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

Abstract

Heparin-induced thrombocytopenia (HIT) is associated with severe and 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 induced ROS-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 thrombi in vivo using FcγRIIa+/hPF4+ transgenic mice. Thrombocytopenia in mice remained unaffected by ROS inhibition. Increased ROS production in activated neutrophils were also confirmed using fresh 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.

NETosis and thrombosis in vaccine-induced immune thrombotic thrombocytopenia

“…The requirement or dispensability of dinucleotide phosphate (NADPH) oxidase (NOX) activity, reactive oxygen species (ROS), and calcium influx on VITT IgG-induced NETosis requires further research. We have previously shown that PAD4, ROS and NOX2 play a crucial role in NETosis and thrombosis in HIT 10 . Due to the analogous nature of HIT and VITT, NET formation is likely dependent on both PAD4 and NADPH oxidase, as in HIT.…”

“…In HIT, we have previously shown that thrombosis is driven by NETosis 10 , 11 . Upon activation by pathogens, immune complexes and other stimuli, neutrophils release their granules and decondensed chromatin in the form of a DNA network, termed Neutrophil Extracellular Traps (NETs).…”

Antioxidants as Therapeutic Agents in Acute Respiratory Distress Syndrome (ARDS) Treatment—From Mice to Men

“…However, further research is necessary to understand the underlying principles in detail [ 162 ]. Studies already performed have shown that ROS mainly contribute to a proinflammatory response, which is associated with activation of platelets and subsequent trapping in the microcirculation leading to thrombocytopenia [ 163 , 164 ]. Nox2 activation is critically involved in platelet activation and thrombosis in response to oxidative stress [ 165 ].…”

“…The contact factor FXIIa, activated by polyphenols released from activated platelets, is an important component in clot formation [ 166 , 167 ], and TF promotes neutrophil extracellular trap (NET) formation, a process which is called NETosis [ 168 ], and disease progression [ 169 ]. NETosis is directly associated with ROS release, as shown in mice where NADPH oxidase inhibition blocked NETosis and improved thrombosis [ 163 ]. Complement activation, a characteristic event in infection, is involved in thrombosis and disseminated intravascular coagulation (DIC).…”

PFKL, a novel regulatory node for NOX2-dependent oxidative burst and NETosis

“…The assembled NOX complex generates ROS by transferring one electron from NADPH to the cytosol O 2 . NOX enzymes are widely distributed across different cells and tissues, while NOX2 is highly expressed in neutrophils and is rapidly activated to generate large quantities of ROS in response to external stressors (Leung et al, 2021).…”