The micro-clot finding in Long Covid — implications for the possible aetiology of ME/CFS

Mij said:
Do you know if micro clotting is the result from a persistent infection?
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Microclotting occurs in subacute bacterial endocarditis. It may occur in other infections but nothing obvious comes to mind immediately.

Remember that what has been reported by Pretorius et al is clotting in a tube rather than clotting in a patient. Clotting occurs in acute severe Covid19 but I have not seen evidence that would go with microblogging in patients in LongCovid. There seems to be an increased risk of some thrombotic events like venous thrombosis of pulmonary embolism but that may just relate to inactivity and generally being unwell.
 
Jonathan Edwards said:
The idea that it might take that long is conceivable. I just cannot at the moment conceive of any mechanism that is likely to apply.

One of the key aspects of credibility of anecdotal evidence from cases or pilot studies is seeing the therapeutic dynamic follow an expected path. Like a dose response curve it turns a single observation into a shape that is far less likely to have occurred by chance.
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Yes — some conjecture:

What we see is a mixture of anecdotal reports of some people recovering and some not recovering, but from what I've read many do report symptomatic improvement on this combined anticogualant / antiplatelet regimen. I think it's important to note that the combination may be having a positive symptomatic effect, but that people (including on S4ME) report no benefit to long-term ME on a single agent, eg "I was on aspirin or warfarin or rivaroxiban" etc.

I think what this is all pointing to is that the microclotting observation is a downstream effect. It may be genuinely reflecting hypercoagulability, but as Jo says this is clotting in a tube and we don't know if microclots are happening in vivo. It may be a specific feature of LC, or a lesser feature of ME, or present in both but simply something that largely resolves with time even as the underlying ME/LC persists.

I'm running with the idea that "something in the blood" is generated or upregulated — maybe not a lot (so we haven't properly taken notice), but constantly — enough to have major physiological effect over sufficient time. That something has a wide area-of-effect: say the entire vascular lining resulting in endothelial dysfunction; and impairments to some contents of the blood, eg lymphocytes and platelets. That results in many deleterious effects, just one of which might be hypercoagulability which is symptomatically, and maybe also pathogenically, cumulative.

Reducing the hypercoagulability might help with the symptoms, something the patients notice. But it doesn't of itself stop the generation of the something-in-the-blood. That process may simply resolve with time in some, but the treatment regimen is erroneously given credit for this rather than simply some symptomatic relief. (If hypercoagulability somehow also up-regulates the something-in-the-blood as a feed back, then yes it may also be helping).

Aside: I see the team put out a preprint earlier in the week, where they demonstrate the effect via an imaging cytometer. However, I don't think this advances things much or even at all. They again used platelet-poor plasma in citrate tubes, with Thioflavin T and were correlating their prior method with the new one. They suggest that a standard lab (non-imaging) cytometer could also be used as a high throughput assay (counting objects of x-y size), which I presume would be in the absence of ThT. I don't know if that could work with citrated whole blood. They say this is the first attempt to use a cytometer to analyse a non-cell.

Jonathan Edwards said:
A thrombus in a capillary will block it completely - whether child or adult. This is visible. I still don't know what people think the actual pathology is.
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I think their idea is that generally flow is impaired rather than blocked blocked? They describe the microclots as resistant to breakdown, but I think also nebulous or at least deformable. Then they would be a bit like a red or white cell that adjusts to fit through the smaller capillaries. Over the capillary network, general vascular flow and flow control might then be degraded (adding to the probably more important NO reduction / endothelial dysfunction).

They always say that the capillaries are blocked, preventing oxygen getting to the tissues - QED. I don't think so simple, although maybe it's contributing. I think the metabolic reprogramming is the primary event — eg the Warburg effect of aerobic glycolysis and reduced fatty acid oxidation etc across many tissues, but perhaps in particular affecting endothelium and lymphocytes.

Jonathan Edwards said:
Indeed, where is the Budd-Chiari liver enlargement or the ventilation perfusion mismatch or reduced GFR?
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I haven't looked at liver, but for lungs and kidneys:

Pulmonary Dysfunction after Pediatric COVID-19 (2022, Radiology)

A total of 54 participants after COVID-19 infection (mean age, 11 years ± 3 [SD]; 30 boys [56%]) and nine healthy controls (mean age, 10 years ± 3; seven boys [78%]) were included: 29 (54%) in the COVID-19 group had recovered from infection and 25 (46%) were classified as having long COVID on the day of enrollment. Morphologic abnormality was identified in one recovered participant. Both ventilated and perfused lung parenchyma (ventilation-perfusion [V/Q] match) was higher in healthy controls (81% ± 6.1) compared with the recovered group (62% ± 19; P = .006) and the group with long COVID (60% ± 20; P = .003). V/Q match was lower in patients with time from COVID-19 infection to study participation of less than 180 days (63% ± 20; P = .03), 180–360 days (63% ± 18; P = .03), and 360 days (41% ± 12; P < .001) as compared with the never-infected healthy controls (81% ± 6.1).
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Persistent 129Xe MRI Pulmonary and CT Vascular Abnormalities in Symptomatic Individuals with Post-acute COVID-19 Syndrome (2022, Radiology)

Together, the abnormal MRI and CT findings were consistent with abnormal gas exchange stemming from the alveolar tissue barrier and pulmonary vascular compartments. Similar to previous reports of post-COVID coagulation and emboli, it is possible that we were measuring micro-embolic or micro-thrombotic obstruction of small capillaries which explained the abnormal RBC signal. Other vascular changes, such as vascular injury, vascular remodelling or shunting may also be possible and has previously been hypothesized post-COVID-19. Post-mortem micro-CT imaging of COVID-19 infection supports these interpretations as abnormal alveolar-level structures and occluded capillaries were observed.
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Kidney Outcomes in Long COVID (2021, J Soc Am Nephrology) —

Beyond the acute illness, 30-day survivors of COVID-19 exhibited a higher risk of AKI (aHR, 1.94; 95% CI, 1.86 to 2.04), eGFR decline ≥30% (aHR, 1.25; 95% CI, 1.14 to 1.37), eGFR decline ≥40% (aHR, 1.44; 95% CI, 1.37 to 1.51), eGFR decline ≥50% (aHR, 1.62; 95% CI, 1.51 to 1.74), ESKD (aHR, 2.96; 95% CI, 2.49 to 3.51), and MAKE (aHR, 1.66; 95% CI, 1.58 to 1.74).
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The mechanism or mechanisms of increased risk of AKI, eGFR decline, ESKD, and MAKE in the post-acute phase of COVID-19 infection are not clear. Although initial observations suggested that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) may have kidney tropism, more recent evidence does not endorse the earlier assessment. Other potential explanations include dysregulated immune response or autoimmunity, persistent inflammation, disturbances in endothelial function and the coagulation system, and disturbances in the autonomic nervous system.
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"But the nature of the biological evidence remains contentious. Jeffrey Winters, a pathologist and chair of the division of transfusion medicine at the Mayo Clinic, said the way Pretorius and Kell describe amyloid clots is confusing to scientists who view amyloids in a different context. “I don’t understand how they are using this term,” he said. “I assume that what they are referring to is not what I refer to as amyloids,” which he describes as various proteins that deposit into organs and don’t ordinarily float around in the blood. “I don’t understand what they are testing, and I don’t understand what they are seeing.” The dye used in the test, thioflavin T, can and does stain other things that are not amyloids, Winters said in an email"
 
Only read some of it - my reaction was to think --- why don't they [Long COVID] run a large GWAS [DecodeME size]? I think Jonathan posted (previously) that there was some evidence of abnormalities in clotting proteins - sort of theory GWAS would test.
 
John Mac said:
https://undark.org/2024/05/20/bad-blood-microclots-long-covid/

Bad Blood? The Uncertainty Around Microclots and Long Covid

Scientists are debating whether microscopic blood clots are responsible for the wide range of symptoms in long Covid.
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The blood thinner arm of the U.K.’s Stimulate-ICP study is testing a single drug — rivaroxaban — rather than the triple therapy administered by Vaughn and other doctors.
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It's only an N=1, but I've been on rivaroxaban for 8 years and warfarin for 10 years previous to that and my ME/CFS has actually gotten worse while I've been on anti-coagulants. The one I'm more agnostic about is enoxaprin (Lovenox) as I did experience a significant improvement during the 10 days I was on it. However, there were several other chemical confounders during my use of that to be able to know if it was indeed the enoxaprin that caused the improvement.
 
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Months on, where have we got to with this hypothesis?
I think it's now clear that these "microclots" exist in processed plasma. We've seen them identified in a couple of different ways.

Some of the questions that come to mind are
* are these particular amyloid microclots actually in the blood? I still don't think we have seen any evidence of that.
Why are proponents of the microclot theory so sure that there are microclots in the blood? Surely there is a method that would 'see' them in a living person if they were there? There was that study that had amyloid fluorescing, but not in capillaries - so it didn't seem that the microclots were there.

* Has anyone taken rat blood (from rats with and without an infection) and seen if they could find ex-vivo microclots in the plasma?

* is the prevalence of microclots ex vivo significantly affected by small changes in blood storage and processing? Are the prevalence levels reliable, replicable? (i.e. if different labs calculate prevalence from the same blood in a blinded fashion, do they get the same result? Do you get the same result from someone's blood sampled each day for a week?) Could something about the donor somewhat unrelated to their pathology or downstream of the pathology (e.g. dehydration, high blood sugar) affect whether microclots form ex vivo?

* does higher fibrinogen in the blood correlate with the presence of microclots ex vivo? Or with other proteins/ other substances in the blood? Is there something in the blood that makes ex vivo microclots more likely to form?
google said:
Fibrinogen is a glycoprotein complex that circulates in the blood of all vertebrates. When tissue or blood vessels are injured, thrombin enzymatically converts fibrinogen into fibrin, which forms a blood clot to stop bleeding.

Elevated fibrinogen levels can be caused by a number of conditions, including acute infections, cancers, heart disease, stroke, and trauma. These elevated levels are usually temporary and return to normal after the underlying condition is resolved.
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* is the microclots ex vivo prevalence associated with thrombotic events in the various diseases?

*are microclots relevant to the pathology of ME/CFS?
Digital holo-tomographic 3D maps of COVID-19 microclots in blood to assess disease severity, 2023, Bergaglio noted that
Currently, it is estimated that approximately 30% of individuals with COVID-19 continue to suffer from a variety of different symptoms involving specific or multiple organ systems, with neurological, neuropsychiatric, and cardiorespiratory clinical presentations (8, 9), a condition known as post-acute sequelae of COVID (PASC) or long COVID (8, 10, 11).
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So Long covid cohorts are a very mixed bag and there is likely to be people with overt some tissue damage and resulting thrombosis (ie normal clots). I don't think the studies suggesting higher levels of microclots in the processed plasma of people with ME/CFS and LC ME/CFS are terribly convincing yet. (?)

I don't think we have seen higher levels of thrombotic events in non-LCME/CFS. There was that interesting study, I think from the UK Biobank? that presented odds ratios for a whole range of health conditions for people with/without ME/CFS. I'm not sure where we are with replicated findings of molecules like fibrinogen in ME/CFS.


I'd really appreciate it if people who believe that microclots are important in ME/CFS could answer some of my questions. Does anyone know if there is a study planned/underway of the favoured anti-coagulation treatment?
 
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I also have a question. I admit I haven't been able to keep up with the research, so it may have been answered.

I gather Pretorius was studying microclots in other diseases before Covid came along, is that correct? If so, what were the clinical and biomedical findings in these other diseases that were attributed to microclots?

Edit:
I found this paper from 2019 by Pretorius et al.
Parkinson’s Disease: A Systemic Inflammatory Disease Accompanied by Bacterial Inflammagens
 
Hutan said:
Surely there is a method that would 'see' them in a living person if they were there? There was that study that had amyloid fluorescing, but not in capillaries - so it didn't seem that the microclots were there.
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Seeing particles in blood in living people is not easy, although you can see red cells moving in retinal vessels with the right equipment I think. (You can see them in yourself with a clever trick with a light shining on to your eyeball from the side while you wiggle it.)

But a reasonable ask would be to see these particles in freshly drawn whole blood, without any processing.

I may be wrong but I think one can argue like this:
If these particles are able to block capillaries they will be of the same size as white blood cells or larger and would be visible on a Coulter counter. The Coulter count shines a fine light beam on to a stream of cells and measures the size and character of each cell separately using forward scatter and side scatter. You can get more information by staining cells with fluorescent dyes but that introduces potential artefacts. Let's stick with Coulter. The Coulter machine counts cells of different character and plots them out on a chart in two dimensions (potentially more). If microclots were big enough to block they should show up in a new part of the plot or scattered across regions - just not in a typical place for usual cells.

If the clots are slowing blood flow because they make it more viscous but are too small to block capillaries they would need to be present in high enough concentration to have more effect on viscosity than the usual cells. That is a tall order. About 45% of blood is made of normal red cells - making it much more viscous than plasma. To increase viscosity further noticeably you would need microclots in numbers sufficient to produce a pretty major fog of tiny particles on the Coulter plot. You would also expect to see a band of microclots sitting on top of the red cells when blood was left to settle or centrifuged.

So to produce any clinical effect you would expect the microclots to be barn door observable on routine clinical kit.

There is an interesting question as to whether these are in vitro artefacts that reflect a change on plasma soluble proteins rather than lumps. Both fibrinogen and amyloid proteins are standard acute phase reactants - which means they go up in inflammatory disease. The fibrinogen is present in enough quantity to cause a raised ESR and plasma viscosity. But we do not see those, or acute phase response proteins in ME/CFS or LC.

Some unusual protein might form amyloid lumps but there aren't that many options and such proteins should have been identifiable using standard immunochemistry.

So to me it is all very mysterious how on earth there can be anything there of clinical significance.

The muscle tissue that showed amyloid type fluorescence was not showing clots. The bright objects were 'vermiform' lines and dots and I am pretty certain they were nerves. Nerve endings are known to pick up that stain. It may be important that more staining was seen but I don't think it has anything to do with microclots.
 
Jonathan Edwards said:
There is an interesting question as to whether these are in vitro artefacts that reflect a change on plasma soluble proteins rather than lumps. Both fibrinogen and amyloid proteins are standard acute phase reactants - which means they go up in inflammatory disease. The fibrinogen is present in enough quantity to cause a raised ESR and plasma viscosity. But we do not see those, or acute phase response proteins in ME/CFS or LC.
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I may be being stupid, but …

Is a reproducible difference in vitro being found in the presence of micro clots in the processed plasma between people with Long Covid or ME and healthy controls? If this is the case but it is looking like such micro clots are not an issue in vivo, does this mean that there is a real as yet unidentified anomaly in the patients’ blood though the micro clots themselves are not the cause of any pathology rather a downstream effect seen after processing samples.
 
Thanks Jonathan
Jonathan Edwards said:
If these particles are able to block capillaries they will be of the same size as white blood cells or larger and would be visible on a Coulter counter. The Coulter count shines a fine light beam on to a stream of cells and measures the size and character of each cell separately using forward scatter and side scatter. You can get more information by staining cells with fluorescent dyes but that introduces potential artefacts. Let's stick with Coulter. The Coulter machine counts cells of different character and plots them out on a chart in two dimensions (potentially more). If microclots were big enough to block they should show up in a new part of the plot or scattered across regions - just not in a typical place for usual cells.
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I think we have seen one study that did something like this. There was a PCA type chart, with some groups of oddly shaped particles off to the right. I'll see if I can find it. I can't recall how fresh the blood was.

Jonathan Edwards said:
If the clots are slowing blood flow because they make it more viscous but are too small to block capillaries they would need to be present in high enough concentration to have more effect on viscosity than the usual cells. That is a tall order. About 45% of blood is made of normal red cells - making it much more viscous than plasma. To increase viscosity further noticeably you would need microclots in numbers sufficient to produce a pretty major fog of tiny particles on the Coulter plot. You would also expect to see a band of microclots sitting on top of the red cells when blood was left to settle or centrifuged.
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To play devil's advocate in a poorly thought through way, what if the particles have an electrostatic force or something? So, you don't necessarily have swarms of them, but they encourage other things to clump together or they loosely attach to the endothelium? A lot of people with ME/CFS do say that there is trouble when blood samples are taken, that the blood flows slowly. But maybe everyone has that happen from time to time, when the phlebotomist misses the mark a bit.
 
I recall that an independent laboratory in the north of England, (independent of Pretorius et al), was going to try to replicate her work. I think the lead researcher was a haematologist. That was some months ago and not seen the results on here

My concern with this theory is there is no clear pathogenesis from having these fibrin-amyloid "micro clots" and the pathology that they cause. I think most is supposition. All I have read is somehow these particles are inflammatory and work on the endothelium or clotting cascades etc or on brain tissue and speculation this causes neuroinflammation.

But I have yet to see the necessary research to show pathogenesis, even in LC. I hoped we would have more information by now. Although Pretorius has done her study in South Africa, a few years ago, showing these "micro-clots" in people with ME. I think it would be useful to know how frequent microclots are in the general population. So a much larger study.

I don't think these microclots are blocking/impairing the microcirculation. Wouldn't it be pretty easy to attach some sort of radioactive particle to these microclots and do radiographic studies of them going around in the circulation? eg to see if they are impairing the microcirculation or activating something in the endothelium or in brain cells?

Could they test to see if there is some sort of dose response? i.e. the more micro-clots you have - the more severe the illness? but that doesn't really answer the question that is most needed - what are these fibrin amyloid particles actually doing?

I would be concerned that people with ME were being given anticoagulant treatment on the basis of their research. I am not sure how convincing the research is for LC either.
 
Jonathan Edwards said:
If these particles are able to block capillaries they will be of the same size as white blood cells or larger and would be visible on a Coulter counter. The Coulter count shines a fine light beam on to a stream of cells and measures the size and character of each cell separately using forward scatter and side scatter. You can get more information by staining cells with fluorescent dyes but that introduces potential artefacts. Let's stick with Coulter. The Coulter machine counts cells of different character and plots them out on a chart in two dimensions (potentially more). If microclots were big enough to block they should show up in a new part of the plot or scattered across regions - just not in a typical place for usual cells.
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Hutan said:
I think we have seen one study that did something like this. There was a PCA type chart, with some groups of oddly shaped particles off to the right. I'll see if I can find it. I can't recall how fresh the blood was.
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It's not yet published, but hopefully at least in preprint by the end of this year (Kräter, Scheibenbogen) but see the presentation here —



hibiscuswahine said:
Could they test to see if there is some sort of dose response? i.e. the more micro-clots you have - the more severe the illness?
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This is unpublished.
 
SNT Gatchaman said:
Still in preprint, but see thread for Increased fibrinaloid microclot counts in platelet-poor plasma are associated with Long COVID (2024, Preprint: MedRxiv)
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Thank you, I missed that one. It is good to see they have replicated her work but still unclear the clinical significance which may be made clearer in the preprint you mentioned on a dose response. Could it be a situation of ongoing active inflammatory response in some people with LC? (something I think is less clear in ME/CFS)

It appears from the Youtube video with Dr Martin Krater, that he believes that some people with LC are not able to clear these fibrin-amyloid micro clots from the circulation and that might be the basis of the pathology. The electron microscopy is interesting, I would like to see if research can tie the presence of these particles to specific symptoms and clinical signs of poor perfusion of tissues or increased viscosity of the blood etc.
 
Hutan said:
To play devil's advocate in a poorly thought through way, what if the particles have an electrostatic force or something?
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So we are now really talking about non-covalent chemical interaction in solution. Biological chemistry gets complicated at these intermediate scales but an electrostatic effect without a 'lump' effect really boils down to a non covalent chemical effect. That is what fibrinogen and immunoglobulin normally do very well. They increase plasma viscosity (and ESR) by attracting red cells together. But these have not been reported as abnormal in ME/CFS or 'ME/CFS-type' LC. Autoantibodies could do it but they do not form amyloid aggregates.
 
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