Identification of the antithrombotic protein S as a potential target of the SARS-CoV-2 papain-like protease.

A well-documented complication associated with severe cases of COVID-19 is the development of a hypercoagulable state. Although described as reminiscent of disseminated intravascular coagulation (DIC) and thrombotic microangiopathy, its clinical characteristics, referred to as thromboinflammation or MicroCLOTS, appear unique with a multifaceted and incompletely elucidated etiology [1]. The most consistent abnormality is a significant elevation of D-dimer, which is observed in up to 50% of infected patients and serves as a negative prognostic indicator. Serum fibrinogen is commonly elevated, and a moderate reduction in platelet count and prolonged prothrombin time are also typical findings. Notably, venous thromboembolism (VTE), especially pulmonary embolism, is observed in a high percentage of COVID-19 ICU patients, and the thrombotic complications contribute to multiple organ damage. Described here is the hypothesis that the papain-like protease (PLpro) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2 or CoV2), the causative agent of COVID-19, may contribute to the coagulation and thrombotic dysregulation by the promiscuous cleavage of protein S, a pleiotropic factor with antithrombotic and immunomodulatory properties.

Vitamin K-dependent protein S (PROS1) is a plasma glycoprotein synthesized in endothelial cells, hepatocytes, and megakaryocytes. It is perhaps most recognized as a cofactor of activated protein C (APC), promoting an antithrombotic state by facilitating the cleavage of activated factors Va and VIIIa. It also serves as a cofactor for tissue factor pathway inhibitor (TFPI), aiding suppression of coagulation by inhibiting factor Xa, and it has additional anticoagulant effects via direct interaction with other coagulation factors [2]. Loss of PROS1 function, such as due to heterozygous mutations in the PROS1 gene, predisposes an individual to VTE.

PROS1 also serves as an activating ligand for the TAM family of receptor tyrosine kinases (RTKs), a role that ties PROS1 to the “cytokine storm” observed in severe cases of COVID-19 [3]. Signaling via these RTKs, TYRO3, AXL, and MER, negatively regulates immune cell function and inhibits the innate immune response. PROS1 is believed to primarily activate TYRO3 and MER, attenuating the response of dendritic cells and macrophages to pathogens and reducing the secretion of proinflammatory cytokines such as interleukin-6 (IL-6), tumor necrosis factor α and type 1 interferons. Loss of PROS1 would blunt this inhibitory mechanism and promote the hyperactive immune response seen with COVID-19.

The replicase of all SARS coronaviruses, including CoV2, SARS-CoV-1 (SARS) and Middle East CoV (MERS), encodes two cysteine proteases, 3C-like protease (3CLpro; also referred to as the “main” protease, Mpro) and PLpro. Together, the two are responsible for cleaving the initially translated viral polyproteins pp1a and pp1ab into 16 mature nonstructural proteins (nsps), an intracellular, cytoplasmic process [4]. A review of the three N-terminal cleavage sites recognized by the PL proteases of the SARS viruses reveals a highly conserved LXGG motif (Fig. 1A and B). A BLAST search query using the 10 amino acids spanning the nsp3/4 cleavage site of CoV2 (IALKGG/KIVN) returns an eight amino acid “hit” to PROS1, including the core LXGG sequence (Fig. 1C). The “X” in the CoV2 polyprotein, a lysine, is replaced by a highly conserved arginine in PROS1; studies indicate that this is the only amino acid substitution at P3 that retains high substrate specificity for PLpro of both CoV2 and SARS [5]. Other hits are returned when the cleavage site sequences recognized by CoV2 PLpro are used as BLAST search queries; many do not contain the required LXGG motif or are to proteins in cell types not known to be infected by CoV2. The identification of PROS1 in this manner was noteworthy given the clinical observations of hypercoagulopathy. Other identified host proteins may be viable targets of promiscuous cleavage by CoV papain-like protease(s).

Fig. 1

A. Comparison of the consensus amino acid sequence recognized by the papain-like proteases of the three SARS coronaviruses with the identified amino acid sequence in the first laminin G domain of PROS1. The height of each letter is proportional to the occurrence of the corresponding amino acid in the nine sites (B) used to generate the alignment (https://weblogo.berkeley.edu/logo.cgi); the sequence of the prospective cleavage site in PROS1 is shown below. C. Alignment returned by a protein-protein BLAST search (blastp) using the CoV2 nsp3/4 cleavage site sequence as query and restricting the search to Homo sapiens (taxid 9606). The position numbers for PROS1 reflect those in the NCBI reference sequence (accession number NP_000304.2). A “+” indicates chemical similarity. Note the arginine at position P3 is the only amino acid identified as promoting high substrate specificity by CoV PLpro other than lysine [5].

In addition to the match found using BLAST, the on-line, open access computational tools PROSPER, the Protease Specificity Prediction Server (https://prosper.erc.monash.edu.au/home.html) and Procleave both identified the prospective cleavage site in PROS1 as a cysteine protease target. The available homology model for PROS1 indicates the cleavage site should be spatially accessible (Fig. 2 ).

Fig. 2

Homology model of PROS1, based on growth-arrest-specific protein, the other known agonist for TAM RTKs. The lysine at P1′ in the prospective recognition site L R G G/K I E V is circled. The model was downloaded from https://swissmodel.expasy.org/repository/uniprot/A0A0S2Z4K3?template=1h30.1.A&range=266-673.

PROS1 is synthesized as a 676 amino acid precursor protein which is processed to a mature multimodular protein of 635 amino acids. It circulates in both free form (~40% of total) and complexed to the complement regulator C4BP. The PROS1 N-terminal region includes the Gla domain, important for binding to phosphatidylserine, a thrombin-sensitive region, and four epidermal growth factor domains, followed by a C-terminal region comprised of a sex hormone binding globule (SHBG) encompassing two tandem laminin G (LG) domains. The prospective cleavage site of PLpro at G357 is in the first laminin G domain and would separate approximately 80% of the SHBG from intact PROS1, significantly denigrating its cofactor activity with both APC and TFPI [2]. The LG domains are also necessary for the binding of PROS1 to TAM receptors, and cleavage potentially promotes the cytokine storm [3].

For such a mechanism to be operative PROS1 and PLpro would have to be in spatial proximity. A primary site of PROS1 synthesis is endothelial cells, which also express ACE2, the cell surface receptor used by CoV2. Infection of and subsequent damage to the endothelium is a major contributor to the coagulation and immune response dysregulation seen with COVID-19. CoV2 has also been detected in hepatocytes [6], another significant site of PROS1 synthesis. Although PROS1 is a secreted protein, it must undergo post-translational processing, including gamma-carboxylation in the endoplasmic reticulum (ER). Coronavirus replication complexes are located in the ER, perhaps increasing the likelihood of intracellular contact between PLpro and PROS1 [4,7].

Megakaryocytes, the parent cells of platelets, also synthesize PROS1, and it is becoming increasingly clear that COVID-19 dysregulates multiple platelet functions. Platelet PROS1 limits venous thrombin formation and the dissemination of activated platelets. The RNA of CoV2 has been detected in platelets [8], possibly enhancing viral dissemination and platelet activation. Whether this puts platelet PROS1 and an active PLpro in potential contact is unclear.

The mechanisms leading to the hypercoagulation seen in COVID-19 are obviously multifactorial. Activation of endosomal NADPH oxidase, the complement cascade, and platelets have been advanced as contributors. A role for PROS1 has also been proposed, based on its consumption due to excessive coagulation with concomitant loss of TAM signaling. The authors note that infection of the vasculature with CoV2 may deplete PROS1, and that measurement of circulating levels of PROS1 is not routine [9]. The hypothesis described here may operate in tandem with this mechanism of PROS1 depletion and adds an additional impetus to better characterize PROS1 levels during severe COVID-19 infections. It may also help clinically differentiate the coagulation pathologies observed between CoV2, SARS, and MERS [10] as the abilities of the cognate PL proteases to cleave PROS1 would likely vary.