A central role for amyloid fibrin microclots in long COVID/PASC: origins and therapeutic implications

Abstract

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.

Keywords: COVID; amyloid; clotting.

Figure 1.. Many proteins can adopt more than more thermodynamically stable microstate with no change in primary structure (sequence), in which the more stable contains an ordered β-sheet ‘amyloid’ structure.

Normally, however, it is present in a less stable state that is kinetically more accessible during and following its synthesis. The more stable (labeled PrP^Sc) is separated from the initial state (PrP^C) via a large energy barrier. This is true for amyloid proteins generally, and is illustrated here for classical prion proteins. Redrawn from a CC-BY publication at [26].

Figure 2.. Representation of the classical blood-clotting cascades, ending in the removal of two fibrinopeptides from fibrinogen, and its self organization to produce fibrin (that may then be cross-linked).

The eventual result of fibrinolysis, that acts as a record of its extent, is d-dimer. Redrawn from a CC-BY publication at [26].

Figure 3.. Self organization of fibrinogen into fibrin under the action of thrombin in removing two fibrinopeptides.

Redrawn from a CC-BY publication at [26].

Figure 4.. Fluorescence microscopy of representative micrographs showing microclots in the circulation of controls (A) and in patients with Long COVID (B–D).

Absence of significant amyloid microclots in the plasma of ‘normal’ individuals, and their significant presence in the plasma of individuals with long COVID. Platelet-poor plasma was produced by centrifugation at 3000×g for 15 min, stained with 5 µM thioflavin T, and imaged in a fluorescence microscope (Zeiss Axio Observer 7 with a Plan-Apochromat 63×/1.4 Oil DIC M27 objective (Carl Zeiss Microscopy, Munich, Germany). Wavelengths were Exc 450–488 nm/emission 499–529 nm, all as in [108].

Figure 5.. d-dimer production by fibrinolysis.

It is believed to be similar when produced from fibrinaloid clots, but the rate is considered to be slower. Image created with BioRender (https://biorender.com/).

Figure 6.. d-dimer levels reflect both the rate of production and rate of degradation of clots, whether the clots are ‘normal’ or fibrinaloid in nature.

Image created with BioRender (https://biorender.com/.

Figure 7.. A simplified diagram to explain microclot formation that might either be resolved via fibrinolytic processes after acute COVID-19 or, in some patients, result in a failed fibrinolytic process.

Image created with BioRender (https://biorender.com/).

Figure 8.. TEG® traces with the main parameters visualized.

(A) Healthy (normocoagulable) trace; (B) Hypercoagulable trace (seen during early stages of acute COVID-19) and (C) Hypocoagulable trace. Image created with BioRender (https://biorender.com/). In B and C the dotted lines represent the normocoagulable case. R represents the time taken to initiate clot formation, α and K reflect the rate and extent of clot formation (e.g. [52]).

Figure 9.. Platelet interactions with viruses.

Figure adapted from [259]. Various platelet receptors can mediate binding to viral particles, where pattern recognition receptors recognize viral signals, viral products and also modulate platelet function. Platelets mediate viral attack by secreting virucidal proteins and by engulfing viral particles, as well as by interacting with immune cells and enhancing the immune response. Virus-platelet aggregates and platelets with a viral load are targeted by leukocytes, and platelets are ultimately cleared from the circulation (Figure created with https://biorender.com/).

Figure 10.. Some of the sequelae of fibrinaloid microclot formation in the symptomology of Long COVID.

Many others, such as a role for auto-antibodies, are not shown.