There is no global consensus among researchers and clinicians on a definition for long COVID, no adequate diagnostic test for the debilitating condition, and the causes of patients progressing to long COVID are, at this stage, theoretical.
However, writes Adele Baleta for Spotlight, Professsor Resia 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 this puzzle.
She says scientific collaboration is intensifying to find answers to long COVID which affects 43% or 100m people globally post-infection, according to a meta-analysis and systematic review.
Pretorius says she 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. There are 40 to 50 researchers globally who talk to each other regularly. We are going to crack this. We have to.”
Causes of long COVID
Scientists are pursuing three leading theories in efforts to decipher the effects of post-COVID-19 infection – leading to various 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 the virus lingers in a 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 body’s vascular and coagulation systems.
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.
Pretorius says 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 long COVID patients 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 people, 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 people with long COVID, the body’s ability to break down the clots is significantly undermined. The blood circulation clogs up.
Microclots are generally not found in people who do not have long COVID. Pretorius says you can find them in some other conditions, like diabetes, “but the difference is the number and the extreme presence of the clots with long COVID: that’s what’s making the difference”.
Another difference is that the long COVID clots are insoluble. When Pretorius tried to dissolve them 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 made blood smears, looking 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.
In early 2021, she says, “I got a report from Harvard collaborators and others to say 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 these, and lo and behold, we found the clots and they were fully persistent. I was not surprised. I knew that 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.”
In 2020, she recalls, 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.
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.”
She adds: “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.”
Pretorius says there are no general pathology tests readily available to diagnose people with long COVID.
“People who are desperately ill – bedridden or in wheelchairs – are often given generalised blood tests. They are told their pathology test results are within normal to healthy ranges. Many are told their symptoms are possibly psychological, all in their head, and told to get some rest and to stop stressing. Meanwhile, they are very ill.”
She says the main reason 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 – just a simple microscopy test that is a useable diagnostic method to see if microclots are present.”
Microscopy methods are not readily available at pathology labs. However, she adds: “We 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 to be used in the typical pathology labs. So we hope to have a diagnostic that will be readily available in a couple of months.”
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 are planning to publish their UK cohort results soon. They are also correlating long COVID severity to microclot presence, Pretorius says.
“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 of the issues is just so sad.”
But she remains determined to help “crack” long COVID.
Persistent clotting protein pathology in Long COVID/Post-Acute Sequelae of COVID-19 (PASC) is accompanied by increased levels of antiplasmin
Etheresia Pretorius, Mare Vlok, Chantelle Venter, Johannes Bezuidenhout,
Gert Jacobus Laubscher, Janami Steenkamp & Douglas Kell
Published in Cardiovascular Diabetology on 23 August 2021
Severe acute respiratory syndrome coronavirus 2 (SARS-Cov-2)-induced infection, the cause of coronavirus disease 2019 (COVID-19), is characterised by acute clinical pathologies, including various coagulopathies that may be accompanied by hypercoagulation and platelet hyperactivation. Recently, a new COVID-19 phenotype has been noted in patients after they have ostensibly recovered from acute COVID-19 symptoms. This new syndrome is commonly termed Long COVID/Post-Acute Sequelae of COVID-19 (PASC). Here we refer to it as Long COVID/PASC. Lingering symptoms persist for as much as 6 months (or longer) after acute infection, where COVID-19 survivors complain of recurring fatigue or muscle weakness, being out of breath, sleep difficulties, and anxiety or depression. Given that blood clots can block microcapillaries and thereby inhibit oxygen exchange, we here investigate if the lingering symptoms that individuals with Long COVID/PASC manifest might be due to the presence of persistent circulating plasma microclots that are resistant to fibrinolysis.
We use techniques including proteomics and fluorescence microscopy to study plasma samples from healthy individuals, individuals with Type 2 Diabetes Mellitus (T2DM), with acute COVID-19, and those with Long COVID/PASC symptoms.
We show that plasma samples from Long COVID/PASC still contain large anomalous (amyloid) deposits (microclots). We also show that these microclots in both acute COVID-19 and Long COVID/PASC plasma samples are resistant to fibrinolysis (compared to plasma from controls and T2DM), even after trypsinisation. After a second trypsiniaation, the persistent pellet deposits (microclots) were solubilised. We detected various inflammatory molecules that are substantially increased in both the supernatant and trapped in the solubilised pellet deposits of acute COVID-19 and Long COVID/PASC, versus the equivalent volume of fully digested fluid of the control samples and T2DM. Of particular interest was a substantial increase in α(2)-antiplasmin (α2AP), various fibrinogen chains, as well as Serum Amyloid A (SAA) that were trapped in the solubilised fibrinolytic-resistant pellet deposits.
Clotting pathologies in both acute COVID-19 infection and in Long COVID/PASC might benefit from following a regime of continued anticlotting therapy to support the fibrinolytic system function.
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