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. Author manuscript; available in PMC: 2010 Nov 1.
Published in final edited form as: Arthritis Rheum. 2009 Nov;60(11):3455–3464. doi: 10.1002/art.24935

Dabigatran, a direct thrombin inhibitor, blocks differentiation of normal fibroblasts to a myofibroblast phenotype and demonstrates anti-fibrotic effects on scleroderma lung fibroblasts

Galina S Bogatkevich, Anna Ludwicka-Bradley, Richard M Silver
PMCID: PMC2837365  NIHMSID: NIHMS178432  PMID: 19877031

Abstract

Myofibroblasts are the principal mesenchymal cells responsible for tissue remodeling, collagen deposition, and the restrictive nature of lung parenchyma associated with pulmonary fibrosis. We previously reported that thrombin activates protease-activated receptor (PAR)-1 thereby inducing normal lung fibroblasts to differentiate to a myofibroblast phenotype resembling scleroderma lung myofibroblasts. Here we demonstrate that the thrombin inhibitor dabigatran inhibits in a dose-dependant manner thrombin's induction of myofibroblasts. Dabigatran inhibits thrombin-induced cell proliferation, α-smooth muscle actin (α-SMA) expression and organization, and the production of collagen and connective tissue growth factor (CTGF). Moreover, when treated with dabigatran scleroderma lung myofibroblasts produce less CTGF, α-SMA, and collagen type I. We conclude that dabigatran restrains important profibrotic events in lung fibroblasts and that this oral direct thrombin inhibitor warrants study as a potential anti-fibrotic drug for the treatment of fibrosing lung diseases, e.g. scleroderma lung disease and idiopathic pulmonary fibrosis.

Introduction

Thrombin is a multi-functional serine protease and a key enzyme of blood coagulation, catalyzing the conversion of fibrinogen to fibrin (1). In addition to its essential role in coagulation, thrombin has several important functions at a cellular level, both in normal health and in multiple disease processes (2). The majority of the cellular responses to thrombin are mediated via the G protein-coupled receptor PAR-1 (protease-activated receptor 1)(3, 4). In previous studies we demonstrated that PAR-1 expression is dramatically increased in patients with pulmonary fibrosis associated with scleroderma (systemic sclerosis associated interstitial lung disease, SSc-ILD), notably in lung parenchyma associated with inflammatory and fibroproliferative foci (5). PAR-1 is co-localized with myofibroblasts in SSc-ILD tissue and appears to decrease during later stages of pulmonary fibrosis when a decreased number of myofibroblasts is observed (5).

Pulmonary fibrosis is the end stage of many chronic lung diseases including SSc-ILD and idiopathic pulmonary fibrosis (IPF). The molecular mechanisms underlying the pathogenesis and progression of lung fibrosis in these diseases are not entirely clear. The conceptual process of fibrogenesis involves tissue injury and activation of the coagulation cascade, the release of various fibrogenic factors, and the induction of myofibroblasts culminating in enhanced extracellular matrix deposition (6, 7). Cells with a myofibroblast phenotype appear in the early stages of fibrosis (8) and are characterized by an increased proliferative capacity and abundant expression of α-SMA, collagens and other extracellular matrix proteins (5, 7 - 9). Myofibroblasts can be cultured from bronchoalveolar lavage (BAL) fluid of SSc-ILD patients, and thrombin activity is also significantly greater in BAL fluid from SSc-ILD patients compared with healthy controls (10, 11). Thrombin is mitogenic for lung fibroblasts (5, 11, 12) and enhances the proliferative effect of fibrinogen on fibroblasts (13). Thrombin is also a potent inducer of fibrogenic cytokines, such as transforming growth factor-β (TGF-β) (14), connective tissue growth factor (CTGF) (15, 16), platelet-derived growth factor-AA (PDGF-AA) (11), chemokines (17, 18), and ECM proteins such as collagen, fibronectin, and tenascin in various cells, including lung fibroblasts (19 - 21).

Dabigatran, N-[2-(4-Amidinophenylaminomethyl)-1-methyl-1H-benzimidazol-5-ylcarbonyl]-N-(2-pyridinyl)-β-alanine, is a selective direct thrombin inhibitor that reversibly binds to thrombin and prevents the cleavage of Arg-Gly bonds of fibrinogen needed for the formation of fibrin (22). Previously we demonstrated that thrombin differentiates normal lung fibroblasts to a myofibroblast phenotype via the PAR-1/PKC pathway (12, 23). The present study was undertaken to investigate whether dabigatran interferes with signal transduction in human lung fibroblasts induced by thrombin and mediated via PAR-1. We demonstrate herein that dabigatran inhibits thrombin-induced differentiation of normal lung fibroblasts to the myofibroblast phenotype. Additionally, scleroderma lung fibroblasts treated with dabigatran produce less CTGF, α-SMA, and collagen as compared to non-treated fibroblasts. However, dabigatran does not appear to affect the signaling of the PAR-1 agonist (PAR-1 selective activating peptide, PAR1-AP).

Materials and Methods

Materials

Dabigatran was obtained from Boehringer Ingelheim Pharma GmbH & Co KG, Biberach, Germany. Thrombin from human plasma was purchased from Calbiochem, La Jolla, CA. We obtained PAR-1 selective activating peptide (PAR1-AP), protease inhibitor cocktail, anti-α-SMA and anti-β-actin antibodies from Sigma-Aldrich (St. Louis, MO); type I collagen from rat tail tendon from BD Bioscience (Bedford, MA), anti-type I collagen antibody from SouthernBiotech (Birmingham, AL). 4′-6-Diamidino-2-phenylindole (DAPI) and BCA™ protein assay were purchased from Pierce (Rockford, IL). Anti-CTGF antibody and FITC-conjugated goat anti-mouse antibody were obtained from Santa Cruz Biotechnology (Santa Cruz, CA); Quick Cell Proliferation Assay was acquired from BioVision Research Products (Mountain View, CA).

Cell culture

Lung fibroblasts were derived from lung tissues obtained at autopsy from three scleroderma patients and from three age-, race-, and sex-matched normal subjects. Lung tissue was diced (0.5 × 0.5 mm pieces) and cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco, Grand Island, NY) supplemented with 10% fetal calf serum (FCS), 2 mM l-glutamine, 50 μg/ml of gentamicin sulfate, and 5 μg/ml of amphotericin B at 37°C in 10% CO2. Medium was changed every three days to remove dead and non-attached cells until fibroblasts reached confluence. Monolayer cultures were maintained in the same medium. Lung fibroblasts were used between second and fourth passages in all experiments. Purity of isolated lung fibroblasts was determined by crystal violet staining and by immunofluorescent staining (monoclonal antibody against human fibroblasts as described previously (10) followed by FITC-conjugated goat anti-mouse IgG staining).

Quick Cell Proliferation Assay and Cell Counting

Lung fibroblasts (104/well) were cultured in 96-well plates in a final volume of 100 μl/well DMEM in the absence or presence of thrombin, PAR-1-AP, and dabigatran. After 24 hours of incubation, 10 μl/well tetrazolium salt WST-1 in Electro Coupling Solution (ECS) was added, and cells were incubated for another two hours under standard culture conditions. Plates were then placed for 1 minute on a shaker, and the optical density (OD) of each well was determined using a micro plate reader set to 450 nm.

For cell counts lung fibroblasts were detached from six-well plates, and a 0.5ml aliquot of cell suspension was diluted in 9.5ml of Isoton II solution for counting in Z1 Coulter particle counter (Coulter Electronics, Hialeah, FL). The number of cells per dish was calculated on the basis of a dilution factor that was identical for all groups.

Preparation of cell extracts and immunoblotting

Normal and SSc lung fibroblasts on 100-mm dishes were washed with ice-cold PBS and lysed with ice-cold lysis buffer (10 mM Tris, 10 mM EDTA, 1% Nonidet P-40, 0.5% deoxycholate, 0.1% SDS, pH = 7.4). Protein concentration was determined by BCA™ protein assay as described previously (24). For each sample, 40 μg of protein was denatured, subjected to SDS-polyacrylamide gel electrophoresis and analyzed by immunoblotting with appropriate antibodies. The immunoblots were then stripped and re-blotted with anti-β-actin antibody as a loading control. For CTGF assays, cells were harvested with PBS and solubilized in heparin-sepharose binding buffer containing 100mM Tris, pH 7.4; 10mM trisodium citrate; 100mM NaCl; 1% Triton, and protease inhibitor cocktail. Cell lysates were cleared by centrifugation and CTGF was selected from 500 μg of cell lysate per sample using heparin sepharose (GE Healthcare Bio-Sciences Corp, Piscataway, NJ) and subjected to 4-20% gradient SDS-polyacrylamide gel electrophoresis. Western blotting was performed using anti-CTGF antibody in accordance with the manufacturer's instructions.

Floating and Fixed Collagen Gel Contraction Assays

Collagen lattices were prepared with type I collagen from rat tail tendon adjusted to a final value of 2.5 mg/ml with 0.01% acetic acid. Lung fibroblasts at a concentration of 2.5 × 105 cells/ml were suspended in collagen (1.25 mg/ml of collagen) and aliquoted into 24 well plates (300 μl/well). Then collagen lattices were polymerized for 45 minutes in a humidified 10% CO2 atmosphere at 37°C followed by incubation with DMEM containing 10% FCS for 4 hours. For floating gel experiments, polymerized gels were gently released from the underlying culture dish followed by overnight incubation in serum-free medium and stimulation with 0.5 U/ml of thrombin or PAR1-AP (10μM) in the presence or absence of dabigatran (1ng/ml - 1μg/ml, added 30 min prior to the addition of thrombin or PAR1-AP). For fixed gel experiments, gels remained attached to tissue culture dishes for the duration of the experiment. To determine the degree of collagen gel contraction, pictures were taken after 24 and 48 hours with a digital camera. Measurement of the diameter of each gel in mm was recorded as the average values of the major and minor axes. Calculation of gel contraction was presented as difference between diameters of wells and contracted gels. In some experiments, collagen gels were collected, digested with collagenase and analyzed by Western blot using anti-SM-α actin and β-actin antibodies.

α-SMA Expression and Organization Assay

Human lung fibroblasts were cultured to subconfluence on glass slides in DMEM containing 10% FCS, after which medium was changed to serum-free medium. Cells were stimulated with or without thrombin (0.5 U/ml) or PAR1-AP for 24 hours in the presence or absence of dabigatran, added 30 min prior to the addition of thrombin. After incubation, cells were washed with cold PBS, fixed in methanol at −20°C for 4 min and washed with cold PBS twice followed by incubation with α-SMA antibody (1:500) for 1 hour at room temperature. Cells were washed three times with cold PBS, incubated with Alexa Fluor 488 anti-mouse IgG (1:200) and DAPI (1:10 000) for 1 hour at room temperature, then washed with cold PBS, air-dried, covered and sealed. Images were acquired with an Olympus IX71 fluorescence microscope equipped with objective ×60/1.42 and Olympus Slidebook 4.1 software.

Statistical Analysis

Statistical analyses were performed with KaleidaGraph 4.0 (Synergy Software, Reading, PA). All data were analyzed using ANOVA with Tukey HSD post-hoc testing. The results were considered significant if p<0.05.

Results

Effects of dabigatran on thrombin- and PAR1-AP-induced lung fibroblast proliferation

Thrombin is a well known mitogen and has been shown to induce human lung fibroblast proliferation. We measured the effect of dabigatran on thrombin-induced lung fibroblast proliferation using a quick cell proliferation assay. This method is based on cleavage of a tetrazolium salt, WST-1, to formazan by cellular mitochondrial dehydrogenases. Expansion of the number of viable cells results in an increase in the activity of the mitochondrial dehydrogenases leading to an increase in the amount of formazan dye detected by spectrometry. Basal levels of viable cells were in a range of between 0.38 and 0.51 OD. Thrombin increased cell proliferation 1.8-fold within 24 hours (Figure 1). Dabigatran alone had no significant effect on lung fibroblast proliferation, although a trend to an inhibition with dabigatran at concentrations of 50ng/ml and higher was observed. Dabigatran did inhibit thrombin-induced cell proliferation in a dose-dependant manner, and at concentrations of 50, 100, 500ng/ml and 1μg/ml significantly decreased thrombin-induced proliferation of lung fibroblasts (Figure 1A). Interestingly, the PAR-1 selective activating peptide PAR1-AP increased proliferation of lung fibroblasts similar to thrombin, yet dabigatran had no effect on PAR1-AP-induced cell proliferation even at high concentrations (Figure 1B). Additionally, we measured time-dependent effects of thrombin and dabigatran on lung fibroblast proliferation by cell counting. Lung fibroblasts were stimulated with thrombin (0.5U/ml) with or without dabigatran (0.5μg/ml) for 24h, 48h, 72h, and 96h. Lung fibroblasts incubated with 1% FCS were used as a control. Effect of dabigatran alone was similar to that observed in control cells (data not shown). We observed that normal and SSc lung fibroblasts respond differently to thrombin treatment. The proliferation rate of normal lung fibroblasts stimulated with thrombin was increased 4-fold (Figure 1C, upper panel), while the proliferative response of SSc lung fibroblasts to thrombin was significantly lower (2.5-fold) (Figure 1C, lower panel). Thrombin's mitogenic effect for normal lung fibroblasts was observed for 72 hours, while in SSc lung fibroblasts it lasted for 48 hours (Figure 1C). Dabigatran significantly inhibited thrombin-induced proliferation at all time points measured.

Figure 1. Effect of dabigatran on lung fibroblast proliferation.

Figure 1

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A, Dabigatran inhibits thrombin-induced cell proliferation in a dose-dependent manner. B, Dabigatran does not affect cell proliferation induced by the PAR-1 selective agonist PAR-1-AP. Serun-starved lung fibroblasts were incubated with or without thrombin (Thr), PAR-1-AP, and dabigatran (Dabi, 1- 1000ng/ml) in 96-well plates for 24 hr, and then subjected to Quick Cell Proliferation Assay. Data are presented as optical density (OD) determined at 450 nm. C, Time-dependant effects of thrombin and dabigatran on lung fibroblast proliferation. Normal and SSc lung fibroblasts in 1% FCS were incubated with thrombin (0.5U/ml) alone or with dabigatran (0.5μg/ml) for time indicated. At 24 hour intervals, cells were trypsinized and counted using a Coulter particle counter. Experiments were performed three times and mean values ± SD are presented. The asterisk represents statistically significant (p<0.05) differences between cells stimulated with thrombin versus cells stimulated with thrombin and dabigatran.

Effects of dabigatran on thrombin- and PAR1-AP-induced α-SMA expression and organization

Previously we reported that within 24 hours of exposure thrombin increases the amount of highly organized α-SMA in normal lung fibroblasts (12, 23). Here we again show that thrombin (0.5U/ml) significantly induces α-SMA expression (Figure 2A lane 6). Dabigatran had no effect on the basal level of α-SMA (Figure 2A lanes 1-5), and dabigatran decreased thrombin-induced α-SMA expression in a dose-dependant manner. Pretreatment of lung fibroblasts with 100ng/ml of dabigatran significantly inhibited thrombin-induced α-SMA expression; in a concentration of 1μg/ml dabigatran completely prevented thrombin-induced expression of α-SMA (Figure 2A lanes 9 and 10). Immunoblots were analyzed with NIH Images software and the results are presented in Figure 2B. PAR1-AP at a concentration of 10μM increased α-SMA to a similar extent as thrombin (0.5U/ml), but was not blocked by dabigatran (Figure 3A).

Figure 2. Dabigatran inhibits α–smooth muscle actin (α–SMA) expression in lung fibroblasts.

Figure 2

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A, cells were serum-deprived overnight and then treated with thrombin and/or dabigatran for 24 hr. Cell lysate proteins (40μg) were separated by SDS-polyacrylamide gel electrophoresis and immunoblotted with anti- α–SMA antibody. The immunoblots were then stripped and re-blotted with anti-β-actin antibody as a loading control. B, The images were scanned and analyzed with NIH Imaging software. Densitometric analysis of immunoblots from 3 independent experiments is presented. The asterisk represents statistically significant differences (p<0.05) between cells stimulated with thrombin and dabigatran versus cells treated with thrombin alone.

Figure 3. Effect of dabigatran on thrombin and PAR-1-AP-induced α–SMA expression and organization in lung fibroblasts.

Figure 3

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Cells were incubated with or without thrombin (0.5U/ml), PAR-1-AP (10μM), and dabigatran (1μg/ml) for 24 hr. A, Cell lysates were separated by SDS-polyacrylamide gel electrophoresis and immunoblotted with anti- α–SMA antibody followed by immunoblotting with anti-β-actin antibody as a loading control. B, Cells were labeled with anti-α–SMA mouse monoclonal antibody followed by anti-mouse antiserum tagged with Alexa Fluor 488 (green) and DAPI staining for nuclei (blue). Images were acquired with an Olympus IX71 fluorescence microscope, objective ×60/1.42. Image 1 represents control cells in serum-free medium, 2 represents cells treated with dabigatran, 3 represents cells treated with thrombin, 4 represents cells treated with PAR-1-AP, 5 represents cells treated with dabigatran and thrombin, and 6 represents cells treated with dabigatran and PAR-1-AP.

In addition to α-SMA expression, thrombin and PAR1-AP promote α-SMA organization. Dabigatran at a concentration of 1μg/ml inhibited thrombin's effect on α-SMA organization (Figure 3B, panels 3 and 5); however, dabigatran had no effect on α-SMA organization induced by PAR1-AP (Figure 3B, panels 4 and 6).

Dabigatran and collagen gel contraction

We previously observed that thrombin induces collagen gel contraction by normal lung fibroblasts in a dose-dependent manner with a maximal effect at 0.5 U/ml (12). Thrombin induced contraction of floating collagen gels from 15 mm in diameter to less than 4 mm in diameter within 24 hours (10.95±0.61 mm contraction). In contrast, cells in serum-free medium contracted only ~ 3 mm. Cells treated with dabigatran alone contracted to a similar extent as cells in serum-free medium (2.98 mm to 3.57 mm). Dabigatran was found to inhibit thrombin-induced collagen gel contraction in a dose-dependent manner. The significant inhibition of thrombin-mediated contraction by dabigatran was observed starting at 50ng/ml (6.12±0.55 mm), achieving maximal inhibition at concentrations of 0.5 μg/ml (3.17±0.22 mm) and 1μg/ml (2.98±0.15 mm) (Figure 4, A and B). Collagen gel contraction induced by PAR1-AP was not, however, affected by dabigatran (Figure 4C).

Figure 4. Effect of dabigatran on collagen gel contraction induced by thrombin and PAR-1-AP in lung fibroblasts.

Figure 4

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A, Dabigatran inhibits thrombin-induced collagen gel contraction in lung fibroblasts in a concentration-dependent manner. B, the degree of collagen gel contraction was determined as the difference between diameters of well itself and released gels. C, dabigatran does not affect PAR-1-AP-induced collagen gel contraction. The experiment was performed on floating gels seeded with lung fibroblasts stimulated with thrombin and/or dabigatran for 24h. Data are presented as mean values ± SD of three tests. The asterisk represents statistically significant differences (p<0.05) between cells stimulated with thrombin and dabigatran versus cells treated with thrombin alone.

To further investigate the effects of dabigatran on collagen gel contraction we used floating and fixed collagen gel assays with normal and SSc lung fibroblasts treated with and without thrombin or dabigatran for 48 hours. SSc lung fibroblasts inherently contain higher levels of α-SMA and readily contracted both floating and fixed collagen gels (Figure 5). Dabigatran significantly reduced collagen gel contraction in SSc lung fibroblasts and α-SMA in both floating and fixed collagen gels; however, thrombin only slightly induced α-SMA and did not significantly affect collagen gel contraction by SSc lung fibroblasts. We observed notable differences for floating and fixed collagen gels seeded with normal lung fibroblasts when stimulated with thrombin. We found that thrombin stikingly contracted floating collagen gels within 48 hours in a similar manner as within 24 hours; in contrast, thrombin only slightly affected fixed collagen gels. Similarly, α-SMA was induced to a much higher extent by thrombin in floating gels as compared to fixed gels. In contrast, dabigatran inhibited collagen gel contraction and α-SMA not only in floating but also in fixed collagen gels.

Figure 5. Inhibition of floating and fixed collagen gel contraction by dabigatran in lung fibroblasts.

Figure 5

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Normal and SSc lung fibroblasts were subjected to collagen gel contraction assay as described in Materials and Methods. Cells were stimulated with thrombin (Thr, 0.5U/ml) and dabigatran (Dabi, 1μg/ml) for 48 h. A, left panel - floating gels; right panel – fixed gels. Data are presented as mean values ± SD of three experiments. The asterisk represents statistically significant differences (p<0.05) between cells stimulated with thrombin and dabigatran versus cells treated with thrombin alone. B, Collagen gels were digested with collagenase and analyzed by Western blot using anti-SM-α-actin and anti-β-actin antibodies. Note that thrombin promotes contraction and induces SM-α-actin in floating gels; dabigatran inhibits SM-α-actin and contraction in both floating and fixed collagen gels.

Effects of dabigatran on collagen type I, CTGF, and α-SMA in normal and scleroderma lung fibroblasts

Normal lung fibroblasts naturally produce collagen type I and CTGF in very low concentrations. Thrombin and PAR1-AP notably increased the production of these proteins within 48 hours. Pre-treatment of lung fibroblasts with dabigatran (1μg/ml) prevented the accumulation of collagen type I and CTGF induced by thrombin, but not by PAR1-AP (Figure 6A).

Figure 6. Effect of dabigatran on collagen type I, CTGF, and α–SMA in normal and scleroderma lung fibroblasts.

Figure 6

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A, Dabigatran inhibits thrombin- but not PAR-1-AP-induced collagen type I and CTGF. Serum-starved normal lung fibroblasts were incubated with or without thrombin (0.5U/ml), PAR-1-AP (10μM), and dabigatran (1μg/ml) for 48 hr. Cells were then collected with lysis buffer and analyzed by Western blot using anti-type I collagen antibody and anti-CTGF antibody. Anti-α-actin antibody was used as sample loading control. B, Dabigatran inhibits collagen type I, CTGF, and α–SMA expression in scleroderma lung fibroblasts. Confluent cultures of scleroderma lung fibroblasts were serum-starved for 24hr followed by incubation for 24, 48, 72 and 96hr with dabigatran (1μg/ml). C, Dabigatran inhibits α–SMA expression and organization in scleroderma lung fibroblasts. Scleroderma lung fibroblasts cultured on glass slides were incubated with dabigatran (1μg/ml) for 72 hr. Expression and organization of α–SMA were studied with an Olympus IX71 fluorescence microscope equiped with Olympus Slidebook 4.1 software. The experiments were repeated three times in three different cell lines and representative immunoblots and images are presented.

Lung fibroblasts derived from SSc-ILD patients express considerably higher levels of α-SMA, CTGF, and collagen type I when compared with normal lung fibroblasts (12, 16). To establish whether dabigatran would interfere with the expression of these markers of fibrosis we incubated scleroderma lung fibroblasts with dabigatran (1μg/ml) for 24, 48, 72, and 96 hours. We observed that the addition of dabigatran for 72 and 96 hours reduced the levels of α-SMA and CTGF; however, treatment of cells with dabigatran for 48 and 24 hours had little or no effect (Figure 6B). In contrast, the level of collagen type I was not significantly affected by dabigatran even after 72 hours. Yet after 96 hours of incubation with dabigatran SSc lung fibroblasts expressed significantly less collagen type I. To investigate whether, in addition to α-SMA expression, α-SMA organization would also be affected by dabigatran in SSc lung fibroblasts, we performed fluorescence microscopy studies. We observed that prolonged incubation of SSc lung fibroblasts with dabigatran indeed results in decreased α-SMA expression and organization (Figure 6C).

Discussion

Tissue injury with activation of the coagulation cascade and increased thrombin activity with deposition of fibrin are characteristic features of pulmonary fibrosis – the end result of a heterogeneous group of disorders that includes IPF and SSc-ILD. Characterized by microvascular injury and inflammation, SSc-ILD culminates in excessive deposition of extracellular matrix proteins, often resulting in severe lung dysfunction and death (25, 26). Although cyclophosphamide treatment may stabilize lung function in some patients, long-term treatment is required and significant toxicity may occur (27, 28). There is, therefore, a great need for new therapeutic approaches that would be more effective and less toxic than current treatments. In recent years, increasing evidence has accumulated to implicate involvement of the coagulation system in various fibrotic diseases, including SSc-ILD (reviewed in 29). Activation of coagulation proteases, e.g. thrombin, is one of the earliest events following tissue injury. Thrombin modulates tissue repair responses by altering vascular permeability, stimulating fibroblast and neutrophil migration, and promoting adhesion and spreading of endothelial cells and fibroblasts (2, 4). It also activates various cell types and induces secretion of several pro-immune, profibrotic, and angiogenic factors (2, 4, 29). Activation of these cells by thrombin is a likely mechanism for the development and progression of pulmonary fibrosis in general, and SSc-ILD in particular.

It is likely that similar pathophysiologic events occur in a multitude of other lung diseases characterized by diffuse alveolar damage and tissue injury; thus, thrombin inhibition might have broad applications for treating a variety of fibrosing pulmonary diseases. Modulation of the coagulation cascade, in particular targeting the coagulation protease thrombin, might be principally useful in a disease such as SSc-ILD where there is widespread vascular injury and over-expression of thrombin and its receptor. Several groups have recently reported results of studies in these directions with encouraging outcomes. Günther et al. demonstrated in a rabbit model of pulmonary fibrosis that aerosolization of heparin or urokinase prevents bleomycin-induced lung fibrosis (30). In bleomycin-treated rats Howell et al. found that the direct thrombin inhibitor UK-156406 attenuates lung collagen accumulation by lowering the profibrotic effects of thrombin and suppressing CTGF synthesis (31). Later, the same group demonstrated that mice lacking the PAR-1 thrombin receptor are significantly protected from bleomycin-induced lung fibrosis by reduction in CCL2 and CTGF expression and TGF-β immunoreactivity (32). In human studies, anticoagulation treatment with warfarin and low-molecular-weight heparin has been associated with significant beneficial outcomes on survival in IPF patients (33).

Dabigatran is a direct thrombin inhibitor that reversibly binds to the active site of thrombin preventing the conversion of fibrinogen to fibrin (22). Here we demonstrate that binding of dabigatran to thrombin prevents cleavage of the extracellular N-terminal domain of the PAR-1 receptor. Normally, thrombin binds to the PAR-1 receptor and cleaves the peptide bond between residues Arg-41 and Ser-42, thereby unmasking a new amino terminus, SFLLRN, which then can bind to the second extracellular loop of PAR-1 and initiate receptor signaling (3). Dabigatran-bound thrombin is unable to cleave and activate PAR-1.

In the current study we demonstrate that dabigatran effectively blocks development of the myofibroblast phenotype from thrombin-activated normal lung fibroblasts and, moreover, the thrombin inhibitor reverses the myofibroblast phenotype expressed by lung fibroblasts of SSc-ILD patients. In contrast, the synthetic agonist for PAR-1, PAR1-AP, was not affected by dabigatran, confirming that dabigatran binds selectively to thrombin and not to the thrombin receptor PAR-1. PAR1-AP has the sequence SFLLRN-NH2, identical to the tethered ligand of PAR-1. To induce thrombin receptor signaling, PAR1-AP binds directly to the second extracellular loop of PAR-1 in a manner identical to that of the unmasked tethered ligand SFLLRN. Ligand-bound PAR-1 couples to Gq, G12/13, or Gi proteins and initiates several intracellular signal transduction pathways. Evidently, dabigatran acts upstream of the PAR-1 thrombin receptor by directly binding and inactivating thrombin per se.

The presence of myofibroblasts has been extensively documented in active fibrotic lesions in many diseases, including SSc-ILD(7-9, 33). Myofibroblasts appear to diminish with the progression of pulmonary fibrosis, and in the late stages of fibrosis myofibroblasts are not observed (8). We previously reported that PAR-1 is significantly increased in lung tissues from SSc-ILD patients, mainly in lesions containing inflammatory and fibroproliferative foci (5). We noted that PAR-1 expression tends to decrease in the later stages of SSc-ILD, suggesting that its primary role occurs early in the development of lung fibrosis. PAR-1, which is responsible for most cellular events induced by thrombin, co-localizes with myofibroblasts in scleroderma lung tissue, lending additional support for thrombin in the process of lung fibroblast differentiation.

The precise sources of myofibroblasts are still not well known. Relative contributions from circulating mesenchymal stem cells or from local trans-differentiation of epithelial cells to fibroblasts have been reported (reviewed in 9). It has become generally accepted that lung fibroblasts may differentiate to a myofibroblast phenotype under the influence of local growth factors and cytokines, such as TGF-β, endothelin-1, and thrombin (9, 12, 34). Interestingly, thrombin itself has been demonstrated to induce the secretion of TGF-β and endothelin-1(14, 35). Several studies have demonstrated a correlation of fibrosis with α-SMA-expressing myofibroblasts (6 - 8, 36), and myofibroblasts isolated from various fibrotic tissues, including lungs, are believed to be the primary source of collagen and other ECM proteins (10, 36, 37). Previously, we identified four histological stages of SSc lung fibrosis (8). We found that the total population of fibroblasts was increased in the early, active phases of fibrosis (stages I and II) with myofibroblasts and excessive ECM deposition dominating in stage II of SSc pulmonary fibrosis. It is well accepted now that myofibroblasts containing significant amounts of α-SMA are characterize by a reduced rate of proliferation (37). In agreement with this, we observed that the thrombin-induced mitogenic effect in SSc lung fibroblasts with increased expression of α-SMA was significantly lower than observed in normal lung fibroblasts exposed to thrombin. Nevertheless, dabigatran completely blocked proliferation of both normal and scleroderma lung fibroblasts upon thrombin stimulation.

A characteristic feature of activated lung fibroblasts or “myofibroblasts” is an increase in contractile activity (12, 23, 34). Contractile forces of the myofibroblast are generated by α-SMA, which is extensively expressed in stress fibers and by large fibronexus adhesion complexes connecting intracellular actin with extracellular fibronectin fibrils (36). Fibroblasts cultured in collagen gel matrices provide an in vitro model of fibrocontractility and fibrosing diseases such as scleroderma and IPF (38). When cultured within collagen gels, fibroblasts recognize collagen fibers leading to contraction of the gels. This is believed to reflect the in vivo phenomenon of wound contraction and extracellular remodeling in connective tissue. In lung fibrosis it might also reflect the pathologic stiffness observed in SSc-ILD and other restrictive lung diseases. Lung fibroblasts from SSc-ILD patients express abundant and highly organized α-SMA (12). In contrast, normal lung fibroblasts contain relatively small amounts of α-SMA which is not fully organized (12, 34). We observed that dabigatran blocks thrombin-induced α-SMA and contraction of floating gels in normal lung fibroblasts. Contraction of floating collagen gels is considered to resemble more closely the initial phase of wound contraction and reflects the induction of the myofibroblast phenotype by various growth factors (34, 38). In contrast, attached or fixed collagen gels serve as a model of the late phase of excessive scarring observed in contractures and reflect the direct ability of proteins to enhance contraction of already formed α-SMA through mechanical stress (34, 38). The significance of this study is that dabigatran inhibits α-SMA and contraction in both floating and fixed collagen gels, thus blocking differentiation to a myofibroblast phenotype, as well as reversing the already existing myofibroblast phenotype.

Over-production of collagen with increased expression of CTGF is considered to be a molecular hallmark of fibrosis (37). Thrombin increases the expression of collagen type I and CTGF (15, 16, 20). Here we demonstrate for the first time that the direct thrombin inhibitor dabigatran restrains thrombin-induced accumulation of collagen type I and CTGF in human lung fibroblasts. Next, we sought to investigate whether dabigatran would affect collagen and CTGF in SSc lung fibroblasts, which demonstrate a myofibroblast phenotype in vivo and in vitro, in the absence of exogenous thrombin stimulation. We observed that incubation of SSc-ILD fibroblasts with dabigatran for 24 hours had no effect on these cells. However, upon longer exposure to dabigatran (72 hours) considerable inhibition of CTGF and α-SMA expression/organization was observed. Yet more time (96 hours) was required for dabigatran to significantly decrease collagen type I, suggesting that down-regulation of collagen by dabigatran in SSc lung fibroblasts occurs after inhibition of CTGF and α-SMA. It was reported that CTGF induces collagen I by stimulating transcription and promoter activity (39, 40), and diminishing CTGF by small interfering RNA lowers collagens I and IV in rats (41). Similarly, the inhibition of α-SMA by the NH2-terminal peptide of α-SMA results in reduction of collagen gene expression (42). Therefore, our data suggest that dabigatran may contribute to collagen type I down-regulation secondarily via reduced expression of CTGF and α-SMA.

In conclusion, dabigatran is a novel thrombin inhibitor that modulates the coagulation cascade (43) and inhibits thrombin-induced and PAR-1-mediated profibrotic signaling. Importantly, antifibrotic effects of dabigatran in lung fibroblasts were observed at clinically relevant plasma concentrations (44). Clinical studies have demonstrated that oral dabigatran etexilate is well tolerated and effective for the prevention of thromboembolic events after orthopaedic surgery (43, 45). Phase III testing with chronic administration in the treatment of thromboembolic events or prevention of stroke in patients with atrial fibrillation are currently ongoing. Our studies suggest that dabigatran might also prove to be a promising drug for the treatment of fibrosing conditions, e.g. SSc-ILD and IPF, where there is evidence for tissue injury with over-expression of thrombin. Any future studies of thrombin inhibition for the treatment of SSc-ILD would need to demonstrate a positive benefit: risk ratio taking into account potential risks such as gastrointestinal tract hemorrhage. Further studies on bleomycin-induced pulmonary fibrosis in mice are required and currently in progress in our laboratory. They are aimed to establish the effective dose of dabigatran for the treatment of fibrosis and will determine if such a dose will be below that with a significant risk of hemorrhage. The studies in mice, if successful, will be followed by clinical trials for SSc-ILD.

Acknowledgements

The authors would like to thank Joanne van Ryn (Boehringer Ingelheim Pharma GmbH & Co KG) for her critical reading of the manuscript and valuable comments. We also thank C. Beth Singleton for her excellent technical work.

Grants

This work was supported in part by a career award from National Institutes of Health K01AR051052 and Boehringer Ingelheim International GmbH (to GSB).

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