Coagulation factors directly cleave SARS-CoV-2 spike and enhance viral entry.
Edward R. Kastenhuber
1
, Javier A. Jaimes
2
, Jared L. Johnson
1
, Marisa Mercadante
1
, Frauke
Muecksch
3
, Yiska Weisblum
3
, Yaron Bram
4
, Robert E. Schwartz
4,5
, Gary R. Whittaker
2
and
Lewis C. Cantley
1,*
Affiliations
1. Meyer Cancer Center, Department of Medicine, Weill Cornell Medical College, New York,
NY, USA.
2. Department of Microbiology and Immunology, Cornell University, Ithaca, New York, USA.
3. Laboratory of Retrovirology, The Rockefeller University, New York, NY, USA.
4. Division of Gastroenterology and Hepatology, Department of Medicine, Weill Cornell
Medicine, New York, NY, USA.
5. Department of Physiology, Biophysics and Systems Biology, Weill Cornell Medicine, New
York, NY, USA.
*Correspondence: lcantley@med.cornell.edu
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The copyright holder for this preprint (whichthis version posted July 30, 2021. ; https://doi.org/10.1101/2021.03.31.437960doi: bioRxiv preprint
Summary
Coagulopathy is a significant aspect of morbidity in COVID-19 patients. The clotting
cascade is propagated by a series of proteases, including factor Xa and thrombin. While certain
host proteases, including TMPRSS2 and furin, are known to be important for cleavage
activation of SARS-CoV-2 spike to promote viral entry in the respiratory tract, other proteases
may also contribute. Using biochemical and cell-based assays, we demonstrate that factor Xa
and thrombin can also directly cleave SARS-CoV-2 spike, enhancing viral entry. A drug-
repurposing screen identified a subset of protease inhibitors that promiscuously inhibited spike
cleavage by both transmembrane serine proteases as well as coagulation factors. The
mechanism of the protease inhibitors nafamostat and camostat may extend beyond inhibition of
TMPRSS2 to coagulation-induced spike cleavage. Anticoagulation is critical in the management
of COVID-19, and early intervention could provide collateral benefit by suppressing SARS-CoV-
2 viral entry. We propose a model of positive feedback whereby infection-induced
hypercoagulation exacerbates SARS-CoV-2 infectivity.
Keywords:
SARS-CoV-2, COVID-19, coronavirus, entry, factor Xa, thrombin, coagulopathy, anticoagulants,
camostat, nafamostat,
was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (whichthis version posted July 30, 2021. ; https://doi.org/10.1101/2021.03.31.437960doi: bioRxiv preprint
Introduction
SARS-CoV-2 emerged into the human population in late 2019 and has evolved into a
devastating global health crisis. Despite the recent success of vaccines (Baden et al., 2020;
Polack et al., 2020), the limited world-wide vaccine distribution (Kwok et al., 2020; Lin, Tu, &
Beitsch, 2020; Nhamo, Chikodzi, Kunene, & Mashula, 2020; So & Woo, 2020), the emergence
of viral variants (Wang et al., 2021; Weisblum et al., 2020), and the repeated SARS-like
zoonotic outbreaks over the last 20 years (Cheng, Lau, Woo, & Yuen, 2007; Ge et al., 2013;
Menachery et al., 2015) underscore the urgent need to develop antivirals for coronavirus
(Consortium et al., 2020).
In addition to attachment to specific receptors on target cells, coronaviruses require
proteolytic processing of the spike protein by host cell proteases to facilitate membrane fusion
and viral entry (Glowacka et al., 2011; J. A. Jaimes, J. K. Millet, & G. R. Whittaker, 2020; Walls
et al., 2020). In SARS-CoV-2, host cell proteases act on two sites residing at the S1/S2 subunit
boundary and at the S2’ region proximal to the fusion peptide (Belouzard, Chu, & Whittaker,
2009; Hoffmann, Kleine-Weber, et al., 2020; J. Jaimes, J. Millet, & G. Whittaker, 2020; Millet &
Whittaker, 2014). S1/S2 site cleavage opens up the spike trimer and exposes the S2’ site, which
must be cleaved to allow for the release of the conserved fusion peptide (Benton et al., 2020;
Madu, Roth, Belouzard, & Whittaker, 2009). While the prevailing model suggests that furin
cleaves the S1/S2 site and TMPRSS2 cleaves the S2’ site (Bestle et al., 2020), it remains
unclear to what extent other proteases may be involved (Hoffmann et al., 2021; Ou et al., 2020).
TMPRSS2 is an important host cell factor in proteolytic activation across multiple
coronaviruses (Hoffmann, Kleine-Weber, et al., 2020; Jaimes, Millet, Goldstein, Whittaker, &
Straus, 2019). TMPRSS2 knockout or inhibition reduces infection in mouse models of SARS
and MERS (Iwata-Yoshikawa et al., 2019; Y. Zhou et al., 2015). More recently, TMPRSS2 has
been highlighted as a drug target for SARS-CoV-2 (Hoffmann, Kleine-Weber, et al., 2020;
Hoffmann, Schroeder, et al., 2020).
Furin activity is not essential to produce infectious particles (Tang et al., 2021) and furin
is not necessary for cell fusion (Papa et al., 2021), but deletion of the S1/S2 site attenuates
SARS-CoV-2 in vivo (Johnson et al., 2021). Proteolytic activation of envelope proteins
presumably coordinates target cell engagement and envelope conformational changes leading
to fusion. Furin cleavage during viral biogenesis, before release of viral particles, may render
SARS-CoV-2 spike less stable in solution and reduce the likelihood to reach and interact with
target cells (Amanat et al., 2021; Berger & Schaffitzel, 2020; Wrobel et al., 2020). Although the
was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (whichthis version posted July 30, 2021. ; https://doi.org/10.1101/2021.03.31.437960doi: bioRxiv preprint
S1/S2 site is often referred to as the “furin site” (Johnson et al., 2021), the full spectrum of
proteases that catalyze biologically relevant activity in the lung remains incompletely defined.
Proteases also orchestrate the coagulation pathway, via a series of zymogens that are
each activated by a chain reaction of proteolytic processing. Coagulopathy and thromboembolic
events have emerged as a key component of COVID-19 pathogenesis (McGonagle, O'Donnell,
Sharif, Emery, & Bridgewood, 2020). Comorbidities associated with severe COVID-19 are also
linked to dysregulated blood clotting (F. Zhou et al., 2020). Patients with a history of stroke prior
to infection have nearly twice the risk of in-hospital mortality (Qin et al., 2020). Upon hospital
admission, elevated D-dimer levels (an indicator of fibrinolysis and coagulopathy) and low
platelet counts (an indicator of consumptive coagulopathy) are predictive biomarkers of severe
disease and lethality in COVID-19 patients (F. Zhou et al., 2020). Systemic activity of clotting
factors V, VIII, and X are elevated in severe COVID-19 disease (Stefely et al., 2020). While
early phase disease is typically restricted to a local pulmonary hypercoagulable state, late stage
disease may be accompanied by systemic DIC, stroke and cardio-embolism (Huang et al.,
2020; Kipshidze et al., 2020; McGonagle et al., 2020; Tsivgoulis et al., 2020). Ischemic stroke
occurred in approximately 1% of hospitalized COVID-19 patients, and strikingly, a fraction of
them experienced stroke even prior to onset of respiratory symptoms (Yaghi et al., 2020).
In a drug repurposing effort to target TMPRSS2, we observed that multiple direct-acting
anticoagulants have anti-TMPRSS2 off-target effects. We subsequently investigated overlap in
substrate specificity between TMPRSS2, factor Xa and thrombin. Circulating proteases involved
in blood clotting can cleave and activate SARS-CoV-2 spike, enhancing viral entry. We propose
that the serine protease inhibitor nafamostat may incorporate a combined mechanism in the
treatment of COVID-19 through inhibition of TMPRSS2 and coagulation factors.
was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (whichthis version posted July 30, 2021. ; https://doi.org/10.1101/2021.03.31.437960doi: bioRxiv preprint
Results
Serine protease inhibitors suppress SARS-CoV-2 entry via inhibition of TMPRSS2
We developed a fluorescence resonance energy transfer (FRET)-based protease
enzymatic assay based on peptides containing either the S1/S2 or S2’ cleavage sites of SARS-
CoV-2 spike (Fig. 1A, S1). Upon cleavage, the liberated 5-FAM emits fluorescent signal
proportional to the quantity of product (Fig. S1A). Camostat and nafamostat resulted in strong
inhibition of TMPRSS2 (Fig. 1B), as expected (Hoffmann, Kleine-Weber, et al., 2020; Hoffmann,
Schroeder, et al., 2020). We also identified that otamixaban and the active form of dabigatran
(but not its prodrug dabigatran etexilate) inhibit TMPRSS2 enzymatic activity in vitro (Fig. 1B-
C).
To explore these candidates in a cell-based functional assay of spike protein, SARS-
CoV-2 S-pseudotyped HIV-1 particles were employed to infect human lung Calu3 cells (Fig. 1D)
(Schmidt et al., 2020). Consistent with the TMPRSS2 enzymatic assay, camostat, nafamaostat,
otamixaban, and dabigatran etexilate suppressed pseudoviral entry, as indicated by
nanoluciferase luminescent signal (Fig. 1E). No effects on relative cell growth were observed at
the same timepoint in Calu3 (Fig. 1F) or A549 cells (data not shown), confirming that reduced
luminescent signal was not due to cytotoxicity. A dose-response experiment with select
protease inhibitors revealed a sub-micromolar IC50 for camostat and nafamostat and IC50s in
the 10-20 µM range for otamixaban and dabigatran in Calu3 cells (Fig. 1G).
Using A549 cells with or without ectopic ACE2 expression, we confirmed that HIV-
1
NL
/SARS-CoV-2 pseudovirus infection is dependent on ACE2, while infection with HIV-1
NL
pseudotyped instead with VSV G envelope protein is not ACE2 dependent (Fig. S2). Caco2
cells, which endogenously express ACE2 and TMPRSS2, show greater susceptibility to SARS-
CoV-2 S-pseudotyped HIV-1
NL
, but equivalent susceptibility to VSV G-pseudotyped HIV-1
NL
,
when compared to A549/ACE2 cells (Fig. S2).
To further validate these results in an alternative pseudovirus system, we used
recombinant G protein-deficient vesicular stomatitis virus (rVSV∆G) pseudotyped with SARS-
CoV-2-S (Fig. S3A), yielding pseudovirus dependent on spike for cell entry (Fig. S3B). The
antiviral effects of the four candidate protease inhibitors were confirmed in the VSV pseudovirus
system in multiple cell lines, and response was associated with TMPRSS2 expression (Fig.
S3C-F).
We aimed to determine whether the effects of camostat and nafamostat are indeed
TMPRSS2-dependent, or if other unidentified cellular proteases can compensate for TMPRSS2
was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (whichthis version posted July 30, 2021. ; https://doi.org/10.1101/2021.03.31.437960doi: bioRxiv preprint