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Novel NADPH Oxidase 1-selective Inhibitors Impair Collagen-dependent Platelet Activation and Thrombus Formation in vitro and Ferric Chloride-induced Carotid Occlusion in vivo with Negligible Effects on Thrombin Responses and Bleeding Time
ISTH Academy. Pula G. Jul 10, 2019; 274116; OC 78.1
Dr. Giordano Pula
Dr. Giordano Pula
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OC 78.1

Novel NADPH Oxidase 1-selective Inhibitors Impair Collagen-dependent Platelet Activation and Thrombus Formation in vitro and Ferric Chloride-induced Carotid Occlusion in vivo with Negligible Effects on Thrombin Responses and Bleeding Time

D. Vara1, A. Tarafdar1, M. Celikag1, D. Souto Patinha1, L. Caggiano2, M. Koeners1, G. Pula3
1University of Exeter, Exeter, United Kingdom, 2University of Bath, Bath, United Kingdom, 3University of Exeter, Exeter Medical School, Exeter, United Kingdom

Main Topic: Platelets and Megakaryocytes
Category: Platelet Antagonists & Novel Therapeutics

Background: NADPH oxidases (NOXs) produce reactive oxygen species in platelets and their enzymatic activity is essential for platelet activation.
Aims: Despite its poor pharmacokinetic properties, the NOX1-selective inhibitor 2-acetyl phenothiazine (2-APT) can inhibit collagen-dependent platelet responses in vitro. We synthesised a series of novel chemical derivatives with improved physicochemical profile compared to 2-APT and tested their ability to affect platelet responses in vitro and in vivo.
Methods: We utilised turbidimetry, flow cytometry, thrombus formation under flow or electron paramagnetic resonance to measure platelet activation or oxidative stress in vitro and ferric chloride-induced carotid occlusion or tail bleeding assays to assess thrombosis or haemostasis in vivo.
Results: Amongst the newly synthesised compounds, 2APT-PD06, showed significantly higher potency as collagen-selective inhibitor than 2-APT in vitro. When administered in mice, 2APT-PD06 inhibited collagen-dependent aggregation in platelet rich plasma (PRP) and thrombus formation under flow in whole blood. Carotid occlusion time was also significantly increased, while tail bleeding time was only marginally affected. The effect of 2APT-PD06 reproduced the functional phenotype that we observed for genetically silenced NOX1-/- mice (reduced collagen response in vitro and reduced carotid occlusion in vivo). The administration of 2APT-PD06 in NOX1-/- mice did not reduce carotid occlusion further, confirming the NOX1-/- selectivity of this compound.
Conclusions: This study describes the generation of novel NOX1-selective inhibitors able to target collagen-dependent platelet responses in vitro and carotid occlusion in vivo. The limited effect on thrombin-induced responses in vitro and tail bleeding time in vivo, supports the hypothesis that NOX1 is dispensable for platelet activation in response to thrombin and for the haemostatic response induced by acute vascular injury, while it is essential for collagen-dependent platelet activation and vascular occlusion induced by endothelial dysfunction. Taken together, these data indicate NOX1 as an interesting target for the development of selective antiplatelet drugs with limited bleeding side effects.

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