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Acute induction of anomalous blood clotting by molecular amplification of highly substoichiometric levels of bacterial lipopolysaccharide (LPS)

Etheresia Pretorius, Sthembile Mbotwa, Janette Bester, Christopher Robinson, Douglas B. Kell
doi: https://doi.org/10.1101/053538
Etheresia Pretorius
1Department of Physiology, Faculty of Health Sciences, University of Pretoria, Arcadia 0007, South Africa
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Sthembile Mbotwa
1Department of Physiology, Faculty of Health Sciences, University of Pretoria, Arcadia 0007, South Africa
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Janette Bester
1Department of Physiology, Faculty of Health Sciences, University of Pretoria, Arcadia 0007, South Africa
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Christopher Robinson
2Faculty of Life Sciences, The University of Manchester, 131, Princess St, MANCHESTER M1 7DN, Lancs, UK
4The Manchester Institute of Biotechnology, The University of Manchester, 131, Princess St, MANCHESTER M1 7DN, Lancs, UK
5Centre for Synthetic Biology of Fine and Speciality Chemicals, The University of Manchester, 131, Princess St, MANCHESTER M1 7DN, Lancs, UK
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Douglas B. Kell
3School of Chemistry, The University of Manchester, 131, Princess St, MANCHESTER M1 7DN, Lancs, UK
4The Manchester Institute of Biotechnology, The University of Manchester, 131, Princess St, MANCHESTER M1 7DN, Lancs, UK
5Centre for Synthetic Biology of Fine and Speciality Chemicals, The University of Manchester, 131, Princess St, MANCHESTER M1 7DN, Lancs, UK
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ABSTRACT

It is well known that a variety of inflammatory diseases are accompanied by hypercoagulability, and a number of more-or-less longer-term signalling pathways have been shown to be involved. In recent work, we have suggested a direct and primary role for bacterial lipopolysaccharide in this hypercoagulability, but it seems never to have been tested directly. Here we show that the addition of tiny concentrations (0.2 ng.L−1) of bacterial lipopolysaccharide (LPS) to both whole blood and platelet-poor plasma of normal, healthy donors leads to marked changes in the nature of the fibrin fibres so formed, as observed by ultrastructural and fluorescence microscopy (the latter implying that the fibrin is actually in an amyloid β-sheet-rich form. They resemble those seen in a number of inflammatory (and also amyloid) diseases, consistent with an involvement of LPS in their aetiology. These changes are mirrored by changes in their viscoelastic properties as measured by thromboelastography. Since the terminal stages of coagulation involve the polymerisation of fibrinogen into fibrin fibres, we tested whether LPS would bind to fibrinogen directly. We demonstrated this using isothermal calorimetry. Finally, we show that these changes in fibre structure are mirrored when the experiment is done simply with purified fibrinogen and thrombin (± 0.2 ng.L−1 LPS). This ratio of concentrations of LPS:fibrinogen in vivo represents a molecular amplification by the LPS of more than 108-fold, a number that is probably unparalleled in biology. The observation of a direct effect of such highly substoichiometric amounts of LPS on both fibrinogen and coagulation can account for the role of very small numbers of dormant bacteria in disease progression, and opens up this process to further mechanistic analysis and possible treatment.

Significance statement Most chronic diseases (including those classified as cardiovascular, neurodegenerative, or autoimmune) are accompanied by long-term inflammation. Although typically mediated by ‘inflammatory’ cytokines, the origin of this inflammation is unclear. We have suggested that one explanation is a dormant microbiome that can shed the highly inflammatory lipopolysaccharide LPS. Such inflammatory diseases are also accompanied by a hypercoagulable phenotype. We here show directly (using 6 different methods) that very low concentrations of LPS can affect the terminal stages of the coagulation properties of blood and plasma significantly, and that this may be mediated via a direct binding of LPS to a small fraction of fibrinogen monomers as assessed biophysically. Such amplification methods may be of more general significance.

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    Posted May 16, 2016.
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    Acute induction of anomalous blood clotting by molecular amplification of highly substoichiometric levels of bacterial lipopolysaccharide (LPS)
    Etheresia Pretorius, Sthembile Mbotwa, Janette Bester, Christopher Robinson, Douglas B. Kell
    bioRxiv 053538; doi: https://doi.org/10.1101/053538
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    Acute induction of anomalous blood clotting by molecular amplification of highly substoichiometric levels of bacterial lipopolysaccharide (LPS)
    Etheresia Pretorius, Sthembile Mbotwa, Janette Bester, Christopher Robinson, Douglas B. Kell
    bioRxiv 053538; doi: https://doi.org/10.1101/053538

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