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 : Coagulopathy is among the most alarming drivers of COVID-19-induced pathology. COVID-19 can result in blockade of the microvasculature in the lungs with microthrombi. While most of the studies concern COVID-19 impact on plasma coagulation, platelet dysfunction has been reported as well. However, the mechanism of platelet malfunctioning in COVID-19 has not been described yet. Aims : To determine the mechanism of COVID-19 induced platelet disfunction. Methods : 46 patients with confirmed COVID-19 (11 non-ICU, 26 ICU, and 9 ECMO) and 26 healthy volunteers were studied (independent ethics committee of NMRC PHOI No 3/2020). Citrated whole blood samples were diluted in Tyrode's buffer and activated by collagen and TRAP-6. Platelets of healthy donors were additionally washed and pre-treated by 0.5 nM of thrombin. Samples were analyzed using BC Navios flow cytometer. Additionally, light transmission aggregometry (AP-2110 SOLAR) of citrated platelet-rich plasma (PRP) with TRAP-6 and fucoidan as activators was conducted. Results : Platelet forward scattering parameter (FSC-A) for COVID-19 patients was significantly increased compared to healthy donors. This parameter reversibly correlated with mild thrombocytopenia observed in some patients. The amount of Annexin-V positive platelets was increased in all patients as well. Relative CD42b and CD62p binding upon activation were decreased, being statistically different in non-ICU and ICU patients. Both of the parameters also correlated to CRP concentration in patient blood plasma. Platelet aggregation was reduced as well. Altogether, platelets demonstrated refractoriness resembling desensitization of receptors. To prove this hypothesis, we performed thrombin pre-treatment of healthy donor platelets, which resulted in a phenotype resembling the COVID-19. Conclusions : Platelets of COVID-19 patients demonstrate refractoriness to activation through PAR1 receptor, probably because of a previous activation with thrombin in circulation. Together with their increased size and fraction of necrotic platelets, this suggests that in COVID-19 platelets encounter thrombin in circulation.
ABSTRACT
One of the most dangerous features of the new coronavirus infection caused by the SARS-CoV-2 virus is the tendency of the hemostasis system of patients to excessive thrombus formation. Among the possible causes of this pathology, both the activation of vascular endothelial cells, leading to the exposure of tissue factor by these cells, and direct activation of the plasma hemostasis were named. Besides, there is a significant change in platelet responses to activation, which is not accompanied by significant thrombocytopenia. The mechanism of platelet dysfunction is rather controversial. On the one hand, there are suggestions that platelets can act as a direct “container” for the virus, thus spreading it throughout the body. On the other hand, the presence of viral RNA in platelets has been demonstrated in only one study, while other authors have obtained the opposite result. Another mechanism of the virus's direct effect on platelets is the penetration of the virus into megakaryocytes and the subsequent violation of thrombocytopoiesis. However, three of the four published works show that platelets from patients with SARS-CoV-2 are in an activated state (the so-called platelet pre-activation). This phenomenon can be caused by the direct influence of the virus and the effect of thromboinflammation in the lungs on platelet functions. Here we review the known data and possible causes of the platelet functionality changes observed in patients with SARS-CoV-2.