Abstract
Post-acute sequelae of SARS-CoV-2 infection (PASC) is recognized as a syndrome marked by persistent thromboinflammation, even in individuals who experienced only mild acute illness. While few studies have reported presence of elevated extracellular vesicles (EVs) and tissue factor (TF) in PASC, their mechanistic contribution to coagulation activation and thrombosis in vivo is poorly characterized. We hypothesized that plasma from PASC individuals contains elevated levels of procoagulant TF and EVs that contribute to accelerated thrombin generation and in vivo thrombosis. To test this hypothesis, we enrolled 71 adult PASC patients (2 months to 2.7 years post-infection) from a dedicated Post-COVID Clinic, along with 72 healthy, asymptomatic controls.
Plasma from PASC patients showed elevated levels of fibrinogen and thrombin-antithrombin complex consistent with ongoing coagulation activation. Towards determining the mechanisms of coagulation activation, we first performed thrombin generation assay in Calibrated Automated Thrombography, in presence of exogenous TF and phospholipids (PLP). We observed that PASC plasma had significantly shortened lag time, increased endogenous thrombin potential (ETP), higher peak thrombin, and reduced time to peak compared to controls. These differences persisted when thrombin generation was triggered by PLP alone, implicating elevated endogenous TF activity in PASC plasma. TF-neutralizing antibodies normalized the differences between two groups, confirming a causal role for TF. Given that plasma TF is predominantly EV-associated, we next assessed EV abundance and its contribution to procoagulant and prothrombotic state. Nanoparticle tracking analysis demonstrated significantly increased EV particle count in PASC plasma. Thrombin generation potential triggered by TF alone remained elevated in PASC samples, suggesting a synergistic contribution from EVs. EV depletion from PASC plasma resulted in slower and less-robust generation of thrombin and normalization of differences between the groups, further confirming a functional role for EVs in maintaining procoagulant state in PASC. To evaluate in vivo thrombogenicity, EVs were isolated from equal plasma volumes of PASC and control subjects and infused into mice following partial inferior vena cava (IVC) ligation. All mice receiving PASC-EVs developed IVC thrombi after 48 hrs. of ligation, whereas only 50% of mice infused with control-EVs formed thrombi. Furthermore, thrombi from mice receiving PASC-EVs were significantly longer and heavier.
In summary, utilizes multiple assays, and complementary approaches to dissect the roles of TF and EVs, we demonstrate that TF and EVs from non-hospitalized PASC patients are functionally procoagulant and promote venous thrombosis in vivo. This mechanistic evidence underscores the need for ongoing thrombotic risk surveillance in PASC populations.
