ABSTRACT
Background: Neutrophil extracellular traps (NETs) release is the one of the main mechanisms behind hypercoagulability and disease severity in severe acute respiratory syndromes. The identification of drugs capable of inhibiting this pathological mechanism is mandatory. Aim(s): Neutrophil extracellular traps (NETs) release is the one of the main mechanisms behind hypercoagulability and disease severity in severe acute respiratory syndromes. The identification of drugs capable of inhibiting this pathological mechanism is mandatory. Method(s): Healthy neutrophils (20 x 103/well) were stimulated with phorbol myristate acetate (PMA) or sera from severe COVID-19 patients (n = 16) in the presence or absence of dipyridamole (10 muM), aspirin (1 mM) and heparin (50 mug/mL). Neutrophils nuclei were stained with nuclear red and incubated with a medium containing the non-permeable cell membrane marker Sytox Green. Cell images were obtained using IncuCyte ZOOM and the number of cells that suffered netosis was monitored over time. NETs release was determined after 1 h of incubation and the percentage of NETs was calculated dividing the number of green cells by the total number of cells per well. Result(s): COVID-19 induced NETs was lower in neutrophils pretreated with heparin (median 2.6%, IQR 2.6-2.9) than in non-treated neutrophils (median 3.6%, IQR 3.2-4.0, p < 0.0001). Pretreatment with dipyridamole and aspirin did not change the effect of COVID-19 sera in inducing NETs. A similar pattern of inhibition was observed with PMA stimulation, in which heparin decreased NETs by 3 times (NETs after PMA 43.2% and NETs after PMA and heparin 14.8%) while dipyridamole and aspirin did not significantly affect the release of PMA-induced NETs (Figure 1). Figure 2 illustrates the identification of NETs. Conclusion(s): Heparin was capable of inhibiting in vitro NETs release induced by COVID-19, while dipyridamole and aspirin had no significant effect on this process. Such findings are in line with evidence that heparin use can improve COVID-19 prognosis. (Figure Presented).
ABSTRACT
Background : Hypercoagulability in COVID-19 has been attributed to immunothrombosis, a process that involves the formation of neutrophils extracellular traps (NETs). The moment of the COVID-19 evolution in which immunothrombosis mechanisms are triggered is not established. Aims : To describe the association of the kinetics of NETs release during COVID-19 hospitalization with death and thrombosis. Methods : We quantified markers of NETs (citrullinated H3) and inflammatory cytokines (TNF-α, IL-6) on 4 time points during COVID-19 hospitalization (admission, day 4, day 8 and last day) between May and July 2020. The association between changes in these markers levels and clinical outcomes was determined. Results : Table 1 summarizes the patients characteristics. 101 patients were included, 59% were critically ill, 11% had a thrombotic event and 21% died. Figure 1 illustrates the changes in citH3, IL-6 and TNF-α levels during hospitalization. IL-6 levels were high on admission in survivors (median 25.32, IQR 24.19-28.15) and nonsurvivors (median 24.19, IQR 12.51-27.19), but gradually decreased after day 4 in survivors. TNF-α levels remained 2 times higher in non-survivors than in survivors during the entire hospitalization period. CitH3 levels were similar between non-survivors and survivors until day 4. On day 8, citH3 increased by 3-fold (median 3.80, IQR 1.98-10.15) in non-survivors and 2-fold (median 2.60, IQR 1.22-5.01) in survivors. While IL-6 and TNF-α levels were similar between patients with and without thrombosis, citH3 levels increased shortly before the occurrence of a thrombotic event. Conclusions : Markers of inflammation and immunothrombosis were associated with poor outcomes in COVID-19;however, these disorders were detected in different moments during COVID-19 course. While an increased inflammatory response was observed since the beginning of hospitalization, markers of immunothrombosis arose latter during the course of the disease. Acknowledgment of the timecourse of immunothrombosis development in COVID-19 is important for planning therapeutic strategies against this pathological process.