| 1. |
- Saravanan, Rathi, et al.
(author)
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Cell-Free DNA Promotes Thrombin Autolysis and Generation of Thrombin-Derived C-Terminal Fragments
- 2021
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In: Frontiers in Immunology. - : Frontiers Media SA. - 1664-3224. ; 12
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Journal article (peer-reviewed)abstract
- Cell-free DNA (cfDNA) is the major structural component of neutrophil extracellular traps (NETs), an innate immune response to infection. Antimicrobial proteins and peptides bound to cfDNA play a critical role in the bactericidal property of NETs. Recent studies have shown that NETs have procoagulant activity, wherein cfDNA triggers thrombin generation through activation of the intrinsic pathway of coagulation. We have recently shown that thrombin binds to NETs in vitro and consequently can alter the proteome of NETs. However, the effect of NETs on thrombin is still unknown. In this study, we report that DNA binding leads to thrombin autolysis and generation of multiple thrombin-derived C-terminal peptides (TCPs) in vitro. Employing a 25-residue prototypic TCP, GKY25 (GKYGFYTHVFRLKKWIQKVIDQFGE), we show that TCPs bind NETs, thus conferring mutual protection against nuclease and protease degradation. Together, our results demonstrate the complex interplay between coagulation, NET formation, and thrombin cleavage and identify a previously undisclosed mechanism for formation of TCPs.
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| 2. |
- Lim, Chun Hwee, et al.
(author)
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Thrombin and plasmin alter the proteome of neutrophil extracellular traps
- 2018
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In: Frontiers in Immunology. - : Frontiers Media SA. - 1664-3224. ; 9:JUL
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Journal article (peer-reviewed)abstract
- Neutrophil extracellular traps (NETs) consist of a decondensed DNA scaffold decorated with neutrophil-derived proteins. The proteome of NETs, or "NETome," has been largely elucidated in vitro. However, components such as plasma and extracellular matrix proteins may affect the NETome under physiological conditions. Here, using a reductionistic approach, we explored the effects of two proteases active during injury and wounding, human thrombin and plasmin, on the NETome. Using high-resolution mass spectrometry, we identified a total of 164 proteins, including those previously not described in NETs. The serine proteases, particularly thrombin, were also found to interact with DNA and bound to NETs in vitro. Among the most abundant proteins were those identified previously, including histones, neutrophil elastase, and antimicrobial proteins. We observed reduced histone (H2B, H3, and H4) and neutrophil elastase levels upon the addition of the two proteases. Analyses of NET-derived tryptic peptides identified subtle changes upon protease treatments. Our results provide evidence that exogenous proteases, present during wounding and inflammation, influence the NETome. Taken together, regulation of NETs and their proteins under different physiological conditions may affect their roles in infection, inflammation, and the host response.
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| 3. |
- Lim, Chun Hwee, et al.
(author)
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Thrombin-derived host defence peptide modulates neutrophil rolling and migration in vitro and functional response in vivo
- 2017
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In: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 7:1
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Journal article (peer-reviewed)abstract
- Host defence peptides (HDPs) derived from the C-terminus of thrombin are proteolytically generated by enzymes released during inflammation and wounding. In this work, we studied the effects of the prototypic peptide GKY25 (GKYGFYTHVFRLKKWIQKVIDQFGE), on neutrophil functions. In vitro, GKY25 was shown to decrease LPS-induced neutrophil activation. In addition, the peptide induced CD62L shedding on neutrophils without inducing their activation. Correspondingly, GKY25-treated neutrophils showed reduced attachment and rolling behaviour on surfaces coated with the CD62L ligand E-selectin. The GKY25-treated neutrophils also displayed a dampened chemotactic response against the chemokine IL-8. Furthermore, in vivo, mice treated with GKY25 exhibited a reduced local ROS response against LPS. Taken together, our results show that GKY25 can modulate neutrophil functions in vitro and in vivo.
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| 4. |
- Petrlova, Jitka, et al.
(author)
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Selective protein aggregation confines and inhibits endotoxins in wounds : Linking host defense to amyloid formation
- 2023
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In: iScience. - : Elsevier BV. - 2589-0042. ; 26:10
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Journal article (peer-reviewed)abstract
- Bacterial lipopolysaccharide (LPS) induces rapid protein aggregation in human wound fluid. We aimed to characterize these LPS-induced aggregates and their functional implications using a combination of mass spectrometry analyses, biochemical assays, biological imaging, cell experiments, and animal models. The wound-fluid aggregates encompass diverse protein classes, including sequences from coagulation factors, annexins, histones, antimicrobial proteins/peptides, and apolipoproteins. We identified proteins and peptides with a high aggregation propensity and verified selected components through Western blot analysis. Thioflavin T and Amytracker staining revealed amyloid-like aggregates formed after exposure to LPS in vitro in human wound fluid and in vivo in porcine wound models. Using NF-κB-reporter mice and IVIS bioimaging, we demonstrate that such wound-fluid LPS aggregates induce a significant reduction in local inflammation compared with LPS in plasma. The results show that protein/peptide aggregation is a mechanism for confining LPS and reducing inflammation, further emphasizing the connection between host defense and amyloidogenesis.
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