Toll-like receptor 4-dependent and Independent platelet-dependent thrombosis in SARS-CoV-2 infection

ABSTRACT

Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is associated with an increased risk of venous and arterial thrombosis but the underlying mechanism if still unclear. Aim(s) The study would identify a mechanism implicated in platelet activation and thrombus growth during SARS-CoV-2 infection. Method(s) We performed a cross-sectional analysis of platelet function in 30 SARS-CoV-2 and 20 healthy subjects (HS) by measuring Nox2-derived oxidative stress and thromboxane (Tx) B2 and investigated if administration of monoclonal antibodies against the Spike(S) protein of SARS-CoV-2 affects platelet activation. Furthermore, we investigated in vitro if the Spike(S) protein of SARS-CoV-2 or plasma from SARS-CoV-2 enhanced platelet activation. Result(s) Ex vivo studies showed enhanced platelet Nox2-derived oxidative stress and TxB2 biosynthesis and under laminar flow platelet-dependent thrombus growth in SARS-CoV-2 compared to controls;both effects were lowered by Nox2 and Toll-like receptor 4(TLR4) inhibitors. Two hours after administration of monoclonal antibodies a significant inhibition of platelet activation was observed in SARS-CoV-2 patients compared to untreated ones. In vitro study showed that S protein functionally interacts with platelet TLR4, and a docking simulation analysis suggested that TLR4 binds to S protein via three receptor-binding domains;furthermore, in platelets from SARS-CoV-2 S protein co-immunoprecipitated with TLR4. Plasma from SARS-CoV-2 patients incubated with normal platelets enhanced platelet activation and Nox2-related oxidative stress, an effect blunted by TNF-alpha inhibitor;this effect was recapitulated by an in vitro study documenting that TNF-alpha alone promoted platelet activation and amplified the platelet response to S protein via p47phox up-regulation. Conclusion(s) The study identifies two TLR4-dependent and independent pathways promoting platelet-dependent thrombus growth and suggests inhibition of TLR4 or p47phox as a tool to counteract thrombosis in SARS-CoV-2.