Contribution of Neutrophil Extracellular Traps (NETs) and platelet activation to Immunothrombosis in COVID-19 and inhibitory effect of substances that increase intracellular levels of cyclic nucleotides

Abstract

Approximately 14% of patients with Coronavirus Disease 2019 (COVID-19) have severe disease and 6% develop critical symptoms. The lethality of COVID-19 is around 4%. Severe symptoms manifest after approximately 7 to 8 days from the beginning of the disease, and progression to the critical form occurson average after the 11th day of symptoms. The pathogenesis of COVID-19 is related to the invasion of the Severe Acute Respiratory Syndrome Coronavirus - 2 (SARS-CoV-2) in the lung epithelial cells combined with the hosts immune reaction. The uncontrolled systemic inflammatory response is themain mechanism behind Severe Acute Respiratory Syndrome and hypercoagulability. The latter is attributed to Immunothrombosis, whose main participating cells are monocytes, which express tissue factor, neutrophils, which release extracellular nets composed of DNA, enzymes and histones (NETs) and platelets, through degranulation and aggregation. The aim of this project is to describe the association between the kinetics of NETS formation and platelet activation during hospitalization by COVID-19 and the occurrence of thromboembolic events, need for mechanical ventilation and death. We will also evaluate the capacity of patients sera to trigger NETS release and platelet activation and identify drugs capable of inhibiting the Immunothrombosis process in vitro. For this, we will aprospectively follow 100 patients with COVID-19 and will dose markers of NETs (MPO-DNA and H3citrus), platelet activity (PF4 and RANTES) and inflammatory cytokines (TNF-±, IL-17a, IL-6, and IL-8) at 4 time points during hospitalization (admission, day +4, day +8 and last day of hospitalization). We will evaluate the association of the change in the pattern of these markers with clinical outcomes. We will perform culture assays on normal neutrophils with serum from patients and will measure the concentration of free DNA in the medium. We will perform platelet stimulation assays using patients era and will measure the capacity of the stimulated platelets to aggregate and degranulate. Next, we will evaluate the inhibitory potential on NETs release and platelet activation of drugs that increase intracellular cAMP and cGMP concentrations, aspirin and enoxaparin. The results will allow: 1. to know the kinetics of the Immunothrombosis process throughout the period of hospitalization by COVID-19, to identify the moment in the evolution of the disease in which there is the greatest alteration of this phenomenon and to associate it to the occurrence of adverse clinical outcomes; 2. to identify drugs with the potential to inhibit the release of NETs and platelet activation in vitro, whose therapeutic efficacy may be tested in the future for the treatment of COVID-19 and other diseases associated to Immunothrombosis. (AU)