1. Platelet-derived extracellular vesicles express NADPH oxidase-1 (Nox-1), generate superoxide and modulate platelet function
- Author
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Joanne L. Mitchell, Jonathan M. Gibbins, Giordano Pula, Renato Simões Gaspar, and Plinio Ferreira
- Subjects
Blood Platelets ,Platelets ,0301 basic medicine ,Short Communication ,CRP, collagen-related peptide ,ERK, extracellular signal-regulated kinases ,GAPDH, glyceraldehyde 3-phosphate dehydrogenase ,NADPH Oxidase ,Biochemistry ,Collagen receptor ,PRP, platelet-rich plasma ,Extracellular Vesicles ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,NTA, nanoparticle tracking analysis ,PMA, phorbol-12-myristate-13-acetate ,Superoxides ,Physiology (medical) ,PKC, protein kinase C ,EV, extracellular vesicles ,TRAP-6, thrombin receptor activator peptide 6 ,Platelet ,Platelet activation ,Redox biology ,chemistry.chemical_classification ,Reactive oxygen species ,NADPH oxidase ,biology ,Chemistry ,Superoxide ,NADPH Oxidases ,Fibrinogen binding ,NADPH, nicotinamide adenine dinucleotide phosphate ,Platelet Activation ,Cell biology ,WP, washed platelets ,030104 developmental biology ,GPVI, Glycoprotein VI ,PDEVs, platelet-derived extracellular vesicles ,NADPH Oxidase 1 ,biology.protein ,GPVI ,Reactive Oxygen Species ,030217 neurology & neurosurgery - Abstract
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., Graphical abstract Image 1, Highlights • Activated platelets have increased NADPH oxidase-1 (Nox-1) exposure on the outer membrane. • Platelet-derived extracellular vesicles (PDEVs) express Nox-1 and generate superoxide in a process mediated by Nox-1. • PDEVs bind and activate platelets in a Nox-1-dependent manner.
- Published
- 2021