key: cord-0804381-0by0bkgs
authors: Colarusso, Chiara; Terlizzi, Michela; Pinto, Aldo; Sorrentino, Rosalinda
title: A lesson from a saboteur: high molecular weight kininogen (HMWK) impact in COVID‐19
date: 2020-06-04
journal: Br J Pharmacol
DOI: 10.1111/bph.15154
sha: 33c0169e0afcd8036a92870b7a30e62ff9b40801
doc_id: 804381
cord_uid: 0by0bkgs

Severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) is a newly identified coronavirus which has spread from China to the rest of the world causing the pandemic coronavirus disease 19 (COVID‐19). It has fatality rate that floats from 5 to 15% and the symptoms are fever, cough, myalgia and/or fatigue up to dyspnea, responsible for hospitalization and in most of the cases of artificial oxygenation. In the attempt to understand how the virus spreads and how to pharmacologically abolish it, it was highlighted that SARS‐CoV‐2 infects human cells by means of angiotensin converting enzyme 2 (ACE2), transmembrane protease serine 2 (TMPRSS2) and SARS‐CoV‐2 main protease (M(pro)). Once bound to its receptor ACE2, the other two proteases, in concert with the receptor‐mediated signaling, allow virus replication and spread throughout the body. Our attention has been focused on the role of ACE2 in that its blockade by the virus increases Bradykinin and its metabolites, well known to facilitate inflammation in the lung (responsible for cough and fever), facilitate both the coagulation and complement system, three mechanisms that are typical of angioedema, cardiovascular dysfunction and sepsis, pathologies which symptoms occur in COVID‐19 patients. Thus, we propose to pharmacologically block the kallikrein‐kinin system upstream bradykinin and the ensuing inflammation, coagulation and complement activation by means of lanadelumab, which is a clinically approved drug for hereditary angioedema.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a newly identified coronavirus which emerged for the first time in the city of Wuhan and rapidly spread through China to cause a disease known as coronavirus disease 19 (http://www.who.int/csr/don/12-january-2020-novel-coronavirus-china/en/). Because the outbreak of COVID-19 has rapidly spread worldwide, affecting millions of people, the World Health Organization (WHO) has declared SARS-CoV-2 as a global pandemic (https://www.who.int/dg/speeches/detail/who-director-general-s-opening-remarks-at-themedia-briefing-on-covid-19-11-march-2020).

SARS-CoV-2 is a new beta-coronavirus belonging to the same sub-group as Severe Acute Respiratory Syndrome-CoV (SARS-CoV) and the Middle East Respiratory Syndrome-CoV (MERS-CoV) which caused SARS and MERS outbreak in 2002 and 2012, respectively (Chen, Liu and Guo, 2020) . Several studies have identified a sequence homology of 79.5% between SARS-CoV-2 and SARS-CoV Wu et al. 2020) . Therefore, SARS-CoV-2 genome sequencing was rapidly performed, leading to the rapid availability of real-time PCR diagnostic test which is actually used to identify infected subjects allowing the epidemiologic tracking (Corman et al. 2020) . SARS-CoV-2 is a single-stranded RNA virus characterized by an envelope-anchored Spike glycoprotein (S) which drives virus entry into target cells by binding membrane receptors of sensitive cells and leading to viral replication (Xu et al. 2020b) .

Epidemiological data indicate that SARS-CoV-2 infection progresses through human-tohuman contact, which is predominantly realized via droplet transmission (Ong et al. 2020) .

As reported by WHO, the incubation period for SARS-CoV-2 is 2-14 days, although a longer period may be at the basis of asymptomatic and subclinical infection (https://www.who.int/docs/default-source/coronaviruse/who-china-joint-mission-on-covid-19-final-report.pdf), whereas illness establishment mainly occurs in 10 days (Guan et al. 2020) . Although the estimated case fatality rate (CFR) of COVID-19 floats from 5 to 15%, the number of deaths is very high.

Several reports have summarized the clinical and epidemiological features of patients affected by COVID-19. In the first published cohort of 41 laboratory-confirmed cases infected with SARS-CoV-2 (Huang et al. 2020) , it was reported that infected patients had a median age of 49.0 years and 73% of them were men. The common symptoms are fever This article is protected by copyright. All rights reserved.

(98%), cough (76%), myalgia and/or fatigue (44%); dyspnea occurs within 8 days from the establishment of the pathology in 55% of patients. Very few COVID-19 patients have gastrointestinal symptoms and prominent upper respiratory tract signs and symptoms, indicating that the target cells might be located in the upper and lower airways. All hospitalized patients show abnormalities in chest computed tomography (CT) images, which are characterized by grinding glass-like and consolidation areas in 98% of the cases reporting bilateral lungs impairment at the basis of bilateral interstitial pneumonia. Because of respiratory complications, around 32% of COVID-19 patients are admitted to intensive care unit (ICU). The morbidity is mainly due to respiratory failure typical of acute respiratory distress syndrome (ARDS), but the mortality underlies multiple organ failure due to coagulation alteration with ensuing thrombosis and embolism, consequences of septic shock and/or cardiovascular alterations (Huang et al. 2020 ).

One key discovery in understanding the secrets of SARS-CoV-2 infection involves the viral S protein, which binds to the host Angiotensin-Converting Enzyme 2 (ACE2) via the recognition of the receptor binding domain (RBD) Sriram and Insel 2020) , similar mechanism that is used by SARS-CoV to mediate infection Sriram and Insel 2020) . The viral attachment to ACE2 is the first of a multi-step process in that the next one is mediated by cleavage by cellular proteases of S protein at S1/S2 and S2 site (Letko et al. 2020; . As in the case of SARS-CoV (Li et al. 2005) , the RBD comprised in S1 subunit directly interacts with the peptidase domain (PD) of ACE2 providing for tighter and higher binding of the virus to the host cell. So far, three mutations (V367F, W436R, and D364Y) of the RBD on SARS-CoV-2 have been correlated to higher human ACE2 affinity, ensuing higher infectivity . Therefore, very relevant is the localization of ACE2 to identify the viral route to be hosted (Sriram and Insel 2020) .

Besides type II pneumocytes , other organs, i.e. heart, esophagus, kidney, bladder, ileum, oral cavity and testes, express ACE2, explaining why some COVID-19 patients also exhibit non-respiratory symptoms. To date, in the attempt to find a potential drug against COVID-19, human recombinant soluble ACE2 (hrsACE2) was proposed to prevent viral attachment (Monteil et al. 2020; Sriram and Insel 2020) , however, phase 1 and This article is protected by copyright. All rights reserved.

phase 2 clinical trial results demonstrated a lack of therapeutic effect in COVID-19, most likely due to its biological nature or because ACE2 is just the tip of the iceberg.

Another key event for virus entrance into the host is represented by the cellular transmembrane protease serine 2 (TMPRSS2) that drives the S protein priming (Hoffmann et al., 2020) . TMPRSS2 is a cell surface protein of the serine protease transmembrane family type II that is broadly expressed by epithelial cells (Zou et al. 2020; Xu et al. 2020a) and is involved in the cleavage of the SARS-CoV and influenza virus hemagglutinin protein (Böttcher et al. 2006 For this reason, it was speculated that M pro could represent an attractive target for COVID-19 treatment. In this context, two different molecular docking and molecular dynamic simulation studies reveled 4 drugs that could act against M pro : the antibacterial drug talampicillin, the antipsychotic drug lurasidone (Elmezayen et al. 2020) , and the antiviral drug raltegravir and paritaprevir, which were already used in the antiretroviral therapy against the Human Immuno-deficiency Virus (HIV) infections as integrase strand transfer inhibitors (INSTI) (Khan et al. 2020) . M pro also cleaves the 2'-O-Ribose Methyltransferase (2'-O-MTase), a protein that catalyzes the methylation of 5'-terminal cap structure of viral mRNAs (Chen et al. 2011) . Because this reaction is crucial for viral replication and expression in host cells (Menachery et al. 2014 ), 2'-OMTase was suggested as another possible druggable target for COVID-19 treatment (Khan et al. 2020) , although it is still unclear whether 2'-O-MTase, as well as M pro , contributes to SARS-CoV-2 infection. This article is protected by copyright. All rights reserved.

ACE2 is a membrane-associated aminopeptidase and belongs to the angiotensinconverting enzyme family of dipeptidyl carboxydipeptidases and has high homology to human angiotensin converting enzyme 1 (ACE1) (Tipnis et al. 2000) . Secreted ACE2 cleaves angiotensin I into angiotensin-(1-9), and angiotensin II into the vasodilator angiotensin-(1-7) (Patel et al. 2016; Sriram and Insel 2020) . Beyond its role in the cardiovascular system, it plays a role in the regulation of renal function and fertility (Koitka et al. 2008; Pan et al. 2013) . Once SARS-CoV-2 binds to ACE2 , the enzyme is blocked, therefore, leading to what we are actually assisting in terms of high blood pressure in COVID-19 patients and pulmonary edema up to angioedema, which underlies the fact that physiologically ACE2 cleaves several bioactive peptides, among which [des-Arg 9 ]bradykinin ([des-Arg 9 ]BK) (Vickers et al. 2002; Donoghue et al. 2003) (Figure 1 ). Herein, besides the perturbation of the renin-angiotensin system (RAS) Kuba et al. 2005; Sriram and Insel 2020), increasing inflammation and vascular permeability occur, due to the activity of [des-Arg 9 ]BK that binding to bradykinin 1 receptor (B1 receptor) can lead to acute lung inflammation (Sodhi et al. 2018; Sriram and Insel 2020) (Figure 1 ). The activation of the [des-Arg 9 ]BK/B1 receptor axis induces the release of pro-inflammatory chemokines (i.e. CXCL5, CCL2, CXCL1) and cytokines (i.e. TNF-α, IL-1β and IL-6), exacerbating lung inflammation/edema up to organ dysfunction (Sodhi et al. 2018) . Therefore, as already suggested by van de Veerdonk et al., 2020, the cytokine storm observed in COVID-19 may underlie an impaired catabolism of [des-Arg 9 ]BK, paving the way for the pharmacological blockade of B1 receptor signaling.

Instead, in this review we want to focus the Reader's attention on the upstream signaling that leads to BK. The kallikrein-kinin system (KKS) consists of the complex between prekallikrein and high molecular weight kininogen (HMWK) (Hooley, McEwan, and Emsley, 2007) (Figure 2 ). HMWK is a multifunctional single-chain plasma glycoprotein primarily expressed by the liver and secreted into the bloodstream. HMWK consists of 6 different protein domains (Shariat-Madar and Schmaier, 1999) and binds to prekallikrein by means of a sequence in domain 6. The detachment of the domain 4 releases BK (Griffin and Cochrane, 1979) . Kallikreins are serine proteases responsible for the release of kinins, vasoactive peptides that cause vascular smooth muscle relaxation and an increase of vascular permeability (Bhoola, Figueroa, and Worthy, 1992) . It has been found that kallikrein exists in two different forms: kallikrein B1, also known as plasma kallikrein, which cleaves HMWK This article is protected by copyright. All rights reserved.

into BK, which in turn interacts with the constitutive B2 receptor, and tissue kallikrein which processes low-molecular-weight kininogen (LMWK) into Lys-BK. The interaction of BK or Lys-BK with B1 and B2 receptors will increase the activation of both endothelial nitric oxide synthase (eNOS) and inducible NOS (iNOS), with an ensuing release of nitric oxide, potent vasodilator, and of prostaglandin I2 (PGI2) and pro-inflammatory cytokines/chemokines responsible for acute inflammation that is accompanied by vasodilation, pain, cell proliferation and fibrosis (Kuhr et al. 2010; Tsai et al. 2015) , symptoms typical of COVID-19 ( Figure 1; Figure 2 ).

Plasma as well as tissue kallikrein are initially secreted as inactive, but both of them are activated by serine protease activity (Bhoola, Figueroa, and Worthy, 1992) . The reciprocal activation of coagulation factor XIIa (Hageman Factor) and plasma prekallikrein promotes the activation of the kallikrein, which, besides the catabolism of HMWK into BK, initiate the intrinsic pathway of coagulation, influencing fibrinolysis (Figure 2 ). At the same time, tissue pre-kallikrein cleaves low molecular weight kininogen (LMWK) in [des-Arg 10 ]kallidin and [des-Arg 9 ]BK which interact with B1 receptors further enhancing inflammation. The intrinsic pathway of coagulation is then correlated to the extrinsic pathway in that factor XIIa activates coagulation factor XI, which in turn activates coagulation factor IX which subsequently leads into the common pathway by means of coagulation factor X and then thrombin, with fibrin aggregates generation, hence the need to detect D-dimer in COVID-19 patients (Figure 2 ). In this context, studies looking at rat models that express both BK receptors show, in vitro, that BK acting through the B2 receptor on the surface of endothelial cells promotes the expression of procoagulant and antifibrinolytic proteins, such as tissue factor (TF) and plasminogen activator inhibitor 1 (PAI-1) (Kimura et al. 2002) . On the other hand, plasma kallikrein can align pro-urokinase plasminogen activator (uPA) in such close proximity as to drive plasminogen activation into plasmin which degrades fibrin aggregates (Selvarajan et al. 2001) , effects that are widely observed in sepsis, another co-morbidity of COVID-19. However, it has been shown that the complex HMWK and factor XIIa can also bind to another of the three endothelial cell-binding sites, the 33-kDa cell surface receptor for the first component of complement C1q (gC1qR/p33) which has high affinity for HMWK (Ghebrehiwet et al. 2006) . Therefore, the activation of the classical pathway of the complement together with the activation of the plasmin on the conversion of C3 into C3a and C3b induce the activation of both lecithin and extrinsic pathways of the complement with the This article is protected by copyright. All rights reserved. ensuing activation of the humoral immunity, exacerbating the inflammatory process ( Figure   2 ). These events may happen in COVID-19 patients from the early onset up to the severe step of the pathology. To date, the above pathological conditions are typical of angioedema, cardiovascular dysfunction and sepsis, which symptoms occur in COVID-19 patients. But it is obvious to ask the correlation between these symptoms and the viral infection. Why would this happen? As above reported (van de Veerdonk et al., 2020 ) the viral blockade of ACE2 inhibits not only the degradation of angiotensin II, but also the degradation of BK. Therefore, because BK derives from HMWK and because KKS leads to the coagulation and complement activation, we believe that the alteration of plasmatic kallikrein could serve as potential pharmacological tool.

In the attempt to identify the effective anti-SARS-CoV-2 therapy, many therapeutic approaches have been proposed. In particular, ongoing clinical trials are focusing on two big branches, the antiviral drugs, which aim to diminish viral replication, and the diseasemodifying antirheumatic drugs (DMARDs) and immunotherapeutic agents to hijack the cytokine storm that the virus is able to induce. Encouraging clinical trials indicate that remdesivir (Grein et al. 2020 ) and neutralizing monoclonal antibodies (mAbs: i.e. tocilizumab, sarilumab) (Xu et al. 2020c; http://www.news.sanofi.us/2020-03-16-Sanofi-and-Regeneron-begin-global-Kevzara-R-sarilumab-clinical-trial-program-in-patients-with-severe-COVID-19 ) are a promise for fighting COVID-19.

It has to be pointed out that all the ongoing clinical trials include monitoring of coagulation parameters, such as D-dimer, which is a metabolite of fibrin aggregates. Although there are no published case series reporting abnormal coagulation parameters in hospitalized severe COVID-19 patients, in a multicenter retrospective cohort study in China, elevated D-dimer levels (> 1 g/L) were strongly associated with in-hospital deaths, therefore to severe COVID-19 (Zhou et al. 2020a) . To date, low molecular weight heparin (LMWH), enoxaparin, has been proposed for these patients either to avoid thromboembolism events (Tang et al. 2020) or to inhibit the cytokine storm (Shi et al. 2020) , due to non-anticoagulant fraction of enoxaparin suppresses in vitro IL-6 and IL-8 release from human pulmonary epithelial cells This article is protected by copyright. All rights reserved. (Shastri et al., 2015) . Moreover both in vitro and in vivo experimental studies have shown that human coronaviruses utilize heparin sulfate proteoglycans for attachment to target cells (Milewska et al. 2014) . Indeed, interaction between the SARS-CoV-2 Spike S1 protein receptor binding domain (SARS-CoV-2 S1 RBD) and heparin has been recently showed, suggesting a role for heparin in the therapeutic armamentarium against COVID-19 (Mycroft-West et al. 2020) .

So far, the published clinical observations of biochemical markers in COVID-19 patients include elevated LDH, D-dimer, bilirubin, high levels of pro-inflammatory cytokines that accompany interstitial pneumonia, renal and cardiac injury due to thromboembolic events, which also underlie septic shock that occurs in severe COVID-19 patients. Therefore, based on what described above and cross-linking biochemical with clinical outcomes, in this review we propose another therapeutic approach based on the inhibition of the KKS.

Lanadelumab is a monoclonal antibody against the plasmatic kallikrein, which is important for the cleavage of HMWK into BK and is involved in the coagulation as well as in the induction of the complement system ( Figure 2) . Actually, lanadelumab is used for the treatment of angioedema and has not reported adverse, severe events, other than hypersensitivity, myalgia and hepatic alteration of alanine aminotransferase (ALT) (https://www.ema.europa.eu/en/documents/assessment-report/takhzyro-epar-publicassessment-report_en.pdf).The rationale to suggest lanadelumab is in that this mAb can block the upstream axis that leads to kinin formation (van de Veerdonk et al. 2020) , avoiding the inflammatory and coagulation storm besides the complement system in SARS-CoV-2 infected patients, likely preventing the exacerbation of COVID-19, in parallel with antiviral therapy.

Lanadelumab has never been used to control COVID-19 symptoms, however very recently an open controlled trial entitled 'Lanadelumab for treatment of COVID-19 disease' was registered, in order to generate the proof of concept (https://www.clinicaltrialsregister.eu/ctrsearch/trial/2020-002472-12/NL#summary). In particular, one of the goals of this trial is to demonstrate the safety the dose of 300 mg, injected intravenously. Most likely, the choice of this dose is based on the positive results in that to prevent acute angioedema attacks in This article is protected by copyright. All rights reserved.

In conclusion, we believe that the blockade of ACE2 increases not only the activity of angiotensin II on the cardiovascular system, but also the levels of [des-Arg 9 ]BK derived by HMWK. Therefore, the hypothesis to block the production of [des-Arg 9 ]BK upstream by blocking the metabolism of HMWK could be another option to face this tremendous pandemic event that affected whole world lifestyle obliging to social limitations and stay-athome politics.

A rational roadmap for SARS-CoV-2/COVID-19 pharmacotherapeutic research and development. IUPHAR Review 29

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MasR) leading to reduced inflammation and vasodilation. ACE2 also cleaves [des-Arg 9 ]bradykinin ([des-Arg 9 ]BK), a bioactive kinin derived from kininogen pathway, into inactive metabolites. ACE2 is the cell entry receptor for SARS-CoV-22 (TMPRSS2), leads to SARS-CoV-2 infection. The binding of SARS-CoV-2 downregulates ACE2 expression, leading to a reduction of its enzymatic activity and the ensuing increase of Angiotensin II and

BK concurs to inflammation by binding bradykinin 1 receptor (B1 receptor), resulting in severe lung injury, pulmonary inflammation and edema, increased coagulation, hypertension and cardiac hypertrophy

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No conflict of interest to disclose. This article is protected by copyright. All rights reserved.

This article is protected by copyright. All rights reserved. conformational change upon heparin binding. bioRxiv. doi:This article is protected by copyright. All rights reserved. (grey arrows) pathways, both resulting in activation of the coagulation factor X (FX), which subsequently leads to thrombin and fibrin generation (common pathway; blue arrows). The coagulation cascade is also a starting point for the complement system (pink box and arrows).FXIIa binds C1q component of the complement triggering the classic pathway; moreover, plasmin activation, which is also promoted via B2 receptor signalling, triggers C3 cleavage inducing the activation of both lecithin and extrinsic pathways of the complement.