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

Heparin-induced thrombocytopenia (HIT) is a severe immune reaction to heparin, an anticoagulant. The condition manifests with thrombocytopenia (low blood platelet count) and arterial or venous thrombosis. If not treated, HIT can cause heart attack, stroke, pulmonary embolism, arterial blockage, limb gangrene, and death.

In HIT, an antibody recognizes and forms a complex with platelet factor 4 (PF4) and heparin or polyanions. This immune complex interacts with FcᵧRIIa receptors, causing platelet activation and aggregation. Platelet activation results in the release of factors that stimulate coagulation and perpetuate the cycle.

But other factors lead to thrombosis in HIT, such as neutrophil activation. The engagement of HIT immune complex with FcᵧRIIa receptors on neutrophils induces the release of neutrophil extracellular traps (NETs) and NETosis, a regulated cell death process.

Until now, the molecular mechanisms of NET formation in HIT are not entirely understood. Specific agonists like dinucleotide phosphate (NADPH), oxidase (NOX), and oxygen species (ROS) may be involved. Researchers at the University of New South Wales (Sydney, AU) investigated whether ROS and NOX were needed in HIT-induced thrombosis to understand these mechanisms better.

Study Overview

HIT samples

The researchers collected blood samples from healthy volunteers and HIT patients.

Microfluidic studies

The group conducted microfluidics studies using the Vena8 Fluoro+ biochip microchannels.

First, they coated the microchips with fibrinogen or von Willebrand factor (WWF) at 4C overnight. After washing and blocking the microchips with bovine serum, they incubated fresh citrate-anticoagulated whole blood from healthy donors with DPI (a ROS inhibitor). They treated it with purified IgG (HIT or normal) and heparin.

Neutrophil-targeted ROS-inhibition assays

The researchers used neutrophil-depleted blood treated with or without DPI for this experiment. They labelled the samples with Hoechst 33342, Sytox Green, anti-CD41 or AP2, anti-CD15, and CellROX. Then, they perfused the blood at a shear stress of 67 dyne/cm2 (1500 s-1) for up to 30 minutes.

To visualize the biochip, they used a Q-Imaging EXi Blue camera connected to a fluorescent microscope driven by VenaFluxAssay software (Cellix Ltd), followed by quantitative analysis.