ABSTRACT
Background: Neutrophil extracellular traps (NETs) are suggested to be the key driver in COVID-19 related immunothrombosis. Increased levels of soluble NETs markers are shown to correlate with COVID-19 severity and outcome. However, limited data is available on the impact of drug therapy on NETs level and the possibility to determine NETs in blood smears. Aims: To determine the possible role of NETs observed in blood smears during COVID-19. Methods: 46 patients with confirmed COVID-19 (11 non-ICU, 26 ICU, 9 ECMO) and 53 healthy volunteers were studied (independent ethics committee of NMRC PHOI No 3/2020). NETs were investigated in standardized thin blood smears produced of citrated whole blood and stained by May-Grünwald-Giemsa method. NETs percentage to number of neutrophils was calculated (%NETs). %NETs median levels between groups are compared using Mann-Whitney U-Test. Results: %NETs was 3.3 times higher in COVID-19 patients compared to healthy donors ( p <0.0001) and 1.3 times higher in ECMO patients ( p <0.0001). Drugs intake was shown to decrease %NETs: tocilizumab -1.6-fold ( P = 0.0066) glucocorticoids -1.2-fold ( P = 0.00087) omeprazole -1.3-fold ( p <0.0001), antibiotic therapy with any antibiotics -1.44-fold ( p <.0001). Interestingly, levofloxacin showed higher decrease in %NETs -1.76-fold. %NETs negatively correlated with platelets refractoriness to activation. %NETs and platelets count were analyzed in those 23 patients (13 deceased), who was observed two last days. %NETs was 2 times higher ( P = 0.47) and the platelets count was 3.3 times lower ( P = 0.0024) in deceased patients. A negative correlation was found between %NETs and platelets count (Spearman ' s correlation R=-0.414 P = .049). Conclusions: COVID-19 severity and outcome correlated with increased level of NETs, determined in blood smears. Intake of several drugs, known to inhibit NETosis or neutrophil activation, lead to decrease of NETs level. Thrombocytopenia and reduced activation capacity of platelets correlated with NETs level, confirming that NETs and platelets together are involved in coagulopathy in COVID-19.
ABSTRACT
Background : Virus infection COVID-19 caused by beta-coronavirus SARS-CoV-2 spread over the majority of countries and continents within several months. Pathophysiology of the disease is directly connected to uncontrollable progression of system inflammation, resulting in developed immunothrombosis of microvasculature and multiple organ dysfunction. Aims : In the present investigation, the effort was made to to clarify several pathophysiological mechanism of COVID-19 development, to determine diagnostic and prognostic criteria of disease severity and outcome. Methods : The blood of 87 patients with COVID-19 was examined using routine hemostasiological tests. In parallel, morphological and functional features of living functioning platelets were evaluated using quantitative phase imaging (QPI) technology at the level of superresolution and 3D visualization of cells and subcellular structures. According to the author ' s technique, the formation of neutrophil extracellular traps during netosis (NETosis) of neutrophils was analyzed in whole blood smears. Results : It was found that patients are characterized by the development of COVID-associated coagulopathy, which correlates with the severity of the disease. We found that, compared with control, the patients had a elevated plasma D-dimer, fibrinogen and FDPs, as well as shortened TT. We identified the increasing by 27% platelets with low and high level of activation (64 ± 6.9% vs 37% in norm) in 83% of patients. The level of NETs was statistically significantly (53.1 ± 7.2%, P < 0.05) higher than healthy volunteers (9.3 ± 3.5%) and progressively increased with the development of acute respiratory distress syndrome. Conclusions : Hemostatic disorders during SARS-CoV-2 infection can be caused by hyperactivity of platelets, hypercoagulation, hyperfibrinolysis and an inadequate immune response due to the formation NETs, finally causing coronavirus-induced coagulopathy. Platelets and neutrophils not only serve as markers of acute infection, but also, as a source of neutrophilic extracellular traps, play a key role in the development of immunothrombosis, which ultimately leads to acute respiratory failure with COVID-19.