A specific proteinase 3 activity footprint in α₁-antitrypsin deficiency

Discussion

This article describes a unique epitope of fibrinogen cleavage providing a footprint to identify and track neutrophil PR3 activity in vivo. In healthy individuals this footprint is almost three times greater than the equivalent NE footprint and would be consistent with the greater amount of PR3 stored in the azurophil granule reported previously [5]. Both enzymes would be released with neutrophil degranulation, leading to an area of obligate enzyme activity until diffusion dilutes each enzyme to equal that of the surrounding α₁-AT [16]. Our data support this concept. The data are also consistent with the greater likelihood of detecting PR3 activity compared with NE activity in the airways of patients with nondeficient COPD [8] where the prevalent lung α₁-AT level is further reduced due to restricted diffusion from plasma by the tight epithelial cell junctions [17] and the lack of a significant local inhibitor of PR3 [7].

There are several factors that likely have a greater influence on the in vivo activity of PR3 in patients with α₁-AT deficiency. These include the low concentration of α₁-AT, the higher PR3 concentration in the neutrophil, the reduced association rate constant compared with NE [7] as well as the lack of local lung inhibitors [8]. The net result is reflected in a ∼17-fold greater PR3 footprint than that of NE. In addition, although the PR3 and NE footprints correlate, the data do not extrapolate to the origin, suggesting significant PR3 activity would persist even after NE had been inhibited in α₁-AT deficiency due to the reasons outlined earlier.

Although both proteinase footprints correlate, only the PR3 footprint related cross-sectionally to lung function and lung densitometry in the PiZZ cohort. The data suggest, at least superficially, that the PR3 footprint would be a better biomarker for the study of lung disease in α₁-AT deficiency. However, the relationship between lung disease and biomarkers is complex [18]. Correlations raise the issue of cause and effect, and in our previous study in PiZZ patients the NE footprint (although stable) did not relate to subsequent progression in those with established COPD (i.e. the activity of the disease), supporting the concept that it reflected, at least in part, the effect of COPD rather than the cause [19]. This was consolidated further by association of the NE footprint with subsequent progression only in α₁-AT-deficient patients without COPD, suggesting that other factors play a role in established COPD [10].

In the nondeficient COPD cohort studied here, the PR3 footprint was lower than for both the PiZZ and PiSZ subgroups. This would be expected due to the normal α₁-AT levels in nondeficient patients and was not different to that in healthy smokers. However, only a proportion of the COPD patients remained currently smoking. The ex-smokers had a lower median PR3 footprint, suggesting it is, at least in part, a smoking-related signal in nondeficient COPD.

The PR3 footprint (like the NE footprint) in α₁-AT deficiency (although highly variable between individuals) is stable, suggesting it has reached equilibrium and therefore it remains possible that pre-inhibition PR3 activity is a major mediator of damage in established COPD related to α₁-AT deficiency. Interpretation of this baseline relationship is, however, further complicated by the fact that many α₁-AT-deficient patients studied here were ex-smokers, which would likely have had an amplifying effect on current lung status prior to PR3 footprint assessment. Further studies will therefore be required to determine future disease progression in α₁-AT-deficient never- or ex-smokers and any relationship to baseline PR3 footprint activity.

Augmentation therapy with human α₁-AT leads to a major reduction in the PR3 footprint (as seen previously for the lower NE footprint) [10] almost, although not quite, back to normal, indicating that this treatment limits the detectable in vivo activity of both enzymes (prior to inhibition and complex formation with α₁-AT), but suggests that a higher dose may be required to normalise PR3 activity. The data support, at least in part, the benefits of such therapy on emphysema progression in α₁-AT deficiency [20–22] and a role for Aα-Val⁵⁴¹ as a biomarker for this treatment effect.

The results for the PiSZ patients are worthy of further comment. There remains uncertainty about the susceptibility of this α₁-AT genotype to developing COPD and hence any role for augmentation therapy. The NE footprint in these patients was slightly raised compared with the healthy controls, as was the PR3 footprint and the NE/PR3 footprint ratio. However, the NE footprint in PiSZ patients was similar to that seen in PiZZ patients where lung disease severity and progression are greater This also suggests that NE may be less important in lung elastin destruction than PR3 in this patient group, and that the lower than normal α₁-AT level and decreased association rate constant of PiZZ α₁-AT for PR3 [7] result in a mild imbalance of PR3 activity compared with usual nondeficient COPD (indicated by the ratio of the two footprints) and may be a physiological mediator of increased tissue damage, and hence a target for treatment in PiSZ subjects. Clearly, further exploration of this issue is indicated.

The current study has both strengths and some weaknesses. The validation of the Aα-Val⁵⁴¹ assay confirms its specificity for the cleavage peptide and suggests the predominance of a PR3 signal in all cohorts consistent with its greater concentration than NE in the azurophil granule. In addition, the even greater PR3 footprint signal in α₁-AT deficiency confirms the potential importance of this enzyme with low concentrations of α₁-AT compounded by its lower association rate constant and the lack of another local lung PR3 inhibitor [7].

However, there is some controversy concerning the amount of PR3 in the azurophil granule (see Introduction); if the absolute amount of PR3 in the neutrophil is similar to or lower than NE, the difference seen in the footprint assays requires consideration for other possibilities. Both enzymes are promiscuous and can potentially have multiple cleavage sites. For this reason our initial digest and plasma analyses identified a fibrinogen cleavage product produced by PR3 present in the plasma of α₁-AT-deficient subjects and not generated by NE. Carboxyl-terminal sequencing was consistent with a PR3 cleavage site exposing a neo-epitope, and our subsequent analyses indicated no or only very weak binding by NE-cleaved fibrinogen and no cross-reactivity with our NE-specific cleavage sequence (the Aα-Val³⁶⁰ peptide).

It remains possible that the respective half-lives of these two fibrinogen cleavage products differ, producing some cross-sectional differences in the quantity of both, although this should not vary with α₁-AT genotype producing the major differences observed here. In addition, augmentation brings these two signals together, suggesting the neo-epitopes generated are predominantly related to activity rather than half-life. Finally, compared with NE, PR3 activity may be preferentially located at the neutrophil membrane where it is less susceptible to inhibition by α₁-AT [5], facilitating its local pericellular activity and hence fibrinogen cleavage. These issues can only be addressed by complex in vitro competition assays, but would require simultaneous pharmacokinetics analysis of patient samples from those receiving both α₁-AT augmentation and the inclusion of future specific and effective anti-NE or -PR3 therapies.

However, whatever the reason, we have studied several independent cohorts including both a severe α₁-AT-deficient group (PiZZ), a mild α₁-AT-deficient group (PiSZ) and a normal α₁-AT COPD group to consolidate the relationship of the marker to α₁-AT deficiency and its severity in the stable state, indicating clear differences in the ratio of the two footprints. Whether the PR3 marker predicts future progression of disease in α₁-AT deficiency is important, and this now requires longitudinal monitoring of disease progression in both the presence and absence of α₁-AT augmentation therapy. This has important implications for the management of α₁-AT deficiency as, although α₁-AT will inhibit both PR3 and NE, the pursuit and development of specific small-molecule inhibitors of NE that do not have broad activity to control both serine proteinases may be inappropriate or only provide partial protection, especially in α₁-AT deficiency. However, inhibiting NE by an alternative route may paradoxically release enough endogenous α₁-AT to provide extra PR3 cover. Clearly, such studies need to be undertaken and will provide important insights into neutrophil proteinase biology. The PR3 signal is high and certainly sensitive to augmentation, and may therefore prove essential as a treatment biomarker in future proteinase therapeutic strategies, including dose-ranging studies.

The fibrinogen footprint of PR3 activity is also likely to be an indirect marker of elastin degradation considered central to the pathophysiology of emphysema. Since our results were generated, a recent article using a similar methodology to measure a PR3-specific elastin cleavage product in healthy controls and a COPD cohort indicates similar increased PR3 compared with an NE cleavage product to that seen here [23]. Direct comparison of both assays should, however, be undertaken in a single α₁-AT-deficient cohort for final confirmation.

We describe a unique in vivo footprint of PR3 activity that is present in greater quantities than an NE footprint in subjects with normal α₁-AT, but especially in the presence of α₁-AT deficiency. The data suggest that PR3 may play a greater role in lung damage than previously thought and is sensitive to antiprotease therapy. As such, it may provide a critical role as a biomarker for dose ranging of antiprotease therapy. The specificity of the PR3 assay suggests it may also be useful in the assessment of other PR3-dependent diseases such as autoimmune vasculitis.