Inhibition of NADPH oxidase blocks NETosis and reduces thrombosis in heparin-induced thrombocytopenia.

Abstract Heparin-induced thrombocytopenia (HIT) is associated with severeand potentially lethal thrombotic complications. NETosis was recently shown to be an important driver of thrombosis in HIT. We investigated the role of reactive oxygen species (ROS) and NADPH oxidase 2 (NOX2) and their contributions to thrombus development in HIT. We showed that neutrophil activation by HIT immune complexes inducedROS-dependent NETosis. Analysis of thrombi formed in a microfluidics system showed ROS production in both platelets and neutrophils, and abundant NETs and ROS distributed throughout the clot. Neutrophil-targeted ROS inhibition was sufficient to block HIT-induced NETosis and thrombosis using human blood. Inhibition of NOX2 with diphenyleneiodonium chloride or GSK2795039 abrogated HIT-induced thrombiin vivo using FcγRIIa+/hPF4+ transgenic mice. Thrombocytopenia in mice remained unaffected by ROS inhibition. Increased ROS production in activated neutrophils were also confirmed usingfresh blood from patients with active HIT.Our findings show that ROS and NOX2 play a crucial role in NETosis and thrombosis in HIT. This enhances our understanding of the processes driving thrombosis in HIT and identifies NOX2 as a potential new therapeutic target for antithrombotic treatment for HIT.