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

Rathi Saravanan,Artur Schmidtchen,Mariena J A Van Der Plas,Yeu Khai Choong,Sven Kjellström,Sunil S Adav,Siu Kwan Sze,Jitka Petrlova

doi:10.1038/s41598-017-13197-3

Rathi Saravanan, Artur Schmidtchen + Show 6 more

Open Access

https://doi.org/10.1038/s41598-017-13197-3

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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.

Highlights

Conditions, such as arterial and venous insufficiency, diabetes, or excessive pressure all predispose to the formation of non-healing ulcers
Thrombin susceptibility to endogenous human proteases secreted by neutrophils during wounding was assessed by analyzing the in vitro peptide patterns generated by human neutrophil elastase (HNE) and cathepsin G (HCG)
Similar to the findings with HNE and human neutrophil cathepsin G (HCG), the results indicate that bacterial proteases cleave thrombin distinctly, yielding unique peptide patterns (Fig. 3)

Summary

Introduction

Conditions, such as arterial and venous insufficiency, diabetes, or excessive pressure all predispose to the formation of non-healing ulcers. Non-healing ulcers are characterized by the presence of an elevated level of proteases, both endogenous and of bacterial origin, which disrupts the delicate balance between acute inflammation and progression of wound healing[8] These wounds are often characterized by high inflammatory activity, excessive proteolytic processing of matrix molecules and immune mediators and impaired wound closure[9,10,11]. We have previously demonstrated that proteolysis of thrombin generates host defense peptides (HDPs), derived from the C-terminal part of the molecule[14] These thrombin-derived C-terminal peptides (TCPs) bind to bacterial lipopolysaccharide (LPS) and display both anti-microbial and anti-inflammatory activities in vitro, with therapeutic potential against infection and septic shock in vivo[15,16]. Our study demonstrates a proof of principle of the feasibility for defining peptide biomarkers that “report” on distinct protease activities in vivo

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