Automated microscopic measurement of fibrinaloid microclots and their degradation by nattokinase, the main natto protease

Grixti, Justine M.; Theron, Chrispian W.; Salcedo-Sora, J. Enrique; Pretorius, Etheresia; Kell, Douglas B.

doi:10.1101/2024.04.06.588397

Cited by 3 publications

(1 citation statement)

References 315 publications

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“…Since the acceptance of this article, three significant papers on fibrinaloid microclots have been published. One [429] describes the importance of microclots in intensive care unit morbidity and disseminated intravascular coagulation, while two others describe an automated microscope method for the detection of microclots [430,431] and their degradation by nattokinase [431].…”

Section: Final Discussion and Conclusion And A Forward Lookmentioning

confidence: 99%

Fibrinaloid Microclots and Atrial Fibrillation

Kell,

Lip,

Pretorius

2024

Biomedicines

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Atrial fibrillation (AF) is a comorbidity of a variety of other chronic, inflammatory diseases for which fibrinaloid microclots are a known accompaniment (and in some cases, a cause, with a mechanistic basis). Clots are, of course, a well-known consequence of atrial fibrillation. We here ask the question whether the fibrinaloid microclots seen in plasma or serum may in fact also be a cause of (or contributor to) the development of AF. We consider known ‘risk factors’ for AF, and in particular, exogenous stimuli such as infection and air pollution by particulates, both of which are known to cause AF. The external accompaniments of both bacterial (lipopolysaccharide and lipoteichoic acids) and viral (SARS-CoV-2 spike protein) infections are known to stimulate fibrinaloid microclots when added in vitro, and fibrinaloid microclots, as with other amyloid proteins, can be cytotoxic, both by inducing hypoxia/reperfusion and by other means. Strokes and thromboembolisms are also common consequences of AF. Consequently, taking a systems approach, we review the considerable evidence in detail, which leads us to suggest that it is likely that microclots may well have an aetiological role in the development of AF. This has significant mechanistic and therapeutic implications.

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Section: Final Discussion and Conclusion And A Forward Lookmentioning

confidence: 99%

Fibrinaloid Microclots and Atrial Fibrillation

Kell,

Lip,

Pretorius

2024

Biomedicines

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Flow Clotometry: Measuring Amyloid Microclots in ME/CFS, Long COVID, and Healthy Samples with Imaging Flow Cytometry

Pretorius,

Nunes,

pretorius

et al. 2024

Preprint

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Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) has received more attention since the characterization of Long COVID (LC), a condition somewhat similar in symptom presentation and, to some extent, pathophysiological mechanisms. A prominent feature of LC pathology is amyloid, fibrinolysis-resistant fibrin(ogen) fragments, termed microclots. Despite prior identification of microclots in ME/CFS, quantitative analysis has remained challenging due to the reliance on representative micrographs and software processing for estimations. Addressing this gap, the present study uses a cell-free imaging flow cytometry approach, optimized for the quantitative analysis of Thioflavin T-stained microclots, to precisely measure microclot concentration and size distribution across ME/CFS, LC, and healthy cohorts. We refer to our cell-free flow cytometry technique for detecting microclots as 'flow clotometry'. We demonstrate significant microclot prevalence in ME/CFS and LC, with LC patients exhibiting the highest concentration (18- and 3-fold greater than the healthy and ME/CFS groups, respectively). This finding underscores a common pathology across both conditions, emphasizing a dysregulated coagulation system. Moreover, relating to microclot size distribution, the ME/CFS group exhibited a significantly higher prevalence across all area ranges when compared to the controls, but demonstrated a significant difference for only a single area range when compared to the LC group. This suggests a partially overlapping microclot profile in ME/CFS relative to LC, despite the overall higher concentration in the latter. The present study paves the way for prospective clinical application that aims to efficiently detect, measure and treat microclots.

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Evidence for the Role of the Mitochondrial ABC Transporter MDL1 in the Uptake of Clozapine and Related Molecules into the Yeast Saccharomyces cerevisiae

Theron,

Salcedo-Sora,

Grixti

et al. 2024

Pharmaceuticals

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Clozapine is an antipsychotic drug whose accumulation in white cells can sometimes prove toxic; understanding the transporters and alleles responsible is thus highly desirable. We used a strategy in which a yeast (Saccharomyces cerevisiae) CRISPR-Cas9 knock-out library was exposed to cytotoxic concentrations of clozapine to determine those transporters whose absence made it more resistant; we also recognised the structural similarity of the fluorescent dye safranin O (also known as safranin T) to clozapine, allowing it to be used as a surrogate marker. Strains lacking the mitochondrial ABC transporter MDL1 (encoded by YLR188W) showed substantial resistance to clozapine. MDL1 overexpression also conferred extra sensitivity to clozapine and admitted a massive increase in the cellular and mitochondrial uptake of safranin O, as determined using flow cytometry and microscopically. Yeast lacking mitochondria showed no such unusual accumulation. Mitochondrial MDL1 is thus the main means of accumulation of clozapine in S. cerevisiae. The closest human homologue of S. cerevisiae MDL1 is ABCB10.

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Automated microscopic measurement of fibrinaloid microclots and their degradation by nattokinase, the main natto protease

Cited by 3 publications

References 315 publications

Fibrinaloid Microclots and Atrial Fibrillation

Fibrinaloid Microclots and Atrial Fibrillation

Flow Clotometry: Measuring Amyloid Microclots in ME/CFS, Long COVID, and Healthy Samples with Imaging Flow Cytometry

Evidence for the Role of the Mitochondrial ABC Transporter MDL1 in the Uptake of Clozapine and Related Molecules into the Yeast Saccharomyces cerevisiae

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