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\begin{document}

\title{A lesson from an old friend: high molecular weight kininogen (HMWK)
impact in COVID-19.}



\author[1]{Chiara Colarusso}%
\author[1]{Michela Terlizzi}%
\author[1]{Aldo Pinto}%
\author[1]{Rosalinda Sorrentino}%
\affil[1]{University of Salerno}%


\vspace{-1em}



  
  \date{May 23, 2024}


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\selectlanguage{english}
\begin{abstract}
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) 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 symtoms 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-CoV2 infects
human cells by means of angiotensin converting enzyme 2 (ACE2),
transmembrane protease serine 2 (TMPRSS2) and 3-chymotrypsin-like
protease (3CLpro), also known as Mpro. 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 kallicrein-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.%
\end{abstract}%



\sloppy




\textbf{Abbreviations:}

2'-O-MTase: 2'-O-Ribose Methyltransferase

3CLpro: 3-chymotrypsin-like protease

AAK1: AP2-associated protein kinase 1

ALT: alanine aminotransferase

ARDS: acute respiratory distress syndrome

BKB1R: bradykinin receptor B1

Bradykinin: BK

CFR: case fatality rate

cGAS: cyclic guanosine monophosphate--adenosine monophosphate

cGAMP: cyclic guanosine monophosphate--adenosine monophosphate

CoV: coronavirus

COVID-19: coronavirus disease 19

CT: computed tomography

CXCL1: C-X-C motif chemokine 1

CXCL5: C-X-C motif chemokine 5

DABK: des-Arg9bradykinin

DMARDS: disease-modifying antirheumatic drugs

EMA: European medical agency

eNOS: endothelial NOS

HIV: Human Immuno-deficiency Virus

HMWK: high molecular weight kininogen

hrsACE2: human recombinant soluble ACE2

ICU: intensive care unit

iNOS: inducible NOS

INSTI: integrase strand transfer inhibitors

KC: keratinocyte-derived chemokine

KKS: kallikrein-kinin system

LMWK: low-molecular-weight kininogen

Lys-BK: lysyl-bradykinin

mAbs: monoclonal antibodies

MERS: Middle East Respiratory Syndrome

MERS-CoV: Middle East Respiratory Syndrome-CoV

MHC: major histocompatibility complex

MIP2: macrophage inflammatory protein-2

NLRP3: Nod-like receptor protein 3

PAI-1: plasminogen activator inhibitor 1

PAR: protease-activated receptors

PD: peptidase domain

PGI2: prostaglandin I2

RAS: renin-angiotensin system

RDB: receptor binding domain

RdRp: RNA-dependent RNA polymerase

S: Spike

SARS: Severe Acute Respiratory Syndrome

SARS-CoV: Severe Acute Respiratory Syndrome-CoV

SARS-CoV2: Severe acute respiratory syndrome coronavirus 2

scRNA-seq: single-cell RNA sequencing

STING: stimulator of interferon genes

TF: tissue factor

TLR: Toll-like receptor

TMA: thrombotic microangiopathy

TMPRSS2: transmembrane protease serine 2

u-PA: urokinase-type plasminogen activator

WHO: World Health Organization

\textbf{1.} \textbf{Introduction}

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) 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 (COVID-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-CoV2 as a global pandemic
(https://www.who.int/dg/speeches/detail/who-director-general-s-opening-remarks-at-the-media-briefing-on-covid-19---11-march-2020).

SARS-CoV2 is a new \emph{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-CoV2 and SARS-CoV (Zhou \emph{et al} . 2020b; Wu \emph{et al} .
2020). Therefore, SARS-CoV2 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 \emph{et al} . 2020). SARS-CoV2 is a
single-stranded RNA virus characterized by an envelope-anchored Spike
glycoprotein which drives virus entry into target cells by binding
membrane receptors of sensitive cells and leading to viral replication
(Xu \emph{et al} . 2020b).

Epidemiological data indicate that SARS-CoV2 infection progresses
through human-to-human contact, which is predominantly realized via
droplet transmission (Ong \emph{et al} . 2020). As reported by WHO, the
incubation period for SARS-CoV2 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 occurs mainly in 10 days (Guan \emph{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. Indeed, as of
May 4th 2020, the virus has infected over 3.4 million individuals in 215
countries, and 238198 people have lost their lives
(https://www.who.int/emergencies/diseases/novel-coronavirus-2019).

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\emph{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 were fever
(98\%), cough (76\%), myalgia and/or fatigue (44\%), and dyspnea
occurred within 8 days from the establishment of the pathology in 55\%
of patients. Very few COVID-19 patients had 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 showed abnormalities in chest computed
tomography (CT) images, which were 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
complications, 32\% of patients were admitted to an intensive care unit
(ICU), among which 15\% worsened in a short period of time. Most of them
died of respiratory failure, but it is not excluded that death was due
to organ failure, coagulation alteration with ensuing thrombosis and
embolism as a consequence of blood clotting due to septic shock and/or
cardiovascular complications (Huang \emph{et al} . 2020). An
exacerbation of SARS-CoV2-induced acute respiratory distress syndrome
(ARDS) is characterized by thrombosis and ischemic events so that the
check for coagulation parameters are daily needed.

It is well-known that in the early stages of ARDS, fluid from the
pulmonary capillaries start to leak into the lungs, making oxygenation
very difficult
(https://www.lung.org/lung-health-diseases/lung-disease-lookup/ards/learn-about-ards).
CT images from COVID-19 patients showed the presence of widespread
ground-glass opacities in the lung, which represent the most common
evidence of pulmonary edema (Hewitt \emph{et al} . 2014) associated to
bilateral diffuse alveolar damage due to high levels of pro-inflammatory
concentrates typical of ARDS, as revealed by biopsy lung specimens from
COVID-19 affected patients (Xu \emph{et al} . 2020d).

\textbf{2. Biological targets for SARS-CoV2}

One key discovery in understanding the secrets of SARS-CoV2 infection
involves virus structure, especially viral spike (S) protein, which
facilitates viral entry into target cells by binding host-cell receptor
and then by fusing viral and host membranes (Li, 2016). SARS-CoV2
specifically recognizes Angiotensin-Converting Enzyme 2 (ACE2) as the
receptor binding domain (RDB) for its S protein to mediate viral entry
and infection (Zhou \emph{et al} . 2020b). Based on the fact that
SARS-CoV2 engages the same receptor used by SARS-CoV to mediate
infection, and that both virus share sequence similarities of 80\%
between their S proteins (Zhou \emph{et al} . 2020b), it was suggested
that they could act in a similar way. It has to be pointed out that the
viral attachment to host cell membrane via ACE2 is the first of a
multi-step process involved in coronavirus infection; indeed, after the
ligation to ACE2, indispensable for infection, the next step is the
priming of S protein by cellular proteases, which consists of S protein
cleavage at the S1/S2 and the S2 site, which allows fusion of viral and
cellular membranes proteases on the host cell (Letko \emph{et al} .
2020; Chen \emph{et al} . 2020). As in the case of SARS-CoV (Li \emph{et
al} . 2005a), the S1 subunit, which contains the RBD, directly interacts
with the peptidase domain (PD) of ACE2 providing for tight and higher
affinity binding between virus and the host cell. Based on the fact that
RBD of SARS-CoV2 is the critical determinant of viral tropism and
infectivity, it was demonstrated that its mutations could alter the
affinity to the binding receptor, leading to increased viral load
(Ou\emph{et al} . 2020). In particular, three mutants of SARS-CoV2 RBD
(V367F, W436R, and D364Y) are correlated to higher human ACE2 affinity,
ensuing higher infectivity. This discovery provides insights into
SARS-CoV2 evolution and highlights how an increased affinity for human
ACE2 due to RDB mutations could further favor COVID-19 diffusion.
Because ACE2 is the receptor that SARS-CoV2 uses to anchor host cell, it
is obvious to speculate that its expression could be correlated to viral
infection susceptibility. Therefore, scientific efforts are focused on
the study of ACE2 localization, in order to identify the possible route
of viral infection, spread and replication throughout the body. ACE2
expression in the lung is concentrated in a small population of type II
pneumocytes, which also express other genes positively correlated to
SARS-CoV2 reproduction and transmission (Zhao \emph{et al} . 2020),
suggesting that alveolar pneumocytes could be a potential site of
entrance of this virus, and prove a possible explanation for rapid lung
viral expansion and pulmonary manifestations typical of COVID-19
patients. If on one side, ACE2-expressing lung cells may be the main
target cells for coronavirus infection, on the other, Hamming \emph{et
al} . 2004 have already reported that other organs express ACE2, maybe
explaining why some COVID-19 patients also exhibit non-respiratory
symptoms. According to the single-cell RNA sequencing (scRNA-seq) and
protein datasets, apart from lung and type II alveolar cells, heart,
esophagus, kidney, bladder, ileum, oral cavity and testes are the organs
at risk due to higher ACE2 expression (Zou \emph{et al} . 2020;
Xu\emph{et al} . 2020a). To date, in the attempt to find a potential
drug against COVID-19, human recombinant soluble ACE2 (hrsACE2), which
has already been tested in phase 1 and phase 2 clinical trials for ARDS
and COVID-19 (Haschke \emph{et al} . 2013; Khan \emph{et al} . 2017;
https://clinicaltrials.gov/ct2/show/NCT00886353), can reduce viral
growth in Vero E6 cells, most probably by acting as a decoy receptor and
preventing viral binding to the natural membrane-bound ACE2
(Monteil\emph{et al} . 2020).

Beyond ACE2, it was recently found that SARS-CoV2 also uses the cellular
transmembrane protease serine 2 (TMPRSS2) for S protein priming, another
key event for virus entrance into host cell (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\emph{et al} . 2020; Xu \emph{et al} . 2020a) and is involved in the
cleavage of the SARS-CoV and influenza virus hemagglutinin protein
(B\selectlanguage{ngerman}öttcher \emph{et al} . 2006). As other member of its family, TMPRSS2
favors the entry of the virus into the lungs leading to respiratory
infections (Shulla \emph{et al} . 2011). This protease was already
described by Gierer \emph{et al} . (2013) and Matsuyama \emph{et al} .
(2010) as the enzyme responsible for SARS-CoV infection. Hoffman et al.,
(2020) found that, SARS-CoV2 uses both TMPRSS2 and endosomal cysteine
proteases cathepsin B and L (CatB/L) to enter host cells. The inhibition
of TMPRSS2 by means of Camostat mesylate, an TMPRSS2 inhibitor,
partially blocked SARS-CoV2 entry, suggesting CatB/L involvement
(Kawase\emph{et al.} 2012). Moreover, the same authors found that
co-treatment with Camostat mesylate and E-64d, an inhibitor of CatB/L,
completely abrogated virus entry in the same cells, indicating that the
virus can use both CatB/L as well as TMPRSS2 for S protein priming in
these cell lines. In contrast, the sole Camostat mesylate was not able
to block SARS-CoV-2 entry into the TMPRSS2 knock-down 293T cells,
confirming that the S protein of SARS-CoV-2 could employ TMPRSS2 for its
priming.

Other lines of research are focusing their attention on the coronavirus
3-chymotrypsin-like protease (3CLpro), also known as Mpro, a cysteine
protease present in the Coronavirus replicase polyprotein (Zhou \emph{et
al} . 2019). This protease plays a critical role both in the immune
regulation and in viral replication in that it regulates the proteolytic
cleavage of some polyprotein. 3CLpro drives the cleavage of polyproteins
pp1a and pp1ab, which in turn are responsible for the generation of
functional proteins such as RNA polymerase, endoribonuclease and
exoribonuclease (Khan \emph{et al} . 2020). For this reason, it was
speculated that 3CLpro 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 3CLpro: the antibacterial drug talampicillin, the antipsychotic
drug lurasidone (Elmezayen \emph{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\emph{et
al} . 2020). 3CLpro 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 \emph{et al} . 2011). Because this
reaction is crucial for viral replication and expression in host cells
(Menachery \emph{et al} . 2014), 2'-OMTase was suggested as another
possible druggable target for COVID-19 treatment (Khan \emph{et al} .
2020), although it is still unclear whether 2'-O-MTase, as well as
3CLpro, contributes to SARS-CoV2 infection.

\textbf{3. ACE2 and Bradykinin}

ACE2 is a membrane-associated aminopeptidase and belongs to the
angiotensin-converting enzyme family of dipeptidyl carboxydipeptidases
and has high homology to human angiotensin 1 converting enzyme (ACE1)
(Tipnis \emph{et al} . 2000). A region of the extracellular portion of
ACE2 that includes the first \selectlanguage{greek}α-\selectlanguage{english}helix and lysine 353 and proximal
residues of the N terminus of \selectlanguage{greek}β-\selectlanguage{english}sheet 5 interacts with high affinity to
the receptor binding domain of the SARS-CoV S glycoprotein (Li \emph{et
al} . 2005b). The secreted ACE2 catalyzes the cleavage of angiotensin I
into angiotensin 1-9, and angiotensin II into the vasodilator
angiotensin 1-7, explaining the negative regulation activity exerted on
angiotensin II-induced increase of blood pressure (Patel \emph{et al} .
2016). Beyond its role in the cardiovascular system, it plays a role in
the regulation of renal function and fertility (Koitka \emph{et al} .
2008; Pan \emph{et al} . 2013). Of recent outbreak, it was demonstrated
that ACE2 plays as a functional receptor for the S glycoprotein of the
human coronavirus SARS-CoV2 (COVID-19 virus) (Imai \emph{et al} . 2005;
Zhou \emph{et al} . 2020b). Once the virus binds with its glycoprotein
moiety of the extracellular S protein to ACE2, it is endocytosed into
the host cell allowing its reproduction, leading to ARDS- and
SARS-induced pulmonary edema. The ligation of ACE2 by the virus provides
ACE2 blockade which systemically translates into higher hypertensive
activity of Angiotensin II, which is not catabolized into angiotensin
1-7 or angiotensin 1-9, promoting not only the well-known hypertensive
and hypertrophic activity on the cardiovascular system, but also the
leakage of pulmonary blood vessels, as demonstrated in an \emph{in
vivo}model (Imai \emph{et al} . 2005) (Figure 1). This likely leads 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 ACE2 cleaves a single-terminal residue of several
bioactive peptides, such as neurotensin, dynorphin A (1-13), apelin-13,
and des-Arg\textsuperscript{9}bradykinin (DABK) (Vickers \emph{et al} .
2002; Donoghue \emph{et al} . 2003). Herein, once ACE2 is blocked by
SARS-CoV2, besides the perturbation of the pulmonary renin-angiotensin
system (RAS) (Imai \emph{et al} . 2005; Kuba \emph{et al} . 2005),
increasing inflammation and vascular permeability occur, due to the
activity of DABK that binding to bradykinin receptor B1 (BKB1R) can lead
to acute lung inflammation (Sodhi \emph{et al} . 2018) (Figure 1). In
support, in a mouse model of endotoxin inhalation, the absence of ACE2
led to the activation of the DABK/BKB1R axis, release of
pro-inflammatory chemokines such as C-X-C motif chemokine 5 (CXCL5),
macrophage inflammatory protein-2 (MIP2), C-X-C motif chemokine 1
(CXCL1), also known as keratinocyte-derived chemokine (KC), and TNF-\selectlanguage{greek}α
\selectlanguage{english}from airway epithelia, increasing neutrophilia and inflammation with an
ensuing lung injury. To date, the same authors showed that endotoxin
administration in mice induced ACE2 attenuation in the lung partly due
to NF-\selectlanguage{greek}κ\selectlanguage{english}B signaling, which is constantly activated during an acute
inflammatory process, especially by IL-1\selectlanguage{greek}β, \selectlanguage{english}as well as IL-6, and other
pro-inflammatory cytokines, exacerbating lung inflammation/edema up to
organ dysfunction (Sodhi \emph{et al} . 2018).

Therefore, if we consider the clinical outcome of COVID-19 patients, we
could speculate that the blockade or under-expression of ACE2 by
SARS-CoV2 and the ensuing pro-inflammatory mediators (i.e. IL-1\selectlanguage{greek}β \selectlanguage{english}and
IL-6) contribute to the pathogenesis of lung inflammation because of an
impaired catabolism of DABK, leading to an enhanced BKB1R signaling,
resulting in what is actually called ``cytokine storm'' during the early
onset of COVID-19. Bradykinin receptors are B1, an induced receptor
during inflammatory conditions, and B2, a constitutive and ubiquitous
receptor (Marceau, Hess, and Bachvarov, 1998). B2 receptor mediates the
action of BK and lysyl-bradykinin (Lys-BK), the first set of bioactive
kinins formed in response to injury from kininogen precursors through
the action of plasma and tissue kallikreins; whereas B1 receptor
mediates the action of DABK and Lys-DABK, the second set of bioactive
kinins formed through the action of carboxypeptidases on BK and Lys-BK,
respectively (Couture \emph{et al} . 2001). The B2 receptor is
ubiquitous and constitutively expressed, whereas the B1 receptor is
expressed at very low levels in healthy tissues but it is induced
following injury by various pro-inflammatory cytokines such as IL-1\selectlanguage{greek}β
\selectlanguage{english}(Marceau , Hess, and Bachvarov, 1998). Both receptors act through
G\selectlanguage{greek}α\selectlanguage{english}\textsubscript{q} to stimulate phospholipase C\selectlanguage{greek}β \selectlanguage{english}followed by
phosphoinositide hydrolysis and intracellular free
Ca\textsuperscript{2+} mobilization, and through G\selectlanguage{greek}α\selectlanguage{english}\textsubscript{i} to
inhibit adenylate cyclase and stimulate the MAPK pathways
(Leeb-Lundberg, 2004) (Figure 2). Although little is known about the
cross-talk between B1 and B2 receptors, it is well established that B2
signalling can mediate B1 upregulation via MAPK- and NF-kB-dependent
pathways, and that the expression of both receptors can be induced by
pro-inflammatory cytokines (i.e. TNF\selectlanguage{greek}α \selectlanguage{english}and IL-1\selectlanguage{greek}β\selectlanguage{english}), creating a
``\emph{catch-22 loop} '' (Brechter \emph{et al} . 2008). This molecular
mechanism/s translated into clinical outcomes underlie vascular
permeability and dilation, bronchoconstriction (cough) and pain
(hyperalgesia, muscular pain) with ensuing fever due to the cytokine
storm, all symptoms of COVID-19. (Figure 1).

An important issue is that BK is produced by the kallikrein-kinin system
(KKS). The KKS consists of prekallikrein in complex with 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 proteic domains (Shariat-Madar and Schmaier, 1999) and
binds to prekallikrein by means of a sequence in domain 6. The
detachment of the domain 4 liberates 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: plasma
kallikrein, which cleaves HMWK 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 onto B1 and B2 receptors will increase the activation of both
endothelial NOS (eNOS) and inducible NOS (iNOS), with an ensuing release
of nitric oxide, potent vasodilator, of prostaglandin I2 (PGI2) and
pro-inflammatory cytokines and chemokines responsible for acute
inflammation that is accompanied by vasodilation, pain, cell
proliferation and fibrosis (Kuhr \emph{et al} . 2010; Tsai \emph{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 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-kallidin and des-Arg\textsuperscript{9}-BK
which interact with B1 receptors further enhancing inflammation. The
intrinsic pathway of coagulation is then correlated to the extrinsic
pathway of the coagulation in that Factor XIIa activates Factor XI,
which leads to the activation of factor IX which subsequently leads into
the common pathway by the activation of 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, \emph{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 \emph{et al} . 2002). On the other hand, plasma kallikrein can
align pro-urokinase plasminogen activator (u-PA) in such close proximity
as to drive plasminogen activation into plasmin which degrades fibrin
aggregates (Selvarajan \emph{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 \emph{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 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, pathologies 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? Our hypothesis is that the infection
by SARS-CoV2 that ``uses'' ACE2 to enter the host, blocks the activity
of the degradation of angiotensin II, but at the same time the
degradation of BK is altered and impaired in that to trigger and enhance
all the above described clinical events.

\textbf{4. Approaching therapies for COVID-19 patients.}

In the attempt to identify the effective anti-SARS-CoV2 therapy some
clinical trials are still ongoing. In particular, the therapeutic
approach can be classified in two big branches: the antiviral, which
aims to diminish virus replication, and the anti-inflammatory agents to
hijack the cytokine storm that the virus is able to induce. In
particular, the antiviral drug that is currently proving of efficacy in
COVID-19 is remdesivir, which tightly binds and inhibits the virus
RNA-dependent RNA polymerase (RdRp) (Elfiky, 2020). In a cohort of
patients hospitalized for severe COVID-19 who were treated with
compassionate-use, remdesivir proved of clinical improvement in 36 out
of 53 patients (68\%) (Grein \emph{et al.} 2020). Instead, lopinavir and
ritonavir, anti-HIV drugs, showed disappointing results beyond standard
care in that the viral load and the mortality were not altered
(Cao\emph{et al.} 2020).

On the other hand, disease-modifying antirheumatic drugs (DMARDS), such
as chloroquine and hydroxychloroquine, as well as immunotherapeutic
agents, such as monoclonal antibodies (mAbs), are being used. In
particular, chloroquine as well as hydroxychloroquine, antimalarial
drugs, can interfere with lysosomal activity and autophagy, interact
with membrane stability and alter signalling pathways and
transcriptional activity, which can result in inhibition of cytokine
production and modulation of immune co-stimulatory molecules
(Schrezenmeier and D\selectlanguage{ngerman}örner, 2020). Thus, they can inhibit lysosomal
activity, preventing major histocompatibility complex (MHC) class
II-mediated antigen presentation. Moreover, they can accumulate in
endosomes and bind to double-stranded DNA, inhibiting both Toll-like
receptor (TLR) signaling (i.e. TLR7 and TLR9) (Kuznik \emph{et al.}2011)
and the nucleic acid sensor cyclic guanosine monophosphate--adenosine
monophosphate (cGMP-AMP or cGAMP) synthase (cGAS) (Zhang \emph{et al.}
2014). By preventing TLR signalling and cGAS--stimulator of interferon
genes (STING) signalling, hydroxychloroquine can reduce the production
of pro-inflammatory cytokines (van den Borne \emph{et al.} 1997).
However, the adverse effects need to be taken into consideration,
especially in regards to the alteration of heart rhythm which cautiously
limits their use.

Importantly, immunotherapeutic agents, such as tocilizumab or sarilumab,
which are mAbs against IL-6 signalling, highly released during the
interstitial pneumonia, have proved an effective treatment in severe
patients of COVID-19 to calm the inflammatory storm and reduce mortality
(Xu \emph{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).
This encouraging clinical trial indicates that neutralizing mAbs against
other pro-inflammatory cytokines may also be of use, with potential
targets including IL-1, IL-17 and their respective receptors. However,
tocilizumab, as well as sarilumab, can induce hepatotoxicity,
neutropenia, tumorigenesis, hypersensitivity, opportunistic infections
(\emph{https://www.accessdata.fda.gov/drugsatfda\_docs/label/2016/125276s107\_125472s018lbl.pdf};\emph{https://www.accessdata.fda.gov/drugsatfda\_docs/label/2018/761037s001lbl.pdf});
therefore, mAbs targeting IL-6 signalling cannot be administered to all
patients, because of co-morbidities to COVID-19 need carefully to be
taken into consideration. Nevertheless, they represent the promise for
blocking cytokine storm-related immunopathology of moderate to severe
COVID-19.

Another clinical trial for stable COVID-19 patients is on the activity
of colchicine, an anti-gout drug, which blocks the mitotic cells in
metaphase, but is also able to block Nod-like receptor protein 3 (NLRP3)
inflammasome inhibiting the release of IL-1-like cytokines
(\emph{https://clinicaltrials.gov/ct2/show/NCT04322565}), such as IL-1\selectlanguage{greek}β,
\selectlanguage{english}that was in the attempt to be blocked by means of anakinra in another
clinical trial (https://clinicaltrials.gov/ct2/show/NCT04366232).
Additionally, emapalumab, a monoclonal antibody against IFN-\selectlanguage{greek}γ,
\selectlanguage{english}associated to anakinra, has been proposed
(https://clinicaltrials.gov/ct2/show/NCT04324021). However, the
inhibition of such an important anti-viral cytokine could be on one side
important to block the cytokine storm, but on the other can be hijacked
by the virus due to the absence of one of the most important army
against viral infections, creating further opportunistic pathologies.

Baricitinib has been identified as a molecule potentially useful in
COVID-19 because of a double action to down-modulate the inflammatory
storm and reduce the entry of the virus into type II pneumocytes due to
the blockade of the AP2-associated protein kinase 1 (AAK1), a regulator
of the endocytosis of the virus (Richardson \emph{et al.} 2020).
Moreover, baricitinib also binds to the cyclin G-associated kinase,
another regulator of endocytosis. Thus, baricitinib may be useful for
both reducing inflammatory response and viral endocytosis.

It has to be pointed out that all the above 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 (\textgreater{} 1 g/L) were strongly associated
with in-hospital deaths, therefore to severe COVID-19 (Zhou \emph{et
al.} 2020a). To date, low molecular weight heparin (LMWH), enoxaparin,
has been proposed for these patients either to avoid thromboembolism
events (Tang \emph{et al.} 2020a) or to inhibit the cytokine storm
(Shi\emph{et al.} 2020), due to non-anticoagulant fraction of enoxaparin
suppresses \emph{in vitro} IL-6 and IL-8 release from human pulmonary
epithelial cells (Shastri \emph{et al.} , 2015). Moreover both \emph{in
vitro} and \emph{in vivo} experimental studies have shown that human
coronaviruses utilize heparin sulfate proteoglycans for attachment to
target cells (Milewska \emph{et al.} 2014). Indeed, interaction between
the SARS-CoV2 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 \emph{et
al.} 2020).

Another immunotherapeutic agent that was suggested is eculizumab, a mAb
against C5 complement. Diffuse microvascular thrombi in multiple organs
in COVID-19 non-survivors have been announced and even more important,
thrombotic microangiopathy (TMA) can occur in many different clinical
scenarios including pathogenic complement activation (Campbell and
Kahwash, 2020). Altered complement system occurs in a number of
pathologic settings, leading to diffuse thrombotic microangiopathy
(TMA), microangiopathic hemolytic anemia, thrombocytopenia, and acute
renal failure up to organ dysfunction. If given early, eculizumab
therapy can reverse both renal and cardiac dysfunction (Campbell and
Kahwash, 2020). Campbell and Kahwash (2020) suggest that complement
inhibition could be a promising treatment for severe COVID19 by reducing
the innate immune-mediated consequences of severe coronavirus infection,
and it would pair well with direct anti-viral therapy.

Another suggested approach includes the off-label use of Camostat or
Nafamostat mesylate, inhibitors of the host cell protease TMPRSS2, that
could arrest coronavirus infections by controlling viral entry into the
human cells. It has to be noted that, if on one side TMPRSS2 inhibitors
could prevent SARS-CoV2 replication by blocking the fusion of the virus
envelope with host cell surface membranes, they could also be effective
in controlling pathological conditions correlated to COVID-19, such as
coagulation and inflammation, based on their pharmacological properties.
As it is well-known, Nafamostat mesylate has been used as a short-acting
anticoagulant in patients with disseminative blood vessel coagulation,
hemorrhagic lesions, and hemorrhagic tendencies (Maruyama \emph{et
al.}2011; Choi \emph{et al.} 2015) due to its ability to competitively
inhibit various enzyme systems, such as coagulation and fibrinolytic
systems (thrombin, Xa, and XIIa), the KKS, the complement system,
pancreatic proteases and activation of protease-activated receptors
(PARs) (Kim \emph{et al.} 2016). Similarly, Gabexate mesylate, binds and
inhibits kallikrein, plasmin and thrombin (Tamura \emph{et al.} 1977).
Therefore, it was suggested the use of these drugs to prevent thrombosis
and disseminated intravascular coagulation typical of COVID-19 patients
(Tang \emph{et al.} 2020b; Cui \emph{et al.} 2020). Beyond their
anticoagulant propriety, both Gabexate and Nafamostat mesylate show
anti-inflammatory effects, which could be useful in COVID-19
uncontrolled inflammation (Tay \emph{et al.} 2020). In particular,
Gabexate mesylate decreases the production of inflammatory cytokines,
such as TNF-\selectlanguage{greek}α \selectlanguage{english}by attenuating NF-\selectlanguage{greek}κ\selectlanguage{english}B and JNK pathway activity, most
probably through the proteolytic destruction of I\selectlanguage{greek}κ\selectlanguage{english}B (Yuksel \emph{et
al.} 2003); Nafamostat mesylate shows an anti-inflammatory
effect\emph{in vitro} , where it mediates the inhibition of
lipopolysaccharide-induced nitric oxide production, apoptosis, IL-6 and
IL-8 production in cell cultures (Kang \emph{et al.} 2015; Choi \emph{et
al.} 2016). In this context, Camostat mesylate could be also useful to
reduce the production of inflammatory cytokines due to SARS-CoV2
infection. Indeed, it was already found that Camostat mesylate reduces
the release of IL-6 and TNF-\selectlanguage{greek}α \selectlanguage{english}into cell supernatants infected with
influenza virus (Yamaya \emph{et al.} 2016).

\textbf{5.} \textbf{Further therapeutic hypotheses.}

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 both BK receptors and HMWK.
Icatibant is an antagonist of B2 receptor blocking the activity of the
BK avoiding both the pro-inflammatory cytokine storm and cell
proliferation; it is a drug approved by the European medical agency
(EMA) for the treatment of angioedema in both children and adults
(https://www.ema.europa.eu/en/documents/assessment-report/firazyr-epar-public-assessment-report\_en.pdf).
No specific adverse events have been reported, unless urticaria, nausea
and headache, though, specific attention to be paid in patients with
compromised cardiovascular system (i.e. ischemia and angina pectoris).
However, preclinical studies did not show any genotoxic activity,
alteration of the cardiac conduction and ischemia events or hemodynamic
parameters. Nevertheless, it has been demonstrated that icatibant highly
binds to B2 receptor, while the affinity to the analogous B1 receptor is
at least 100 times lower. It has to be pointed out, though, that little
is known about B1 receptor, which is the inducible receptor during
inflammatory conditions, although several pre-clinical and phase I/II
trials have been performed to evaluate possible use of agents targeting
B1 receptor for inflammation-related diseases (Qadri and Bader, 2018).

In addition, another drug to point the attention on could be
lanadelumab, which 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-public-assessment-report\_en.pdf).
Differently from icatibant, lanadelumab could block upstream the
activity of BK, avoiding the inflammatory and coagulation storm besides
the complement system in SARS-CoV2 infected patients, likely preventing
the exacerbation of COVID-19, in parallel with antiviral therapy.

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 DABK derived by HMWK. Therefore, the hypothesis to block
the production of DABK upstream by blocking the metabolism of HMWK could
be another option to face this tremendous pandemic event that affected
whole world life style obliging to social limitations and stay-at-home
politics.

\textbf{Acknowledgements:}

N/A

\textbf{Conflict of Interest:}

No conflict of interest to disclose.

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\textbf{Figures and Figure Legends}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image1/image1}

\end{center}
\end{figure}

\textbf{Figure 1. ACE2 function and its regulation in SARS-CoV2
infection.}

ACE2 is a carboxypeptidases which catalyzes and inactivates Angiotensin
I and Angiotensin II, respectively, into the vasodilator peptides
Angiotensin 1--9 and Angiotensin 1--7, which bind Mas receptor (MasR)
leading to reduced inflammation and vasodilation. ACE2 also cleaves
des-Arg\textsuperscript{9}bradykinin (DABK), a bioactive kinin derived
from kininogen pathway, into inactive metabolites. ACE2 is the cell
entry receptor for SARS-CoV2; the binding of viral spike glycoprotein
with ACE2 and the priming of the spike through the transmembrane
protease serine 2 (TMPRSS2), leads to SARS-CoV2 infection. The binding
of SARS-CoV2 downregulates ACE2 expression, leading to a reduction of
its enzymatic activity and the ensuing increase of Angiotensin II and
DABK levels. Angiotensin II takes its deleterious effect by binding the
Angiotensin II type 1 receptor (AT1R), whereas DABK concurs to
inflammation by binding BK receptor B1 (B1R), resulting in severe lung
injury, pulmonary inflammation and edema, increased coagulation,
hypertension and cardiac hypertrophy, which are all features of COVID-19
patients.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image2/image2}

\end{center}
\end{figure}

\textbf{Figure 2. Crosstalk between Kallikrein-Kinin System (KKS),
coagulation, fibrinolysis and complement cascade.}

Kallikrein-Kinin system (KKS) (black box and arrows) consists of tissue
and plasma kallikrein which act on high molecular weight kininogen
(HMWK) and low molecular weight kininogen (LMWK) to generate bradykinin
(BK) and kallidin (Lys-BK). BK and Lys-BK, and their metabolites
(Lys-des-Arg\textsuperscript{9}-BK and DABK) act via two G-coupled
receptors, B1R and B2R, resulting in increased vascular permeability,
vasodilation, edema formation and ultimately hypotension. Plasma
kallikrein, which is induced by the reciprocal activation of the Factor
XIIa (FXIIa) and plasma prekallikrein, also influences the fibrinolytic
pathway by activating plasminogen into plasmin and leading to fibrin
degradation and D-dimer generation (yellow box and arrows). Beyond its
role in KKS, FXIIa starts the intrinsic coagulation pathway (red
arrows). Blood coagulation consists of an intrinsic and extrinsic (grey
arrows) pathways, both resulting in activation of 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 signalling, triggers C3
cleavage inducing the activation of both lecithin and extrinsic pathways
of the complement.\selectlanguage{english}
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\includegraphics[width=0.70\columnwidth]{figures/Figure-1/Figure-1}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
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\includegraphics[width=0.70\columnwidth]{figures/Figure-2/Figure-2}

\end{center}
\end{figure}

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