg·ml−1). n = 8. * P < .05, significant effect of plasmin or time. (h) Plasmin stimulates the activity of a human mutant (αβγ5A) for plasmin cleavage (K189 and 178RKRK181 were substituted with alanine). Measurements of whole‐cell currents were performed 1 and 24 hr after exposure to plasmin. n = 22, * P < .05 significantly different from before addition of plasmin. (i) Western blot assay of biotinylated plasma membrane proteins an anti‐V5 monoclonal antibody. Plasma membrane proteins from noninjected oocytes (NI), cells expressing αβγ ENaC (Control), and cells pretreated with plasmin (Plasmin) were loaded on a 7.5% SDS‐PAGE gel. Furin and plasmin‐cleaved proteins are marked with black and white rightward arrowhead respectively. Similar results were observed in four blots
FIGURE 6
FIGURE 6
Potential proteolysis domains for plasmin beyond the putative cleaving motif (131–138) in γ ENaC subunit. (a) In silico prediction with the database for plasmin‐specific motifs. Left, a combination (logo) of reported 92 substrate motifs for plasmin from P4 to P4′. Right, a logo of input substrate sequences for plasmin from P3 to P2′. Top 10 predicted sites and the size of C‐terminal fragments by both strategies are listed below. Default set‐up was used for running the SitePrediction server (Gosalia et al., 2005; Hervio et al., 2000). Penalty: 0.1; sort order: average score. Similarity score: 100; specificity, >95%. (b) Effects of plasmin on αβγΔ131–138 + 3A (K168A, R178A, and K179A) channel activity in 1 and 24 hr. n = 18. * P < .05, significantly different from before; NS, not significant. (c) Immunoblotting assays of αβγΔ131–138 and αβγΔ131–138 + 3A channel proteins. These results are a representative blot of three experiments. (d) Regulation of single point mutants (γK168A, γR178A, and γK179A) derived from αβγΔ131–138 by plasmin. n = 12. NS, not significantly different from αβγΔ131–138 + 3A (3A) construct. (e) Detection of plasmin‐cleaved fragments of three single point mutants (from left to right are αβγΔ131–138, αβγΔ131–138 + K168A, αβγΔ131–138 + R178A, and αβγΔ131–138 + K179A). (f) Cleavage efficacy (cleaved band/uncleaved band). n = 3
FIGURE 7
FIGURE 7
Prediction and validation of putative plasmin cleaved sites preceding γK168. (a) In silico predicted four potential cleavage sites (K126, R135, K136, and R153) and the peptide size of C‐terminal fragments. (b) Effects of plasmin on the channel activity of αβγΔ178–193 (n = 14) and a mutant combining γΔ178–193 and four predicted sites (+4A) (n = 9) that were replaced with alanine. NS, not significantly different from αβγΔ178–193 mutant. (c) Immunoblotting assays. This blot represents four experiments with similar results. (d) Contribution of four predicted cleavage sites. n = 4. (e) Summary of newly identified and validated cleavage sites for plasmin in γ‐ENaC subunit. Three putative proteolytic regions for truncation mutants are marked with cylinders; four identified amino acid residues are labelled in purple font and balls
FIGURE 8
FIGURE 8
Validation of plasmin cleaved sites by MS. (a) Sequences of three synthesized peptides with identified plasmin cleavage sites (red font). Three peptides with a continuous sequence were separated by two gaps. The numbers above the cleaved sites are the frequency of the fragments with an end of this amino acid residue identified by MS. The underlining indicates the sequences of the fragments for panels b to h from left to right. (b–h) MS1 spectrum for the representing fragments. The size and charge are labelled for each isotope. Corresponding sequences of each fragment is shown as insets. n =1
FIGURE 9
FIGURE 9
Location of deletion and single or multiple point mutants in 3D model and gating mechanism. (a) 3D structure of truncated human ENaC subunits, adapted from Noreng et al. (2018). (b) Location of deleted regions (Δ1, Δ 2, and Δ 3) for both α and γ‐ENaC subunits. (c) Positions of identified amino acid residues for plasmin cleavage in human γ subunit. (d–f) Schematic mechanism for plasmin to cleave and gate ENaC protein complexes. Plasmin cleaves full‐length ENaC (d) to remove the GRIP domain (e). The finger domain will subsequently fall down to hit the thumb to increase the width of channel pore, allowing Na+ ions to go through channel pore faster as shown by increased currents. However, for some mutants, for example, Δ3 deletion mutants of α and γ subunits, the link between the finger and the thumb disappears so that downward movement of the finger cannot reach the thumb to enlarge the channel pore (f)
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