The Occurrence of Hyperactivated Platelets and Fibrinaloid Microclots in ME/CFS, 2022, Nunes, Pretorius et al

Abstract
We have previously demonstrated that platelet poor plasma (PPP) obtained from patients with LongCovid/Post-Acute Sequelae of COVID-19 (PASC) is characterized by a hypercoagulable state reflected in hyperactivated platelets and the presence of considerable numbers of fibrin(ogen) amyloid microclots or fibrinaloid microclots. Due to substantial overlap in symptoms and aetiology between PASC and ME/CFS, we investigated whether coagulopathies, platelet hyperactivation and/or fibrin amyloid formation differed between individuals exhibiting ME/CFS and gender- and age-matched healthy controls. ME/CFS patients were statistically far more hypercoagulable as judged by thromboelastography of both whole blood and platelet-poor plasma.

The area of plasma images containing fibrinaloid microclots was commonly more than 10-fold greater in untreated platelet-poor plasma from individuals with ME/CFS than in that of healthy controls. A similar difference was found when the plasma samples were treated with thrombin. Using fluorescently labelled PAC-1, which recognizes glycoprotein IIb/IIIa, and CD62P, which binds P-selectin, we observed massive hyperactivation and spreading of platelets in samples from individuals with ME/CFS. Using a quantitative scoring system, this was found to have a score of 2.72 ± 1.24 vs 1.00 (activation with pseudopodia formation) for healthy controls.

We conclude that ME/CFS is accompanied by substantial and measurable changes in coagulability, platelet hyperactivation, and fibrinaloid microclot formation. However, fibrinaloid microclot load was not as prevalent as was previously noted in PASC. Fibrinaloid microclots, in particular can provide a ready explanation, via (temporary) blockage of microcapillaries and hence ischaemia, for many of the symptoms, such as fatigue, seen in patients with ME/CFS. The discovery of these biomarkers pointing to significant and systemic endothelial inflammation, represents an important development in ME/CFS research. It also points at novel treatment strategies using known drugs and/or nutraceuticals that target systemic vascular pathology and endothelial inflammation.

Preprint

Now published:
https://mdpi-res.com/d_attachment/p...armaceuticals-15-00931.pdf?version=1658922364
Non-PDF version, https://www.mdpi.com/1424-8247/15/8/931

 

"ME/CFS patients (n=25; 20 females; 5 males) were recruited via the ME/CFS Foundation of South Africa, and only included in this study if they had not previously been infected with the COVID-19 virus. Participants had to have been diagnosed for longer than 6 months, and were still asked to complete the International Consensus Criteria (ICC) questionnaire 48 to gain an understanding of their perspective of disease severity."

 

Would this not show up on blood work in a lab? I can't count the number of blood tests I've had done over the past four years and surely this would have shown up in at least one of the tests as my blood has been very well tested for many things.

 

Yes, I agree. I have been wrong before but these results do not look real to me.
And I don't think they make any sense in terms of the clinical picture of ME.

 

I don't think so, not in the NHS (which I realize doesn't routinely operate in Canada ).

I've had a nurse shake a vial of fresh blood (mine) in front of me and ask me if it looked right, it was thick, dark, not red, viscous, and had taken her minues to obtain, and looked like tar.

I am unaware of any results being sent back to my GP which suggested anything out of the ordinary.

They only test the things they are asked to, and even then, if anything is too out of range, they seem to assume it's an error and don't bother mentioning it at all.

 

I hope this means that there will soon be a replication attempt. It does look like the kind of finding that would help us a lot in making sense of what is going on.

In my opinion, microclots that impair microcirculation to some degree seems like it could fit with an inability to tolerate activities. Activities become abnormally stressful for the body in a weird, hard to describe way.

 

https://twitter.com/user/status/1534628098339131395

 

Small numbers but very interesting. Quite an effect…

I wonder where I could get this testing procedure done in NZ? @SNT Gatchaman (for my own scientific exploration, will pay for it as likely to be experimental)

I do think this warrants further investigation by other specialties like haematology

Will be good to see what critical discussion arises within the scientific community.

 

Interestingly, 40% had comorbid leaky gut/gut dysbiosis so could suggest (big suggest) it might be related to pwME that have prominent bowel symptoms as part of their presentation (speculation on my part).

 

Trish, do you recall how many of the 25 had these clots? Thanks.

 

Having thought about this, I don't care if it makes no sense or doesn't fit with established knowledge. We need a replication to confirm or refute this.

ME/CFS seems to be an illness where established knowledge is of limited use because it seems to be in its own distinct category, with some separation between other categories of illness that are better understood.

 

And how does one heal this? A million protocols for this have been suggested, and no result. Thank you.

 

Not a direct replication, but the Roy et al paper on ATG13 did find similar results, also showing quite a strong effect, in a small sample (7 patients and 7 controls) using Thioflavin T as well. This is pretty similar to the first part of what Pretorius did in this paper (though not the addition of thrombin or the platelet part). The main difference was Pretorius used platelet poor plasma and Roy was using serum with ammonium sulfate and a longer time period (though they saw significant differences between patients and controls at 30 minutes as well). It seems reasonably likely to me that they are observing the same phenomenon — so the question may be more what it means.

 

I had a SED, Fibrinogen, Thrombin Antithrombin Complexes, Soluble Fibrin Monomer, CD62, CD62+ADP, Platelet Activation Index, Prothrombin Fragment 1+2 and Antithrombin Activity test done in 2001 and they all came back normal.

 

If this turns out to be a solid finding, I wonder how much we'll see it in other diseases as well. The same authors have previously done work that looks at least superficially similar in rheumatoid arthritis. It might be a quite general finding in the end.

Regarding the first experiment: "A nonparametric Mann-Whitney test was performed, which indicated a significant difference (p<0.0001) between the control (0.10 ± 0.54) and ME/CFS (1.37 ± 3.05) mean % area amyloid."

1.37 ± 3.05 definitely indicates that a significant number of the ME/CFS patients had near zero results similar to controls. Somewhere between 5-10 out of 25 maybe?

 

Basic coagulation screening is normal. I expect it was normal in their patient group too. TEG (which was non-universally abnormal in their sample) is not done as part of standard tests of coagulation — it's typically an ICU/theatre procedure. I also note @Mij's more advanced tests from 2001 above were normal (esp platelet activation). Some 20 year old techniques may well have been superseded.

One idea I had was that fibrinogen levels might be truly elevated but reading as normal range. This might be because the Clauss clinical assay estimates fibrinogen levels by its ability to produce clots in the presence of more-than-enough thrombin. Abnormally glycated fibrinogen (eg poorly controlled diabetic patients) does not clot so well, so reads artificially low. A similar thing might be happening here: more fibrinogen but it doesn't clot as effectively — i.e. nearly net zero for quantity x quality, otherwise LC patients would all be dropping dead of PE etc.

Have had this exact experience too.

I share your concerns. "Microclots" may be circulating, but they may simply form up once the blood is out, non-circulating and handled. I think hypercoagulation is very important but it's probably only part of the whole explanation. Other signals, such as the autophagy/ATG13 finding recently, might map better with dysfunctional mitochondria. I think we need to look at all these aspects as fully as possible.

Interesting to note that the findings in this small study were not universal. Also much less than their previous LC studies. That suggests to me that LC might have a "double effect" whereby immune dysregulation causes abnormal clotting in some/many/all, but the spike protein itself also causes this and amplifies the effect dramatically. This might be why LC patients have a much higher rate of late thromboembolic complications than ME patients (as far as seems to be reported in both groups).

I'd be interested to run this same evaluation on patients known to be hypercoagulable for other reasons, eg cancer patients. Do they show this amyloid signal or are they just clotty in the standard way? However, there might be just far too much going on in the blood of say an ovarian cancer patient to be able to make sense of observations. And platelets become hyperactive very easily.

Very doubtful it could become available here or other countries, even ad hoc. There would need to be a high throughput quantitative assay. Research needs to move further with this concept but different tools are required. We need the upstream pathology with markers of immunothrombosis etc.

Agree, but no-one seems prepared to accept that they could be on the edge of a paradigm shift. This is par for the course for paradigm shifts ("Paradigm shifts arise when the dominant paradigm under which normal science operates is rendered incompatible with new phenomena, facilitating the adoption of a new theory or paradigm", "Paradigm shifts tend to be most dramatic in sciences that appear to be stable and mature").

Sure, it may all be artefact and amount to nothing, but if amyloid clotting is real that could tie in to many chronic disease processes. Unfortunately as noted by Jo previously, everyone in the field looks at the word "microclot" and says "nope, not worth reading, microclots can't persist in the circulation". I think amyloid clotting is a valid concept and it might be ideal to try and separate that out from "microclots". Ie explore abnormal fibrinogen in its amyloid form and park the idea that microclots are intermittently blocking capillaries etc.

 

If I have this right, this article is basically about viscous blood and microclots.

It looks like it's coming from a different angle - some different function or process in the body, but David Berg, and before him Dr.Leslie Simpson researched viscous blood many years ago. I recall heparin as a suggestion for this issue in the 2000's. At about the time I was diagnosed, in 1991, evening primrose oil was being suggested.

It would be good to see this research replicated.

 

I have factor V Leiden. This is often described as sticky blood or blood which is more liable to clot in certain circumstances such as immobility following surgery or during long haul air travel. A blood thinner, low molecular weight heparin, is often prescribed in these circumstances to prevent the possibility of clotting.

I have noticed on many occasions that the fatigue/ exhaustion that accompanies ME is significantly better after taking heparin in order to fly, allowing me to function at a higher level than expected. I don't know how or even whether this links with microclots but heparin improves my functioning.

It is a shame Dr Simpson's work from the 90s was not followed up, and that the OMF has not seemed to complete its work on red cell deformability. The latter might not link to microclots but blood related research has been started before and then abandoned. Resia Pretorius has been working very hard on microclots; I hope she has the resources to complete her efforts.