Potent inhibition of matriptase by serpin A5 is abrogated by heparin-binding.

Serpin A5 (SA5), also known as protein C inhibitor and plasminogen activator inhibitor-3 (PAI-3), is a serine protease inhibitor principally reported as a heparin-dependent inhibitor of proteases involved in coagulation and fibrinolysis, to include thrombin and factor Xa. High concentrations of SA5 (3-4 mM) in human seminal plasma and inhibition of kallikrein-like protease, prostate specific antigen (PSA) and acrosin indicate a further role in the regulation of fertilisation. Given its broad specificity and wide tissue distribution, the aim of this study was to investigate the ability of SA5 to inhibit other physiologically relevant trypsin-like proteases, such as the type II transmembrane serine proteases (TTSPs).
Recombinant human TTSPs (human airway trypsin-like protease (HAT), hepsin and matriptase) as well as prostasin, a glycophosphatidylinositol-anchored membrane protease, were screened in the absence and presence of heparin using peptide-based fluorogenic substrate activity assays. Novel interactions were further probed using SDS-PAGE and western blot analysis, supported by molecular modelling studies.
SA5 was found to be an effective inhibitor of HAT, hepsin and prostasin however, in contrast to thrombin for which the pIC50 increased from 5.9 to 7.2, no further enhancement of inhibitory activity against these proteases was observed in the presence of heparin. Of note, potent inhibition of matriptase (pIC50 7.5) by SA5 was observed (complex formation confirmed by SDS-PAGE and Western blotting), which was abolished when SA5 was first bound by heparin. Several structural differences between thrombin and matriptase related to heparin binding and identified by molecular modelling help explain these results.
This is the first report of SA5 as a potent inhibitor of matriptase. It is possible that physiological regulation by heparin serves as a molecular switch that shifts the inhibitory activity of the protein from one protease target to another.

This study was supported by the Borders and Regions Airways Training Hub project (BREATH; INT-VA/045) funded by the European Union (EU), under the INTERREG VA Programme, managed by the Special EU Programmes Body.