Proteinase 3 activity in sputum from subjects with alpha-1-antitrypsin deficiency and COPD

Abstract

Chronic obstructive pulmonary disease (COPD) is associated with tissue damage believed to result from an imbalance between serine proteinases and their inhibitors. Although the role of neutrophil elastase (NE) has been studied, it is likely that other proteinases play a role. The importance of proteinase 3 (PR3) has not been established, as specific substrates have only recently been available.We studied clinically stable subjects with either alpha-1-antitrypsin (A1AT) deficiency or usual COPD with chronic bronchitis. Sol phase sputum was analysed for PR3 activity and concentration, NE activity and concentration, concentrations of airway inhibitors (A1AT, secretory leukoproteinase inhibitor and elafin) and markers of neutrophilic inflammation. 12 patients were also studied during exacerbations.PR3 activity was present in most sputum samples and greater than NE activity (which was largely undetectable) in both subject groups (A1AT deficiency median PR3 128 nM, interquartile range (IQR) 33-558 nM; NE 0 nM, IQR 0-0 nM; p50.0043; COPD PR3 22 nM, IQR 0-103 nM; NE 0 nM, IQR 0-0 nM; p50.015). PR3 activity was greater during exacerbations than in the stable state (p50.037) and correlated with markers of neutrophilic inflammation.The regular identification of PR3 activity in sputum from stable subjects with A1AT deficiency or usual COPD suggests it may play a greater role in the pathophysiology than previously thought.

α-1-Antitrypsin variants and the proteinase/antiproteinase imbalance in chronic obstructive pulmonary disease

“…Pure PR3 (Merck, Feltham, United Kingdom; EC number 3.4.21.76) and pure NE (Athens Research and Technology, Athens, GA; EC number 3.4.21.37) were active site titrated against pure M variant A1AT (Athens Research and Technology), as described previously (44).…”

“…The A1AT concentration was measured in the serum samples using a locally developed ELISA, which has been described elsewhere (44). The A2M concentrations of serum samples were determined using a commercially available A2M ELISA kit (Universal Biologicals, Cambridge, United Kingdom), according to the manufacturer's instructions.…”

“…Although many studies to date have focused on the importance of NE in the proteinase/antiproteinase imbalance (45,50), there is increasing evidence that other NSPs such as PR3 may also play an important role (44). PR3 is the most abundant serine proteinase in the neutrophil with each cell being estimated to store 3.0 and 1.1 pg of PR3 and NE, respectively (8).…”

Recent advances in α-1-antitrypsin deficiency-related lung disease

“…PR3 is also a serine proteinase found in the azurophil granules of neutrophils which also causes emphysema in animal models [33]. PR3 activity has been shown to be greater than that of NE in sputum of A1AT-deficient individuals especially during exacerbations, suggesting a role in the pathophysiology of COPD in these individuals [34]. Matrix metalloproteinases (MMPs; particularly MMP-12) and cysteine proteinases such as cathepsin B have also been implicated as having a direct role in proteolytic alveolar destruction [35,36].…”

Inhibitors and Antibody Fragments as Potential Anti-Inflammatory Therapeutics Targeting Neutrophil Proteinase 3 in Human Disease

“…We recently developed a selective biotinylated pNA substrate, Bt-Pro-Tyr-Asp-Ala-pNA (k cat /K m = approximately 30 mM 21 s 21 ), for PR3 over HNE (Guarino et al, unpublished data). Structural differences between PR3 and HNE at S3, S2, S19, and S29 have been exploited to develop highly sensitive and specific synthetic FRET peptide substrates for human PR3 that allow its detection in complex biologic samples (Hajjar et al, 2006;Korkmaz et al, 2007;Popow-Stellmaszyk et al, 2013;Sinden and Stockley, 2013;Hinkofer et al, 2015). In addition, a cell-permeable selective PR3 substrate, O 2 Oc-K(HMC)-YYAbu-Orn(CM3), was recently developed (Wysocka et al, 2012).…”