Protease-Antiprotease Imbalance in Bronchiectasis.

Airway inflammation plays a central role in bronchiectasis. Protease-antiprotease balance is crucial in bronchiectasis pathophysiology and increased presence of unopposed proteases activity may contribute to bronchiectasis onset and progression. Proteases' over-reactivity and antiprotease deficiency may have a role in increasing inflammation in bronchiectasis airways and may lead to extracellular matrix degradation and tissue damage. Imbalances in serine proteases and matrix-metallo proteinases (MMPs) have been associated to bronchiectasis. Active neutrophil elastase has been associated with disease severity and poor long-term outcomes in this disease. Moreover, high levels of MMPs have been associated with radiological and disease severity. Finally, severe deficiency of α1-antitrypsin (AAT), as PiSZ and PiZZ (proteinase inhibitor SZ and ZZ) phenotype, have been associated with bronchiectasis development. Several treatments are under study to reduce protease activity in lungs. Molecules to inhibit neutrophil elastase activity have been developed in both oral or inhaled form, along with compounds inhibiting dipeptydil-peptidase 1, enzyme responsible for the activation of serine proteases. Finally, supplementation with AAT is in use for patients with severe deficiency. The identification of different targets of therapy within the protease-antiprotease balance contributes to a precision medicine approach in bronchiectasis and eventually interrupts and disrupts the vicious vortex which characterizes the disease.

Evaluation of active neutrophil elastase in sputum of bronchiectasis and cystic fibrosis patients: A comparison among different techniques.

Neutrophil elastase (NE) is a crucial marker of neutrophilic inflammation. We aimed to compare different techniques to detect active NE in sputum samples of 50 Bronchiectasis (BE) and 50 Cystic Fibrosis (CF) patients. Three methods including a ProteaseTag® Active NE Immunoassay (ELISA) and two enzymatic digestion assays (chromogenic -CS- and fluorogenic -FS- substrate) were compared. Results of active NE were also correlated with clinical data. The three methods provided statistically different values for NE activity in the same sputum samples in both cohorts. In the BE cohort, the highest correlations between NE activity and Bronchiectasis Severity Index (rho = 0.40, P < 0.0001), sputum purulence (AUC = 0.79), and chronic infections due to any pathogen (AUC = 0.76) and P. aeruginosa (AUC = 0.80) were found when NE was measured through the activity-based immunoassay. In the CF cohort, the highest correlations between NE activity and sputum quantity (rho = 0.71) and FEV1% (rho = 0.42, P = 0.03) were observed when the FS method was used, while similar correlations with chronic P. aeruginosa infection were identified with the FS and ELISA methods. NE activity in sputum correlates with clinical variables in both diseases. However, different methods to evaluate active NE in sputum lead to significantly different results, also in terms of correlation with clinical data.

Neutrophil elastase in bronchiectasis.

The role of neutrophil elastase (NE) is poorly understood in bronchiectasis because of the lack of preclinical data and so most of the assumptions made about NE inhibitor potential benefit is based on data from CF. In this context, NE seems to be a predictor of long-term clinical outcomes and a possible target of treatment. In order to better evaluate the role of NE in bronchiectasis, a systematic search of scientific evidence was performed.Two investigators independently performed the search on PubMed and included studies published up to May 15, 2017 according to predefined criteria. A final pool of 31 studies was included in the systematic review, with a total of 2679 patients. For each paper data of interest were extracted and reported in table.In this review sputum NE has proved useful as an inflammatory marker both in stable state bronchiectasis and during exacerbations and local or systemic antibiotic treatment. NE has also been associated with risk of exacerbation, time to next exacerbation and all-cause mortality. This study reviews also the role of NE as a specific target of treatment in bronchiectasis. Inhibition of NE is at a very early stage and future interventional studies should evaluate safety and efficacy for new molecules and formulations.

The Role of Neutrophil Elastase Inhibitors in Lung Diseases.

In many respiratory diseases characterized by an intense inflammatory response, the balance between proteolytic enzymes (proteases, including elastases) and their inhibitors (proteinases inhibitors) is not neutral. Excess activity of neutrophil elastase (NE) and similar proteases has been reported to cause tissue damage and to alter the remodeling process in many clinical conditions such as pneumonia, respiratory distress, and acute lung injury (ALI). Several experimental NE inhibitors have been tested in preclinical and clinical studies of different conditions of inflammatory lung injury such as ALI and pneumonia, with contrasting results. This study reviews the literature regarding NE inhibitors in the field of respiratory diseases and reflects on possible future developments. In particular, we highlight potential gaps in the scientific evidence and discuss potential strategies for focusing investigation on antielastases in clinical practice through the selection of targeted populations and proper outcomes.

Identification of a novel SERPINA-1 mutation causing alpha-1 antitrypsin deficiency in a patient with severe bronchiectasis and pulmonary embolism.

Deficiency in the serine protease inhibitor, alpha-1 antitrypsin (AAT), is known to cause emphysema and liver disease. Other manifestations, including airway disease or skin disorders, have also been described. A 44-year-old woman presented to our emergency department with dyspnea and respiratory insufficiency. She had never smoked, and had been diagnosed with COPD 9 years earlier. Three months previously, she had suffered a pulmonary embolism. Chest computed tomography scan revealed severe cystic bronchiectasis with destruction of the lung parenchyma. The sweat test was normal and there was no evidence of the cystic fibrosis transmembrane conductance regulator (CFTR) mutation. Capillary zone electrophoresis showed a decrease of alpha-1 globin band and AAT levels were below the quantification limit (<25 mg/dL). No S or Z mutation was identified, but sequencing analysis found a homozygous cytosine and adenine (CA) insertion in exon 2 of the SERPINA-1 gene, probably leading to a dysfunctional protein (PI Null/Null). This mutation has not been previously identified. The atypical presentation of the patient, with severe cystic bronchiectasis, highlights AAT deficiency as a differential diagnosis in bronchiectasis. Further, awareness should be raised regarding a possible increased risk of thromboembolism associated with AAT deficiency.

Matrix metalloproteinase activation by free neutrophil elastase contributes to bronchiectasis progression in early cystic fibrosis.

Neutrophil elastase is the most significant predictor of bronchiectasis in early-life cystic fibrosis; however, the causal link between neutrophil elastase and airway damage is not well understood. Matrix metalloproteinases (MMPs) play a crucial role in extracellular matrix modelling and are activated by neutrophil elastase. The aim of this study was to assess if MMP activation positively correlates with neutrophil elastase activity, disease severity and bronchiectasis in young children with cystic fibrosis.Total MMP-1, MMP-2, MMP-7, MMP-9, tissue inhibitor of metalloproteinase (TIMP)-2 and TIMP-1 levels were measured in bronchoalveolar lavage fluid collected from young children with cystic fibrosis during annual clinical assessment. Active/pro-enzyme ratio of MMP-9 was determined by gelatin zymography. Annual chest computed tomography imaging was scored for bronchiectasis.A higher MMP-9/TIMP-1 ratio was associated with free neutrophil elastase activity. In contrast, MMP-2/TIMP-2 ratio decreased and MMP-1 and MMP-7 were not detected in the majority of samples. Ratio of active/pro-enzyme MMP-9 was also higher in the presence of free neutrophil elastase activity, but not infection. Across the study cohort, both MMP-9/TIMP-1 and active MMP-9 were associated with progression of bronchiectasis.Both MMP-9/TIMP-1 and active MMP-9 increased with free neutrophil elastase and were associated with bronchiectasis, further demonstrating that free neutrophil elastase activity should be considered an important precursor to cystic fibrosis structural disease.

Matrix metalloproteinases vary with airway microbiota composition and lung function in non-cystic fibrosis bronchiectasis.

RATIONALE: Despite growing evidence for the roles of airway remodeling and bacterial infection in the progression of non-cystic fibrosis bronchiectasis, relationships between collagen-degrading proteases and chronic airway infection are poorly understood. OBJECTIVES: The aim of this study was to determine which matrix metalloproteinases (MMPs) are elevated in bronchiectasis, whether these MMP levels vary based on patients' dominant infective microbe, and how these levels correlate with clinical measures of disease severity. METHODS: We determined concentrations of nine MMPs and four tissue inhibitors of metalloproteinases (TIMPs) in induced sputum from 86 patients with bronchiectasis and 8 healthy control subjects by Luminex protein assay. Concentrations were then assessed in relation to lung function, inflammatory markers, and airway microbiota composition, determined by 16S rRNA gene amplicon sequencing. Airway microbiota composition was classified as Pseudomonas aeruginosa-dominated, Haemophilus influenzae-dominated, or dominated by another species. MMP-8 and MMP-9 activity levels were also measured in a subset of patients. MEASUREMENTS AND MAIN RESULTS: MMP-1, -3, -7, -8, and -9 and TIMP-2 and -4 levels, as well as MMP-8/TIMP-1 and MMP-9/TIMP-1 ratios, were significantly higher in patients with bronchiectasis than in healthy control subjects (all: P < 0.001, except MMP-7: P < 0.05). Patients with bronchiectasis with H. influenzae-dominated airway infections demonstrated higher MMP-2 levels (P < 0.01) and MMP-8 activity (P < 0.05) than those with P. aeruginosa-dominated airway infections. Among patients with bronchiectasis, there were significant inverse correlations between FEV1 as a percentage of predicted value, MMP-8 and MMP-1 levels, and MMP-8/TIMP-1 and MMP-9/TIMP-1 ratios (P < 0.01). CONCLUSIONS: Increased MMP levels (particularly MMP-8 and MMP-1) and MMP/TIMP ratios in patients with bronchiectasis compared with healthy control subjects correlated with lower lung function and higher levels of inflammatory markers. Further, MMP profiles differed in patients with bronchiectasis according to the dominant pathogen determined by gene sequencing, raising the possibility of differential airway remodeling according to airway microbiology.

Neutrophil elastase gene polymorphisms: modulators of response to therapy in childhood bronchiectasis?

BACKGROUND: The aim of this study was to investigate polymorphisms in the promoter region of the neutrophil elastase (ELANE) gene as potential modulators of the therapeutic response in children with idiopathic bronchiectasis. METHODS: The study included 48 children between 5 and 17 years old who were diagnosed with idiopathic bronchiectasis based on high-resolution computed tomography of the thorax. In all patients therapy included administration of antibiotics, anti-inflammatory drugs, expectorants, and postural drainage. Response to therapy was evaluated by the change in FeNO levels before and after administration of therapy. The ELANE promoter region polymorphisms were analyzed by PCR-direct DNA sequencing. RESULTS: According to the predicted activity of ELANE genotypes, subjects were divided into two groups: low/intermediate activity (n = 18) and high activity (n = 30). Subjects in the group with high-activity genotype had higher initial FeNO levels and this difference was statistically significant (t = 2.906; p = 0.006). The difference between FeNO levels before and after therapy was also statistically significantly higher in children with high-activity genotype (t = 3.329; p = 0.002). Statistically significant correlation was observed between the change in FeNO levels and ELANE genotypes (r = 0.350; p = 0.015). CONCLUSION: Children with high-activity genotype had higher initial FeNO levels and showed better response to therapy than children with low/intermediate-activity genotypes.

Significance of serum peptidylarginine deiminase type 4 in ANCA-associated vasculitis.

OBJECTIVE: To investigate the clinical significance of peptidylarginine deiminase type 4 (PAD4) in the pathogenesis of antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV). To make a primary observation on the relationship of chronic bronchitis and bronchiectasis (CB) with the pathogenesis of AAV by PAD4. METHODS: The sera from 13 patients with AAV, 13 patients with CB, 11 patients with rheumatoid arthritis (RA), 11 patients with primary chronic kidney disease (CKD) and 12 normal controls were collected. Serum PAD4 was detected using commercial ELISA kits. The serum levels of PAD4 were compared not only among the different groups but also between the activity and remission stage of the same disease. The associations between serum PAD4 and the Birmingham Vasculitis Activity Score (BVAS) of AAV were further investigated. RESULTS: (1) The serum levels of PAD4 in patients with AAV, RA and CB at activity stage were all higher than that in the normal controls (P<0.001, respectively, α'=0.007). The serum level of PAD4 in patients with CB at remission stage and that in CKD group were not found elevated compared with the normal controls (P=0.02, P=0.085, respectively, α'=0.007). (2) At activity stage, among the groups of simple AAV, AAV with a long history of CB and CB without AAV, no significant difference was detected. While at remission stage among the 3 groups, the serum level of PAD4 was at the lowest level in CB group without AAV. (3) The serum level of PAD4 in some patients with CB without AAV were found still higher at remission stage. (4) The serum level of PAD4 in AAV with renal damage at activity stage was positively correlated with BVAS (the activity score of AAV, r=0.71, P=0.02). CONCLUSION: PAD4 is involved in the pathogenesis of AAV. Whether some patients with CB might progress to AAV by the link with PAD4 still need further investigation.

Shed syndecan-1 restricts neutrophil elastase from alpha1-antitrypsin in neutrophilic airway inflammation.

Persistent proteolytic imbalance in chronic inflammatory diseases has been ascribed to neutrophil elastase (NE)/antielastase imbalance in wound fluids. In sputum sols of patients with bronchiectasis, we found unopposed NE activity, despite overwhelming excess of the physiological antielastase, alpha(1)-antitrypsin (alpha(1)-AT). Western blot analysis found NE in a supramolecular complex with shed ectodomains of syndecan (Syn)-1 in sputum sol samples and, as such, inhibition of NE activity was incomplete, even with addition of exogenous alpha(1)-AT. To confirm that NE binding to heparan sulfate (HS) components of Syn-1 limits the antielastase effect, recombinant human Syn-1 was recovered from stable Syn-1 transfectants of a human B-lymphoid cell line (ARH-77). Western ligand blot confirmed that NE bound to HS moieties and alpha(1)-AT to the core protein of the recombinant product. Inhibition of NE activity by standard additions of alpha(1)-AT was incomplete unless Syn-1 had been deglycanated by heparitinase treatment. Surface plasmon resonance analysis revealed that NE binding to HS (equilibrium dissociation constant, approximately 14 nM) could be outcompeted by heparin variants. We conclude that the HS moiety of shed Syn-1 binds and restricts NE from inhibition by alpha(1)-AT.

Matrix metalloproteinases gene variants in idiopathic disseminated bronchiectasis.

BACKGROUND: The excess of matrix metalloproteinases (MMPs) might be associated with the airways destruction or dilatation in bronchiectasis. The functional promoter polymorphisms of MMP1 and MMP9 genes, involved in the extracellular matrix remodeling, might increase the expression of MMPs leading to the development of bronchiectasis. METHODS: Detection of MMP1 G-1607GG and MMP9 C-1562T gene variants was performed on 37 patients with idiopathic disseminated bronchiectasis and 102 control subjects. We also described a novel method for simple and rapid detection of MMP1 G-1607GG polymorphism. RESULTS: The frequency of -1607GG allele was significantly higher in the group of patients than in control subjects (P = 0.014). The heterozygote genotype showed association with bronchiectasis (odds ratio, 5.3; 95% confidence intervals, 1.4-20.0). The association was even stronger in homozygotes for -1607GG allele (odds ration, 8.7; 95% confidence intervals, 1.9-41.0). The allelic and genotype frequencies of MMP9 C-1562T variant did not show significant differences between the groups. CONCLUSIONS: This is the first report concerning a role of MMP1 G-1607GG and MMP9 C-1562T variants in pathogenesis of idiopathic disseminated bronchiectasis. The results of our study revealed the association of -1607GG allele and the lack of association of MMP9 C-1562T variant with the disease.

Lack of association between matrix metalloproteinase 8 promoter polymorphism and bronchiectasis in Koreans.

The observation that human matrix metalloproteinase (MMP)-8 is over-expressed in ectatic bronchi in patients with bronchiectasis suggests that polymorphisms altering the expression of MMP-8 may contribute to the susceptibility to development of bronchiectasis. We evaluated the association between the presence of bronchiectasis in a Korean population and two single nucleotide polymorphisms (SNPs) (-799C/ T and -381A/G) on the promoter region of the MMP-8 gene that are reported to alter the promoter activity and thereby the gene expression. Genotyping through polymerase chain reaction (PCR) and subsequent automatic sequencing was done in 167 patients with bronchiectasis and their age-, sex-matched healthy controls to reveal that only -799C/T is polymorphic among Koreans. In the patient group with bronchiectasis, the frequency of -799C/C, C/T, and T/T genotypes were 41.9%, 49.7%, and 8.4%, respectively. A similar distribution was observed in the control group: C/C (49.7%), C/T (43.1%), and T/T (7.2%) (p=0.36). In subgroup analysis, no significant difference was observed among the patients according to; the extent of disease (p=0.76), colonization of microorganisms (p=0.56), or association of mycobacteria (p=0.17). From these results, we conclude that -799C/T on the promoter region of MMP-8 lacks association with development of bronchiectasis in Koreans.

Sputum sol neutrophil elastase activity in bronchiectasis: differential modulation by syndecan-1.

The persistently dominant activity of neutrophil elastase in bronchial secretions replete with antielastases is crucial to the pathogenesis of bronchiectasis. We hypothesize that components in the bronchial secretions bind neutrophil elastase and compromise the inhibitory efficiency of prevailing antielastases. Zymographic analysis of sputum sols from patients with bronchiectasis found elastase activity in a polydisperse, alcian blue-stained zone of high molecular mass. This suggested that neutrophil elastase was complexed with polyanionic partners. Western blot analysis found not only the polyanionic partner, heparan sulfate/syndecan-1, but also the physiological antielastases, secretory leukoproteinase inhibitor and alpha1-antitrypsin, in the complex. Both dissociative density gradient ultracentrifugation and heparin displacement revealed that elastase dissociated from heparan sulfate/syndecan-1 was fully inhibited by the endogenous antielastases. This contrasts with the effects of exogenous antielastases on sputum neutrophil elastase activity-that of alpha1-antitrypsin was limited, but that of secretory leukoproteinase inhibitor was facilitated. Similarly, complexed elastase on blots of sputum sol zymographs was bound and inhibited by exogenous secretory leukoproteinase inhibitor but not by exogenous alpha1-antitrypsin. Taken together, the results bring a new focus to heparan sulfate/syndecan-1 complexed with neutrophil elastase in inflamed bronchial secretions as a target for modulating elastase susceptibility to physiological antielastases.

Soluble membrane-type 1 matrix metalloproteinase (MT1-MMP) and gelatinase A (MMP-2) in induced sputum and bronchoalveolar lavage fluid of human bronchial asthma and bronchiectasis.

The aim of this study was to investigate the involvement of the MT1-MMP/MMP-2 cascade in induced sputum (IS) and bronchoalveolar lavage fluid (BALF) from bronchial asthma (BA) and bronchiectasis (BE) patients and healthy controls. The molecular forms and cellular origins of MT1-MMP and MMP-2 were determined by Western immunoblotting, immunohistochemistry and in situ hybridization. Elevated levels of soluble activated and autocatalyzed MT1-MMP species as well as activated forms of MMP-2 in IS and BALF samples from BA and BE patients were evidenced. The activation degrees of soluble MT1-MMP and MMP-2 were significantly correlated in BA and BE IS and BALF. Only low levels of both these MMPs were observed in healthy control IS and BALF. The co-expression of MMP-2 with MT1-MMP was evidenced by double immunostaining in bronchial epithelial cells, submucosal glandular cells, smooth muscle cells and monocyte/macrophages. The MT1-MMP/MMP-2 cascade is present and active in human inflammatory lung disease fluid and tissue samples. This cascade seemingly reflects the active destructive phases of these chronic lung diseases.

In vivo collagenase-2 (MMP-8) expression by human bronchial epithelial cells and monocytes/macrophages in bronchiectasis.

The purpose of this study was to determine whether other cellular sources than neutrophils can express matrix metalloproteinase (MMP)-8 protein and mRNA in bronchiectatic (BE) lung. The molecular forms of MMP-8 in the BE bronchoalveolar lavage fluid (BALF) and healthy control BALF were analysed by western immunoblotting. MMP-8 expression was demonstrated by immunohistochemistry and in situ hybridization in BE lung tissue and by immunohistochemistry in control lung tissue. In the BE BALF, different MMP-8 species were detected: 70-80 kD MMP-8 apparently of polymorphonuclear leukocyte (PMN) origin and also 40-60 kD MMP-8 from non-PMN cellular sources, such as bronchial epithelial cells, glandular cells or monocytes/macrophages. Both of these MMP-8 species were elevated and converted to a significant extent to activated forms in BE BALF compared with healthy control BALF. The levels of high molecular weight (>80 kD) MMP-8 complexes, evidently representing MMP-8 trapped by endogenous MMP inhibitors and/or MMP-8 dimers, were significantly elevated in BE BALF compared with healthy control BALF. In BE lung tissue, the MMP-8 protein and mRNA expression was found in bronchial ciliated epithelial cells, glandular cells, neutrophils, and monocytes/macrophages infiltrating the bronchial epithelial area. Minimal MMP-8 expression was observed in neutrophils, monocytes/macrophages, and epithelial cells in control lung tissues. In this study, new potential cellular sources have been demonstrated for MMP-8 in the inflamed lung. MMP-8 from multiple cellular sources, including inflamed lung epithelium, was activated to a significant extent in the BE BALF, indicating a major role for MMP-8 in the destruction of lung and bronchial tissues.

Validation of assays for inflammatory mediators in sputum.

Commercially available immune assays are being used with increasing frequency in the study of lung inflammation. However, their performance in complex biological fluids is rarely assessed. The authors wished to assess their reliability to determine whether the results obtained in sputum samples can be easily interpreted. The reproducibility of several such assays was therefore determined together with their ability to recover known amounts of pure reagent. Sputum sol phase was obtained from several patients with chronic lung disease and used together with the reagents in a series of "spiking" and dilutional experiments. Results confirmed that the enzyme assay for myeloperoxidase and the immune assays for interleukin-8, leukotriene B4 and secretory leukoproteinase inhibitor were all reproducible (intra-assay coefficient of variation 3.8-7.7%). Furthermore, each of these assays gave >85% recovery of a "spike" with pure reagent. However, the immune assay for myeloperoxidase (although reproducible) gave poor recovery and was dependent on the degree of sample dilution and elastase content. These studies confirm that the reliabilities of fluid phase measurements should be assessed before being widely applied to complex biological samples.

Sputum elastase in steady-state bronchiectasis.

STUDY OBJECTIVES: To study the correlations between sputum elastase output with clinical and sputum inflammatory and microbial factors in steady-state bronchiectasis. DESIGN: Prospective recruitment of patients with bronchiectasis (17 women; 48.5 +/- 16.5 years old; FEV(1)/FVC, 1.3 +/- 0.6/2.1 +/- 0.9) for assessment of 24-h sputum output of elastase, bacteria, leukocytes, interleukin (IL)-1beta, IL-8, tumor necrosis factor-alpha, and leukotriene B(4). Clinical variables assessed concomitantly included 24-h sputum volume, lung spirometry, number of lung lobes affected by bronchiectasis, and exacerbation frequency. SETTING: Consecutive recruitment of outpatients (n = 30) in steady-state bronchiectasis. MEASUREMENTS AND RESULTS: Twenty-four-hour sputum elastase output correlated with 24-h sputum volume (r = 0.79, p = 0.0001); number of bronchiectatic lung lobes (r = 0.54, p = 0.0026); percent predicted FEV(1) (r = -0. 48, p = 0.0068); percent predicted FVC (r = -0.49, p = 0.001); and leukocyte output (r = 0.75, p = 0.0001). There was no correlation between the sputum output of bacteria with either inflammatory or enzymatic factors (p > 0.05). CONCLUSION: Our data highlight the importance of elastase and the possibility of independent roles for enzymatic, inflammatory, and microbial components in the pathogenesis of bronchiectasis. Further research on novel therapy targeting each of these components should be pursued.

[Fibrinogenolytic activity of a novel trypsin-like enzyme found in human airways].

We previously reported finding a novel trypsin-like enzyme in sputum samples from patients with chronic airway diseases, and named it human airway tryptase (HAT). We also obtained data suggesting that HAT is secreted from submucosal serous glands onto the mucous membrane of the airway, and that fibrinogen is cleaved by HAT. We studied whether HAT can act as an anticoagulant in the airway by breaking down fibrinogen. The concentration of fibrinogen in sputum samples was measured by enzyme-linked immunosorbent assay. The concentration was higher in mucoid sputum from patients with bronchial asthma (6.3 +/- 5.5) than in mucoid sputum from patients with chronic bronchitis (1.9 +/- 1.1), and it was higher in purulent sputum from patients with bronchiectasis (18.8 +/- 8.8) than in mucoid sputum from patients with asthma. The trypsin-like activity in sputum (milliunit/ml) was 23.4 +/- 18.0 in mucoid sputum from patients with chronic bronchitis, 46.9 +/- 43.9 in mucoid sputum from patients with asthma, and 14.9 +/- 8.23 in purulent sputum from patients with bronchiectasis. The effect of HAT on human fibrinogen at concentrations from 4 to 2000 micrograms/ml was examined at pH 7.4 and 8.6, with purified HAT at concentrations from 0.5 to 10 milliunit/ml. SDS-polyacrylamide gel electrophoresis showed that HAT can cleave fibrinogen. especially the alpha-chain, at low concentrations (4 to 16 micrograms/ml) and at a high concentration (2000 micrograms/ml) of fibrinogen. Exposure of fibrinogen to HAT resulted in the loss of thrombin-induced clotting capacity; the strength of that effect depended on the duration of exposure to HAT and on the concentration of HAT. From these results we postulate that HAT acts at an anticoagulant in the airways, especially on the mucous membrane, by cleaving fibrinogen transported from blood.

Potentiative effects of neutral proteinases in an inflamed lung: relationship of neutrophil procollagenase (proMMP-8) to plasmin, cathepsin G and tryptase in bronchiectasis in vivo.

We attempted to study the possible relationships between neutrophil-type procollagenase/pro-matrix metalloproteinase (MMP-8) and the serine proteinases plasmin, cathepsin G and tryptase in bronchiectasis. The presence of the plasmin/plasminogen system and plasmin-, cathepsin G- and tryptase-like activities were compared to the activity of endogenously activated MMP-8 in bronchoalveolar lavage (BAL) fluid in 38 bronchiectasis patients and in 14 healthy controls by means of immunohistochemistry, Western-blot and substrate-based functional assays. In contrast to cathepsin G- and tryptase-like activities, the plasmin/plasminogen activator system in BAL fluid was observed to have a relatively weak activation stage and no correlation with disease severity. Neither plasmin-like activities nor concentrations of plasminogen activators from the bronchiectatic patients differed significantly from the values of healthy controls. Immunolocation of plasminogen activator inhibitor-1 showed a marked, but not significant, increase in bronchiectatic lung as compared to controls. In contrast to cathepsin G- and tryptase-like activities, with their strong and significant correlation with endogenously activated collagenase (r=0.9; p=0.0001; and r=0.6; p=0.03, respectively), no correlations were observed between plasmin-like and endogenously activated collagenase (r=0.3; p=0.2) in bronchiectasis. These findings suggest that cathepsin G- and tryptase-like activities may act as potent pro-matrix metalloproteinase-8 activators in patients with bronchiectasis, whereas the plasminogen activator/plasmin cascade was shown to be down-regulated.