Blood clots in severely ill COVID-19 patients caused by abnormal antibody response



Since the pandemic has unfolded, we have learned more about different symptoms affecting COVID-19 patients, such as a cough and shortness of breath to loss of smell or taste. Symptoms vary depending on the severity of the disease; such as reports of inflammation and fatal blood clotting in critically ill COVID-19 patients. However, it is now believed that these latter symptoms of inflammation and blood clotting may be the result of an excessive immune response rather than the direct actions of the virus itself.


A new paper published in the journal Blood includes work by researchers at the University of Reading which focused on thrombi formation (blood clots) in severely ill COVID-19 patients and importantly, they provide evidence of how to treat this condition, [1].


Study Overview


Researchers successfully isolated monoclonal antibodies from samples of severely ill COVID-19 patients, [2]. They cloned and analysed these antibodies and found that they had low levels of fucosylation and elevated galactosylation (Low Fuc High Gal) in the Fc domain; thus illustrating qualitative and quantitative differences compared to those with mild illness.


Gibbins et al., Fig. 1 Ci: Volume of thrombi formed on vWF with immune complexes containing spike and either WT IgG or IgG with modified glycosylation; and Fig. 1 Cii: representative images of thrombi stained with DiOC6.

The study then further examined how this affected platelet-mediated thrombus formation. The study revealed that potent activation of platelets by immune complexes containing SARS-CoV-2 spike and anti-spike IgG only occurs when the IgG expressed both Low Fuc and High Gal in the Fc domain and an additional prothrombotic signal, vWF, is also present.



Gibbins et al., Fig. 5: Aberrant glycosylation of anti-spike IgG in immune complexes act in concert with vWF to enhance platelet thrombus formation.

Enhanced thrombus formation was measured in vitro using Cellix’s Vena8 Fluoro+ biochips. Researchers also found that thrombus formation was sensitive to Fc-gamma-RIIA inhibition and small molecule inhibitors of Syk, Btk and P2Y12, providing a potential treatment strategy to reduce blood clots in critically ill COVID-19 patients.




Professor Jon Gibbins, Director of the Institute for Cardiovascular and Metabolic Research at the University of Reading said, [3]:

"Until now, we have only had assumptions about why platelets involved in clotting were being activated during Covid-19 infection.

"One way to think of what is happening is that the immune response that is designed to protect you from the infection in some cases, particularly in severely ill patients, actually causes more damage. In this case, the antibodies that are produced to stop Covid-19 from spreading trigger infected cells to induce platelet activity which causes clotting even though there is no wound that needs healing.

"We are particularly excited because our studies of platelets in the laboratory establishes important mechanisms that explain how and why dangerous blood clots may occur in severely ill Covid-19 patients, and importantly, also provides clues as to how this may be pr


In vitro thrombus formation assays with Cellix’s Vena8 Fluoro+ biochips

vWF facilitates platelet adhesion to vascular endothelium under high shear stresses similar to those found in arteries and arterioles. Researchers replicated these conditions using Cellix’s Vena8 Fluoro+ biochips which enabled thrombus formation experiments.


Vena8 Fluoro+ biochips were coated with IgG-spike immune complexes (recombinant SARS-CoV-2 spike protein, FCS and COVA1-18 antibodies) and citrated whole blood with vWF was perfused through the microchannels of the biochips at 1000s-1 for 6 minutes before staining and acquiring images, as shown in Fig. 2Bii.


Gibbins et al., Fig. 2Bi and 2Bii: Platelet activation by low fucose, high galactose IgG1 immune complexes is dependent on Fc-gamma-RIIA and functions at low and high shear.

Results showed a significant increase in thrombus volume formed on vWF in the presence of Low Fuc High Gal IgG immune complexes suggesting that anti-spike IgG with aberrant glycosylation of the Fc domain synergises with vWF to enhance thrombus formation.


The volume of thrombi formation under perfusion was subsequently inhibited by treatment with R406, ibrutinib or Cangrelor as shown in Fig. 3A and 3B.


Gibbins et al., Fig. 3A and 3B: Prothrombotic activity of low fucose, high galactose IgG1 immune complexes is inhibited by R406 (Syk inhibitor), Ibrutinib (Ibr: Bruton tyrosine kinase inhibitor, Btk) or Cangrelor (P2Y12 inhibitor).

Next steps: on-going clinical trials

The findings of this study supported the scientific basis of a clinical trial called MATIS. Co-author of this study, Nichola Cooper, reader at Imperial College London and consultant haematologist at Imperial College Healthcare NHS Trust, also designed and leads the MATIS clinical trial. This trial is testing some of these inhibitors in COVID-19 patients at hospitals in the UK to determine whether serious clotting can be reduced. Nichola Cooper said, [3]:


"Early on in the COVID-19 pandemic it was clear that the infection was causing an overwhelming immune response, including blood clotting, and that many of the more severe cases and deaths were related to this.

"Having been involved in early research around blood clotting related to inflammation, it occurred to me that the drugs we already use for other disorders could be easily accessible treatments for COVID-19. We are yet to see results from the MATIS trial so we do not yet know how these drugs will work in patients, but our hope is that we can both inhibit the inflammatory response and prevent severe disease and blood clots. It is exciting to see our collaboration with Reading backing our theory already and providing a solid scientific basis for clinical trials."


Consider the advantages of the Vena8 Fluoro+ Biochip

Cellix biochips simulate human capillaries to aid the accurate and the Vena8 Fluoro+ biochip is ideal for platelet adhesion and aggregation shear flow experiments, particularly at high shear rates. The chip is robust, simple, and easy to use.






Here's how it works:

The microchannel (which simulates a blood vessel) is coated with an adhesion molecule such as vWF, collagen, fibrinogen, etc. The biochip is then placed on a microscope frame and connected to the Mirus Evo pump. The prepared cell sample (whole blood or purified

platelets) is then pumped through the microchannel at the user’s defined shear rate, which may be ramped up or down. The platelets engage with receptors on the microchannel wall resulting in adhesion, aggregation and thrombi formation.


Biochip Details:

  • Microchannel Dimensions: 400µm (width) x 100µm (depth) x 20mm (length).

  • 8 channels per biochip = 8 assays with lots of different possibilities - for example, you can coat each channel with a different adhesion molecule.

Features and Benefits of Vena8 Fluoro+

  • Compatible with confocal microscopy and fluorescence immunostaining.

  • Plug and play - Compact with easy to connect tubing.

  • Thrombosis, platelet adhesion, and aggregation flow-based assays with shear rate range from 50 - 10,000 s−1 with ExiGo or Mirus Evo pumps.

  • Reagent savings - only 10µL ligand/protein coating per channel (e.g. VWF, collagen, etc.) with a standard pipette.

  • Low sample volume even at high shear rates with whole blood or cell suspensions.

Click here to find out more information.


References

1. Bye, Alexander P et al. “Aberrant glycosylation of anti-SARS-CoV-2 IgG is a pro-thrombotic stimulus for platelets.” Blood, blood.2021011871. 28 Jul. 2021, doi:10.1182/blood.2021011871

2. Brouwer, Philip J M et al. “Potent neutralizing antibodies from COVID-19 patients define multiple targets of vulnerability.” Science (New York, N.Y.) vol. 369,6504 (2020): 643-650. doi:10.1126/science.abc5902

3. University of Reading. "Blood clots in people with severe COVID-19 may be related to abnormal antibody response." ScienceDaily. ScienceDaily, 28 July 2021. <www.sciencedaily.com/releases/2021/07/210727212832.htm>.




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