In recent years there has been increasing evidence implicating involvement of the coagulation system in various fibrotic diseases, including idiopathic pulmonary fibrosis (IPF) and the interstitial lung fibrosis associated with systemic sclerosis (SSc) (1, 2). Activation of the coagulation cascade is one of earliest events following tissue injury, including lung injury (3). This complex and highly regulated system leads to the generation of insoluble, crosslinked fibrin to form plugs at the site of tissue injury. This process is critically dependent on the action of the serine protease thrombin (4).
In addition to its essential role in coagulation, thrombin has several important functions at the cellular level, both in normal health and in multiple disease processes (5). The majority of the cellular responses to thrombin are mediated via the G protein–coupled receptor protease-activated receptor 1 (PAR-1) (2–6). Previously, we demonstrated that thrombin differentiates normal lung fibroblasts to a myofibroblast phenotype via PAR-1 and a protein kinase C–dependent pathway (7). Thrombin is mitogenic for lung fibroblasts (7–9) and enhances the proliferative effect of fibrinogen on fibroblasts (10). It is also a potent inducer of fibrogenic cytokines, such as transforming growth factor β (TGFβ) (11), connective tissue growth factor (CTGF) (12, 13), and platelet-derived growth factor AA (PDGF-AA) (9). In addition, thrombin increases expression of proinflammatory chemokines (14, 15) and extracellular matrix (ECM) proteins, such as collagen, fibronectin, and tenascin in various cells, including lung fibroblasts (16–18). Activation of these cells by thrombin is a likely mechanism for the development and progression of pulmonary fibrosis in general, and in particular SSc-associated interstitial lung disease (SSc-ILD), in which endothelial injury and activation of the coagulation cascade is widespread.
Activation of the coagulation cascade with generation of thrombin has also been shown to occur in an animal model of bleomycin-induced lung injury and fibrosis (1, 2, 19). Howell et al demonstrated in such a model that direct thrombin inhibition attenuates CTGF and lung collagen accumulation by lowering the profibrotic effects of thrombin (19). Additionally, increased thrombin activity and PAR-1 expression, similar to that which we have demonstrated in SSc-ILD (8, 9), have been observed in bleomycin-induced lung fibrosis (19, 20).
Dabigatran is a direct thrombin inhibitor that reversibly binds to the active site of thrombin, preventing the conversion of fibrinogen to fibrin (21). Recently we demonstrated that binding of dabigatran to thrombin prevents cleavage of the extracellular N-terminal domain of PAR-1 (22). In the absence of dabigatran, thrombin binds to PAR-1 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 (23). Dabigatran-bound thrombin is unable to cleave and activate PAR-1 (22). Further, we have shown that dabigatran inhibits thrombin-induced differentiation of normal lung fibroblasts to the myofibroblast phenotype and decreases CTGF, α-smooth muscle actin (α-SMA), and type I collagen in lung fibroblasts from patients with SSc (22).
In this study we investigated dabigatran etexilate, the oral prodrug of dabigatran. The prodrug does not have antithrombin activity; however, after oral administration, dabigatran etexilate is rapidly converted by ubiquitous esterases to the active moiety, dabigatran (21, 24). The present study was designed to determine whether the oral direct thrombin inhibitor dabigatran etexilate has preventive and/or therapeutic effects on bleomycin-induced pulmonary fibrosis in mice.
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Our recent in vitro studies demonstrated that the direct thrombin inhibitor dabigatran blocks thrombin-induced and PAR-1–mediated profibrotic signaling (22). Dabigatran inhibited thrombin-induced collagen and CTGF production in normal and SSc lung fibroblasts, blocked development of the myofibroblast phenotype from thrombin-activated normal lung fibroblasts, and reversed the myofibroblast phenotype expressed by lung fibroblasts from patients with SSc-ILD (22). These data suggest that dabigatran may serve as a novel and attractive therapeutic agent for pulmonary fibrosis. The present study was designed to determine whether dabigatran etexilate exhibits therapeutic and/or preventive effects on bleomycin-induced pulmonary fibrosis in mice. We demonstrate here for the first time that dabigatran etexilate attenuates bleomycin-induced pulmonary fibrosis by lowering thrombin activity to physiologic levels and by decreasing proinflammatory and profibrotic factors, supporting the idea that it has potential for use in the treatment of pulmonary fibrosis.
Thrombin is a multifunctional protease that promotes a wide range of cellular responses in addition to its central function in thrombosis and hemostasis (1–6). It mediates a variety of inflammatory and tissue repair responses associated with vascular injury (5). Additionally, thrombin stimulates secretion of mediators involved in the pathogenesis of fibrosis, such as TGFβ1 and PDGF (9, 11). TGFβ1 and PDGF are each elevated in BAL fluid of patients with IPF and SSc-ILD, as well as in BAL fluid in animal models of pulmonary fibrosis (9, 31–36). Utilizing a model of bleomycin-induced lung injury and fibrosis, we have demonstrated that dabigatran etexilate significantly decreases inflammatory cell numbers and protein content as well as levels of TGFβ1 in BAL fluid. It also significantly reduces CTGF and α-SMA expression in lung tissue of bleomycin-treated mice, suggesting that it exerts both antiinflammatory and antifibrotic effects.
There is compelling evidence that thrombin is an important mediator of interstitial lung disease including both IPF and SSc-ILD (1, 2, 5). We and others have demonstrated dramatically increased thrombin activity in BAL fluid from patients with SSc-ILD compared to normal subjects (9, 37). Elevated thrombin activity has also been observed in bleomycin-induced pulmonary fibrosis. Howell et al found that in bleomycin-induced lung fibrosis, the direct thrombin inhibitor UK-156406 attenuates collagen accumulation in the lung by lowering the profibrotic effects of thrombin and suppressing CTGF synthesis (19). Later, the same group demonstrated that mice lacking PAR-1 are significantly protected against bleomycin-induced lung fibrosis, with reductions in CCL2 and CTGF expression and TGFβ immunoreactivity (20). Recent studies by Thuillier et al (38) demonstrated that inhibition of thrombin reduced chronic kidney graft fibrosis and significantly improved survival rates in ischemia-reperfusion injury (38, 39).
Different models of pulmonary fibrosis have been developed over the years. Most mimic some but never all features of IPF and SSc-ILD, especially the progressive and irreversible condition (40). This may partially explain why some drugs effective in the treatment of bleomycin-induced pulmonary fibrosis may not demonstrate the same efficacy in pulmonary fibrosis in humans.
In this study, we utilized a single intratracheal administration of bleomycin, which is the most frequently used method for inducing pulmonary fibrosis in animal models. We studied both early and late treatment with dabigatran etexilate to distinguish antiinflammatory and antifibrotic effects of this drug. We observed that dabigatran etexilate markedly improved bleomycin-altered histopathology and reduced interstitial infiltration. It also reduced the thickness of alveolar septa and decreased the accumulation of ECM proteins. The collagen, CTGF, and α-SMA contents of the lung after bleomycin injury were significantly lower in dabigatran etexilate–treated mice than in placebo-treated mice. Importantly, we found that with both early treatment and late treatment, dabigatran etexilate was able to inhibit bleomycin-induced pulmonary fibrosis; however, the inhibition was more profound with early administration. The efficacy of early treatment with dabigatran etexilate was higher because it targeted the inflammatory stage of fibrosis, whereas late treatment was introduced at a stage when the disease was more established.
The role of inflammation in the pathogenesis of progressive pulmonary fibrosis remains a matter of controversy. Administration of bleomycin causes a severe acute inflammatory response followed by chronic inflammation and fibrosis. It has been repeatedly shown that the degree of inflammation in bleomycin-induced lung injury is associated with the intensity of fibrosis (40).
The concentration of dabigatran etexilate used in these experiments yielded plasma levels that are slightly higher than those achieved in patients treated with dabigatran etexilate for various thrombotic diseases (∼180 ng/ml peak levels achieved with 150 mg twice-daily dose) (41). The dose used in this study resulted in an ∼2-fold elevation of the APTT in mice and an ∼10-fold elevation of the TT. This trend is consistent with findings in human plasma, where it has also been shown that the TT is more sensitive to dabigatran than the APTT (41). Although it is not possible to directly relate findings regarding plasma levels and/or anticoagulation from mice to humans, the antifibrotic effects observed in our study were achieved with plasma levels and pharmacologic effects consistent with human dosing.
It is important to note that dabigatran etexilate in the concentrations used in this study significantly reduced, but did not completely inhibit, thrombin activity in BAL fluid. We did not observe any bleeding side effects during the study, suggesting that levels of dabigatran in mouse plasma were not sufficient to completely eliminate thrombin from the normal hemostatic process. However, dabigatran etexilate in the doses tested ameliorated lung fibrosis even after it was established, indicating that it could safely be administered in chronic forms of lung fibrosis, at least in mice.
Pulmonary fibrosis as seen in both IPF and SSc-ILD is often a progressive and irreversible process that leads to respiratory failure and death (32, 42). There is an urgent need for new therapeutic approaches that would delay or reverse pulmonary fibrosis. Blocking of a single profibrotic factor has thus far not been proven to be an effective treatment method. A therapeutic strategy designed to act upstream of multiple fibrogenic pathways, e.g., altering procoagulant activity, might theoretically prove to be more efficacious. Indeed, anticoagulant therapy with warfarin or low molecular weight heparin in combination with prednisolone has been demonstrated to improve survival in patients with IPF (43). Dabigatran etexilate represents the first synthetic oral reversible direct inhibitor of thrombin with a very favorable biochemical and pharmacologic profile that translates into clinical efficacy and safety in patients with coagulation disorders (44). The current study provides important preclinical information about the feasibility and efficacy of dabigatran etexilate for the treatment of fibrotic diseases, including IPF and SSc-ILD, in which there is evidence of tissue injury with overexpression of thrombin. Future studies of thrombin inhibition for the treatment of SSc-ILD would need to demonstrate a positive risk/benefit ratio, taking into account potential risks such as gastrointestinal tract hemorrhage.
- Top of page
- MATERIALS AND METHODS
- AUTHOR CONTRIBUTIONS
- ROLE OF THE STUDY SPONSOR
All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. Dr. Bogatkevich had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.
Study conception and design. Bogatkevich, Ludwicka-Bradley, van Ryn, Silver.
Acquisition of data. Bogatkevich, Akter, van Ryn.
Analysis and interpretation of data. Bogatkevich, Ludwicka-Bradley, Nietert, Akter, van Ryn, Silver.