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

TY - JOUR

T1 - Platelet-derived extracellular vesicles express NADPH oxidase-1 (Nox-1), generate superoxide and modulate platelet function

AU - Gaspar, Renato Simões

AU - Ferreira, Plinio

AU - Mitchell, Joanne L

AU - Pula, Giordano

AU - Gibbins, Jonathan M

N1 - Copyright © 2021. Published by Elsevier Inc.

PY - 2021/3

Y1 - 2021/3

N2 - BACKGROUND: Platelets 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.OBJECTIVES: We aimed to investigate whether PDEVs contain Nox-1 and whether this is relevant for PDEV-induced platelet activation.METHODS: PDEVs 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.RESULTS: Nox-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.CONCLUSIONS: PDEVs 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.

AB - BACKGROUND: Platelets 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.OBJECTIVES: We aimed to investigate whether PDEVs contain Nox-1 and whether this is relevant for PDEV-induced platelet activation.METHODS: PDEVs 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.RESULTS: Nox-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.CONCLUSIONS: PDEVs 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.

U2 - 10.1016/j.freeradbiomed.2021.01.051

DO - 10.1016/j.freeradbiomed.2021.01.051

M3 - SCORING: Journal article

C2 - 33548451

VL - 165

SP - 395

EP - 400

JO - FREE RADICAL BIO MED

JF - FREE RADICAL BIO MED

SN - 0891-5849

ER -