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Uncovering the Hidden Biology of Fibrinaloid Microclot Complexes in Complex, Inflammatory Diseases

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06 May 2026

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07 May 2026

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Abstract
Blood can clot into anomalous, fibrinolysis-resistant forms that arise from prothrombotic seeding areas, including damaged cellular debris and membrane-derived surfaces, giving rise to what we have termed fibrinaloid microclot complexes (colloquially: microclots).Their proteolytic resistance is due to the fact that they are amyloid in nature, and they can also entrap inhibitors of proteolysis. They consist of a variety of proteins besides the expected fibrin, and are highly enriched for other amyloidogenic proteins (in contrast to normal clots, whose proteome largely reflects the soluble plasma proteome). They also contain DNA in the form of neutrophil extracellular traps (NETs). Importantly, fibrinaloid microclot complexes are heterogeneous structures comprising multiple phenotypic forms, including those that nucleate and grow on cellular debris such as damaged membranes, microparticles, and immune-derived material. These debris-associated complexes act as catalytic scaffolds that recruit fibrin(ogen) and inflammatory molecules, thereby amplifying amyloidogenic transformation and prothrombotic activity. Fibrinaloid microclot complexes have been reported in a widening range of chronic inflammatory and thrombo-inflammatory diseases in which they have been sought, and are highly enriched for amyloidogenic proteins. Additionally, the thrombi extracted from ischaemic stroke also contain proteins in an amyloid form, implying that such macroclots can form via the accretion of microclots that already contain amyloid. We here show that these microclots exhibit a classical ‘apple-green’ birefringence when stained with the dye Congo red. The urgent task now is to find means of inhibiting the transition to amyloid forms during the clotting process.
Keywords: 
amyloid
;  
microclots
;  
inflammation
;  
oxidative stress
;  
microcirculation
Subject: 
Medicine and Pharmacology  -   Hematology
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Introduction

Blood clotting is traditionally understood as a thrombin-driven process in which fibrin polymers form structured networks that are susceptible to regulated fibrinolysis; however, under inflammatory conditions, clotting can also originate from prothrombotic seeding areas that give rise to structurally distinct, fibrinolysis-resistant assemblies. In healthy haemostasis these fibres form branching networks whose architecture is influenced by fibrinogen concentration, thrombin generation, flow and cross-linking. In the electron microscope these appear like spaghetti [1,2].
However, blood can also clot into anomalous forms that are significantly resistant to fibrinolysis and that in the electron microscope appear instead as ‘dense matted deposits’ that take on the appearance of parboiled spaghetti that has been allowed to congeal (e.g. [3,4,5]).
It transpired that that this form was in fact amyloid in nature [6], involving the kind of cross-b sheets seen in prion diseases and classical amyloidoses [7]. These observations suggest that pathological clotting does not simply reflect dysregulated thrombin activity, but rather emerges from surface-driven nucleation processes in which damaged biological interfaces act as catalytic scaffolds for amyloidogenic fibrin(ogen) assembly. We have referred to these microclot complexes as fibrinaloid, and they typically possess a diameter in the range 2-200 mm. They also contain neutrophil extracellular traps (NETs [8]). Importantly, these complexes represent heterogeneous phenotypic forms, reflecting their origins on distinct seeding substrates such as membrane debris, microparticles, NET-derived nucleoprotein scaffolds, and plasma protein aggregates.
The fibrinaloid microclot complexes are typically observed using fluorogenic amyloid stains such as thioflavin T [9,10,11] or the oligothiophene ‘Amytracker’ dyes [12,13,14]. As is well known, amyloids are much more resistant to normal proteolysis than the soluble forms formed at the ribosome. Figure 1 shows examples of representative microclot complexes stained with Thioflavin T and detected with imaging flow cytometry. Together, these findings define a mechanistically distinct mode of clot formation in which amyloidogenic, surface-seeded complexes precede and potentially drive the development of larger, clinically relevant thrombotic structures.
Fibrinaloid microclot complexes have now been reported across a widening range of inflammatory and thrombo-inflammatory conditions [16] (Table 1), including acute COVID-19, Long COVID, sepsis, type 2 diabetes, Parkinson’s disease, rheumatoid arthritis and Alzheimer-related syndromes, with support coming both from our own studies and from independent groups. However, while the presence of fibrinaloid microclot complexes has been observed across these diverse conditions, detailed mechanistic characterization of their phenotypes and their origins from specific prothrombotic seeding areas has not been uniformly established.
To date, such phenotypic resolution, including the identification of distinct seeding substrates such as cellular debris, microparticles, and nucleoprotein complexes, has been most clearly demonstrated in COVID-19, Long COVID, and type 2 diabetes. In contrast, in other disease settings, fibrinaloid microclots have largely been described at the level of presence, morphology, or proteomic enrichment, without explicit discrimination of their underlying phenotypic diversity or seeding mechanisms.
The thrombi causing ischaemic stroke also display amyloid [17,18], and the fibrinaloid microclots can be formed using just purified fibrinogen, thrombin, and a suitable trigger molecule [6,19].
Several triggers of amyloidogenic clotting have been observed experimentally, including ferric ions [4,54,55,56], bacterial lipopolysaccharide (LPS) [6], lipoteichoic acids [14], and the SARS-CoV-2 spike protein [57]. These have all been found to trigger the amyloidogenic clotting, often at minuscule levels [6].
The SARS-CoV-2 spike protein is itself highly amyloidogenic [58,59], its amyloidogenicity (in different strains of SARS-CoV-2) being related to the virulence of the strains [60]. Taken together, these findings are consistent with the view that clot amyloidogenesis can lie on an important disease pathway.
It is recognised that thioflavin T, while seen as a gold standard amyloid stain [11,61], is not entirely specific for amyloid [37]. To this end, we have also employed the more selective oligothiophene ‘Amytracker’ dyes, again showing that they stained fibrinaloid microclot complexes (e.g. [12,13,14,62]). Another ‘classical’ amyloid dye is Congo Red, that shows an ‘apple-green’ birefringence when illuminated with polarised light (e.g. [63,64,65], cf. [66]). We here show, for the first time, fibrinaloid microclots assessed in this way with Congo Red.
Figure 2 shows images of a fibrinaloid microclot created in vitro (as per [6,67], stained with the classical amyloid dye Congo Red [68] and assessed using a polarising microscope as described in Methods, below. It displays the classical ‘apple green’ birefringence, providing further evidence for the amyloid nature of the microclots.
In Long COVID, increased FMC burdens have been reported in platelet-poor plasma by more than one group [23,24,25,26], although with inter-individual variability and continuing debate over assay standardisation and interpretation. These observations are consistent with the broader concept that persistent thrombo-inflammation, endothelial activation and impaired fibrinolysis may contribute to symptom burden in at least a subset of patients. Within this framework, microcirculatory impairment provides one plausible route by which diverse symptoms may arise, although the degree of causal contribution is still being defined.
The effect of these insoluble FMCs is potentially to partially block or interfere with blood flow in microcapillaries (the ‘microcirculation’) and hence O2 transfer to tissues. Taking Long COVID as an example, such a blockage can straightforwardly and self-consistently explain a great many symptoms that accompany this highly variable syndrome, including fatigue [27], post-exertional malaise [28], autoimmunity [29], atrial fibrillation [69], fibromyalgia [70], POTS [71], and tinnitus [72].
Emerging evidence also indicates that FMCs represent a spectrum of structurally and mechanistically distinct entities rather than a single homogeneous clotting product. While earlier work has focused primarily on fibrin(ogen)-derived amyloid formation, it is now clear that a substantial proportion of these complexes originate from prothrombotic seeding areas, including cellular debris, membrane fragments, microparticles, and nucleoprotein immune structures. These debris-associated complexes are fundamentally different from classical fibrin networks in that they do not form through thrombin-driven polymerisation, do not integrate into organised fibrin fibre architectures, and are largely resistant to conventional plasmin-mediated fibrinolysis. In parallel, a related but distinct process occurs in which soluble inflammatory and amyloidogenic molecules interact with fibrinogen and, upon thrombin activation, become incorporated into fibrin networks, giving rise to the dense matted deposits previously described. These observations support the existence of at least two overlapping but mechanistically distinct pathways: (i) surface-seeded formation of debris-associated fibrinaloid complexes, and (ii) thrombin-mediated formation of structurally altered fibrin networks incorporating inflammatory components. This distinction is critical, as conventional coagulation and fibrinolysis assays are likely to capture only the latter, thereby underestimating or entirely missing the presence of debris-associated fibrinaloid structures and contributing to ongoing discrepancies in the literature.
We have used the Amylogram program [73,74] to assess the amyloidogenicity of individual proteins (the output is a score between 0 and 1, with anything over ca 0.75 being significantly and experimentally amyloidogenic [75]. The proteome of normal thrombi [76] largely reflects that of the soluble plasma proteome. However, the proteomes of the various fibrinaloid microclot complexes are very different, with the proteins that are enriched in them being themselves highly amyloidogenic [77,78,79]. Table 2 shows proteins enriched in the microclots, a dataset [77] to which we have added their Amylogram scores. Each of these exceeds 0.75, some by a considerable margin. It is likely that this is caused by cross-seeding of these amyloidogenic proteins catalysing the transition to the amyloid form, and that the macroclots in ischaemic stroke and other cardiovascular diseases can form via accretion of these microclots. Of course their amyloid nature again straightforwardly explains their resistance to the normal fibrinolysis that would be catalysed by plasmin. A pictorial summary is given in Figure 3.
Although the broader disease-spanning framework has been developed largely through our own work, recent reports from independent groups in sepsis and Long COVID indicate that the phenomenon is reproducible beyond a single laboratory context. At the same time, the field would benefit greatly from further methodological standardisation, head-to-head comparison of assays, and additional external replication across disease settings.
In particular, the next stage is to develop methods that are not simply anticoagulating (though such can be efficacious [80]), but that can inhibit the transition to the amyloid form.

Methods

To create and analyse fibrinaloid microclots with Congo Red in vitro, 30 ng/mL of bacterial LPS were added to purified FBG at 2.6 mg/mL. To this, Congo Red was added at 0.3 mM, followed by 7U of Thrombin. All concentrations stated are final. Images were taken on an Axio Observer.Z1 (Zeiss, Jena, Germany) microscope using a halogen lamp (HAL100) and a polariser filter with a GXCAM-3 (GT Vision, Wickhambrook, UK). Images were acquired with the software GXCam version 7.3. All images were taken with a Plan-APOCHROMAT FLUAR 20x 0.75 DIC and saved in .tif format. The methods and data for Figure 1 were obtained using imaging flow cytometry, the image being taken from a CC-BY 4.0 publication [15].

Conclusions

FMCs have now been reported across a widening range of inflammatory and thrombo-inflammatory conditions (Kell and Pretorius 2018a) (Table 1), including acute COVID-19, Long COVID, sepsis, type 2 diabetes, Parkinson’s disease, rheumatoid arthritis and Alzheimer-related syndromes, with support coming both from our own studies and from independent groups. However, while the presence of fibrinaloid microclot complexes has been observed across these diverse conditions, detailed mechanistic characterization of their phenotypes and their origins from specific prothrombotic seeding areas has not been uniformly established, although our group has identified the need for better phenotyping [36]. To date, such phenotypic resolution, including the identification of distinct seeding substrates such as cellular debris, microparticles, and nucleoprotein complexes, has been most clearly demonstrated in COVID-19, Long COVID, and type 2 diabetes. In contrast, in other disease settings, fibrinaloid microclots have largely been described at the level of presence, morphology, or proteomic enrichment, without explicit discrimination of their underlying phenotypic diversity or seeding mechanisms.
This distinction is critical, as it indicates that FMCs are not a single pathological entity but a spectrum of mechanistically distinct, surface-seeded assemblies, and that resolving their phenotypic diversity and seeding origins is essential for understanding their role in disease pathogenesis, as well as for developing targeted diagnostic and therapeutic strategies.

Author Contributions

Conceptualization, DBK and EP; Resources, DBK and EP; Data generation for Figure 2 JMG; Writing – Original Draft Preparation, DBK; Writing – Review & Editing, all authors; Visualization, EP; Funding Acquisition, DBK and EP.

Funding

We thank the Balvi Foundation for financial support. The content and findings reported and illustrated are the sole deduction, view and responsibility of the researchers and do not reflect the official position and sentiments of the funders. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Acknowledgments

The authors gratefully acknowledge Marco Marcello of the LIV-SRF Centre for Cell Imaging https://www.liverpool.ac.uk/research/facilities/shared-research-facilities/centre-for-cell-imaging/ for assistance with the polarised light microscopy.

Declaration of Competing Interests

EP holds a patent application for the use of fluorescence microcopy in microclot imaging.

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Figure 1. Fibrinaloid microclot complexes stained with Thioflavin T (ThT) as detected in platelet poor plasma (PPP) using imaging flow cytometry. Image taken from the CC-BY 4.0 publication [15].
Figure 1. Fibrinaloid microclot complexes stained with Thioflavin T (ThT) as detected in platelet poor plasma (PPP) using imaging flow cytometry. Image taken from the CC-BY 4.0 publication [15].
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Figure 2. A fibrinaloid microclot created using purified fibrinogen and thrombin as in Methods, stained with 0.3 mM Congo Red, and imagied in a polarising microcope as in Methods. The classical ‘apple-green’ birefringence is observed in particular around the periphery of the microclot.
Figure 2. A fibrinaloid microclot created using purified fibrinogen and thrombin as in Methods, stained with 0.3 mM Congo Red, and imagied in a polarising microcope as in Methods. The classical ‘apple-green’ birefringence is observed in particular around the periphery of the microclot.
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Figure 3. A summary of fibrinaloid microclot complexes. This figure (and also the graphical abstract) was generated on 23/4/2026 with the assistance of the artificial intelligence tool NotebookLM (https://notebooklm.google/), using author-provided text and figure concepts; all scientific content, interpretation, and final design decisions were verified and approved by the authors.
Figure 3. A summary of fibrinaloid microclot complexes. This figure (and also the graphical abstract) was generated on 23/4/2026 with the assistance of the artificial intelligence tool NotebookLM (https://notebooklm.google/), using author-provided text and figure concepts; all scientific content, interpretation, and final design decisions were verified and approved by the authors.
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Table 1. (above). Summary of representative evidence for fibrinaloid microclot complexes across selected disease settings. The table distinguishes disease context, study provenance, material analysed, analytical modality, and the type of supporting evidence. It is intended as an evidence map rather than an exhaustive systematic review.
Table 1. (above). Summary of representative evidence for fibrinaloid microclot complexes across selected disease settings. The table distinguishes disease context, study provenance, material analysed, analytical modality, and the type of supporting evidence. It is intended as an evidence map rather than an exhaustive systematic review.
Condition Representative independent studies Representative Kell/Pretorius studies Sample/material Readout Amyloid-selective stain Proteomics NET association Clinical link
Acute COVID-19 [20] [21,22] PPP/plasma microscopy / staining yes limited variable coagulopathy
Long COVID [23,24,25,26] [15,27,28,29,30,31,32,33,34,35,36,37,38] PPP/plasma IFC / microscopy / proteomics yes yes yes symptom burden
Sepsis [39] Presaged in [40] plasma amyloid-fibrinogen aggregates yes no not central DIC / mortality
Stroke thrombi — [41,42] retrieved thrombi amyloid staining / proteomics yes yes not central fibrinolysis resistance
T2D — [12,13,22,43,44,45] PPP/fibrin microscopy / stains yes limited no abnormal clotting
Parkinson’s / Alzheimer’s — [2,12,46,47,48,49,50] plasma/fibrin correlative LM/EM, stains yes limited no chronic inflammation
Rheumatoid arthritis — [51] plasma/fibrin EM no no no chronic inflammation
ME/CFS [28,36,52,53] Plasma/fibrin Microscopy /stains Yes Yes No chronic inflammation
Table 2. Proteins enriched or lowered in fibrinaloid microclots relative to normal clots [77], with their Amylogram scores.
Table 2. Proteins enriched or lowered in fibrinaloid microclots relative to normal clots [77], with their Amylogram scores.
Protein Uniprot ID Amylogram score
Adiponectin Q15848 0.833
Kallikrein P03952 0.769
LBLC1/BNIB1/BNIFB1/LPLUNC1 Q8TDL5 0.918
Platelet factor 4 P02776 0.778
Periostin Q15063 0.914
Thrombospondin-1 P07996 0.863
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