Sex-Specific Differences in the Pathogenesis, Endothelial Dysfunction, and Hypercoagulability of Sars-Cov-2 Infection in K18-hACE2 Mice

The pathogenesis of coronavirus disease 2019 (COVID-19) complications in critically ill patients involve virus-mediated cellular damage in infected tissue and secondary organ damage associated with a hyperinflammatory response. The proinflammatory milieu can result in endothelial dysfunction, predisposing patients to thrombosis leading to a life-threatening condition known as COVID-19-associate coagulopathy (CAC). CAC is driven by cellular and molecular mechanisms that are still unclear. We report a mouse model based on the widely used K18-hACE2 transgenic mouse recapitulating many of the CAC-associated pathological findings observed in human patients offering a reliable animal model for the study of SARS-CoV-2 pathogenesis. We show that infection with a low dose of SARS-CoV-2 prolongs the symptomatic phase of the infection, leading to a 50% mortality rate. At this dose, male mice appear more susceptible to developing a severe disease showing increased mortality, weight loss, and decreased body temperature. Disease characteristics showed striking similarities with reported human COVID-19 cases, including severely reduced O₂saturation, leukopenia, moderate to absent thrombocytopenia, and hemolytic anemia. Lung’s histological analysis shows an earlier influx of immune cells in males reaching a higher level of infiltration, tissue damage, and alveolar space reduction. Plasma analysis of endothelial activation markers showed upregulation of soluble Thrombomodulin (sTM), and von Willebrand Factor (VWF). While the upregulation of sTM is visible only in females, where the severity of the disease is limited, VWF upregulation appears only in males correlating with the increased susceptibility to a severe infection. Alterations in the lung’s endothelial barrier function are noticeable in both sexes with an increase in vascular leakage on day 5 post-infection that persists up to day 7 in females. At the same time, vascular permeability drops in males replaced by the appearance of focal hemorrhagic lesions potentially associated with local thrombotic events. The infection-associated procoagulant phenotype appeared as early as day 3 post-infection. Prothrombin time (PT) showed a 1/3 reduction in the normal clotting time in males. The overall procoagulant state is also reflected in the alteration of all the measured coagulation factors' activity. FVIII and FX activity increased to ~150% compared to uninfected controls only in male K18-hACE2 mice while remaining unaltered in females. FIX showed the most significant alteration starting at ~200% of the normal activity on day 3 post-infection and gradually increasing over time reaching ~350%. We also observed a male-exclusive increase in the activity of FV starting 3 days post-infection at ~200/220% of the normal level and persisting unchanged up to day 7 post-infection. As reported during human infections, FV activity increased without significant changes in the protein concentration or FVa levels. Natural anticoagulants and fibrinolytic mechanisms are also dysregulated during SARS-CoV-2 infection resulting in a profound imbalance in thrombus formation and resolution. Infected K18-hACE2 mice showed downregulation of the plasmatic concentration of protein C which is not associated with a concomitant alteration of liver function. While the total concentration of protein S remained unaltered, the male-specific downregulation of the tissue factor pathway inhibitor (TFPI) could play a role in the increased procoagulant activity in this sex. In this prothrombotic microenvironment, regulation of fibrin degradation is pivotal. Fibrin-degradation products, such as D-Dimer, are upregulated only in females indicating the maintenance of an accurate balance between hemostatic pathways. Although males show a significant activation of the fibrinolytic system, measured as increased formation of plasmin-anti-plasmin complexes (PAP), the upregulation of thrombin activatable fibrinolysis inhibitor (TAFI) might drive a fibrinolysis shutdown that, coupled with the increased coagulation factors activity, could be responsible for CAC. Altogether, our results identify the K18-hACE2 transgenic mouse as an essential small animal model to study the molecular mechanisms involved in the derangement of the finely tuned interaction between the immune and coagulation systems associated with severe cases of COVID-19.