Amy Proal - Interview with Dr. Resia Pretorius: LongCovid microclots and Hypercoagulation

Shanti1

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Dr. Resia Pretorius is both the Department Head and a Research Professor in the Physiological Sciences Department, Faculty of Science at Stellenbosch University in South Africa. Her team has used super-resolution and electron microscopy, together with several other methods to identify microclots containing trapped inflammatory molecules in the blood of patients with LongCovid. She has recently partnered with several clinical collaborators to trial if HELP apheresis (a form of blood filtration) can remove these LongCovid microclots and improve patient symptoms. Soon she will be extending her research on blood clotting and possible viral protein-driven platelet hyperactivation to Myalgic Encephalomyelitis (ME/CFS). Here is a link to Resia and team's recent paper where you can see images of microclots identified in LongCovid blood: https://cardiab.biomedcentral.com/art... Here is a link to Resia and team's paper where you can see that platelets (red blood cells) have receptors that recognize a wide range of bacterial, viral and fungal proteins. Sensing such proteins can contribute to platelet hyperactivation and subsequent clotting pathology: https://www.ncbi.nlm.nih.gov/pmc/arti... Also H.E.L.P Apheresis stands for: "Heparin Induced Extracorporeal Lower Density Lipo-Protein (LDL) Precipitation (H.E.L.P.) Apheresis System."
 

Shanti1

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Notes:

Dr. Pretorius has been studying both clotting and platelet activation pathology in individuals with acute and Long-COVID

Her team has looked at a few hundred samples over the past few months from patients diagnosed with Long-COVID. In all of these individuals, they have noted microclotting pathology

Visualization of Microclots (2:39)
The microscope she uses is a fluorescence microscope, but it can be done with a normal light microscope. The fluorescence is used for better visualization.
  1. Use a citrated tube to draw the blood and spin it down to obtain the platelet-poor plasma (PPP)
  2. Add the marker Thioflavin T. It binds to both misfolded fibrin and amyloid protein. Mount 3 uL onto a slide and visualize under the microscope
  3. She then states that you don’t even need the thioflavin marker, you can just mount the sample on a slide in a small thin area and view the microclots on the slide using a regular light microscope with no marker.
Assessment of Platelet Activation (5:35)
  1. Spin down the plasma but use the hematocrit that has been spun down instead of the PPP
  2. Add two markers, P-selectin and a fluorescence antibody for platelets
  3. This is a test for platelet activation and fragility
The team is also working to see if they can’t translate the microscopy work to flow cytometry as that would speed up the process of diagnosis

Properties of Microclots:
  • Many path-labs will not pick up differences in inflammatory markers between Long-COVID and controls. The reason labs are not finding inflammatory markers is because they are trapped within the microclots (the inflammatory markers are released and can be identified when the clot is dissolved in the lab). Microclots are actually “really, really big”.
  • COVID microclots are resistant to breakdown by trypsin (and plasmin) because they contain antiplasmin, which inhibits the enzyme the body uses to break down fibrin. The microclots both block the microcirculation, preventing oxygen delivery by the RBCs. In addition and can directly damage and cause inflammation in the endothelium, further compromising oxygen delivery.
  • When the spike protein is added to healthy blood, you can replicate trypsin-resistant microclots and platelet activation. The spike protein drives a unique clot that tends to be large and resistant to break down.
What is the pathology caused by the microclots?
  • If you look at the 150+ symptoms of Long-COVID that have been identified, they all comes back to a hypoxia of certain organs (brain fog, liver damage, muscle fatigue).
  • Massive fatigue, brain fog, and muscle weakness are key features of Long-COVID and they are related to hypoxia. COVID impacts the lungs, but also the vasculature. This is driven by the spike protein. The clotting and vascular damage should be addressed in the acute COVID stage to prevent Long-COVID.
  • It is possible that some people don’t clear Long-COVID, causing spike protein exposure over time and leading to ongoing symptoms.

Can other pathogens cause microclots?
  • Platelets have receptors to EBV as well as a range of other bacterial and viral pathogens and can become activated by membrane binding of these pathogens. Activated platelets can, in turn, activate WBC and RBC receptors. Other bacteria, viruses, and bacterial endotoxins can bind endothelial receptors and can also cause misfolded fibrinogen. The extent, however, to which spike protein does this is mindboggling and manyfold greater than other pathogens.
  • Pathological clotting happens to some degree in all inflammatory conditions, but the sheer volume and size of the dysfunction is much larger in Acute COVID. Individuals who are already predisposed to clotting and endothelial damage are at greater risk for Long-COVID.
How can clots be removed?
  • Anti-Coagulant therapy (she didn’t say which ones), but said we urgently need a proper trial. We need to be careful to keep it safe and not push people into bleeding. We also need to have individuals properly identified as having microclots and identify which autoantibodies and inflammatory markers are present as part of these trials so that the right medications can be used to target all aspects of clots, antibodies, and platelet activation. If only one aspect of hypercoagulation is targeted, the trial may fail.
  • HELP Apheresis -filters out microclots, fatty deposits, autoantibodies, and inflammatory molecules (She references Dr. Jaeger’s clinic)
  • Dr. Pretorius has confirmed a reduction in microclots from before and after apheresis as well as other parameters of function and hopes to publish the findings with the rest of the scientific team soon
Can centers that perform normal apheresis help Long-COVID patients, or does it have to be HELP apheresis?
  • This is being looked into by scientific teams
  • She is aware of some acute COVID patients treated with normal apheresis, but it isn’t as easy on the body as HELP apheresis
  • She believes the normal apheresis machines do remove some of the inflammatory molecules and autoantibodies, but it is not known how effective they are
Can this work be applied to ME/CFS (56:55)
  • Dr. Pretorius is working with Amy and team to study ME/CFS and clotting pathology
  • They have 60 patients that have been identified with ME/CFS and who have given consent
  • In Jan they will be gathering blood from these individuals to look for microclots and platelet activation. They also hoping to do proteomics with this group, but how much they can do depends on if their grant comes through.
  • Amy comments that clotting pathology could also lead to small fiber neuropathy due to reduced blood flow and oxygen delivery, leading to nerve damage and death.
Dr Pretorius’s message to all of us with ME/CFS and Long-COVID is “Please Don’t Give Up! We are a team of dedicated individuals, we work late into the night, we don’t take breaks, we are working hard and I think we are onto something. Please keep following us and don’t give up”.
 

Countrygirl

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Excellent summary.



Could it really then come down to the spike protein? :monocle:

According to this, we don't need a spike protein @Pyrrhus.....it sounds as if we may have plenty of similar problems of our own. http://www.wisconsinhyperbarics.com/research-pdf/ChronicFatigue443.pdf

(ISAC) Immune System Activation of Coagulation.............if this proves to have mileage, could we adopt this as our new name?

Of a test sample of 54 patients, this shows that greater than 92% of CFS and/or FM patients had a demonstrable hypercoagulable state. What then is the underlying disease process?
 

Mary

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According to this, we don't need a spike protein @Pyrrhus.....it sounds as if we may have plenty of similar problems of our own. http://www.wisconsinhyperbarics.com/research-pdf/ChronicFatigue443.pdf
This is from over 20 years ago . . . and apparently no one has followed up on it? I wonder if Dr. Pretorious would be interested in this study?
(ISAC) Immune System Activation of Coagulation.............if this proves to have mileage, could we adopt this as our new name?
I like this name! Catchier and easier to say and remember than ME/CFS and without the baggage of "chronic fatigue syndrome" --
 

Pyrrhus

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This is not directly related, but it bears mentioning that a portion of the spike protein has been suggested to be a "superantigen". A "superantigen" is a substance that "over-activates" T cells.

Again not necessarily relevant to this conversation. (Unless somehow it is...)

Superantigenic character of an insert unique to SARS-CoV-2 spike supported by skewed TCR repertoire in patients with hyperinflammation (Cheng et al., 2020)
https://doi.org/10.1073/pnas.2010722117
Excerpt:
A hyperinflammatory syndrome reminiscent of toxic shock syndrome (TSS) is observed in severe COVID-19 patients, including children with Multisystem Inflammatory Syndrome in Children (MIS-C). TSS is typically caused by pathogenic superantigens stimulating excessive activation of the adaptive immune system. We show that SARS-CoV-2 spike contains sequence and structure motifs highly similar to those of a bacterial superantigen and may directly bind T cell receptors. We further report a skewed T cell receptor repertoire in COVID-19 patients with severe hyperinflammation, in support of such a superantigenic effect. Notably, the superantigen-like motif is not present in other SARS family coronaviruses, which may explain the unique potential for SARS-CoV-2 to cause both MIS-C and the cytokine storm observed in adult COVID-19.
 

Shanti1

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Could it really then come down to the spike protein? :monocle:
When it comes to the SARS-Cov-2 virus it does seem to mostly revolve around the "superantigen" spike protein as it is primarily responsible for the hypercoagulation, endothelial damage, and overactivation of the immune system which results in organ damage. The spike protein also seems extraordinarily apt at inducing misfolded fibrin to create clots that are particularly resistant to lysis.

As far as Long-COVID goes, Pretorius positions the microclots as the primary factor causing Long-COVID. Other aspects, such as organ damage (esp lungs), vagal nerve activation, microglial activation, immune system dysregulation, viral persistence, latent pathogen reactivation, autoimmune activation, etc, are certainly still on the table as contributors. To me it seems the microclots may be the biggest player. As I Iearn more about ME/CFS, it seems poor oxygenation is an underlying commonality of the various triggers. Due to their ability to induce hypoxia, microclots certainly fit the bill for the ability to cause ME-CFS symptoms in pw Long-COVID.

While other pathogens and inflammatory states have the ability to cause hypercoagulation, it seems none do so to the same degree as SARS-Cov-2. I'm guessing hypercoagulation plays a role to varying degrees in ME/CFS depending on the pathogen involved and the disposition to clotting. There are members on this forum who suffer greatly from coagulopathies, but for others, like me, it doesn't seem to be such a big player. Would be interested in your thoughts if you have the time.
 
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Pyrrhus

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The spike protein also seems extraordinarily apt at inducing misfolded fibrin to create clots that are particularly resistant to lysis.
I'm quoting that specific paper by Resia Pretorius as it's most relevant to this thread:

SARS-CoV-2 spike protein S1 induces fibrin(ogen) resistant to fibrinolysis: implications for microclot formation in COVID-19 (Grobbelaar et al., 2021)
https://dx.doi.org/10.1042/BSR20210611
Abstract:
Severe acute respiratory syndrome coronavirus 2 (SARS-Cov-2)-induced infection, the cause of coronavirus disease 2019 (COVID-19), is characterized by unprecedented clinical pathologies. One of the most important pathologies, is hypercoagulation and microclots in the lungs of patients.

Here we study the effect of isolated SARS-CoV-2 spike protein S1 subunit as potential inflammagen sui generis. Using scanning electron and fluorescence microscopy as well as mass spectrometry, we investigate the potential of this inflammagen to interact with platelets and fibrin(ogen) directly to cause blood hypercoagulation.

Using platelet-poor plasma (PPP), we show that spike protein may interfere with blood flow. Mass spectrometry also showed that when spike protein S1 is added to healthy PPP, it results in structural changes to β and γ fibrin(ogen), complement 3, and prothrombin. These proteins were substantially resistant to trypsinization, in the presence of spike protein S1.

Here we suggest that, in part, the presence of spike protein in circulation may contribute to the hypercoagulation in COVID-19 positive patients and may cause substantial impairment of fibrinolysis. Such lytic impairment may result in the persistent large microclots we have noted here and previously in plasma samples of COVID-19 patients. This observation may have important clinical relevance in the treatment of hypercoagulability in COVID-19 patients.
(spacing added for readability)
 

Pyrrhus

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As far as Long-COVID goes, Pretorius positions the microclots as the primary factor causing Long-COVID. Other aspects, such as organ damage (esp lungs), vagal nerve activation, microglial activation, immune system dysregulation, viral persistence, latent pathogen reactivation, autoimmune activation, etc, are certainly still on the table as contributors. To me it seems the microclots may be the biggest player.
Perhaps that may indeed turn out to be the case in some with Long Covid. In others, the microclots, if any, might not contribute significantly to the symptoms. As always, we need more research, more data, and more open minds.

There are members on this forum who suffer greatly from coagulopathies, but for others, like me, it doesn't seem to be such a big player.
Beside the 1999 study, I haven't heard of any research regarding coagulopathies in ME. Have you?

As I Iearn more about ME/CFS, it seems poor oxygenation is an underlying commonality of the various triggers.
How do you figure? Apart from the known poor oxygenation due to orthostatic intolerance (and aerobic exercise intolerance), I don't see how poor oxygenation can be a significant factor in most of the main symptoms of ME.

According to the International Consensus Criteria, here are the main symptoms of ME:
https://onlinelibrary.wiley.com/doi/10.1111/j.1365-2796.2011.02428.x

Core Symptom:
  1. PENE (better known as PEM): "Post-exertional symptom exacerbation" and "physical/cognitive fatigability in response to exertion". The etiology of PENE/PEM is still unknown, but I don't see how poor oxygenation would explain it. Some people mistakenly assume that PENE/PEM must have something to do with the symptom of exercise intolerance, but "exercise intolerance" only refers to aerobic activity and PENE/PEM results from any exertion, not just from aerobic activity.
3 of the following 4 neurological symptoms:
  1. Cognitive dysfunction: Poor oxygenation of the brain can certainly cause slow thinking, but can it really explain the specific types of cognitive dysfunction that have been documented in ME?
  2. Pain, either headaches or hyperalgesia with widespread pain: Poor oxygenation of muscles and nerves can certainly cause muscle soreness and poor oxygenation of the brain might cause headaches, so poor oxygenation could possibly be a factor here.
  3. Sleep disturbance, either disturbed sleep patterns or unrefreshing sleep: Do we know if poor oxygenation of the brain leads to insomnia or disrupted sleep?
  4. Sensory or motor dysfunction, either sensory hypersensitivity (including blurred vision) or muscle weakness (including fasciculations and balance problems): I can't see how poor oxygenation would play a role here, except possibly by leading to the death of small nerves (small-fiber neuropathy SFN). But the fluctuating nature of these symptoms implicates other types of neuropathy, not the relatively static symptoms of SFN.
3 of the following 5 immune or gastrointestinal symptoms:
  1. Flu-like symptoms, including sore throat, sinusitis, lymph node swelling: These are inflammatory symptoms. I see no way for poor oxygenation to explain these.
  2. Frequent infections: This is an immune symptom. I see no way for poor oxygenation to explain this.
  3. IBS, including nausea and bloating: The bulk of research evidence points to dysautonomia as the cause of IBS. I see no way for poor oxygenation to explain these symptoms, except perhaps the slowed gastromotility in IBS-C. (but certainly not in IBS-D)
  4. Frequent urination: The bulk of research evidence points to hypothalamic dysfunction as the cause of frequent urination. I see no way for poor oxygenation to explain this.
  5. Food sensitivities or MCS: There may be different etiologies at play here, but I don't see poor oxygenation as one of them.
1 of the following 4 "energy production" symptoms:
  1. Orthostatic intolerance, palpitations, or arrythmias: These are generally accepted to be due to dysautonomia, and I can't see how poor oxygenation would explain these types of dysautonomia.
  2. Air hunger: This is generally seen to be a dysfunction of the brainstem. I see no way for poor oxygenation to explain this.
  3. Temperature dysregulation, including night sweats, feverish feeling, cold extremities: The bulk of research evidence points to hypothalamic dysfunction as the cause of temperature dysregulation, immune activation (sickness response) as the cause of night sweats and feverish feeling, and circulatory dysautonomia as the cause of cold extremities. I see no way for poor oxygenation to explain these.
  4. Hot/cold hypersensitivity: There may be different etiologies at play here, but I don't see poor oxygenation as one of them.
Just my 2 cents! :)
 
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Consul

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PEM
Pyrrhus if there are neurons that are poorly oxygenated because of low cerebral blood flow isnt it plausible that overuse (exertion) of these neurons could lead them into a dysfunctional state like PEM? Basically these neurons are barely limping along and then the high demand of exertion drives them further into the ground. The neurons can be activated by physical activity or emotional or mental exertion. A lack of ATP could drive the whole cell into a dysfunctional state lasting e.g a week.

IMMUNE
If you have dysfunctional mitochondria because of low oxygenation then cant that translate into immune dysregulation like excessive inflammation or e.g immune mediators out of control causing flu like symptoms. My understanding is that mitochondria is a central player in immune responces.


*I will also throw in this here:
https://www.ncbi.nlm.nih.gov/labs/pmc/articles/PMC7044650/

[...]the majority of ME/CFS patients (90%) show an abnormal cerebral flow reduction during orthostatic stress testing.[...]
 
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Pyrrhus

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Basically these neurons are barely limping along and then the high demand of exertion drives them further into the ground. The neurons can be activated by physical activity or emotional or mental exertion.
I think you might be on the right track here in trying to describe the physiology of PEM and I am certainly open to the idea that poor oxygenation of these neurons or impaired mitochondria in these neurons can certainly play a role in PEM.

But one would still need to elucidate exactly how such intense firing of a neuron ("exertion") results in such a dramatically dysfunctional neuron, often with a 24-48 hour delay.

If you have dysfunctional mitochondria because of low oxygenation then cant that translate into immune dysregulation like excessive inflammation or e.g immune mediators out of control causing flu like symptoms.
I'm afraid I don't see how. Hypoxia (poor oxygenation) can lead to tissue damage, and there will be some minor inflammation as part of the healing process, but that wouldn't be anything like the type of inflammation one might see with the sickness response (flu-like symptoms).

*I will also throw in this here:
https://www.ncbi.nlm.nih.gov/labs/pmc/articles/PMC7044650/

[...]the majority of ME/CFS patients (90%) show an abnormal cerebral flow reduction during orthostatic stress testing.[...]
Yes, this is the known poor oxygenation of the brain that comes from orthostatic intolerance.

Hope this helps.
 
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I don't see how poor oxygenation can be a significant factor in most of the main symptoms of ME.
Isn't the idea that there are cascade effects among the various factors (with vicious cycles): poor oxygenation => poor/altered energy production => oxidative stress => inflammation + gut dysbiosis => (auto)immune response + vascular damage/dysfunction => poor oxygenation, and so on? And all of this applies to the brain (which has disproportionate energy demands) as much as to the body. So while poor oxygenation doesn't cause all of the symptoms directly, it may be an important link in the chain that does.
 

Pyrrhus

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poor oxygenation => poor/altered energy production => oxidative stress => inflammation
This part makes sense, although the inflammation involved would be relatively minor.

inflammation + gut dysbiosis => (auto)immune response + vascular damage/dysfunction => poor oxygenation, and so on?
This is an interesting hypothesis. I don't know of much evidence supporting it, but it's interesting.
 
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Isn't the idea that there are cascade effects among the various factors (with vicious cycles): poor oxygenation => poor/altered energy production => oxidative stress => inflammation + gut dysbiosis => (auto)immune response + vascular damage/dysfunction => poor oxygenation, and so on? And all of this applies to the brain (which has disproportionate energy demands) as much as to the body. So while poor oxygenation doesn't cause all of the symptoms directly, it may be an important link in the chain that does.
Perhaps gut dysbiosis caused by biofilms? Could Biofilms be in the root of this dysbiosis?