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. 2023 Feb 3;132(3):290-305.
doi: 10.1161/CIRCRESAHA.122.321541. Epub 2023 Jan 13.

Toll-Like Receptor 4-Dependent Platelet-Related Thrombosis in SARS-CoV-2 Infection

Roberto Carnevale #  1   2 , Vittoria Cammisotto #  3 , Simona Bartimoccia  3 , Cristina Nocella  3 , Valentina Castellani  4 , Marianna Bufano  5 , Lorenzo Loffredo  3 , Sebastiano Sciarretta  1   2 , Giacomo Frati  1   2 , Antonio Coluccia  5 , Romano Silvestri  5 , Giancarlo Ceccarelli  6 , Alessandra Oliva  6 , Mario Venditti  6 , Francesco Pugliese  4 , Claudio Maria Mastroianni  6 , Ombretta Turriziani  7 , Martina Leopizzi  1 , Giulia D'Amati  8 , Pasquale Pignatelli  3   9 , Francesco Violi  9
Affiliations
Free article

Toll-Like Receptor 4-Dependent Platelet-Related Thrombosis in SARS-CoV-2 Infection

Roberto Carnevale et al. Circ Res. .
Free article

Abstract

Background: SARS-CoV-2 is associated with an increased risk of venous and arterial thrombosis, but the underlying mechanism is still unclear.

Methods: We performed a cross-sectional analysis of platelet function in 25 SARS-CoV-2 and 10 healthy subjects by measuring Nox2 (NADPH oxidase 2)-derived oxidative stress and thromboxane B2, and investigated if administration of monoclonal antibodies against the S protein (Spike protein) of SARS-CoV-2 affects platelet activation. Furthermore, we investigated in vitro if the S protein of SARS-CoV-2 or plasma from SARS-CoV-2 enhanced platelet activation.

Results: Ex vivo studies showed enhanced platelet Nox2-derived oxidative stress and thromboxane B2 biosynthesis and under laminar flow platelet-dependent thrombus growth in SARS-CoV-2 compared with controls; both effects were lowered by Nox2 and TLR4 (Toll-like receptor 4) inhibitors. Two hours after administration of monoclonal antibodies, a significant inhibition of platelet activation was observed in patients with SARS-CoV-2 compared with untreated ones. In vitro study showed that S protein per se did not elicit platelet activation but amplified the platelet response to subthreshold concentrations of agonists and functionally interacted with platelet TLR4. A docking simulation analysis suggested that TLR4 binds to S protein via three receptor-binding domains; furthermore, immunoprecipitation and immunofluorescence showed S protein-TLR4 colocalization in platelets from SARS-CoV-2. Plasma from patients with SARS-CoV-2 enhanced platelet activation and Nox2-related oxidative stress, an effect blunted by TNF (tumor necrosis factor) α inhibitor; this effect was recapitulated by an in vitro study documenting that TNFα alone promoted platelet activation and amplified the platelet response to S protein via p47phox (phagocyte oxidase) upregulation.

Conclusions: The study identifies 2 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.

Keywords: SARS-CoV-2; oxidative stress; pandemics; platelet activation; thrombosis; thromboxane B2; toll-like receptors.

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