Cell-based high throughput screening to identify FDA/ EMA approved drugs that inhibit the endothelial pro-thrombotic switch during severe COVID19 infection

ABSTRACT

Background: A major complication of COVID19 is severe endothelial injury with micro-and macro-thrombotic disease in the lung and other organs. Several studies have identified high levels of inflammatory cytokines ( cytokine storm ), powerful activators of the endothelium, in plasma of severe COVID19 patients;indeed, COVID19 plasma was shown to activate endothelial cells (EC) in vitro. A consequence of EC activation is loss of anti-coagulant function, with release of pro-thrombotic Von Willebrand Factor (VWF). High levels of plasma VWF in severe COVID19 patients indicate systemic endothelial activation and increased risk of thrombosis. Aim(s) To identify drugs that decrease endothelial activation and VWF release, which may have a therapeutic impact in COVID19 patients. Method(s) We established an in vitro model of endothelial activation driven by 6 cytokines selected because of their high levels in COVID19 plasma. Cells were treated with the 6-cytokine cocktail for 24 hr;endothelial activation was confirmed by a panel of markers including ICAM1, measured by RT-qPCR and immunofluorescence (IF). Result(s) The treatment induced release of VWF and increased VWF-platelet string formation in a platelet flow-based assay. To identify drugs that blocked cytokine-induced VWF release, a high-throughput screening was carried out in human umbilical vein EC (HUVEC);VWF and ICAM1 expression were detected by IF;DAPI was used as nuclear stain. High content imaging screen of 3049 drugs from FDA/EMA-approved drug libraries identified drugs able to decrease VWF release following cytokine treatment. Top hits from several therapeutic classes including anti-inflammatory, anti-viral and hormones were taken forward for validation. Two hits were confirmed to inhibit cytokine-induced VWF release and VWF-platelet string formation. Selected findings were validated in lung microvascular EC. Conclusion(s) This study identified candidate drugs that reduce the enhanced VWF release caused by the cytokine storm typical of severe COVID19;these may be beneficial in the treatment of the pro-thrombotic risk in COVID19 patients.