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 (LPS) 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 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 that on stoichiometric grounds must occur autocatalytically). 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. As the terminal stages of coagulation involve the polymerization 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 in a great many inflammatory conditions, and opens up this process to further mechanistic analysis and possible treatment.
Highlights
‘LPS’ describes a variety of cell wall lipopolysaccharides shed by Gram-negative bacteria; known as ‘endotoxin’, they have been found in various fluids, including whole blood (WB)
LPS from Escherichia coli (100 ng ml21) activated the coagulation system when added to WB, via a complementand CD14-dependent upregulation of tissue factor (TF), leading to prothrombin activation and hypercoagulation [35]; this was noted after 2 h, and it was not an acute process [35]
To investigate our hypothesis that LPS may cause hypercoagulation via an acute and direct binding reaction, we investigated the effect of two LPS preparations from E. coli
Summary
‘LPS’ describes a variety of cell wall lipopolysaccharides shed by Gram-negative bacteria; known as ‘endotoxin’, they have been found in various fluids, including whole blood (WB). LPS from Escherichia coli (100 ng ml21) activated the coagulation system when added to WB, via a complementand CD14-dependent upregulation of TF, leading to prothrombin activation and hypercoagulation [35]; this was noted after 2 h, and it was not an acute process [35] Note that in these studies, the anticoagulant was lepirudin, which prevents thrombin activation such that the effects of thrombin could not be evaluated. It occurred to us that, in addition to changes in TF expression by LPS, the process might involve the direct binding of the lipophilic LPS to circulating plasma proteins, fibrinogen, and that this ( potentially rapid) binding might cause pathological changes in the coagulation process This would be independent of the slower TF activation, and an acute and relatively immediate process (figure 1). A preprint has been lodged at bioRxiv [36]
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