Investigating the Role of SARS-COV-2 ORF8 Protein as a Virulence Factor Driving the COVID-19-Associated Clotting Disorder

ABSTRACT

The formation of small and large blood clots is one of the life-threatening clinical complications of severe COVID-19 and is estimated to affect 10%-47% of hospitalized COVID19 patients. The prevailing hypothesis is that the COVID-19-associated coagulopathy (CAC) results from “immunothrombosis”, a multi-factorial process in which systemic inflammation, vasculitis, and hyperactivated immune cells, together contribute to the formation of a hypercoagulative state, albeit the underlying mechanisms are still not fully elucidated. Of foremost interest here is the imbalance between the pro-thrombotic von Willebrand factor (vWF) and its negative regulator ADAMTS13 (a disintegrin-like and metalloprotease with thrombospondin type 1 motif, member 13), a member of the ADAM/ADAMTS family of metalloproteases (a.k.a. adamalysins). vWF multimerizes and adheres to exposed subendothelial collagen, whereas plasmatic ADAMTS13 depolymerizes large vWF multimers to avoid their accumulation in the blood that will otherwise falsely trigger micro- and macro-thromboses. In COVID-19 patients, the ADAMTS13:vWF ratio was shown to be lowered and negatively correlated with the increased incidence of micro-thrombosis and disease severity, possibly due to the presence of anti-ADAMTS13 antibodies. In contrast to these indirect CAC mechanisms, we hypothesized that one of the viral proteins of SARS-CoV-2, the secreted ORF8 (open reading frame 8) protein - which had already been shown to interact with two adamalysins, ADAM9 and ADAMTS1 - disrupts the regulation of clotting, alters endothelial cell conditions, directly affects blood clotting and inflammation responses, and thereby actively drives the pathogenesis of the CAC. Our central hypothesis is that the secreted ORF8 protein directly interacts with plasmatic ADAMTS13 and other adamalysins and negatively affects their biological function. Our preliminary data demonstrates that recombinant ORF8 and ADAMTS13 proteins interact intracellularly when their genes have been co-transfected, and extracellularly when separately transfected cells are co-cultured. Hence, the overall objective of our project is to fully determine the interaction between ORF8 and ADAMTS13 as well as other adamalysins involved in clotting and vascular biology, such as ADAM28, another vWF regulator, and thereby illuminate the mechanisms with which ORF8 contributes to CAC. In this study, we showed the impact of the ORF8 protein on CAC using three lines of evidence: first, there is substantial amino acid sequence conservation between ORF8, IL17A, and the A2 domain of vWF. Second, the ORF8 protein binds to ADAMTS13 as demonstrated by coincubation of cell supernatants and two-way co-IP using either ORF8 or ADAMTS13 as the bait. Finally, injection of ORF8-expression plasmid induced biomarkers of clotting disease and hyperinflammation, lead to microangiopathic hemolytic anemia, and lead to a severe phenotype of bleeding and clot formation in the lung of an ORF8-injected mouse. Thus, the symptomatology of CAC can be reproduced, at least in the mouse model we used, by ORF8 gene expression alone. These findings are highly significant because they point to ORF8 as a new therapeutic target. The development of novel antiviral drugs, antibodies, and vaccines targeting ORF8 holds great promise for improving individual, public, and global health, which continue to be challenged by circulating SARS-CoV-2 variants.