NADPH oxidase 1 inhibition attenuates platelet activation and thrombosis
in vivo without affecting haemostasis
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.