The impact of pH on proteolytic activity in wound fluid: Implications for acid therapy

Wound healing necessitates a balance between synthesis and breakdown of extracellular matrix components, which is tightly regulated by proteases and their inhibitors. While studies have demonstrated that citric and acetic acid treatments enhance healing in recalcitrant wounds, the underlying proteolytic mechanisms remain elusive. In this study, we systematically evaluated changes in the proteolytic activity of murine wound fluid upon acidification. A library of 228 synthetic peptides served as reporters of protease activity at pH 7.4, pH 5.0, and pH 3.5. The peptide digestion patterns differed at each pH, revealing that proteases active at pH 7.4 are inactivated at pH 3.5. Notably, cathepsin D emerged as the dominant active enzyme at pH 3.5, and its activity was inhibited by pepstatin. Using a fluorogenic substrate, we quantified cathepsin D activity across varying pH levels and demonstrated optimal activity between pH 3.0 and 3.8. This activity was detectable as early as 1 day postinjury and persisted over the following 10 days. Importantly, human wound fluid exhibited the same activity profile, validating the mouse model as a relevant system for studying acid-mediated wound healing processes.