Both lipopolysaccharide and lipoteichoic acids potently induce anomalous fibrin amyloid formation: assessment with novel Amytracker^™stains

Abstract

In recent work, we discovered that the presence of highly substoichiometric amounts (10 -8 molar ratio) of lipopolysaccharide (LPS) from Gram-negative bacteria caused fibrinogen clotting to lead to the formation of an amyloid form of fibrin. We here show that the broadly equivalent lipoteichoic acids (LTAs) from two species of Gram-positive bacteria have similarly (if not more) potent effects. Using thioflavin T fluorescence to detect amyloid as before, the addition of low concentrations of free ferric ion is found to have similar effects. Luminescent conjugated oligothiophene dyes (LCOs), marketed under the trade name Amytracker TM , also stain classical amyloid structures. We here show that they too give very large fluorescence enhancements when clotting is initiated in the presence of the four amyloidogens (LPS, ferric ions and two LTA types). The staining patterns differ significantly as a function of both the amyloidogens and the dyes used to assess them, indicating clearly that the nature of the clots formed is different. This is also the case when clotting is measured viscometrically using thermoelastography. Overall, the data provide further evidence for an important role of bacterial cell wall products in the various coagulopathies that are observable in chronic, inflammatory diseases. The assays may have potential in both diagnostics and therapeutics.

Relative Hypercoagulopathy of the SARS-CoV-2 Beta and Delta Variants when Compared to the Less Severe Omicron Variants Is Related to TEG Parameters, the Extent of Fibrin Amyloid Microclots, and the Severity of Clinical Illness

“…27 Our group has previously shown amyloid fibrin deposits in numerous inflammatory conditions. 46 47 48 49 50 51 52 53 However, the extent of the microclot presence in both acute COVID-19 and long COVID was found to be significantly more than in other chronic systemic inflammatory conditions. 32 36…”

“…CaCl 2 is responsible for the reversal of the anticoagulant action of sodium citrate and, consequently, activation of the coagulation cascade. 46 Samples were loaded into the measuring channels of the TEG 5000 Hemostasis Analyzer System (07–033; Haemonetics, Boston, MA) and allowed to run until the maximum amplitude (MA) was reached. All analyses were performed at 37 °C.…”

A guide to studying protein aggregation

“…However, their inability to identify the prefibrillar species and the growing interest in identifying nonfibrillar oligomers led to the development of negatively charged luminescent conjugated oligothiophenes (LCOs), like pFTAA which identifies several intermediate species occurring during protein aggregation, including the soluble prefibrillar and oligomeric species, amorphous aggregates, as well as mature fibrillar structures [163]. pFTAA has become a popular amyloid-specific dye for investigating amyloid formation properties [27,29,45,128], such as studying the inducing effect of iron, lipopolysaccharides and lipoteichoic acids during plasma protein aggregation [164], or identifying distinct aggregating populations [165][166][167][168][169].…”