Abstract 4144823: Persistent Prothrombotic State in Post-Acute Sequelae of Mild SARS-CoV-2 Infection: Role of Extracellular Histones and Immune Complexes

Post-Acute Sequelae of SARS-CoV-2 infection (PASC) have become a significant healthcare burden. Sustained increases in prothrombotic markers have been reported in hospitalized acute COVID-19 patients. However, whether patients with less severe acute infection also endure a persistent prothrombotic state remains uncertain. We tested for a prothrombotic state in this cohort and examined potential mediators. We enrolled 70 adult patients with prior mild acute SARS-CoV-2 infection and sustained PASC symptoms (per WHO criteria). A control healthy group matched for age and sex was also enrolled who were not previously diagnosed with COVID-19. Markers of platelet activation and platelet-neutrophil aggregates (PNA) were quantified using whole-blood flow cytometry. Markers of extracellular traps (citrullinated histones [H3Cit] and cell-free DNA [cfDNA]), anti-dsDNA IgG, and thrombin generation potential were measured in plasma. At recruitment (6 weeks to 3 years post infection), there was increased potential for thrombin generation in the plasma from PASC compared to control reflected by increased peak and velocity index (P<0.001 vs. control). In whole blood, activation with thrombin receptor activating peptide (TRAP) caused increased surface expression of P-selectin and activation of integrin αIIbβ3 in platelets (P<0.001 and P<0.05, respectively, vs. control). Concurrently, there was an increase in the PNA (P<0.01 vs. control). Plasma levels of H3Cit, cfDNA, as well as anti-dsDNA IgG were found to be elevated (P<0.0001 vs. control for all), suggesting not only an increase in extracellular trap, but also the existence of circulating immune complexes (ICs). While a histone-neutralizing aptamer inhibited P-selectin expression, αIIbβ3 activation, and PNA formation in whole blood, incubation of patient serum with anti-CD32, a FcγRIIa blocker (blocks the interaction of ICs with FcFcγRII on platelets), inhibited the activation of healthy platelets by patient sera. In conclusion, our study revealed a persistent prothrombotic state for months to years in patients who experienced mild acute infection with SARS-CoV-2. Further, our data suggest that the activation of platelet is partly mediated by extracellular histones and ICs, such as anti-dsDNA antibodies. Our findings suggest a model in which extracellular traps activate platelets directly and indirectly through ICs. This study proposes novel therapeutic interventions targeting extracellular histones and ICs-Fcγ in PASC.