Neutrophil elastase and
cathepsin G are abundant intracellular neutrophil
proteinases that have an important role in destroying ingested particles. However, when neutrophils degranulate, these
proteinases are released and can cause irreparable damage by degrading host connective tissue
proteins. Despite abundant endogenous inhibitors, these
proteinases are protected from inhibition because of their ability to bind to anionic surfaces.
Plasminogen activator inhibitor type-1 (PAI-1), which is not an inhibitor of these
proteinases, possesses properties that could make it an effective inhibitor of neutrophil
proteinases if its specificity could be redirected.
PAI-1 efficiently inhibits surface-sequestered
proteinases, and it efficiently mediates rapid cellular clearance of PAI-1-proteinase complexes. Therefore, we examined whether
PAI-1 could be engineered to inhibit and clear
neutrophil elastase and
cathepsin G. By introducing specific mutations in the reactive center loop of wild-type
PAI-1, we generated
PAI-1 mutants that are effective inhibitors of both
proteinases. Kinetic analysis shows that the inhibition of neutrophil
proteinases by these
PAI-1 mutants is not affected by the sequestration of
neutrophil elastase and
cathepsin G onto surfaces. In addition, complexes of these
proteinases and
PAI-1 mutants are endocytosed and degraded by lung epithelial cells more efficiently than either the neutrophil
proteinases alone or in complex with their physiological inhibitors, alpha1-proteinase inhibitor and alpha1-antichymotrypsin. Finally, the
PAI-1 mutants were more effective in reducing the
neutrophil elastase and
cathepsin G activities in an in vivo model of
lung inflammation than were their physiological inhibitors.