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NADPH oxidase 1 inhibition attenuates platelet activation and thrombosis in vivo without affecting haemostasis
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  • Dina Vara,
  • Anuradha Tarafdar,
  • Meral Celikag,
  • Daniela Patina,
  • Christina Gulacsy,
  • Eleanor Hounslea,
  • Zach Warren,
  • Bárbara Ferreira,
  • Maarten Koeners,
  • Lorenzo Caggiano,
  • Giordano Pula
Dina Vara
University of Exeter
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Anuradha Tarafdar
The University of Manchester
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Meral Celikag
University of Exeter
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Daniela Patina
University of Exeter
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Christina Gulacsy
University of Bath
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Eleanor Hounslea
University of Bath
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Zach Warren
University of Exeter
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Bárbara Ferreira
University of Exeter
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Maarten Koeners
University of Exeter
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Lorenzo Caggiano
University of Bath
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Giordano Pula
University Medical Center Hamburg-Eppendorf
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Abstract

Background and Purpose - Growing evidence supports a central role of NADPH oxidases (NOXs) in the regulation of platelets. Experimental Approach - Here, we characterise the NOX inhibitor 2-acetylphenothiazine (2APT) and nine of its chemical derivatives for their selectivity, their effect on platelet activation in response to different stimuli ex vivo, and their modulation of carotid thrombosis and hemostasis in vivo. Key Results - Using Nox1-/- mice, we proved that NOX1 is critical for collagen-dependent platelet aggregation and carotid thrombosis, while it does not affect thrombin-dependent aggregation or haemostasis. 2APT selectively inhibits NOX1 over NOX2 (IC50 141 nM and >10 µM, respectively). In agreement with a central role of NOX1 in collagen signalling, 2APT and its most potent derivative 1-(10H-phenothiazin-2-yl)vinyl tert-butyl carbonate (or 2APT-D6) inhibit collagen-dependent platelet aggregation with negligible effects on thrombin responses. 2APT-D6 displays higher potency compared to 2APT (52 vs 141 nM, respectively). Platelet adhesion to collagen in static and flow conditions, superoxide anion generation and surface maker expression in response to collagen were also inhibited by 2APT and 2APT-D6. Administration of 2APT or 2APT-D6 (200mg/kg) in diet for 48 hours led to inhibition of platelet aggregation, oxygen radical output, and thrombus formation, and carotid occlusion in vivo, while tail hemostasis was not affected. Conclusion and Implications - In summary, this study suggests that NOX1 inhibition by 2APT or 2APT-D6 is a viable strategy to control collagen-induced platelet activation and reduce thrombosis in vivo without acute deleterious effects on hemostasis or other aspects of mouse health.