Proteolytic signatures define unique thrombin-derived peptides present in human wound fluid in vivo

Abstract

The disease burden of failing skin repair and non-healing ulcers is extensive. There is an unmet need for new diagnostic approaches to better predict healing activity and wound infection. Uncontrolled and excessive protease activity, of endogenous or bacterial origin, has been described as a major contributor to wound healing impairments. Proteolytic peptide patterns could therefore correlate and “report” healing activity and infection. This work describes a proof of principle delineating a strategy by which peptides from a selected protein, human thrombin, are detected and attributed to proteolytic actions. With a particular focus on thrombin-derived C-terminal peptides (TCP), we show that distinct peptide patterns are generated in vitro by the human S1 peptidases human neutrophil elastase and cathepsin G, and the bacterial M4 peptidases Pseudomonas aeruginosa elastase and Staphylococcus aureus aureolysin, respectively. Corresponding peptide sequences were identified in wound fluids from acute and non-healing ulcers, and notably, one peptide, FYT21 (FYTHVFRLKKWIQKVIDQFGE), was only present in wound fluid from non-healing ulcers colonized by P. aeruginosa and S. aureus. Our result is a proof of principle pointing at the possibility of defining peptide biomarkers reporting distinct proteolytic activities, of potential implication for improved diagnosis of wound healing and infection.

Structural basis for endotoxin neutralization and anti-inflammatory activity of thrombin-derived C-terminal peptides

“…S1D). Notably, the peptide FYT21, previously identified in infected wounds [6, 23], blocked LPS-induced pro-inflammatory response and displayed LPS neutralization to a similar extent as GKY25, previously used in therapeutic studies (Fig. 2B).…”

“…For illustrative purposes, and to highlight the complete, albeit gradual, formation of such fragments, the generation of the 2 kDa TCP HVF18 HVFRLKKWIQKVIDQFGE, cleaved out by human neutrophil elastase [7], is depicted in Figure 1B. A similar generation of the peptide FYT21 (FYTHVFRLKKWIQKVIDQFGE) has been observed after digestion with the bacterial M4 peptidases Pseudomonas aeruginosa elastase [6, 23] and Staphylococcus aureus aureolysin [23]. Incorporating these endogenous sequences, the prototypic peptide GKYGFYTHVFRLKKWIQKVIDQFGE has been further used in in vitro studies on its mode-of-action [24, 25], as well as in several therapeutic in vivo studies, aimed at targeting endotoxin-mediated inflammatory responses [4, 7].…”

“…1A), comprises a flexible β-strand segment and a compact amphipathic helix. Previous studies have shown that various proteases can generate TCPs derived from this region [23]. For illustrative purposes, and to highlight the complete, albeit gradual, formation of such fragments, the generation of the 2 kDa TCP HVF18 HVFRLKKWIQKVIDQFGE, cleaved out by human neutrophil elastase [7], is depicted in Figure 1B.…”

“…Neutralization of circulating LPS by HDPs has been shown to reduce adverse LPS-induced pro-inflammatory effects in experimental animal models [4, 21, 22]. In this context, a prototypic TCP, GKY25 (GKYGFYTHVFRLKKWIQKVIDQFGE), encompassing sequences of natural TCPs previously identified in human wounds [23], has been shown to protect against P. aeruginosa sepsis and LPS-mediated shock, mainly via reduction of systemic cytokine responses in vivo [4, 24]. These observations, however, disclose neither the exact mode of action, nor whether these peptides can target other molecules apart from LPS.…”

Descriptive vs mechanistic scientific approach to study wound healing and its inhibition: Is there a value of translational research involving human subjects?

“…DFU are also characterized by a significant reduction of miR‐132, which contributes to the deregulation of inflammation‐related pathways such as NF‐κB, NOD‐like receptor, Toll‐like receptor and TNF‐α signalling . Multiple proteomic studies have utilized chronic wound fluid as an accessible source of biomaterial, revealing not only deregulation of secreted host proteases, but also specific peptides from human thrombin derived as result of wound infection . In addition to analysing host response to infection, advances in next‐generation sequencing methods have allowed for the identification and quantification of microbial communities in chronic and acute wound setting (Table ) .…”