Analysis of thrombogenicity under flow reveals new insights into the prothrombotic state of patients with post-COVID syndrome – By Constantinescu-Bercu et al., 2022.
Background
Post-covid syndrome (PCS) refers to symptoms that develop during or following SARS-COV-2 infection, continue for more than 12 weeks, and can’t be explained by alternative diagnosis. Over 60% of people with PCS continue to experience symptoms after the acute infection has passed. On average, each person with PCS has about 13.79 different symptoms. This highlights how PCS can significantly affect a person's quality of life.
Na acute SARS-CoV-2 infection triggers a prothrombotic state in the body. Additionally, it has been shown that acute COVID-19 infection can increase endothelin-1 levels and result in prolonged inflammation. Research has also shown the existence of platelet hyperactivation and anomalous microclots in the platelet-poor plasma of individuals with PCS. Furthermore, an elevated von Willebrand factor antigen (VWF[Ag]): ADAMTS13 ratio has been linked to reduced exercise capacity in patients with PCS. Until now, the mechanisms underlying the different symptoms observed in patients with PCS are unclear. In this study, Constantinescu-Bercu and colleagues investigated the prothrombotic state in patients with PCS using a dynamic microfluidic assay and analysing platelet accumulation, thrombi formation, and geometries in real-time.
Methods
The researchers collected blood from PCS patients who visited a post-COVID clinic from January to April 2022 and from healthy volunteers of similar age. For the microfluidics assay, the researchers used VenaFluoro+ biochips from Cellix. They coated the biochips overnight at 4ËšC with one of three substances: collagen type I; an antibody that targets the VWF A3 domain, or VWF. After that, they blocked them with a 1% (w/v) bovine serum albumin solution in phosphate-buffered saline. Next, the researchers labeled citrated blood with DiOC6 and pumped it through the channels using a Cellix's Mirus Evo Pump at a flow rate of 1800/s for 3 minutes. They completed this step within 2.5 hours of collecting the blood samples. They used an inverted fluorescent microscope from Zeiss and a QImage camera to observe platelet accumulation.
Results
The researchers conducted a microfluidic assay using fresh human whole blood to investigate how the increase in the VWF(Ag): ADAMTS13 ratio affects thrombi formation and platelet binding in PCS. The findings indicate that:
Platelet binding showed a significant increase in patients with PCS compared to controls on both anti-VWF A3 and collagen-coated channels, but not on channels directly coated with VWF (Figure 1 A, B).
Platelets formed microthrombi on collagen channels, while on anti-VWF A3 and VWF-coated channels, they formed small aggregates (Figure 1 A).
Platelet coverage on anti-VWF A3 correlated with VWF levels and VWF(Ag): ADAMTS13 ratio (Figure 1 D), but there was no such connection with platelet coverage on collagen. Conversely, platelet coverage on collagen showed an inverse correlation with ADAMTS13 activity (Figure 1 C).
Other experiments in this study
The researchers examined thrombi geometry on collagen-coated channels to understand better how PCS influences thrombi formation.
Main findings of these experiments
There was no difference in thrombi numbers, but there was a significant increase in the thrombi area, which could be attributed to their length. This correlated with the area under the curve (AUC) in the thrombin generation assay (TGA).
The difference in thrombi length between controls and PCS was noticeable primarily in those with raised AUC.
Thrombi length on collagen also correlated with VWF(Ag): ADAMTS13 ratio, confirming a role for VWF in thrombogenesis.
Conclusion
These data introduce a dynamic assay for exploring the prothrombotic state in PCS. It has the potential to uncover the underlying mechanisms and create new therapeutic approaches to treat this condition.
You can see more details of the experiments here.
How to get started?
Thinking about trying out similar experiments in your lab? Here's what you'll need as a minimum setup:
VenaFlux Pro platform – a semi-automated microfluidic platform designed for conducting studies on cell adhesion, binding, rolling, and migration under shear flow conditions that replicate in vivo flow rates.
Vena8 Fluoro+ Biochips – to mimic human blood vessels and simulate blood clot formation.
Mirus Evo Pump – a microfluidic system equipped with an 8-channel syringe pump for analyzing cells under shear flow, simulating the natural flow conditions within human blood vessels.
Microenvironmental chamber – a temperature-controlled frame that keeps the biochip at 370˚ C.
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 the VenaFlux Pro and Elite options.
Image Pro Cell Analysis software – for image and video analysis.
If you already have some of these items (such as the inverted microscope, camera, or cell analysis software), we recommend the VenaFlux Starter kit. We have options that suit all budgets. You can check them out on our eShop.