Proteomic evidence for amyloidogenic cross-seeding in fibrinaloid microclots, 2024, Kell and Pretorius

[Andy]

Now published: linked here

Preprint
Abstract

In classical amyloidoses, amyloid fibres form through the nucleation and accretion of protein monomers, with protofibrils and fibrils exhibiting a cross-beta motif of parallel or antiparallel beta-sheets oriented perpendicular to the fibre direction. These protofibrils and fibrils can intertwine to form mature amyloid fibres. Similar phenomena occur in blood from individuals with circulating inflammatory molecules (also those originating from viruses and bacteria). In the presence of inflammagens, pathological clotting can occur, that results in an anomalous amyloid form termed fibrinaloid microclots. Previous proteomic analyses of these microclots have shown the presence of non-fibrin(ogen) proteins, suggesting a more complex mechanism than simple entrapment.

We provide evidence against a simple entrapment model, noting that clot pores are too large and centrifugation would have removed weakly bound proteins. Instead, we explore whether co-aggregation into amyloid fibres may involve axial (multiple proteins within the same fibril), lateral (single-protein fibrils contributing to a fibre), or both types of integration. Our analysis of proteomic data from fibrinaloid microclots in different diseases shows no significant overlap with the normal plasma proteome and no correlation between plasma protein abundance and presence in microclots. Notably, abundant plasma proteins like alpha-2-macroglobulin, fibronectin, and transthyretin are absent from microclots, while less abundant proteins such as adiponectin, periostin, and von Willebrand Factor are well represented.

Using bioinformatic tools including AmyloGram and AnuPP, we found that proteins entrapped in fibrinaloid microclots exhibit high amyloidogenic tendencies, suggesting their integration as cross-beta elements into amyloid structures. This integration likely contributes to the microclots' resistance to proteolysis. Our findings underscore the role of cross-seeding in fibrinaloid microclot formation and highlight the need for further investigation into their structural properties and implications in thrombotic and amyloid diseases. These insights provide a foundation for developing novel diagnostic and therapeutic strategies targeting amyloidogenic cross-seeding in blood clotting disorders.

https://www.biorxiv.org/content/10.1101/2024.07.16.603837v1?ct=

 

[SNT Gatchaman]

Some summary passages —

The action of thrombin leads to the serial removal of two fibrinopeptides, which sets in motion a remarkable self-organisation by which the fibrinogen molecules interact to form protofibrils, fibrils, and then fibres of some 50-100nm diameter, implying some hundreds of fibrinogen molecules in each length element of the typical fibrin fibre. In normal clots the direction of the fibrinogen molecules and fibrin protofibrils is parallel to that of the fibres.

The pore sizes of typical clots are of the order 0.5-5 μm when fibrinogen is at its physiological concentrations (the pore diameters can be far lower at massively extraphysiological fibrinogen concentrations), so without specific binding of some kind they are clearly incapable of simply entrapping molecules of globular proteins (with diameters in the low-nm range).

it is of special interest that α-helix-to-β-sheet transitions are a noteworthy feature of such proteins, and particularly, for our present purposes, some in SARS-CoV-2, where several proteins are amyloidogenic. We note too that some alleles of the fibrinogen Aα chain may produce highly amyloidogenic proteolytic fragments, and that fibrinogen can bind to well-established amyloids such as Aβ.

many (and maybe even most) proteins can fold into states that are considerably more stable than the one natively or most commonly adopted as they leave the ribsome, but that this thermodynamically favoured conformation (or, more accurately, the set of isoenergetic conformations) is normally kinetically inaccessible due to a massive energy barrier of some 36-38 kcal.mol-1. A particular class of these more stable conformations involve a cross(ed)-β-sheet motif, and they become insoluble because they tend to aggregate and self-assemble; following their discovery by Virchow in 1854 they are referred to as amyloids.

In contrast to the classical secondary structure of β-sheets, where the rules for their formation in terms of amino acid sequence are broadly polar-apolar-polar-apolar- (etc), the sequence rules for amyloidogenic potential generally, and for cross-β sheet formation in particular, are rather more obscure.

 

[SNT Gatchaman]

On Thioflavin-T —

A continuing and historically important discovery was the fact that the dye thioflavin T binds to a whole series of amyloids, with a concomitant increase in its fluorescence. This occurs because rotation of the normally rotatable single bond between the benzothiazole and dimethylaniline rings allows fluorescence from an excited state to be dissipated and hence quenched. When the ThT is bound appropriately to a macromolecule, no such rotation is possible, fluorescence occurs, and thus ThT is a fluorogenic stain for amyloids. [...] As phrased by Biancalana and Koide, “ThT binds to diverse fibrils, despite their distinct amino acid sequences, strongly suggesting that ThT recognizes a structural feature common among fibrils. Because amyloid fibrils share the cross-β architecture, it is generally accepted that the surfaces of cross-β structures form the ThT-binding sites”.

On microclots —

In the same way that many proteins can adopt an amyloid form, as described above, it was discovered that fibrinogen can polymerise into an anomalous amyloid-like form, that stained very strongly with thioflavin T and indeed other stains such as Amytracker stains [...] The typical size of these clots, that were termed micro-clots is in the range 2-200 μm

On protein entrapment in microclots —

Our own work on the proteomics of fibrinaloid microclots has referred to ‘entrapment’ of non-fibrin proteins in the microclots. However, this cannot be a simple entrapment like a fish in a in a mesh net; the pores are far too big and in any case the centrifugation would have washed soluble proteins away from any weak binding or entrapment. Consequently, the ‘entrapment’ must actually be a forcing of the other proteins to become insoluble, likely by making cross-β sheets and thus joining the tightly-bound-but-noncovalent party and be incorporated into the growing amyloid fibrils.

Evidence for this includes the fact that there is no relationship between what is ‘entrapped’ in the fibrinaloid microclots and the normal plasma abundance of proteins (e.g. albumins and transferrin are pretty well the most abundant and mostly do not appear).

 

[SNT Gatchaman]

On difference between microclot proteome and plasma proteome —

we again see large number of proteins that are high in abundance in the plasma proteome that nevertheless are not ‘entrapped’ in the fibrinaloid microclots, and similarly others in low abundance in the proteome nonetheless appear in the fibrinaloid microclots. The conclusion is very clear: there is a significant selectivity with regard to proteins that are entrapped within fibrinaloid microclots.

If axial or lateral co-aggregation is responsible for the ‘entrapment’ of proteins in fibrinaloid microclots, one would suppose that all the proteins involved would themselves be amyloidogenic. [...] The conclusion is very clear: every single one of the proteins detected in the microclots is highly amyloidogenic, and the microclots evidently involve cross-seeding.

The presence of von Willebrand factor and adiponectin in the fibrinaloid microclots is very inetresting, despite their comparatively low plasma concentration. The former is among the most amyloidogenic proteins in the list, and is notably entrapped and removed by microclots in SARS-CoV-2 infection, while the latter is correlated with amyloid Aβ deposition and may be protective.

thrombospondin-1 is also much over-represented, and it too was neuroprotective against Aβ. A clear pattern emerges.

α-2-antiplasmin (prominent in the findings of), where there is an initial run plus two further prominent peaks. α-2-antiplasmin is of course well known as an inhibitor of fibrinolysis.

A particularly noteworthy observation here is the relatively high amounts of fibronectin seen in normal clots (130 mg/g protein, fibronectin typically being present in plasma at 300-400 mg/L ) as it was not seen in the fibrinaloid microclots. While fibrin is highly amyloidogenic and in vivo can produce insoluble fibrillar components that may be incorporated in the extracellular matrix, fibronectin is somewhat unusual for two reason. First, it is large (2477 residues). Secondly it is relatively thermostable, especially in some of its domains. Together these features can plausibly account for the difficulty of unfolding and incorporating it into an amyloid clot compared to a normal one. Similar comments relate to α-2-macroglobulin (27 mg/g, 1474 residues) and to Factor XIII (12 mg/g, 732 residues), which is in fact inhibited by α-2macroglobulin.

Factor XIII is a transglutaminase (linking glutamate and lysine residues) that is responsible for stiffening normal clots by crosslinking them, mainly via the γ but also partly the α chains, yet does not appear in the fibrinaloid microclot proteomes. This would be entirely consistent with their completely different structures relative to that of normal clots. Lastly, complement factor 3 is fairly well represented in both plasma and in normal clots (12 mg/g) yet is not found in fibrinaloid microclots; consistent with the thrust of our arguments, its total amyloidogenic propensity at just 0.73 is among the lowest

 

[SNT Gatchaman]

Concluding in discussion with —

Seemingly, as with prion proteins, the presence of a small amount of the thermodynamically stabler amyloid form is enough to trigger conversion of a very large number of monomers in the amyloid polymer form in almost an ‘all-or-nothing’ manner

First, their proteome varies strongly with the disease, in a way that cannot reflect changes in the proteome. Secondly, their proteome constitution is far from being related to the concentrations of bulk plasma proteins, with some being excluded and others being highly concentrated. Clearly there must be special mechanisms at work, the most obvious, given their strikingly high amyloidogenicity scores, being a cross-seeding where the various proteins are actually incorporated into the cross-β elements of the fibrils themselves.

This said, there is a consonance in Table 2 between the proteins highlighted as being in fibrinaloid microclots and biological explanations based on their known roles. Most of those that are higher are in the Kruger Long COVID study. Long COVID is of course a chronic disease, and very different from the acute conditions characterising individual in an ICU such as those in the Toh study. It bears relations to myalgic encephalopathy/chronic fatigue syndrome (ME/CFS), however, so it is of interest that thrombospondin and platelet factor 4 are both raised in the plasma of individuals with ME/CFS.

What we propose here is that the massive changes in fibrinogen structure necessary for its conversion to an amyloid form, as also [observed] by others, must then involve cross-seeding, as this provides a simple mechanism that at once accounts for (i) the proteomics, (ii) the resistance to proteolysis, and (iii) the amyloid nature of the mixed clots.

(Quite a few typos to be fixed in the pre-print.)

 

[SNT Gatchaman]

Now published as —

Proteomic Evidence for Amyloidogenic Cross-Seeding in Fibrinaloid Microclots
Kell, Douglas B.; Pretorius, Etheresia

In classical amyloidoses, amyloid fibres form through the nucleation and accretion of protein monomers, with protofibrils and fibrils exhibiting a cross-β motif of parallel or antiparallel β-sheets oriented perpendicular to the fibre direction. These protofibrils and fibrils can intertwine to form mature amyloid fibres. Similar phenomena can occur in blood from individuals with circulating inflammatory molecules (and also some originating from viruses and bacteria). Such pathological clotting can result in an anomalous amyloid form termed fibrinaloid microclots.

Previous proteomic analyses of these microclots have shown the presence of non-fibrin(ogen) proteins, suggesting a more complex mechanism than simple entrapment. We thus provide evidence against such a simple entrapment model, noting that clot pores are too large and centrifugation would have removed weakly bound proteins. Instead, we explore whether co-aggregation into amyloid fibres may involve axial (multiple proteins within the same fibril), lateral (single-protein fibrils contributing to a fibre), or both types of integration.

Our analysis of proteomic data from fibrinaloid microclots in different diseases shows no significant quantitative overlap with the normal plasma proteome and no correlation between plasma protein abundance and their presence in fibrinaloid microclots. Notably, abundant plasma proteins like α-2-macroglobulin, fibronectin, and transthyretin are absent from microclots, while less abundant proteins such as adiponectin, periostin, and von Willebrand factor are well represented. Using bioinformatic tools, including AmyloGram and AnuPP, we found that proteins entrapped in fibrinaloid microclots exhibit high amyloidogenic tendencies, suggesting their integration as cross-β elements into amyloid structures. This integration likely contributes to the microclots’ resistance to proteolysis.

Our findings underscore the role of cross-seeding in fibrinaloid microclot formation and highlight the need for further investigation into their structural properties and implications in thrombotic and amyloid diseases. These insights provide a foundation for developing novel diagnostic and therapeutic strategies targeting amyloidogenic cross-seeding in blood clotting disorders.

Link | PDF (International Journal of Molecular Sciences) [Open Access]