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

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

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Antibodies, Monoclonal / pharmacology
  • Blood Platelets / metabolism
  • COVID-19* / metabolism
  • Cross-Sectional Studies
  • Humans
  • SARS-CoV-2
  • Thrombosis* / etiology
  • Thrombosis* / metabolism
  • Thromboxanes / metabolism
  • Thromboxanes / pharmacology
  • Toll-Like Receptor 4 / metabolism

Substances

  • Antibodies, Monoclonal
  • Thromboxanes
  • Toll-Like Receptor 4
  • TLR4 protein, human