NETosis and thrombosis in vaccine-induced immune thrombotic thrombocytopenia



Vaccine-induced thrombotic thrombocytopenia (VITT) is an uncommon but serious adverse event of adenovirus-based COVID-19 vaccines. The elevated mortality rate (between 23 to 40%) has caused vaccine hesitancy, undermining vaccination efforts in many countries.


The condition presents with thrombocytopenia (low platelets), thrombosis (blood clots), and platelet activation, resulting from the action of the anti-platelet factor 4 (PF4).

VITT resembles heparin-induced thrombocytopenia (HIT), which is driven by NETosis (formation of neutrophil extracellular traps - NETs). When activated by pathogens, the neutrophils release these NETs, activating platelets and blood coagulation pathways.

But we still don´t know how these mechanisms influence thrombus formation in VITT. So, researchers at the University of New South Wales investigated blood samples of vaccinated individuals with VITT. The results were published in a preprint report from 2021.


Study Overview

VIIT samples

The researchers collected blood samples from VITT and HIT patients and healthy donors who had received one dose of the vaccine.


NETosis markers

First, they investigated NETosis markers in VITT patients. For that, they assessed the presence of citrullinated histone H3 (CitH3) and cell-free DNA (cfDNA) concentrations in the plasma and the presence of activated neutrophils in the blood.

Results indicated NETosis in VITT patients:

  • VITT patients had higher levels of CitH3 and cfDNA than healthy controls.

  • The fresh blood of VITT patients contained a significant number of activated neutrophils, neutrophil-platelet aggregates, and neutrophils undergoing NETosis

Thrombosis induction

To test if VITT antibodies induce thrombosis, the researchers treated the healthy donor’s whole blood with VITT IgG or normal IgG. After staining the blood, they flowed it through Cellix’s Vena8 Fluoro+ biochip microchannels coated with von- Willebrand factor (vWf).

They mounted the biochips on a fluorescent microscope, and a camera captured images from different microscopic fields in real-time. The selected samples were fixed and imaged by confocal microscopy.

Results

VITT antibodies induce thrombosis and thrombocytopenia in vitro

Incubation of healthy individual’s blood samples with VITT IgG caused an increase in activated neutrophils and neutrophils undergoing NETosis, which resulted in thrombi formation. Images from the experiments shown below, clearly illustrate that thrombi consisted of platelets, neutrophils and extracellular DNA.

Extended Data Fig 1a & b from Chong et al. (a) VITT IgG and thrombosis. Healthy donors’ blood treated with VITT IgG was flowed in vWf-coated microchannels. Extracellular DNA was stained with Sytox green (green), platelets with anti-CD41 AF647 (magenta) and neutrophils with anti-CD15 AF594 (red). Thrombi were imaged with a confocal laserscanning microscope (Leica TCS SP8 running Leica’s LAS X software) with a 63x oil immersion objective. Scale bar 20 μm. (b) Healthy donors’ blood treated with normal IgG was flowed in vWf-coated microchannels. Total DNA was stained with Hoechst 33342 (blue), platelets with anti-CD41-FITC (green) and neutrophils with anti-CD15 AF594 (red). Scale bar 50 μm. s.d. Statistics: Kruskal-Wallis.

Further analysis of these VITT IgG-induced thrombi showed that they also contained a high amount of fibrin (Fig. 2e) and CitH3 (Fig. 2f) as shown below :

Fig. 2 e & f from Chong et al. Effect of VITT IgG on donor’s blood: (e) VITT IgG induces thrombosis. Healthy donors’ blood treated with VITT IgG was stained for total DNA (blue), platelets (green), fibrin (red) and neutrophils (magenta). Thrombi were imaged with a confocal laser-scanning microscope (overlap of green and red shown as yellow). Scale bar: 10 μm. (f) Thrombi contain CitH3. Thrombi were generated and imaged as in (e), and stained for DNA (blue), platelets (green), CitH3 (yellow) and neutrophils (magenta). Overlap of yellow and green is shown as white.

Finally, they repeated this experiment using blood pre-treated with anti-platelet receptor Fc, IV.3 or DNAse I before VITT IgG incubation. The goal was to confirm the role of this receptor in VITT-induced thrombosis. Blood samples pre-treated with IV.3 strongly inhibited platelet adhesion (Fig. 2g, i) and neutrophil adhesion (Fig. 2g, j). NETosis was not induced illustrated by the lack of DNA release in the presence of IV.3 (Fig. 2g, h). In summary, the results support the hypothesis that anti-platelet factor Fc blockage inhibits NETosis and thrombosis.

Fig. 2 g, h, i & j from Chong et al. (g) IV.3 and DNase I prevent VITT IgG-induced thrombus formation in microfluidics system. Treated blood was stained for DNA (blue), platelets (green) and neutrophils (red). Scale bar: 50 μm. Graphs show area coverage percentage for (h) total DNA, (i) platelets and (j) neutrophils. n=3, mean s.d. Statistics: (h, i, j) One-way ANOVA with Tukey’s correction for multiple comparisons. *P < 0.05; **P <0.01; ***P < 0.001, ****P < 0.0001. LDG, low density granulocytes; ext. DNA, extracellular DNA; Ctrl, control; Pt, patient.

Other experiments in this study:

The researchers also injected VITT IgG into VITT mice and examined thrombosis levels in their lungs. Another experiment consisted in administering anti-platelet receptor Fc and NETosis inhibitors before this evaluation. Furthermore, they tested the contribution of NETosis to thrombosis in mice lacking PAD4 (a NETosis-inductor).


Main finding of in vivo studies

  • VITT IgG caused blood clots in the lungs of VITT mouse models. Treatment with control IgG doesn´t have this effect.

  • Blocking platelet factor Fc receptor or NETosis prevents clot formation in vivo.

  • PAD4 deficient mice treated with VITT IgG had a dramatic reduction in clot formation compared to control.

  • AgIV.3 prevented thrombocytopenia and thrombosis in VITT treated mice. In contrast, NETosis inhibitor and PAD4 knockout did not affect thrombocytopenia.

Conclusion

The researchers concluded that anti-PF4 antibodies are responsible for VITT by inducing platelet and neutrophil activation and NETosis, culminating in thrombosis.

These results can help us understand VITT better and guide other scientists on developing new therapies, improving the outcomes for these patients.


What do I need to get started?

Would you like to run a similar experiment but don´t know where to begin?

Here´s what you´ll need:

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

  • Mirus Evo 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

Chong, Beng, et al. "NETosis and thrombosis in vaccine-induced immune thrombotic thrombocytopenia." (2021).





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