help button home button
AJRCCM
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Published ahead of print on February 20, 2003, doi:10.1164/rccm.200208-905OC
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Online Supplement
Right arrow All Versions of this Article:
200208-905OCv1
167/12/1687    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Fujimoto, H.
Right arrow Articles by Taguchi, O.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Fujimoto, H.
Right arrow Articles by Taguchi, O.
American Journal of Respiratory and Critical Care Medicine Vol 167. pp. 1687-1694, (2003)
© 2003 American Thoracic Society

Original Article

Thrombin-activatable Fibrinolysis Inhibitor and Protein C Inhibitor in Interstitial Lung Disease

Hajime Fujimoto, Esteban Cesar Gabazza, Osamu Hataji, Hisamichi Yuda, Corina Nayade D'Alessandro-Gabazza, Mariko Nakano, Omar Enrique Franco, Tatsuya Hayashi, Koji Suzuki, Yukihiko Adachi and Osamu Taguchi

Respiratory Division of the Third Department of Internal Medicine; Department of Molecular Pathobiology; and Department of Urology, Mie University School of Medicine, Tsu, Mie, Japan

Correspondence and requests for reprints should be addressed to Esteban C. Gabazza, Third Department of Internal Medicine, Respiratory Division, Mie University School of Medicine, Edobashi 2–174, Tsu City, Mie 514–8507, Japan. E-mail: gabazza{at}clin.medic.mie-u.ac.jp


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Intraalveolar activation of the coagulation system due to reduced fibrinolytic function plays a critical role in the pathogenesis of interstitial lung disease. Recently, a new potent inhibitor of fibrinolysis, thrombin-activatable fibrinolysis inhibitor, has been isolated and characterized from human plasma. This study evaluated the levels of thrombin-activatable fibrinolysis inhibitor and protein C inhibitor, another suppressor of fibrinolysis, in the bronchoalveolar lavage fluid from patients with interstitial lung disease. There were 82 patients with interstitial lung disease and 8 normal subjects. The bronchoalveolar lavage fluid levels of thrombin-activatable fibrinolysis inhibitor and protein C inhibitor were significantly higher in all patients with interstitial lung disease than in normal subjects. Both inhibitors of fibrinolysis were significantly and inversely correlated with fibrinolytic activity in all patients. The levels of thrombin-activatable fibrinolysis inhibitor were significantly correlated with those of protein C inhibitor, thrombin–antithrombin complex, and monocyte chemoattractant protein-1. Reverse transcriptase-polymerase chain reaction showed that alveolar macrophages isolated from patients with interstitial lung disease as well as immortalized lung epithelial cell lines express thrombin-activatable fibrinolysis inhibitor antigen. Overall, these findings suggest that thrombin-activatable fibrinolysis inhibitor and protein C inhibitor may play important roles in the mechanism of intraalveolar hypofibrinolysis associated with interstitial lung diseases.

Key Words: lung fibrosis • plasmin • coagulation • activated protein C • metalloproteinases

Interstitial lung disease (ILD) is a group of disorders of the lower respiratory tract that results from injury of the lung parenchyma, increased proliferation of mesenchymal cells, and excessive accumulation of connective-tissue matrix in the lung, causing ultimately lung fibrosis (1). The exact mechanism of lung fibrosis is not clear, but the results of studies performed in experimental animal models suggested that decreased degradation of extracellular matrix caused by deficient function of the alveolar fibrinolysis plays a fundamental role (2). The primary enzyme of the fibrinolysis system is plasmin, a trypsin-like proteinase that is formed when the zymogen plasminogen is cleaved into its two-chain form by either tissue-type plasminogen activator or urokinase-type plasminogen activator. Plasmin can stimulate matrix degradation not only directly by degrading a number of extracellular matrix macromolecules but also indirectly by activating procollagenases and prostromelysins. Plasmin can also rapidly degrade fibrin formed after leakage of proteins and activation of coagulation cascade in the alveolar space (3, 4). Under physiologic conditions, the alveolar space has net fibrinolytic activity because of the presence of urokinase-type plasminogen activator (5, 6). However, during many acute and chronic inflammatory lung disorders, fibrin accumulates in lung tissue (5, 7, 8). The fibrinolytic activity in bronchoalveolar lavage fluid (BALF) from patients with the acute respiratory distress syndrome, idiopathic pulmonary fibrosis (IPF), and sarcoidosis is reduced (8–10). All of these diseases have been associated with the development of pulmonary fibrosis. Depressed fibrinolysis has been also described in a variety of animal models of lung injury and fibrosis (11, 12). The results of these experimental studies suggested that decreased plasmin activity mainly occurs because of overexpression of plasminogen activator inhibitor (PAI)-1, the primary inhibitor of both tissue and urokinase plasminogen activators (12). It has been reported that bleomycin-induced lung fibrosis is more severe in transgenic mice that overexpressed PAI-1 than in PAI-1–deficient mice and that bleomycin-treated PAI-1 deficient mice have enhanced fibrinolysis, less collagen buildup, and longer survival than their PAI-1 expressing counterparts (13). These observations suggest the biologic significance of the fibrinolytic system dysfunction in the pathogenesis of ILD.

Recently, a new potent inhibitor of fibrinolysis, the thrombin-activatable fibrinolysis inhibitor (TAFI) or carboxypeptidase U, has been isolated and characterized from human plasma. TAFI is a (58 kD) glycoprotein synthesized by the liver that can be activated by thrombin-, thrombin–thrombomodulin complex-, plasmin- or trypsin-catalyzed proteolysis to a carboxypeptidase B-like enzyme that inhibits fibrinolysis (14). The thrombin–thrombomodulin complex-catalyzed activation of TAFI may be upregulated by protein C inhibitor (PCI); this serine protease inhibitor, also called PAI-3, is the main suppressor of protein C activation and of the activity of activated protein C (15–17). Activated protein C is a serine protease that stimulates fibrinolysis by binding to plasminogen activator-1 and by inhibiting thrombin generation (17). Thus, PCI may directly inhibit fibrinolysis by suppressing plasminogen activator and also indirectly by blocking the activity of activated protein C. It is well known that C-terminal lysines on cell-surface proteins and partially degraded fibrin enhance fibrinolysis by providing binding sites for plasminogen, which once bound, adopts a more activatable conformation. Activated TAFI inhibits activation of plasminogen to plasmin by removing these C-terminal lysine residues (18). In addition, activated TAFI may also directly inactivate plasmin further impairing fibrinolysis (19). The possibility that TAFI and PCI participate in the fibrinolytic system dysfunction in ILD patients is not known. This study was undertaken to assess the levels of TAFI and PCI in the BALF in patients with ILD. Some of the results of this study have been previously reported in the form of an abstract (20).


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture
The immortalized alveolar epithelial cell line, A549, was obtained from the American Type Culture Collection (Rockville, MD). The primary normal human bronchial epithelial cells were purchased from Clonetics (Walkersville, MD), and the human hepatic HepG2 cells from RIKEN Cell Bank (Ibaraki, Japan; for details, see the online supplement).

Alveolar Macrophage Culture
BALF cells obtained from ILD patients were suspended in RPMI-1640 medium containing 2 mM of glutamine, 10% fetal bovine serum, 50 µg/ml of penicillin, and 50 µg/ml of streptomycin and then seeded in primary cell culture plates. The cells were allowed to adhere for 4 hours in a humidified incubator (5% CO2, 95% air) at 37°C. Nonadherent cells were removed by washing three times with RPMI-1640 medium, and then the cells were used for total RNA preparation.

Reverse Transcriptase-Polymerase Chain Reaction
Total RNA was extracted from cultured cells and tissues by the guanidine isothiocyanate procedure using Trizol Reagent (Invitrogen Life Technologies, Carlsbad, CA). Two micrograms of total RNA were reverse transcribed using oligo-dT primers, and then the cDNA was amplified by polymerase chain reaction (PCR) using the Superscript Preamplification system kit (Invitrogen Life Technologies) following the manufacturer's instructions. Primers and PCR conditions are described in online supplement.

Patient Population
This study comprised 82 newly diagnosed patients with ILD. There were 10 patients (7 males and 3 females) with IPF, 34 (15 males and 19 females) with sarcoidosis, 4 (3 males and 1 female) with eosinophilic pneumonia, 9 (5 males and 4 females) with hypersensitivity pneumonitis, 5 (4 males and 1 female) with cryptogenic organizing pneumonia (COP), and 20 (4 males and 16 females) with collagen vascular disease-associated ILD (CVD-ILD). Among this latter group, there were four patients with rheumatoid arthritis, five with systemic sclerosis, three with systemic lupus erythematosus, five with dermatomyositis, and four with mixed connective tissue disease. The diagnosis of IPF was based on the presence of specific clinical and high-resolution computed tomography findings and evidence of usual interstitial pneumonitis in histologic lung specimens (21). The diagnosis of sarcoidosis was established by histologic evidence of noncaseating granulomas in transbronchial lung biopsy specimens and compatible clinical, serologic, and radiographic findings (20). The diagnosis of CVD-ILD was based on previously reported criteria for each group of CVD and the histologic evidence of pulmonary fibrosis in transbronchial lung biopsy specimens (21). Data were obtained in eight healthy volunteers, who served as control subjects. Written informed consent was obtained from all subjects. The study protocol was approved by the Mie University Hospital Institutional Review Board and was performed following the principles of the Helsinki Declaration.

BALF Sampling
BALF was obtained as previously described (22). A fiberoptic bronchoscope (Olympus 1T10; Olympus, Tokyo, Japan) was wedged into a segmental bronchus of the middle lobe or lingula. Four 50-ml aliquots of warmed sterile normal saline were instilled and were retrieved immediately by suction at low negative pressure adjusted by a vacuum regulator. The pooled BAL was collected on ice and processed within 1 hour. The total amount of recovered fluid was measured, and the recovery rate was calculated. The BALF was filtered through two layers of sterile gauze and was centrifuged at 150 g for 10 minutes at 4°C. The cell-free supernatant was separated and stored in small aliquots at -80°C until analysis. The cellular fraction was resuspended in RPMI-1640 medium (Celox Laboratories, Hopkins, MN), and total cells were counted using a Neubauer hemocytometer counting chamber. Differential cell count was performed in a Giemsa-stained cytocentrifuge preparation at x1,000 magnification.

Laboratory Measurements
The BALF levels of PCI and thrombin–antithrombin complex (TAT) were measured by an enzyme immunoassay using 96-well plate coated with antihuman PCI or TAT monoclonal antibody and, as second antibody, a biotin-labeled antihuman PCI or TAT monoclonal antibody. The interassay and intra-assay coefficients of variability were less than 10%. The concentration of the total antigen levels of TAFI in BALF and plasma was measured using a commercial enzyme immunoassay kit (TAFI-EIA; Kordia Laboratory Supplies, Leiden, Netherlands) following the manufacturer's instructions. A commercial TAFI activity kit (Actichrome TAFI activity kit; American Diagnostica, Greenwich, CT) was used to evaluate whether TAFI is active in BALF of ILD patients. The concentrations of monocyte chemoattractant protein-1 (Biosource International, Camarillo, CA) and PAI-1 (Technoclone, Vienna, Austria) were measured using commercial enzyme immunoassay kits following the manufacturer's instructions. The concentrations of the aminoterminal propeptides of type I and type III procollagen were measured using radioimmunoassay kits (Orion Diagnostica, Oulunsalo, Finland). The intra-assay and interassay variations were less than 10%. The activity of plasminogen activator in BALF was spectrophotometrically measured using the chromogenic substrate S-2444 (Chromogenix, Molndal, Sweden). To measure the balance between coagulation activation and fibrinolytic activity, the ratio of plasminogen activator activity to TAT level in BALF from ILD patients was calculated. Protein concentration in BALF was measured by the Bradford's method using a protein assay kit (Bio-Rad Laboratories, Hercules, CA). Determination of fibrinolytic potential of PAI-1, PCI, and TAFI in BALF was performed as described previously (23) (see the method in the online supplement).

Statistical Analysis
Data are expressed as the mean ± SE unless otherwise specified. The statistical difference between two variables was calculated using the Wilcoxon's rank test. The strength of correlation between variables was calculated by the Pearson product moment correlation or by the Spearman's correlation according to the distribution of the data. Statistical analyses were performed using the StatView 4.1 package software (Abacus Concepts, Berkeley, CA) for the Macintosh; p < 0.05 was considered as statistically significant.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Concentrations of Total Protein, Coagulation, and Collagen Markers in BALF
The BALF concentration of total protein, an indicator of ongoing inflammation, was markedly elevated (p < 0.05) in all patients with ILD (308.713 ± 27.582 µg/ml) as compared with the healthy control group (128.872 ± 36.538 µg/ml). This increase in BALF protein level was statistically significant (p < 0.05) in patients with CVD-ILD (326.361 ± 67.332 µg/ml), hypersensitivity pneumonitis (371.844 ± 72.478 µg/ml), eosinophilic pneumonia (397.359 ± 152.384 µg/ml), and COP (482.011 ± 114.098 µg/ml). BALF levels of TAT were also significantly increased (p < 0.05) in all ILD patients (15.223 ± 2.735 ng/ml) compared with the control group (3.212 ± 0.468 ng/ml) and particularly in patients with CVD-ILD (18.893 ± 5.945 ng/ml), sarcoidosis (17.014 ± 4.949 ng/ml), eosinophilic pneumonia (5.248 ± 0.495 ng/ml), and COP (15.501 ± 7.120 ng/ml). The collagen markers, aminoterminal propeptides of type I procollagen (2.733 ± 0.588 vs. 1.400 ± 0.173 µg/ml) and aminoterminal propeptides of type III procollagen (2.711 ± 1.145 vs. 0 ± 0 µg/ml), were also markedly increased in BALF of all ILD patients compared with healthy control subjects. The BALF concentrations of TAT and total protein were significantly and proportionally correlated (r = 0.3, p < 0.05) in all ILD patients, illustrating the closed relationship between procoagulant activity and inflammation in ILD.

Concentrations of TAFI, PCI, Monocyte Chemotactic Protein-1, and PAI-1 in BALF
Evaluation of the BALF concentration of TAFI disclosed a significant increase (p < 0.001) of this fibrinolysis inhibitor in all ILD patients compared with healthy subjects (Figure 1A) . Analysis by group of disease showed that TAFI is significantly increased (p < 0.05) in patients with CVD-ILD, sarcoidosis, hypersensitivity pneumonitis, IPF, or COP compared with healthy control subjects. BALF concentration of TAFI tended to be increased (p < 0.06) in patients with eosinophilic pneumonia compared with healthy control subjects. The BALF concentrations of TAFI were significantly and proportionally correlated with the BALF levels of TAT (r = 0.4, p < 0.0005; Figure 1B) and total protein (r = 0.4, p < 0.0001) in all ILD patients, suggesting that TAFI is implicated in intraalveolar inflammation and enhanced activation of the coagulation system in ILD patients. In addition, TAFI was significantly and proportionally correlated with the markers of collagen synthesis, aminoterminal propeptides of type I procollagen and aminoterminal propeptides of type III procollagen, in BALF from all ILD patients, suggesting the implication of TAFI in increased collagen formation in the lung (Table 1) .



View larger version (29K):
[in this window]
[in a new window]
  		Figure 1. Bronchoalveolar lavage fluid (BALF) level of thrombin-activatable fibrinolysis inhibitor (TAFI) and its correlation with thrombin–antithrombin complex (TAT) in interstitial lung disease (ILD). TAFI was significantly increased in all patients with ILD, cryptogenic organizing pneumonia (COP), collagen vascular disease (CVD)-associated ILD, hypersensitivity pneumonitis (HP), idiopathic pulmonary fibrosis (IPF), and sarcoidosis compared with healthy control subjects (A). The BALF concentration of TAFI also tended to be high in patients with eosinophilic pneumonia (EP) compared with healthy control subjects. TAFI was significantly correlated with TAT (r = 0.4, p < 0.0005) in all patients with ILD (B). *p < 0.05 and **p < 0.0001 compared with healthy control subjects.

 

View this table:
[in this window]
[in a new window]
  		TABLE 1. Correlation of thrombin-activatable fibrinolysis inhibitor and protein c inhibitor with markers of fibrinolysis and collagen metabolism in bronchoalveolar lavage fluid from all interstitial lung disease patients

 
PCI is the main inhibitor of the activity of activated protein C (24). The antigen levels of PCI were significantly increased (p < 0.05) in all patients with ILD and in particular in those with COP, sarcoidosis, and CVD-ILD (Figure 2A) . The chemokine monocyte chemotactic protein-1 (MCP-1) plays an important role in pulmonary inflammation by promoting the migration of inflammatory cells in the lung (25). The MCP-1 concentration was also markedly increased (p < 0.05) in BALF from all patients with ILD, particularly in patients with COP, IPF, and CVD-ILD (Figure 2B). TAFI was significantly and proportionally correlated with both PCI (r = 0.7, p < 0.0001) and MCP-1 (r = 0.4, p < 0.0007) in BALF from all patients with ILD (Figures 3A and 3B) .



View larger version (22K):
[in this window]
[in a new window]
  		Figure 2. BALF levels of protein C inhibitor (PCI), monocyte chemotactic protein (MCP)-1, and plasminogen activator inhibitor (PAI)-1 in ILD. PCI (A) and MCP-1 (B) were significantly increased in all patients with ILD and in those with COP and CVD-ILD compared with healthy subjects. PCI was also significantly increased in patients with sarcoidosis and MCP-1 in those with IPF. PAI-1 (C) was significantly increased in all ILD patients and in those with CVD-ILD, EP, and sarcoidosis. *p < 0.05 compared with healthy control subjects.

 


View larger version (36K):
[in this window]
[in a new window]
  		Figure 3. Relationship of TAFI with PCI and MCP-1. TAFI was significantly correlated with PCI (A, r = 0.7, p < 0.0001) and MCP-1 (B, r = 0.4, p < 0.0007) in BALF from all ILD patients.

 
PAI-1 is the main inhibitor of plasminogen activator activity. BALF concentration of PAI-1 was significantly increased (p = 0.02) in all ILD patients compared with healthy subjects (Figure 2C). Analysis by group of disease showed that PAI-1 is significantly increased (p < 0.05) in patients with CVD-ILD, sarcoidosis, and eosinophilic pneumonia compared with healthy control subjects. The concentration of PAI-1 was proportionally and significantly correlated with that of TAFI and PCI in BALF from all ILD patients (Table 1).

Fibrinolytic Activity in BALF
To evaluate whether biologically active TAFI is presence in BALF, the active form of TAFI was determined in BALF. The results disclosed that activated TAFI is significantly increased in BALF from all ILD patients compared with healthy control subjects (Figure 4A) . Experimental animals models have shown that lung injury is associated with suppressed fibrinolytic function. In this study, we used the ratio of plasminogen activator activity to TAT level (PA activity/TAT) in BALF as an index of fibrinolytic function. The PA activity/TAT ratio in BALF was significantly higher in healthy control subjects than in all ILD patients (Figure 4B) and particularly in patients with CVD-ILD, IPF, and sarcoidosis (Table 2) . These findings suggest that fibrinolytic function is impaired in ILD. The BALF levels of both TAFI and PCI were found to be inversely and significantly correlated with the PA activity/TAT ratio in all ILD patients (Table 1). Analysis by group of disease showed that the PA activity/TAT ratio is significantly and inversely correlated with TAFI in BALF from patients with CVD-ILD and with PCI and PAI-1 in BALF from patients with CVD-ILD and IPF (Table 2). These results suggest that TAFI, PCI, and PAI-1 are involved in the suppression of fibrinolysis in some groups of ILD patients. Analysis of the antifibrinolytic activity of each fibrinolysis inhibitor showed that PAI-1, PCI, and TAFI have almost the same inhibitory potential in BALF of our ILD patients (Figure 4E).



View larger version (25K):
[in this window]
[in a new window]
  		Figure 4. TAFI activity, PA activity/TAT ratio, and circulating levels of TAFI and PCI in ILD. TAFI activity (A) was significantly increased in all ILD patients compared with healthy control subjects. The PA activity/TAT ratio (B) was significantly decreased in all ILD patients compared with healthy control subjects. Circulating levels of both TAFI (C) and PCI (D) were significantly increased in all ILD patients compared with healthy control subjects. Analysis of the antifibrinolytic activity of each fibrinolysis inhibitor (E) showed that PAI-1, PCI, and TAFI have almost the same inhibitory potential in BALF of our ILD patients. *p < 0.05 compared with healthy control subjects.

 

View this table:
[in this window]
[in a new window]
  		TABLE 2. Plasminogen activity ratio and its correlation with thrombin-activatable fibrinolysis inhibitor, protein c inhibitor, and plasminogen activator INHIBITOR-1 in bronchoalveolar lavage fluid from each group of interstitial lung disease patients

 
Circulating Levels of TAFI and PCI and Expression of TAFI in the Lung
The plasma concentrations of TAFI and PCI were significantly higher in all ILD patients than in healthy control subjects (Figures 4C and 4D). A comparison between the mean values of the BALF and plasma levels of TAFI in all ILD patients showed that the TAFI level is almost twofold higher in BALF than in plasma (2,495.3 ± 211 vs. 1,279.8 ± 106).

It is known that the liver is the primary source of TAFI production (14). In this study, the expression of TAFI by alveolar macrophages isolated from patients with ILD, by an immortalized alveolar epithelial cell line (A549), and by a commercially purchased primary normal bronchial epithelial cells was evaluated by reverse transcriptase-PCR. The results showed that A549 alveolar epithelial cell lines, primary normal bronchial epithelial cells, and alveolar macrophages express TAFI mRNA (600 bp) (Figure 5) . These findings suggest that TAFI may also increase in the intraalveolar space by direct TAFI release from lung cells.



View larger version (49K):
[in this window]
[in a new window]
  		Figure 5. Reverse transcriptase-PCR of TAFI in lung cells. A band (600 bp) corresponding to the reported gene of TAFI could be amplified by reverse transcriptase-PCR in A549 alveolar epithelial cell line, primary normal bronchial epithelial (NHBE) cells, alveolar macrophages, and the HepG2 hepatoma cell lines (positive control).

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Plasmin, the effector enzyme of the fibrinolysis system, impedes excessive accumulation of extracellular matrix in the lung by degrading it directly or by activating the precursors of matrix metalloproteinases (4). Decreased plasmin activity is believed to play a critical role in the augmented deposition of extracellular matrix in the ILD (5). Upregulation of PAI-1 during fibrotic processes is thought to be the explanation for the decreased plasmin activity and, subsequently, for the abnormal extracellular matrix deposition in ILD-associated lung fibrosis (Figure 6) (13). In agreement with these findings, we found in this study that reduced fibrinolytic activity, as estimated by the PA activity/TAT ratio, is associated with increased intra-alveolar concentrations of PAI-1. Recent studies have demonstrated that TAFI is also another potent inhibitor of plasmin generation (19). We found that the total intraalveolar level and the activity of TAFI are increased in patients with ILD. The level of TAFI was sixfold to sevenfold higher than that detected in BALF from healthy subjects. These observations suggest that TAFI may also be an important causative factor of fibrinolytic impairment in ILD (Figure 6). Interestingly, the TAFI level in BALF was not only high in patients with ongoing inflammatory process such as CVD-ILD, COP, or hypersensitivity pneumonitis but also in conditions associated with minor inflammatory components such as IPF (26), suggesting that even under conditions of low degree of inflammation, TAFI may inhibit plasmin formation and thereby promote lung collagen deposition. The fact that the inhibitors of fibrinolysis, TAFI and PCI, were significantly associated with markers of collagen synthesis, aminoterminal propeptides of type I procollagen and aminoterminal propeptides of type III procollagen, supports the assumption that reduced fibrinolytic function promotes collagen deposition in ILD.



View larger version (28K):
[in this window]
[in a new window]
  		Figure 6. Role of TAFI and PCI in the pathogenesis of ILD. PAI-1 has been ascribed to be the main suppressor of plasmin activity and thus of prometalloproteinase activation leading to deposition of extracellular matrix in ILD. This study demonstrated increased concentration of TAFI and PCI in ILD, suggesting that they may be important promoters of extracellular matrix deposition and lung fibrosis in ILD.

 
Another important regulator of the fibrinolysis system and inflammation in the lung is the serine protease activated protein C (27, 28). Activated protein C suppresses coagulation activation by inhibiting factors VIIa and VIIIa and enhances plasmin generation by inhibiting PAI-1 activity (17). Activated protein C also exerts antiinflammatory activity by inhibiting the secretion of several inflammatory cytokines (29). Decreased activated protein C activity is associated with augmented thrombin generation, inflammation, and impaired fibrinolysis in the lung of patients with ILD (30). Furthermore, treatment with activated protein C inhibits bleomycin-induced lung injury and fibrosis in the mouse (11, 22). The mechanism of the activated protein C activity defect in ILD is unclear. In this study, we found that the BALF concentration of PCI, the main inhibitor of activated protein C activity, is increased in all patients with ILD, especially in patients with COP, CVD-ILD, and sarcoidosis. It was previously reported that inhibition of activated protein C by PCI leads to increased TAFI generation by indirectly increasing thrombin generation (15). Thus, increased intraalveolar PCI level may constitute a potential explanation not only for the low activity of activated protein C but also for the increased level of TAFI in ILD. In accord with this, in this study, TAFI was significantly and proportionally correlated with PCI in BALF from ILD patients.

To support further the involvement of both TAFI and PCI in the suppression of fibrinolytic function in the intraalveolar space in lung injury, the relationship of fibrinolytic activity, as estimated by the PA activity/TAT ratio, with these inhibitors of fibrinolysis was evaluated in ILD patients. Fibrinolytic activity was significantly and inversely correlated with TAFI and PCI in all ILD patients. This association was found to be particularly strong in patients with CVD-ILD and IPF. Interestingly, impairment in fibrinolytic activity was also markedly reduced in both CVD-ILD and IPF patients compared with other groups of ILD. These observations suggest that TAFI and PCI may play important roles in the impairment of fibrinolysis, particularly in patients with CVD-ILD and IPF.

After lung injury, there is an exquisite interplay between procoagulant and anticoagulant proteins, cytokines, chemokines, adhesion molecules, and inflammatory cells in an attempt to resolve injury. MCP-1 is one of the chemokines involved in lung inflammation (31). MCP-1 plays a critical role in the early inflammatory response by stimulating the recruitment of inflammatory cells in the lung and in the chronic fibrotic process by promoting the secretion of transforming growth factor-ß1, a potent stimulator of extracellular matrix production (32–34). The products of activation of the coagulation system, thrombin, and fibrin induce MCP-1 secretion (35). Activation of coagulation system occurred in the lung of our ILD patients, as demonstrated by the significant increased BAL concentration of TAT. Thus, enhanced thrombin- and/or fibrin-mediated stimulation of MCP-1 may explain the increased concentration of this chemokine in BALF from our ILD patients. TAFI was significantly correlated with both MCP-1 and TAT, further supporting the participation of TAFI in the procoagulant and inflammatory response after alveolar injury in ILD.

Increased concentration of procoagulant factors in the alveolar milieu may result from a spillover of circulating plasma protein during lung inflammation. However, we found that alveolar macrophages isolated from ILD patients, primary bronchial epithelial cells, and immortalized alveolar epithelial cell lines may themselves express TAFI, suggesting that local production of TAFI may also contribute to matrix deposition by further impairing the plasmin activity in the lung. The fact the concentration of TAFI was markedly higher in BALF than plasma of patients with ILD also suggests that TAFI antigen are locally produced and secreted from lung cells.

In brief, the results of this study showed for the first time that TAFI and PCI concentrations are increased in the intra-alveolar space of patients with ILD and that TAFI antigen may be locally produced by lung cells. These findings suggest that TAFI and PCI may play important roles in the mechanism of reduced plasmin activity in ILD.


   FOOTNOTES
 
Supported by a Grant-in-Aid (No. 13,670,597) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.

This article has an online supplement, which is accessible from this issue's table of contents online at www.atsjournals.org

Received in original form August 20, 2002; accepted in final form February 17, 2003


   REFERENCES
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Canonico AE, Brigham KL. Biology of acute lung injury. In: Crystal RG, Barnes PJ, West JB, Weibel ER, editors. The lung, 2nd edition. Philadelphia: Lippincott-Raven; 1997. p. 2475–2498.
  2. Swaisgood CM, French EL, Noga C, Simon RH, Ploplis VA. The development of bleomycin-induced pulmonary fibrosis in mice deficient for components of the fibrinolytic system. Am J Pathol 2000;157:177–187.[Abstract/Free Full Text]
  3. Hekman CM, Loskutoff DJ. Fibrinolytic pathways and the endothelium. Semin Thromb Hemost 1987;13:514–527.[Medline]
  4. Saksela O, Rifkin DB. Cell-associated plasminogen activation: regulation and physiological functions. Annu Rev Cell Biol 1988;4:93–126.[CrossRef][Medline]
  5. Bertozzi P, Astedt B, Zenzius L, Lynch K, LeMaire F, Zapol W, Chapman HA Jr. Depressed bronchoalveolar urokinase activity in patients with adult respiratory distress syndrome. N Engl J Med 1990;322:890–897.[Abstract]
  6. Idell S, James KK, Levin EG, Schwartz BS, Manchanda N, Maunder RJ, Martin TR, McLarty J, Fair DS. Local abnormalities in coagulation and fibrinolytic pathways predispose to alveolar fibrin deposition in the adult respiratory distress syndrome. J Clin Invest 1989;84:695–705.
  7. Welty-Wolf KE, Carraway MS, Ortel TL, Piantadosi CA. Coagulation and inflammation in acute lung injury. Thromb Haemost 2002;88:17–25.[Medline]
  8. Nakstad B, Lyberg T, Skjonsberg OH, Boye NP. Local activation of the coagulation and fibrinolysis systems in lung disease. Thromb Res 1990;57:827–838.[CrossRef][Medline]
  9. Gunther A, Mosavi P, Heinemann S, Ruppert C, Muth H, Markart P, Grimminger F, Walmrath D, Temmesfeld-Wollbruck B, Seeger W. Alveolar fibrin formation caused by enhanced procoagulant and depressed fibrinolytic capacities in severe pneumonia: comparison with the acute respiratory distress syndrome. Am J Respir Crit Care Med 2000;161:454–462.[Abstract/Free Full Text]
  10. Chapman HA, Allen CL, Stone OL. Abnormalities in pathways of alveolar fibrin turnover among patients with interstitial lung disease. Am Rev Respir Dis 1986;133:437–443.[Medline]
  11. Yasui H, Gabazza EC, Tamaki S, Kobayashi T, Hataji O, Yuda H, Shimizu S, Suzuki K, Adachi Y, Taguchi O. Intratracheal administration of activated protein C inhibits bleomycin-induced lung fibrosis in the mouse. Am J Respir Crit Care Med 2001;163:1660–1668.[Abstract/Free Full Text]
  12. Olman MA, Mackman N, Gladson CL, Moser KM, Loskutoff DJ. Changes in procoagulant and fibrinolytic gene expression during bleomycin-induced lung injury in the mouse. J Clin Invest 1995;96:1621–1630.
  13. Loskutoff DJ, Quigley JP. PAI-1, fibrosis, and the elusive provisional fibrin matrix. J Clin Invest 2000;106:1441–1443.[Medline]
  14. Wang W, Hendriks DF, Scharpe SS. Carboxypeptidase U, a plasma carboxypeptidase with high affinity for plasminogen. J Biol Chem 1994;269:15937–15944.[Abstract/Free Full Text]
  15. Mosnier LO, Elisen MG, Bouma BN, Meijers JC. Protein C inhibitor regulates the thrombin-thrombomodulin complex in the up- and down regulation of TAFI activation. Thromb Haemost 2001;86:1057–1064.[Medline]
  16. Elisen MG, von dem Borne PA, Bouma BN, Meijers JC. Protein C inhibitor acts as a procoagulant by inhibiting the thrombomodulin-induced activation of protein C in human plasma. Blood 1998;91:1542–1547.[Abstract/Free Full Text]
  17. Suzuki K. Protein C. In High KA, Roberts HR, editors. Molecular basis of thrombosis and hemostasis. New York: Marcel Dekker; 1995. p. 393–424
  18. Bajzar L, Manuel R, Nesheim ME. Purification and characterization of TAFI, a thrombin-activatable fibrinolysis inhibitor. J Biol Chem 1995;270:14477–14484.[Abstract/Free Full Text]
  19. Bajzar L. Thrombin activatable fibrinolysis inhibitor and an antifibrinolytic pathway. Arterioscler Thromb Vasc Biol 2000;20:2511–2518.[Abstract/Free Full Text]
  20. Fujimoto H, Gabazza EC, Taguchi O, Yuda Y, Hataji O, Adachi Y. Increased intraalveolar levels of thrombin-activatable fibrinolysis inhibitor (TAFI) in patients with interstitial lung disease [abstract]. Am J Respir Crit Care Med 2002;165:A729.
  21. American Thoracic Society/European Respiratory Society International Multidisciplinary Consensus Classification of the Idiopathic Interstitial Pneumonias. This joint statement of the American Thoracic Society (ATS), and the European Respiratory Society (ERS) was adopted by the ATS board of directors, June 2001 and by the ERS Executive Committee, June 2001. Am J Respir Crit Care Med 2002;165:277–304.[Free Full Text]
  22. Reynolds HY. Bronchoalveolar lavage. Am Rev Respir Dis 1987;135:250–263.[Medline]
  23. He S, Antovic A, Blomback M. A simple and rapid laboratory method for determination of haemostasis potential in plasma: modifications for use in routine laboratories and research work. Thromb Res 2001;103:355–361.[CrossRef][Medline]
  24. Suzuki K, Hayashi T. Cis-elements required for expression of human protein C inhibitor gene in HepG2 cells and its androgen-dependent expression in rat reproductive organs. Semin Thromb Hemost 2000;26:75–83.[CrossRef][Medline]
  25. Hasegawa M, Sato S, Takehara K. Augmented production of chemokines (monocyte chemotactic protein-1 (MCP-1), macrophage inflammatory protein-1alpha (MIP-1alpha) and MIP-1beta) in patients with systemic sclerosis: MCP-1 and MIP-1alpha may be involved in the development of pulmonary fibrosis. Clin Exp Immunol 1999;117:159–165.[CrossRef][Medline]
  26. Selman M, King TE, Pardo A. Idiopathic pulmonary fibrosis: prevailing and evolving hypotheses about its pathogenesis and implications for therapy. Ann Intern Med 2001;134:136–151.[Abstract/Free Full Text]
  27. Joyce DE, Gelbert L, Ciaccia A, DeHoff B, Grinnell BW. Gene expression profile of antithrombotic protein c defines new mechanisms modulating inflammation and apoptosis. J Biol Chem 2001;276:11199–11203.[Abstract/Free Full Text]
  28. Kobayashi H, Gabazza EC, Taguchi O, Wada H, Takeya H, Nishioka J, Yasui H, Kobayashi T, Hataji O, Suzuki K, et al. Protein C anticoagulant system in patients with interstitial lung disease. Am J Respir Crit Care Med 1998;157:1850–1854.
  29. Esmon CT. Protein C anticoagulant pathway and its role in controlling microvascular thrombosis and inflammation. Crit Care Med 2001;29:S48–S51.[CrossRef][Medline]
  30. Tobin MJ. Tuberculosis, lung infections, interstitial lung disease, and socioeconomic issues in AJRCCM 2001. Am J Respir Crit Care Med 2002;165:631–641.[Free Full Text]
  31. Moore BB, Paine R III, Christensen PJ, Moore TA, Sitterding S, Ngan R, Wilke CA, Kuziel WA, Toews GB. Protection from pulmonary fibrosis in the absence of CCR2 signaling. J Immunol 2001;167:4368–4377.[Abstract/Free Full Text]
  32. Smith RE, Strieter RM, Phan SH, Kunkel SL. C–C chemokines: novel mediators of the profibrotic inflammatory response to bleomycin challenge. Am J Respir Cell Mol Biol 1996;15:693–702.[Medline]
  33. Brieland JK, Jones ML, Flory CM, Miller GR, Warren JS, Phan SH, Fantone JC. Expression of monocyte chemoattractant protein-1 (MCP-1) by rat alveolar macrophages during chronic lung injury. Am J Respir Cell Mol Biol 1993;9:300–305.
  34. Gharaee-Kermani M, Denholm EM, Phan SH. Costimulation of fibroblast collagen and transforming growth factor beta1 gene expression by monocyte chemoattractant protein-1 via specific receptors. J Biol Chem 1996;271:17779–17784.[Abstract/Free Full Text]
  35. Szaba FM, Smiley ST. Roles for thrombin and fibrin(ogen) in cytokine/chemokine production and macrophage adhesion in vivo. Blood 2002;99:1053–1059.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 ERRHome page
R. C. Chambers
Abnormal wound healing responses in pulmonary fibrosis: focus on coagulation signalling
Eur. Respir. Rev., December 1, 2008; 17(109): 130 - 137.
[Abstract] [Full Text] [PDF]

Home page
 Eur Respir JHome page
J-F. Cordier
Cryptogenic organising pneumonia.
Eur. Respir. J., August 1, 2006; 28(2): 422 - 446.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Pathol.Home page
H. Fujimoto, E. C. Gabazza, O. Taguchi, Y. Nishii, H. Nakahara, N. E. Bruno, C. N. D'Alessandro-Gabazza, M. Kasper, Y. Yano, M. Nagashima, et al.
Thrombin-Activatable Fibrinolysis Inhibitor Deficiency Attenuates Bleomycin-Induced Lung Fibrosis
Am. J. Pathol., April 1, 2006; 168(4): 1086 - 1096.
[Abstract] [Full Text] [PDF]

Home page
 CLIN APPL THROMB HEMOSTHome page
O. Kaftan, O. S. Balcik, H. Cipil, G. Ozet, N. Bavbek, A. Kosar, and S. Dagdas
Plasma Levels of Thrombin-Activatable Fibrinolysis Inhibitor in Primary and Secondary Thrombocytosis
Clinical and Applied Thrombosis/Hemostasis, October 1, 2005; 11(4): 449 - 454.
[Abstract] [PDF]

Home page
 Am. J. Respir. Crit. Care Med.Home page
M. J. Tobin
Tuberculosis, Lung Infections, Interstitial Lung Disease, Social Issues and Journalology in AJRCCM 2003
Am. J. Respir. Crit. Care Med., January 15, 2004; 169(2): 288 - 300.
[Full Text] [PDF]

Home page
 Am. J. Respir. Crit. Care Med.Home page
R. M. Strieter
To Clot or Not to Clot, That Is the Question in Pulmonary Fibrosis
Am. J. Respir. Crit. Care Med., June 15, 2003; 167(12): 1589 - 1590.
[Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Online Supplement
Right arrow All Versions of this Article:
200208-905OCv1
167/12/1687    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Fujimoto, H.
Right arrow Articles by Taguchi, O.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Fujimoto, H.
Right arrow Articles by Taguchi, O.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Proc. Am. Thorac. Soc. Am. J. Respir. Cell Mol. Biol.
Copyright © 2003 American Thoracic Society