NADPH oxidase 1 is a novel pharmacological target for the development of an antiplatelet drug without bleeding side effects

Dina Vara, Anuradha Tarafdar, Meral Celikag, Daniela Patinha, Christina E. Gulacsy, Ellie Hounslea, Zach Warren, Barbara Ferreira, Maarten P. Koeners, Lorenzo Caggiano, Giordano Pula

Research output: Contribution to journalArticlepeer-review

16 Citations (SciVal)

Abstract

Growing evidence supports a central role of NADPH oxidases (NOXs) in the regulation of platelets, which are circulating cells involved in both hemostasis and thrombosis. Here, the use of Nox1−/− and Nox1+/+ mice as experimental models of human responses demonstrated a critical role of NOX1 in collagen-dependent platelet activation and pathological arterial thrombosis, as tested in vivo by carotid occlusion assays. In contrast, NOX1 does not affect platelet responses to thrombin and normal hemostasis, as assayed in tail bleeding experiments. Therefore, as NOX1 inhibitors are likely to have antiplatelet effects without associated bleeding risks, the NOX1-selective inhibitor 2-acetylphenothiazine (2APT) and a series of its derivatives generated to increase inhibitory potency and drug bioavailability were tested. Among the 2APT derivatives, 1-(10H-phenothiazin-2-yl)vinyl tert-butyl carbonate (2APT-D6) was selected for its high potency. Both 2APT and 2APT-D6 inhibited collagen-dependent platelet aggregation, adhesion, thrombus formation, superoxide anion generation, and surface activation marker expression, while responses to thrombin or adhesion to fibrinogen were not affected. In vivo administration of 2APT or 2APT-D6 led to the inhibition of mouse platelet aggregation, oxygen radical output, and thrombus formation, and carotid occlusion, while tail hemostasis was unaffected. Differently to in vitro experiments, 2APT-D6 and 2APT displayed similar potency in vivo. In summary, NOX1 inhibition with 2APT or its derivative 2APT-D6 is a viable strategy to control collagen-induced platelet activation and reduce thrombosis without deleterious effects on hemostasis. These compounds should, therefore, be considered for the development of novel antiplatelet drugs to fight cardiovascular diseases in humans.

Original languageEnglish
Pages (from-to)13969-13977
Number of pages9
JournalFASEB Journal
Volume34
Issue number10
Early online date26 Aug 2020
DOIs
Publication statusPublished - 29 Sept 2020

Funding

This work was funded by a British Heart Foundation grant (PG/15/40/31522) and Alzheimer Research UK (ARUK-PG2017A-3) to Giordano Pula. Work in Maarten Koeners' laboratory was supported by the British Heart Foundation (FS/14/2/30630 and PG/15/68/31717). The authors would also like to thank the Biological Service Unit of the University of Exeter for the in vivo work, the Clinical Research Facility (CRF) of the University of Exeter and in particular Dr Bridget Knight for blood collections, the Chemical Characterization and Analysis Facility (CCAF) of the University of Bath (especially Dr Anneke Lubben and Dr Shaun Reeksting) for the mass spectrometry analysis, and Dr Bruno Fink from Noxygen Science Transfer & Diagnostics GmbH for the technical support. Open access funding enabled and organized by Projekt DEAL.

Keywords

  • free radical
  • hemostasis
  • NADPH oxidase
  • oxidative stress
  • platelet
  • redox
  • thrombosis

ASJC Scopus subject areas

  • Biotechnology
  • Biochemistry
  • Molecular Biology
  • Genetics

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