Anti-protease Treatments Targeting Plasmin(ogen) and Neutrophil Elastase May Be Beneficial in Fighting COVID-19.

To the Editor: Ji et al. (6) have convincingly shown that elevated plasmin(ogen) is a common risk factor in COVID-19 susceptibility. They highlighted the clinical relevance of plasmin(ogen) in patients who are susceptible to SARS-CoV-2 because of underlying conditions, such as hypertension, diabetes, cardiovascular diseases, cerebrovascular diseases, and chronic renal illnesses. They also pointed to other evidence in the literature that various functions of plasmin(ogen) increase the pathogenicity of COVID-19 (6).

We recently proposed a novel hypothesis (10) concerning COVID-19 pathogenesis which is linked to that of Ji et al. (6). We are convinced that neutrophil extracellular traps (NETs) formation and its byproducts play a key role in COVID-19 pathogenesis (10). NETs are extensive structures released extracellularly from activated neutrophils in response to infection. They are composed of cytosolic protein assembled on a scaffold of released chromatin (10). These structures prevent the dissemination of microorganisms in blood by trapping them mechanically and by exploiting the coagulant function to segregate them within the circulation (5). In addition to this, NET components [DNA, histone, and granule proteins such as myeloperoxidase and elastase (NE)] also contribute to the triggering of an inflammatory process. The dysregulation of NET formation and the consecutive release of NET byproducts is involved in thrombosis and fibrinolysis disorders in autoimmune diseases, as well as non-autoimmune diseases, in particular viral infection (1, 5, 10).

The coagulation system and innate immunity are coordinately activated and highly integrated during venous and arterial thrombus formation and progression (10). Platelet-neutrophil interactions at the site of deep vein thrombosis formation were found to induce NETosis, and to be of substantial relevance for thrombogenesis in the context of deep vein thrombosis in general. Barbosa da Cruz et al. (1) revealed that NE/DNA complexes in NETs play a central role in a mechanism that results in severe fibrinolytic failure. NE forms a tight complex with DNA that strongly impairs its inhibition by the α1-proteinase inhibitor (α1-PI) (1). In this way, NE highly degrades plasminogen without generating plasmin, which in turn leads to the production of antifibrinolytic plasminogen fragments. NETs can therefore serve as a platform for NE-mediated activation of intravascular coagulation in vivo (1, 10).

With regard to the interaction of plasmin with NETs, the serine proteases, thrombin and plasmin, were also found to interact with DNA, and bound to NETs in vitro (1, 9). In this respect, it is interesting to note that thrombin and plasmin, like NE, belong to the vast family of S1 peptidases, which share an overall similar structure and folding (1). Thrombin can induce neutrophil chemotaxis and aggregation at submicromolar concentrations. Moreover, plasmin has been shown to cause neutrophil aggregation and adhesion to the endothelial surface in vitro at submicromolar concentrations. Lim et al. (8) observed reduced histone (H2B, H3, and H4) and neutrophil elastase levels with the addition of thrombin and plasmin. While Ryan et al. (9) did not observe neutrophil lysis after treatment with plasmin, they speculated that thrombin and plasmin displace NE from NETs, thus allowing NE to undergo auto-proteolysis. Alternatively, NE could be directly proteolyzed by thrombin and plasmin, as was observed for the histones. Lim et al. (8) showed that thrombin and plasmin alter the NET proteome and concluded that NETs and their proteic byproducts are regulated according to physiological conditions, which affects their roles in inflammation and host response during viral infection. Further investigations on NETome dynamics are needed to circumscribe these interactions.

Elastase-mediated activation of SARS-CoV-2 was originally reported by Taguchi and co-workers (11), and the potentially significant implications of elastase for viral pathogenesis have been proposed (3, 11). NE is one of the most abundant NET byproduct proteins identified in a recent proteome analysis (8). Human NE is a granular serine protease with broad substrate specificity, expressed and stored in human neutrophils, released upon neutrophil activation, and involved primarily in host defense. Thus NE attacks proteins of invading microorganisms, but enables the hydrolyzation of proteins in the host extracellular matrix, such as collagen-IV and elastin. As a consequence, NE plays a role in degenerative and inflammatory diseases. During the phagocytosis of such foreign substances as pathogen-derived compounds, elastase as other proteins are also excreted into the surrounding extracellular environment, where the activity of elastase is regulated by inhibitors (i.e., α1-PI). NE could be responsible, in part, for the high multiplication of SARS-CoV-2-mediated infection, by means of multiple mechanisms (3, 11).

First, like other proteases (trypsin, TMPRSS2, or plasmin), NE enables S protein cleavage and entry into cells directly from the cell surface (11). Wild-type human SARS-CoV-2 prefers cell-surface entry, in contrast to cathepsin-mediated endosomal cell entry (11). This multiplies its infectivity. Second, NE is a known activator of epithelial Na+ channel (ENaC). Intact furin activity is necessary for full elastase-induced ENaC activation (3). Caldwell et al. (4) showed that NE activates near silent ENaC and increases airway epithelial Na+ transport. NE, which is found in microgram quantities in the lungs of cystic fibrosis patients, activates ENaC via cleavage of the γ subunit, as observed with plasmin. This results in Na+ hyperabsorption, reduced airway surface liquid height, and dehydrated mucus, culminating in inefficient mucociliary clearance (such as found in cystic fibrosis). Third, in addition to its physiological function as a powerful host defense, neutrophil elastase is also known as one of the most destructive enzymes in the body. An overwhelming release of enzymatically active elastase, exceeding the inhibitory potential of the α1-PI, together with simultaneously produced reactive oxygen species, can cause local tissue injury. Once unregulated, NE activity disturbs the function of the lung permeability barrier, and induces the release of pro-inflammatory cytokines, participating in the cytokine storm observed in COVID-19. These symptoms are typical in the pathophysiology of acute lung injury found in COVID-19. As previously reported (7), α1-antitrypsin inhibits ENaC activity by inactivating elastase. In inflammatory conditions, peroxynitrite formed by the interaction of nitric oxide and superoxide inactivates α1-PI by oxidizing a methionine in its active site (7). It is likely that the resulting NE disinhibition could lead to the exacerbation of NE’s detrimental effects, particularly its effect on ENaC regulation, and the endothelial and epithelial tissue damage it causes in COVID-19 inflamed lungs.

Taken as a whole, the extent to which Ji et al.’s observations regarding plasmin functions (6) echo those we ourselves have made concerning NE is remarkable. Both related proteases 1) cleave S proteins, which facilitates SARS-CoV-2 cell entry and enhances its virulence (3, 11); 2) are involved in mechanisms leading to the coexistence of coagulation activation and hyperfibrinolysis (6, 8, 9); and 3) cleave ENaC subunits, which results in hypertension and dehydration of the fluid lining lung airways and alveolar cells (4, 6). Also, in those underlying conditions which increase susceptibility to SARS-CoV-2 infection, such as hypertension, diabetes, cardiovascular diseases, cerebrovascular diseases, and chronic renal illnesses, both elevated plasmin(ogen) and dysregulation of NET formation are common features (10). In addition, our previous work showed that NET dysregulation is involved in other comorbidities such as pulmonary diseases, obesity, inflammatory bowel diseases, sickle cell disease, and rheumatoid arthritis (10).

Factors that influence successful recovery from respiratory viral infection (vs. a lethal outcome) are complex, and are both host- and virus-specific. That said, we have suggested that NET formation can be inhibited by various strategies such as DNase-1, anti-interleukin (IL)-1, or IL-6 treatments. Alternatively, preventing (or at least reducing) the entry of SARS-CoV-2 into respiratory cells by antiproteases may improve the clinical outcome of patients with COVID-19. In view of this, the more specific type of approach proposed by Ji et al. (6), such as targeting plasmin(ogen), may be beneficial. Likewise, tranexamic acid, a synthetic analog that binds lysine receptor sites on plasminogen and prevents its conversion to plasmin, may have therapeutic potential (2, 11). It should be noted that a clinical study is currently ongoing which evaluates the efficacy of tranexamic acid, along with the administration of an anticoagulant, in decreasing the severity of the disease (12). Alternatively, the promise of treatments which target elastase should be also seriously considered. These include the use of sivelestat or alvelestat (small molecule inhibitors of NE) or Bay-678 antibodies, as part of a fast-track drug repurposing strategy for treating COVID-19. The combination of anti-plasmin or anti-elastase approaches with those anti-inflammatory approaches already identified, therefore, deserves serious evaluation in the fight against COVID-19.

GRANTS

A. R. Thierry is supported by INSERM. This work was funded by the “SIRIC Montpellier Cancer Grant INCa_Inserm_DGOS_12553.”

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author.