Tifacogin, a recombinant tissue factor pathway inhibitor eliminates serum-resistant Escherichia coli from blood cultures
Abstract number: P575
Schirm S., Liu X., Jennings L., Jedrzejewski P., Dai Y., Hardy S.
Objectives: Tifacogin tissue factor pathway inhibitor (rTFPI), has demonstrated therapeutic effectiveness in acute inflammatory disease after lethal challenge with Escherichia coli in several animal models of severe sepsis. Tifacogin is being evaluated in a phase 3 clinical trial in patients with severe community acquired pneumonia. The mechanism of action of tifacogin after acute bacterial infection is not well understood. Our current study investigates the effects of tifacogin after acute infection in vitro. The objectives were to show whether fragmented tifacogin had antibacterial activity against E. coli which could be mapped to the C-terminus and to investigate the effect of heparin on the antibacterial activity of fragmented tifacogin.
Methods: 10% donor derived blood or serum were used to culture 3 strains of serum resistant E. coli for 4 hours at 37°C with varied concentrations of fragmented tifacogin prepared using plasma proteases or synthetic C-terminal peptides. Bacterial survival was assessed by plating serial dilutions of the reaction mixes onto tryptase soy agar plates. Colony numbers were determined after overnight incubation at 37°C. Direct interaction between TFPI peptides and E. coli was investigated by fluorescent microscopy using fixed bacteria and fluorescently labeled peptides. Heparin was included in some cultures or binding experiments.
Results: C-terminal tifacogin fragments and synthetic peptides exhibited antibacterial activity in a complement-dependent fashion. While intact tifacogin slightly enhanced E. coli growth, tifacogin digested with plasma proteases dramatically reduced growth. The activity was mapped to a highly positively charged region previously identified within intact TFPI as a heparin binding site. Synthetic peptides including the positive region were highly active (MIC90 1030nM). Antibacterial action involved the classical complement pathway including terminal complex formation since factors C1q, C2, C3, C4, C5, C6 and C9 were required. Direct interaction with the bacterial cell surface of E. coli was seen. Complement mediated killing and cell surface binding were reversed by low amounts of heparin.
Conclusions: Our results show a novel activity of the C-terminus of tifacogin in complement resistant E. coli infection that is released by fragmentation of tifacogin. As tifacogin is rapidly degraded in vivo, our findings suggest that tifacogin may have a therapeutic role in management of disease caused by acute infection.
|Session name:||18th European Congress of Clinical Microbiology and Infectious Diseases|
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