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 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.

Toll-like receptor 4-dependent platelet-related thrombosis in SARS-CoV-2 infection / Carnevale, Roberto; Cammisotto, Vittoria; Bartimoccia, Simona; Nocella, Cristina; Castellani, Valentina; Bufano, Marianna; Loffredo, Lorenzo; Sciarretta, Sebastiano; Frati, Giacomo; Coluccia, Antonio; Silvestri, Romano; Ceccarelli, Giancarlo; Oliva, Alessandra; Venditti, Mario; Pugliese, Francesco; Maria Mastroianni, Claudio; Turriziani, Ombretta; Leopizzi, Martina; D'Amati, Giulia; Pignatelli, Pasquale; Violi, Francesco. - In: CIRCULATION RESEARCH. - ISSN 0009-7330. - 132:3(2023), pp. 290-305. [10.1161/CIRCRESAHA.122.321541]

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

Carnevale, Roberto
Co-primo
;
Cammisotto, Vittoria
Co-primo
;
Bartimoccia, Simona;Nocella, Cristina;Castellani, Valentina;Bufano, Marianna;Loffredo, Lorenzo;Sciarretta, Sebastiano;Frati, Giacomo;Coluccia, Antonio;Silvestri, Romano;Ceccarelli, Giancarlo;Oliva, Alessandra;Venditti, Mario;Pugliese, Francesco;Maria Mastroianni, Claudio;Turriziani, Ombretta;Leopizzi, Martina;D'Amati, Giulia;Pignatelli, Pasquale;Violi, Francesco
Ultimo
2023

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 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.
2023
SARS-CoV-2; oxidative stress; pandemic; thrombosis; thromboxane B2
01 Pubblicazione su rivista::01a Articolo in rivista
Toll-like receptor 4-dependent platelet-related thrombosis in SARS-CoV-2 infection / Carnevale, Roberto; Cammisotto, Vittoria; Bartimoccia, Simona; Nocella, Cristina; Castellani, Valentina; Bufano, Marianna; Loffredo, Lorenzo; Sciarretta, Sebastiano; Frati, Giacomo; Coluccia, Antonio; Silvestri, Romano; Ceccarelli, Giancarlo; Oliva, Alessandra; Venditti, Mario; Pugliese, Francesco; Maria Mastroianni, Claudio; Turriziani, Ombretta; Leopizzi, Martina; D'Amati, Giulia; Pignatelli, Pasquale; Violi, Francesco. - In: CIRCULATION RESEARCH. - ISSN 0009-7330. - 132:3(2023), pp. 290-305. [10.1161/CIRCRESAHA.122.321541]
File allegati a questo prodotto
File Dimensione Formato  
Carnevale_Toll-Like_2023.pdf

solo gestori archivio

Tipologia: Versione editoriale (versione pubblicata con il layout dell'editore)
Licenza: Tutti i diritti riservati (All rights reserved)
Dimensione 6.02 MB
Formato Adobe PDF
6.02 MB Adobe PDF   Contatta l'autore

I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.

Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11573/1667362
Citazioni
  • ???jsp.display-item.citation.pmc??? 9
  • Scopus 13
  • ???jsp.display-item.citation.isi??? 12
social impact