A central role for amyloid fibrin microclots in long COVID/PASC: origins and therapeutic implications, Kell, Laubscher, Pretorius, 2022

[SNT Gatchaman]

A central role for amyloid fibrin microclots in long COVID/PASC: origins and therapeutic implications
Douglas B. Kell, Gert Jacobus Laubscher and Etheresia Pretorius

Post-acute sequelae of COVID (PASC), usually referred to as ‘Long COVID’ (a phenotype of COVID-19), is a relatively frequent consequence of SARS-CoV-2 infection, in which symptoms such as breathlessness, fatigue, ‘brain fog’, tissue damage, inflammation, and coagulopathies (dysfunctions of the blood coagulation system) persist long after the initial infection.

It bears similarities to other post-viral syndromes, and to myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS). Many regulatory health bodies still do not recognize this syndrome as a separate disease entity, and refer to it under the broad terminology of ‘COVID’, although its demographics are quite different from those of acute COVID-19.

A few years ago, we discovered that fibrinogen in blood can clot into an anomalous ‘amyloid’ form of fibrin that (like other β-rich amyloids and prions) is relatively resistant to proteolysis (fibrinolysis). The result, as is strongly manifested in platelet-poor plasma (PPP) of individuals with Long COVID, is extensive fibrin amyloid microclots that can persist, can entrap other proteins, and that may lead to the production of various autoantibodies.

These microclots are more-or-less easily measured in PPP with the stain thioflavin T and a simple fluorescence microscope.

Although the symptoms of Long COVID are multifarious, we here argue that the ability of these fibrin amyloid microclots (fibrinaloids) to block up capillaries, and thus to limit the passage of red blood cells and hence O2 exchange, can actually underpin the majority of these symptoms.

Consistent with this, in a preliminary report, it has been shown that suitable and closely monitored ‘triple’ anticoagulant therapy that leads to the removal of the microclots also removes the other symptoms. Fibrin amyloid microclots represent a novel and potentially important target for both the understanding and treatment of Long COVID and related disorders.

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[SNT Gatchaman]

Selected quotes (minus refs)

We here make the case (with evidence) that much of the aetiology of long COVID can be attributed to the formation of aberrant amyloid fibrin microclots, triggered in particular by the SARS-Cov-2 spike protein, and that by inhibiting the transport of erythrocytes to capillaries, and hence O2 transfer, it is these amyloid microclots that are mainly responsible for the various long COVID symptoms observed. The microclots may also present novel antigens that lead to the production of autoantibodies, that can exacerbate symptoms further.

While we do not yet know the details of the mechanical properties of the fibrin amyloid microclots, it is known that amyloid fibrils can typically exhibit unusually high mechanical strength and resistance to deformation. The stiffness also increases with the thickness of the fibres. This implies strongly that the fibrinaloids are likely to be more prone to ‘getting stuck’ in capillaries.

One source of the continuous production of a stimulus is represented by microbes, including virions, that persist in a largely dormant state (often in intracellular reservoirs) but can occasionally continue to replicate. There is now considerable evidence for the persistence of SARS-CoV-2. Another is the continued release of sequestered microbially derived substances than can act as stimuli for continuing microclot formation. Here, the finding that S1 spike protein can itself persist in CD16+ Monocytes in PASC for up to 15 months post-infection is highly relevant, as the amplification of trigger proteins to make microclots as part of the clotting mechanism means that miniscule (and highly substoichiometric) amounts of suitable triggers can suffice. This alone is sufficient to account for the chronic nature of such diseases.

An extra consequence of the production of microclots that sequester other proteins whose concentration would otherwise appear elevated is that those proteins do not then appear in plasma from which the microclots have been removed (e.g. by centrifugation), and thus they do not manifest, and cannot usefully be used, as biomarkers.

The list of molecules that could induce this anomalous clotting, additional to the bacterial LPS that was then our main focus, included iron ions, oestrogens, lipoteichoic acid, and serum amyloid A.

While the list of the many other molecules that can plausibly effect this anomalous amyloid-type clotting is unclear, such clots may also be observed in the blood of individuals with inflammatory diseases such as Alzheimer’s, Parkinson’s, type 2 diabetes, and rheumatoid arthritis. Similar phenomena have also been observed in the pregnancy disorder pre-eclampsia, where there is also strong evidence for a microbial component.

Importantly, the addition of purified, recombinant SARS-CoV-2 S1 spike protein to coagulation-competent normal plasma is sufficient to induce the formation of anomalous clots that adopt amyloid states that are also resistant to fibrinolysis. Note that the observations of the microclots in (platelet-poor) plasma are performed without the addition of exogenous thrombin; they are naturally there in the circulation of patients with both acute and long COVID. The size of these amyloid microclots, that can be observed microscopically and stained e.g. with thioflavin T (Figure 4; control vs LC plasma), are typically anywhere from 1–200 mm; this means that they can effectively block up, and inhibit blood flow through, all kinds of microcapillaries, thereby strongly lowering the availability of oxygen in tissues.

Importantly, it has already been shown that both acute and Long COVID are accompanied by immunological dysfunction, and by novel antibodies, including in the latter case to (an ‘abnormal’ but unspecified form of) fibrin.

One approach to understanding the mechanisms of long COVID is to analyse epidemiological data, since the characteristics of those experiencing acute COVID differ markedly from those with long COVID. Among these (and in significant contrast with the case of acute COVID), is a striking over-representation of long COVID in women, and especially younger women. From this point of view, it is of considerable interest that female sex hormones can induce anomalous blood clotting.

While it has been somewhat slowed by the generally sluggish recognition of Long COVID, it is to be expected that different strains of SARS-CoV-2 may have different tendencies to induce it. To this end, it is reasonable that if microclots are important to LC their prevalence should also vary with the severity or frequency of LC induced by different strains of SARS-CoV-2. In a sense this would provide a very important kind of ‘control’, since the only thing varying as the stimulus is the strain of SARS-CoV-2.

While we here focus on SARS-CoV-2, we note that all kinds of molecules have been shown to affect the extent of fibrinaloid clot formation, including iron, other amyloids, bacterial cell wall components, etc., and that we have observed them in a variety of chronic, inflammatory diseases including Alzheimer’s, Parkinson’s disease, Type 2 diabetes (where the amyloid protein amylin is of course a well-known player) and rheumatoid arthritis. Although not yet tested directly, we consider it likely that this will also be true for infectious diseases known to be causing similar post-infection syndromes, such as Dengue, Ebola, Lyme, Zika and others where viruses persist and can cause microangiopathies that we suspect are also amyloid in character. There is also likely to be a role for molecules raised in pre-existing inflammatory diseases, as well as substances produced by dyregulation microbiomes, and Leiden factor V.

As well as the post-infection diseases referred to above, the emergence of long COVID has brought to the fore its similarities to other even more widely established syndromes such as myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS), and full details are available in these recent reviews. Berg and colleagues have also highlighted the role of coagulopathies in ME/CFS.

 

[SNT Gatchaman]

However... as much as I like the hypothesis, it still remains to be proven that micro-clots are present in circulation (rather than being an in vitro artefact of hypercoagulation).

 

[Wyva]

I think someone asked about the conflict of interest a couple of weeks ago in another thread and what it really meant in this case, because it wasn't so clear. It seems clearer from this one:

Competing Interests
E.P. is a named inventor on a patent application covering the use of fluorescence methods for microclot detection in Long COVID.​

 

[SNT Gatchaman]

On the competing interests question: I don't personally see this as problematic. Surely it's simply a standard protective mechanism? I.e. to stop a bad actor coming along and patenting the technique and locking up its use by the inventors or any other scientist round the world.

The authors seem very keen (to a fault on Twitter) that everyone use the technique to investigate Long Covid and there is no indication that there is any attempt to profit from this - currently or in the future.

 

[cassava7]

This may have been discussed elsewhere, but I do not understand why the symptoms of long Covid would not be observed in other diseases where the authors claim microclots have been found (bolding mine):

Other causes of amyloid microclots

While we here focus on SARS-CoV-2, we note that all kinds of molecules have been shown to affect the extent of fibrinaloid clot formation, including iron, other amyloids, bacterial cell wall components, etc., and that we have observed them in a variety of chronic, inflammatory diseases including Alzheimer's [37,50,60–62], Parkinson's disease [37,48], Type 2 diabetes [37,38,62,63,213,214] (where the amyloid protein amylin [215–217] is of course a well-known player) and rheumatoid arthritis [65,67]. Although not yet tested directly, we consider it likely that this will also be true for infectious diseases known to be causing similar post-infection syndromes, such as Dengue [218–220], Ebola [221–223], Lyme [224], Zika and others where viruses persist and can cause microangiopathies [139] that we suspect are also amyloid in character. There is also likely to be a role for molecules raised in pre-existing inflammatory diseases, as well as substances produced by dyregulation microbiomes [225], and Leiden factor V [226,227].

More specifically, while the authors explain that SARS-CoV-2 is inductive to (micro)clotting, their proposed pathological mechanism for acute and long Covid does not seem specific to either condition. Wouldn’t it generally apply to other diseases in which microclots are found? (bolding mine)

Ability of amyloid microclots to explain the symptoms of long COVID

While long COVID is a multi-system disorder with multiple symptoms of varying severity, it remains possible that there is in fact a particular major underlying cause (or that a very small number are the main contributors). Our view is as follows: given that amyloid microclots can clog up capillaries and inhibit the transport of O2 to tissues, this alone can in fact more or less self-evidently serve to explain a great many observations, and in particular the symptoms of both acute and long COVID. These obviously include breathlessness due to low O2 directly, and thrombotic events such as acute myocardial infarction [180,181], stroke [181–183], etc due to the microclots. The lack of O2 transport to tissues explains straightforwardly how a nominally respiratory disease also leads to the dysfunction of organs such as the kidney [184,185], PoTS (postural tachycardia syndrome [186]), myalgia in skeletal muscle [187,188], neurological disorders [189], and lactic acidosis [190,191] (the mass of lactate is too low to have been detected in our own COVID untargeted metabolomics experiments [192]), and potentially the benefits of hyperbaric O2 therapy [193]. It is also worth stressing that amyloid structures themselves tend to be cytotoxic, often via membrane disruption [194–198].

It should also be noted that their main hypothesis for the persistence of long Covid is two-fold:

- viral persistence in immune-privileged reservoirs with occasional reactivation

- continued release of inflammatory molecules and autoantibodies entrapped in microclots (see also Dr Pretorius’ tweet below)

The authors cite Bruce Patterson’s findings on the persistence of the S1 spike protein in CD16+ monocytes in long Covid — the basis of his commercial “diagnostic” assay — as “highly relevant”, but these are from a small unreplicated study. (bolding mine)

What makes long COVID long?

For any deterministic system to change its behavior there has to be a change of its parameters [199]. In the case of long COVID we need mechanisms that can explain how something that was initiated a long time ago can somehow persist. One source of the continuous production of a stimulus is represented by microbes, including virions, that persist in a largely dormant state (often in intracellular reservoirs) but can occasionally continue to replicate [1,200]. There is now considerable evidence for the persistence of SARS-CoV-2 [201]. Another is the continued release of sequestered microbially derived substances than can act as stimuli for continuing microclot formation. Here, the finding [202] that S1 spike protein can itself persist in CD16+Monocytes in PASC for up to 15 months post-infection is highly relevant, as the amplification of trigger proteins to make microclots as part of the clotting mechanism means that miniscule (and highly substoichiometric) amounts of suitable triggers can suffice [26,47]. This alone is sufficient to account for the chronic nature of such diseases.

Dr Pretorius’ tweet seems ambiguous with regards to the paper. The latter mentions that microclots preventing O2 supply to tissues is the cause of long Covid symptoms alone, but Dr Pretorius writes that the release of their contents as well as vascular pathology causes long Covid. I am not sure whether she meant that they cause the persistence of the condition or its symptoms.

If the contents of the microclots explain the symptoms in part, and assuming that the said contents vary between diseases in which the microclots are found (e.g. type 2 diabetes and rheumatoid arthritis), this could presumably explain the differences with the symptoms of long Covid.

However, in other diseases, microclots would still be blocking capillaries and causing some of the symptoms of LC unless their sizes and structures do not allow for it. These properties seem to vary among individuals and between diseases:

More on the size and properties of microclots [note: in long Covid]

As mentioned, the fibrinaloid microclots that we observe are typically in the range 1–200 µm on their longest axis. This is consistent with the ‘ground glass’ appearance, It is also true for ‘artificial’ amyloid-type protein structures [28], and for the kinds of amyloid seen as deposits in thrombotic microangiopathies [138–142]. However, the size distribution differs markedly between individuals (Figure 4), and while hard to pin down it does provide a ready general explanation for the very different manifestations of Long COVID (including suggestions that the term covers ‘multiple’ diseases). This is also true for prion diseases, where specific ‘strains’ based on particular conformations can propagate in the same form. In some cases, there can be differential staining of individual amyloids by different dyes [26,49,143–145], which also allows a certain degree of differentiation of the structure of the clots. In the case of Long COVID, we are probably not quite ready for such subtleties.

But the authors argue that, in general, microclots are stiff and resistant and thus “strongly” prone to blocking capillaries:

Mechanical properties of amyloid microclots

While we do not yet know the details of the mechanical properties of the fibrin amyloid microclots, it is known that amyloid fibrils can typically exhibit unusually high mechanical strength and resistance to deformation (e.g. [174–177]. The stiffness also increases with the thickness of the fibres [178]. This implies strongly that the fibrinaloids are likely to be more prone to ‘getting stuck’ in capillaries.

I am not sure what to make of all of this. Overall, the microclot theory is constituted of multiple hypotheses that are yet to be proven robustly. Independently, the diagnostic assay has been patented but no public and replicated data is available on its sensitivity and specificity for now. We will see what the ongoing research efforts from Dr Pretorius’ team yield.

 

[SNT Gatchaman]

A follow-on from Prof Makris' tweets above on this subject. We have previously noted that there seemed little knowledge of these studies by haematologists.

An NHS haematologist replied in-thread.

 

[SNT Gatchaman]

The authors seem very keen (to a fault on Twitter) that everyone use the technique to investigate Long Covid and there is no indication that there is any attempt to profit from this - currently or in the future.

Now contradicted by —

A noteworthy excerpt: “Jaeger has bought a €200 000 microscope for her clinic and pays licence fees to Stellenbosch University for use of its method.”

Dr Jaeger is the doctor who runs the HELP apheresis clinic in Mulheim, Germany. Stellenbosch University is home to Dr Pretorius, whose research now focuses on detecting microclots in long Covid. Her microscope method is patented, hence why Dr Jaeger must pay fees for its use.

Dr Pretorius thus has a significant financial conflict of interest in promoting the microclot theory.

I guess it's possible the licence fee is merely nominal to maintain the patent, but yes that does rather overturn my initial claim.

 

[cassava7]

Dr Pretorius has also said that she wants her test to be deployed “at clinics around the world”:
https://twitter.com/resiapretorius/status/1516638314450898951

 

[cassava7]

Another question: if microclots are resistant to fibrinolysis, as Drs Pretorius and Kell argue, then they should forever persist in the body; how does fit in with the natural course of long Covid (and other post-viral syndromes), which in the vast majority of cases is self-remission after a few months or 1-2 year(s)?

Going by Dr Pretorius’ tweet, a simple explanation would be that all of the “inflammatory molecules and antibodies” entrapped in microclots are eventually released and cleared. But the microclots themselves would still be present as they cannot be broken down. Depending on their sizes, they may still block capillaries and thus oxygen uptake in tissues, so some symptoms would persist (e.g. breathlessness) — this does not fit.

It may be that the microclots lose some of their resistance over time and can eventually be broken down by fibrinolysis, but this raises the question of why some people do not recover from long Covid. Pretorius and Kell may counter-argue, based on the recent Harvard pre-print, that the spike protein of SARS-CoV-2 still circulates in the blood of some long-haulers and continuously drives the formation of new microclots. So it seems we are circling back to the start.

Perhaps the conditions for long Covid recovery are the eventual clearances of:

1) the spike protein from the blood, which may stop the formation of new microclots

2) the remaining microclots — both their contents and the clots themselves, assuming they lose resistance over time and then get broken down by fibrinolysis.

(Edit: I am referring two these two papers:

https://www.s4me.info/threads/prepr...ute-covid-19-sequelae-2022-swank-et-al.28133/

https://www.s4me.info/threads/amyloidogenesis-of-sars-cov-2-spike-protein-2022-nyström-hammarström.27708/)

Once again, I am not sure what to make of it all.