NADPH oxidase 1/4 dual inhibitor setanaxib suppresses platelet activation and thrombus formation

Aims: The production of Reactive Oxygen Species (ROS) by NADPH Oxidase (NOX) is able to induce platelet activation, making NOX a promising target for antiplatelet therapy. In this study, we examined the effects of setanaxib, a dual NOX1/4 inhibitor, on human platelet function and ROS-related signaling pathways.

Materials and methods: In collagen-stimulated human platelets, aggregometry, assessment of ROS and CA²⁺, immunoblotting, ELISA, flow cytometry, platelet adhesion assay, and assessment of mouse arterial thrombosis were performed in this study.

Key findings: Setanaxib inhibited both intracellular and extracellular ROS production in collagen-activated platelets. Additionally, setanaxib significantly inhibited collagen-induced platelet aggregation, P-selectin exposure from α-granule release, and ATP release from dense granules. Setanaxib blocked the specific tyrosine phosphorylation-mediated activation of Syk, LAT, Vav1, and Btk within collagen receptor signaling pathways, leading to reduced activation of PLCγ2, PKC, and CA²⁺ mobilization. Setanaxib also inhibited collagen-induced activation of Integrin αIIbβ3, which is linked to increased cGMP levels and VASP phosphorylation. Furthermore, setanaxib suppressed collagen-induced p38 MAPK activation, resulting in decreased phosphorylation of cytosolic PLA₂ and reduced TXA₂ generation. Setanaxib also inhibited ERK5 activation, affecting the exposure of procoagulant phosphatidylserine. Setanaxib reduced thrombus formation under shear conditions by preventing platelet adhesion to collagen. Finally, in vivo administration of setanaxib in animal models led to the inhibition of arterial thrombosis.

Significance: This study is the first to show that setanaxib suppresses ROS generation, platelet activation, and collagen-induced thrombus formation, suggesting its potential use in treating thrombotic or cardiovascular diseases.

Antiplatelet; Antithrombotic; Dual NOX1/4 inhibitor; Platelet; ROS; Setanaxib.