Investigating the Effect of the SARS-CoV-2 Spike-1 (S1) Protein on Coagulation

Abstract

Infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes the disease referred to as COVID-19. COVID-19 produces variable clinical manifestations, ranging from asymptomatic disease, to severe pneumonia with acute respiratory distress syndrome (ARDS). There remains no specific anti-viral treatment or vaccine to date. However, increasing reports indicate the ability of glycosaminoglycans (GAGs), such as heparin, to bind SARS-CoV-2, with the potential to prevent its entry into cells. Nevertheless, the impact of this interaction on heparin’s anticoagulant effects is not yet known. This study consequently aims to investigate the interaction between the Sars-CoV-2 spike receptor protein and heparin, specifically investigating the hypothesis that the spike-1 (S1) receptor-binding domain (RBD) protein can impair heparin’s anticoagulant effects. The SARS-CoV-2 S1 RBD was produced using recombinant DNA technology, and the secondary structure was analysed by means of circular dichroism. The effect of S1 RBD on coagulation was investigated in an activated partial thromboplastin time (aPTT) assay. The results indicate that the S1 RBD can be successfully expressed and purified from a selected E. coli strain, while the alpha-helices in its secondary structure rapidly degrade if the protein is left standing for 24 hours. Compared to a control, the addition of RBD alone increased coagulation time by 12.4% (1.46mg/ml), 7.1% (2.92 mg/ml) and 22.5% (8.76 mg/ml). Heparin alone, compared to a control, increased coagulation time by 38.3%. The addition of S1 RBD (at a concentration of 0.15 mg/ml) did not significantly alter this effect. Meanwhile, at a final RBD concentration of 0.37 mg/ml and heparin, clotting time increased by 55.6%. In summary, S1 RBD is unstable in vitro and can undergo rapid structural degradation. This has an implication for future studies aimed at identifying candidate therapeutic agents that could bind it. Furthermore, S1 RBD has an unexpected anti-coagulant effect in vitro, both on its own and when bound to heparin. Heparin therapy may therefore be useful for addressing the venous thromboembolism (VTE) risk associated with SARS-CoV-2 infection. Thus, further studies are necessary to investigate the side-effects arising from its anticoagulant effect when bound to an RBD protein.