Platelet-derived extracellular vesicles express NADPH oxidase-1 (Nox-1), generate superoxide and modulate platelet function

Renato Simões Gaspar,Plinio M Ferreira,Joanne L Mitchell,Giordano Pula,Jonathan M Gibbins

doi:10.1016/j.freeradbiomed.2021.01.051

Abstract

BackgroundPlatelets release platelet-derived extracellular vesicles (PDEVs) upon activation – in a process that is regulated by generation of reactive oxygen species (ROS). Platelet NADPH oxidase-1 (Nox-1) contributes to ROS generation and thrombus formation downstream of the collagen receptor GPVI. ObjectivesWe aimed to investigate whether PDEVs contain Nox-1 and whether this is relevant for PDEV-induced platelet activation. MethodsPDEVs were isolated through serial centrifugation after platelet activation with thrombin receptor agonist TRAP-6 (activated PDEVs) or in the absence of agonist (resting PDEVs). The physical properties of PDEVs were analyzed through nanoparticle tracking analysis. Nox-1 levels, fibrinogen binding and P-selectin exposure were measured using flow cytometry, and protein levels quantified by immunoblot analysis. ROS were quantified using DCF fluorescence and electron paramagnetic resonance. ResultsNox-1 was found to be increased on the platelet outer membrane upon activation and was present in PDEVs. PDEVs induced platelet activation, while co-addition of GPVI agonist collagen-related peptide (CRP) did not potentiate this response. PDEVs were shown to be able to generate superoxide in a process at least partially mediated by Nox-1, while Nox-1 inhibition with ML171 (also known as 2-APT) did not influence PDEV production. Finally, inhibition of Nox-1 abrogated PDEV-mediated platelet activation. ConclusionsPDEVs are able to generate superoxide, bind to and activate platelets in a process mediated by Nox-1. These data provide novel mechanisms by which Nox-1 potentiates platelet responses, thus proposing Nox-1 inhibition as a feasible strategy to treat and prevent thrombotic diseases.

Highlights

Upon vascular injury, platelets adhere to the subendothelium to maintain vascular integrity during damage or inflammation
If surface localised NADPH oxidase-1 (Nox-1) levels increased upon activation, it is possible that this is a consequence of binding of platelet-derived extracellular vesicles (PDEVs) to platelets, we assessed if Nox-1 is expressed in PDEVs
Data presented show that PDEVs bind to platelets in a process regulated by Nox-1, which is downstream of the collagen receptor Glycoprotein VI (GPVI)

Summary

Introduction

Platelets adhere to the subendothelium to maintain vascular integrity during damage or inflammation (reviewed in Ref. [1]). Nox-2 was shown to be relevant to collagen receptor (GPVI)-mediated responses and regulate thrombus formation in male mice [8]. This has been disputed by us [9,10] and Abbreviations: CRP, collagen-related peptide; ERK, extracellular signal-regulated kinases; EV, extracellular vesicles; GAPDH, glyceraldehyde 3-phosphate dehy drogenase; GPVI, Glycoprotein VI; NADPH, nicotinamide adenine dinucleotide phosphate; NTA, nanoparticle tracking analysis; PDEVs, platelet-derived extracellular vesicles; PKC, protein kinase C; PMA, phorbol-12-myristate-13-acetate; PRP, platelet-rich plasma; TRAP-6, thrombin receptor activator peptide 6; WP, washed platelets. Conclusions: PDEVs are able to generate superoxide, bind to and activate platelets in a process mediated by Nox1 These data provide novel mechanisms by which Nox-1 potentiates platelet responses, proposing Nox-1 inhibition as a feasible strategy to treat and prevent thrombotic diseases

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Conclusion