SA at forefront of long Covid research, with microclots offering vital clues

27 Jul 2022

Professor Resia Pretorius and her colleagues at the University of Stellenbosch are at the cutting edge of research into one of the most likely causes of long Covid – inflammatory microclots. Adele Baleta spoke to Pretorius about their work.

Professor Resia Pretorius sounds rushed when Spotlight first tracks her down by phone at Heathrow Airport outside London. She is about to board a plane to South Africa after attending a conference, meetings, and symposia in the United Kingdom, all with the purpose of unravelling the complexity of long Covid and how to treat it.

There is no global consensus among researchers and clinicians on a definition for long Covid, there is no adequate diagnostic test for the debilitating condition, and the causes of patients progressing to long Covid are, at this stage, theoretical.

However, Pretorius who heads the department of Physiological Science at the University of Stellenbosch remains upbeat. Her research group is the first to have reported evidence of inflammatory microclots in blood samples from individuals with long Covid, potentially solving an important piece of the long Covid puzzle.

She says scientific collaboration is intensifying to find answers to long Covid which affects 43% or 100 million people globally post-infection, according to a meta-analysis and systematic review.

Later speaking from Stellenbosch, Pretorius describes herself as a “lab person” who has been trying to find the cause of long Covid since 2020.

“I have always been passionate about research. Now, I am working with clinicians and researchers in the UK, the USA, and other parts of the world. I am too worried to miss anything so I am at all of these meetings. There are 40 to 50 researchers globally who talk to each other regularly. We are going to crack this I know. We just have to.”

Dr Resia Pretorius studying long Covid Dr Resia Pretorius in her laboratory looking at microclots - now researched as one of the most likely causes of longCovid. (Photo: Supplied / Spotlight)

Causes of long Covid

As explained in a recent article in the journal Science, there are three leading theories scientists are pursuing in an attempt to decipher the effects of post-Covid-19 infection — which leads to an array of symptoms, including shortness of breath, fatigue, headaches, palpitations, and impairments in mental health and cognition or brain fog.

One theory is that Sars-CoV-2 stubbornly persists in the body, even after the acute infection passes. Studies have shown that the virus lingers in a wide range of body sites, especially in the nerves and other tissues.

Another theory based on blood samples from Covid-19 patients reveals an immune system in disarray even eight months after first testing positive. The body’s cells do not appear to recover.

The third, an area in which Pretorius has distinguished herself internationally, is that Covid-19 is not only a lung disease but significantly affects the vascular (blood flow) and coagulation (blood clotting) systems of the body.

A recent study published in the Cardiovascular Diabetology journal, conducted by Pretorius and colleagues, found that there is significant microclot formation in the blood of both acute Covid-19 and long Covid patients. A microclot is a blood clot that can only be seen through a microscope.

Pretorius explains that in a healthy person clots may form, for example, when you cut yourself. The main clotting protein is a molecule called fibrinogen. “When you’re healthy, it’s in solution. And then when you cut yourself, collagen is exposed, and a little gel called fibrin prevents you from bleeding out. In healthy individuals, the clots are then broken down by a process called fibrinolysis.

Blood samples from patients with long Covid have revealed high levels of various inflammatory molecules trapped in the microclots including fibrinogen and Alpha-2 antiplasmin — a molecule that prevents the breakdown of microclots.

The persistent blood clots essentially result in cells not getting enough oxygen in the tissues to sustain bodily functions. This, Pretorius says, may be central to numerous debilitating symptoms.

In healthy individuals, the body's plasmin-antiplasmin system maintains a fine balance between blood clotting to prevent blood loss after an injury and fibrinolysis which prevents blood clots from forming.

With high levels of alpha(2)-antiplasmin in the blood of acute Covid-19 patients and individuals suffering from long Covid, the body’s ability to break down the clots is significantly undermined. The blood circulation becomes clogged up.

Microclots are generally not found in people who do not have long Covid. Pretorius says you can find them in some other conditions, such as diabetes, “but the difference is the number and the extreme presence of the clots with long Covid, that's what's making the difference,” she says.

Insoluble clots

Another difference is that the clots in long Covid are insoluble. When Pretorius tried to dissolve these clots using an enzyme called trypsin in her laboratory, they would not dissolve. They are resistant to fibrinolysis.

Initially, Pretorius was looking at acute Covid-19 infection. We received blood samples from ICU patients and we made blood smears and looked at them under a scanning electron microscope that can enlarge a sample hundreds of thousands of times. We then added a fluorescent dye or marker called Thioflavin T which lights up when there are misfolded proteins. This happens when, for example, the spike protein binds to the soluble fibrinogen molecule making it insoluble.

The Sars-CoV-2 virus is known to bind to ACE2 receptors and TMPRSS receptors which are found on platelets (blood cells that help with clotting). They are also found on the endothelium (the inner-most lining of the blood vessels). By binding to the platelets and the endothelium, the virus sets off a torrent of clotting causing vascular damage.

Pretorius says in early 2021, “I got a report from Harvard collaborators and others to say that patients do not fully recover post-infection and they referred to this as long Covid.

“I said let’s get the samples. We looked at the blood samples and lo and behold we found the clots and they were fully persistent. I was not surprised to find the clots in long Covid because I knew with acute Covid many people were dying because of clots in the lungs and shortness of breath. But, I did not know the extent to which they were present in long Covid.

“When we did proteomics analysis on the sample, when we looked at the different molecules in the blood, I could not dissolve the sample with typical enzymes. I used a massively abrasive enzyme called Trypsin which dissolves any possible protein. But it could not dissolve these cells. The resilience of these clots, that they simply don’t get dissolved, surprised me,” Pretorius says.

Pretorius recalls that in 2020, several South African clinicians alerted others to Covid-19 not being a typical viral pneumonia but suggested it was also a vascular disease. “At that stage, it was massively controversial with many dismissing this idea saying it’s a virus that affects the lungs and that’s it,” she says.

Pretorius says this was despite papers published overseas in 2020 that concluded Covid-19 was also a vascular disease. “It was made controversial in South Africa but it is now widely accepted that Covid-19 also affects clotting as well as the body’s vasculature (network of blood vessels).”

Pretorius says, “Although the microclot is a theory, it encompasses all of the other suggested causes of long Covid because the spike protein itself can trigger microclots. We have submitted a paper, looking at many more blood samples, where we found inflammatory molecules trapped inside the blood clots which do not break down. We also found antibodies so the theories about immune abnormalities, persistent virus, and microclots are intertwined. All of these can cause organ damage. So if you look at it from a systems biology approach, all of these are valid.”

Images of micro clots as seen under an electron microscope. (Photo: Suplied / Spotlight)

Diagnostics

Pretorius says there are no general pathology tests readily available to diagnose people with long Covid.

“People that are desperately ill — bedridden or in wheelchairs — are often given generalised blood tests. They are told that their pathology test results are within normal to healthy ranges. Many are told that their symptoms are possibly psychological, all in their head, and they are told to get some rest and to stop stressing. Meanwhile, the patients are very ill,” she says.

Pretorius says the main reason the traditional laboratory tests do not pick up any of the inflammatory molecules is that they are trapped inside the insoluble microclots. A typical pathology test looks at the soluble content of the blood, so if the molecules are trapped they will be missed.

“We patented a long Covid test which is just a simple microscopy test that is a useable diagnostic method to see if microclots are present,” Pretorius says.

Microscopy methods are not readily available at pathology laboratories. However, Pretorius says, “We have crowd-funded and received funding from the Polybio Research Foundation in America to buy a flow cytometer for our blood lab to develop a flow cytometry method that can be used in the typical pathology labs. So we hope to have a diagnostic that will be readily available in a couple of months.”

Treatment

Pretorius says colleagues in the United Kingdom have already designed two randomised controlled trials to independently test both coagulation therapy (Clott-UK) and Apheresis (Clott-Apheresis trial) in which microclots and inflammatory molecules are filtered out in a dialysis-type treatment. These trials will study whether anticoagulants and Apheresis give long-lasting relief of symptoms. These trials are being planned and researchers are waiting for ethics approval.

In addition, colleagues from the University of Sheffield Hallam and from the University of Manchester have independently set up microclot testing in their labs and they are planning to publish their UK cohort results soon. They are also correlating long Covid severity to microclot presence, says Pretorius.

“It’s been quite a ride. Seeing the devastation of long Covid, I realise why I decided not to be a clinician... handling and hearing all the issues is just so sad,” says Pretorius.

But, she remains determined to help “crack” long Covid. DM/MC

This article was produced by Spotlight – health journalism in the public interest.

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South Africa

Causes of long Covid

Insoluble clots

Diagnostics

Treatment