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.


Results

Thrombus formation in HIT requires neutrophil-derived ROS

When the researchers analysed the thrombi, they saw abundant platelets, neutrophils, ROS, and released DNA within the clot (Fig 3A). HIT patients' circulating platelets and neutrophils also produced more ROS.


Thrombus formation in HIT requires neutrophil-derived ROS

When the researchers analysed the thrombi, they saw abundant platelets, neutrophils, ROS, and released DNA within the clot (Fig 3A). HIT patients' circulating platelets and neutrophils also produced more ROS.

Fig. 3A from Chong et al. HIT IgG induced NETosis and ROS-containing thrombi. (A) Confocal microscopy images of KKO-induced thrombi in whole blood showing platelets (red), neutrophils (green), ROS (magenta), and DNA release (blue).

Thrombi induced by KKO and HIT IgG showed marked deposition of extracellular DNA, platelets, and neutrophils (Fig 4 A-B). Treating the blood with DPI significantly reduced these components and clot size. This observation confirmed the central role of ROS in HIT-associated thrombosis.

Figure 4A-B from Chong et al. Inhibiting ROS reduced clot formation and NETs released under flow conditions. (A) Fluorescent images of whole blood treated with isotype control and heparin, or KKO and heparin plus vehicle control or DPI. (B) Whole blood treated with normal human IgG (hIgG) and heparin or a representative HIT IgG and heparin plus vehicle control or DPI. Extracellular DNA (blue), neutrophils (green), and platelets (red).

Neutrophil-derived ROS and thrombus formation in HIT

In HIT IgG-treated blood reconstituted with vehicle-treated neutrophils, the researchers observed large clots full of extracellular DNA, neutrophils, and platelets. They didn´t observe these characteristics in reconstituted blood treated with normal IgG (Fig. 5).

Figure 5 from Chong et al. Neutrophil-targeted ROS inhibition blocked NETs and thrombosis in whole blood. (A) Fluorescent images showing extracellular DNA (blue), neutrophils (green), and platelets (red).

Treating the reconstituted blood with DPI significantly reduced DNA, neutrophil, and platelet deposition after HIT IgG treatment.

This data suggests a direct link between neutrophil-derived ROS and thrombus formation in HIT.


Other experiments in this study:


ROS production in neutrophils

The experiment involved stimulating purified neutrophils with HIT patients IgG or KKO, PF4, and heparin. KKO-treated neutrophils showed enhanced ROS generation.


HIT IgG-mediated neutrophil activation and NETosis

This experiment involved treating healthy blood with KKO or HIT IgG in the presence of heparin. The treatment increased neutrophils undergoing NETosis in the whole blood and LDG population compared with controls. DNP and DPI blocked ROS production in HIT IgG-induced LDGs.


In vivo studies

The group also conducted animal studies using double-transgenic FcᵧRIIa+/hPF4+ mice expressing the R131 isoform of human FcᵧRIIa and human PF416. They injected HIT-like monoclonal antibody KKO and heparin to induce HIT in these animals. Then, they administrated ROS inhibitors and analyzed the animals’ lungs and platelet count.


Main findings of these experiments

  • KKO-treated mice without DPI treatment demonstrated a substantial accumulation of luminescence in the lungs. DPI treatment reduced luminescence, indicating ROS inhibition. (Supplemental Fig 5A)

  • DPI-treated HIT mice showed a reduction in circulating activated neutrophils (LDGs) and ROS+ cells, similar to control levels. These results are consistent with the microfluidics observations in which ROS inhibition blocked neutrophil activation. (Supplemental Fig 5D)

  • Mice treated with KKO and heparin showed dense platelet accumulation and thrombosis in the lungs. (Fig 6)

  • ROS inhibition with DPI reduced HIT-induced thrombosis, supporting the microfluidics studies.

  • DPI was not capable of preventing thrombocytopenia and hypothermia.

See this study in more detail here


Conclusion

The researchers concluded that:

  • ROS and NOX2 play a crucial role in NETosis and thrombosis in HIT.

  • NOX2 could be a new therapeutic target for the antithrombotic treatment of HIT.

What do I need to get started?

Did you find these experiments interesting? This is what you´ll need to run similar experiments in your lab:

  • Vena8 Fluoro+ biochip – to mimic human blood vessels and model blood clots, see further details below.

  • Mirus Evo pump or ExiGo Pump – to control shear rates (flow rates) in the biochip; this enables you to set the shear rate at a setting which models flow rates for thrombosis in micropillaries or other vessels.

  • Microenvironmental chamber – this is a temperature-controlled frame, the biochip sits in this and it keeps everything at 370C. The microenvironmental chamber sits on the microscope stage.

  • Inverted microscope – we supply the Zeiss AxioVert A1 with the VenaFlux Pro option or the Zeiss AxioObserver7 with the VenaFlux Elite option.

  • Digital camera – to capture images and video recordings. We supply the Prime BSI Express with both the VenaFlux Pro and Elite options. This is an excellent camera with a high frame rate suitable for thrombosis studies.

  • Image Pro Cell Analysis software – to analyse the images and videos from your experiments.

If your lab already has some of these items (like the inverted microscope, camera, or cell analysis software), we recommend the VenaFlux Starter kit.

Our options suit all budgets. Take a look at them on our eShop.


References

Leung, Halina HL, et al. "Inhibition of NADPH oxidase blocks NETosis and reduces thrombosis in heparin-induced thrombocytopenia." Blood Advances 5.23 (2021): 5439-5451.











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