Factor Xa stimulates fibroblast procollagen production, proliferation, and calcium signaling via PAR1 activation
Introduction
Fibroblast proliferation and extracellular matrix production are central features of normal tissue repair and pathological tissue fibrosis. Tissue fibrosis is the end stage of a heterogenous group of disorders that can affect many organs, including the lung [1], the kidney [2], and the liver [3]. Excessive fibroblast proliferation and collagen deposition are common to these diseases. Many normal responses to tissue injury are recapitulated in tissue fibrosis, but they are deregulated, exacerbated, and abnormally sustained. Eventually, the accumulation of connective tissue, mainly collagen types I and III, compromises the organ's function and often leads to premature death.
In pulmonary fibrosis, increased levels of classical cytokines and growth factors, such as transforming growth factor-β1, platelet-derived growth factor (PDGF), and insulin growth factor-1 promote fibroblast function [4]. However, blood coagulation and the dramatic activation of the coagulation cascade proteinase thrombin have also been extensively documented in association chronic lung injury [5], in pulmonary fibrosis associated with scleroderma, and in interstitial pulmonary fibrosis [6], [7]. Activation of the coagulation cascade is particularly relevant to the lung and organs where the interstitial compartment is in close contact with an extensive microvascular bed. Persistent activation of the coagulation cascade is thought to result from an imbalance between pro- and anticoagulant factors. For instance, bronchoalveolar lavage (BAL) fluid from patients with acute respiratory distress syndrome (ARDS) contains elevated levels of factor VIIa, a factor that initiates blood coagulation [8]. High levels of factor Xa are also generated following lung injury [8], during pulmonary fibrosis [9], [10], and in a variety of fibrotic and infectious conditions [11], [12]. On the other hand, abnormally low levels of anti-coagulant factors such as antithrombin III have been observed in ARDS, resulting in increased procoagulant activity [13].
There is increasing evidence that the coagulation cascade proteinase thrombin contributes to the pathogenesis of lung fibrosis. Thrombin stimulates fibroblast recruitment [14] and proliferation [15], [16], increases procollagen production in vitro [17], and accelerates tissue fibrosis [18]. Thrombin can activate several seven transmembrane domain G protein-coupled cell surface receptors [19], [20] termed proteinase-activated receptors (PAR1, PAR3 and PAR4). PAR1 is the principal receptor for thrombin in fibroblasts [17], [20], [21] and its activation leads to autocrine fibroblast simulation via the production of PDGF [16] and possibly CTGF [22]. We recently obtained the first evidence that direct thrombin inhibition attenuates connective tissue deposition in a model of experimental pulmonary fibrosis [23], supporting the idea that thrombin contributes to tissue fibrosis [24]. However, little is known about the potential role of other coagulation cascade proteinases.
Factor Xa plays a critical role at the point of convergence of the intrinsic and extrinsic coagulation pathways, by converting prothrombin into active thrombin during blood coagulation [11], [12]. We have recently shown that factor Xa is also a potent stimulator of PDGF-A expression and a mitogen for human lung fibroblasts in vitro, and that factor Xa acts independently of thrombin generation [25]. In contrast, coagulation cascade proteinases activated upstream of factor Xa, such as factor IXa and VIIa, had no significant effect at physiological concentrations. We further showed that ligation of effector cell protease receptor-1 (EPR-1) by factor Xa enhances its mitogenic effect. However, the factor Xa receptor that transduces its mitogenic signal remains unknown. In pioneering studies, Riewald and Ruf [26] recently reported that factor Xa can activate PAR1 and PAR2 in heterologous transfection systems. PAR activation by factor Xa was greatly enhanced by cotransfection of tissue factor and the presence of factor VIIa, presumably associating at the cell surface. This group also showed that factor Xa can stimulate gene expression in HeLa cells that express PAR1 and not PAR2 [27].
In this study, we aimed to further investigate the profibrotic effects of factor Xa and begin to characterize the signaling receptors responsible for these effects in fibroblasts. Our experiments showed that factor Xa stimulates fibroblast procollagen production in human and mouse fibroblasts and put in light the essential role of PAR1 in these events, even in the presence of other factor Xa receptors such as PAR2 and EPR-1.
Section snippets
Materials
Purified Russel's viper venom-activated human factor Xa, pertussis toxin (PTX) and dansyl-Glu-Gly-Arg chloromethylketone dihydrochloride (DEGR-ck) were from Calbiochem-Novabiochem UK Ltd. (Nottingham, UK). Catalytically inactivated factor Xa (DEGR-factor Xa) was prepared by Dr C. Goodwin (National Heart and Lung Institute, London, UK) by incubation of factor Xa with DEGR-ck until no proteolytic activity remained. Excess DEGR-ck was removed by extensive dialysis and the purity of DEGR-factor Xa
Factor Xa stimulates fibroblast procollagen production
We determined the effects of factor Xa on procollagen-α1(I) promoter activity and procollagen protein production in human lung fibroblasts. Fig. 1A shows that factor Xa stimulated procollagen protein production by up to 125% in fully confluent and quiesced human fibroblasts in vitro over 48 h. The effect of factor Xa was dose-dependent and peaked at 25 nM (P < 0.01). Thrombin (25 nM) and TGF-β1 (1 ng/ml) stimulated rates of procollagen production by about 200% and 250%, respectively. In
Discussion
Our previous report that factor Xa is mitogenic for human lung fibroblasts suggested that other coagulation cascade factors, and not just thrombin, may exert pro-fibrotic effects. In this study, we investigated further the effects of factor Xa on fibroblast procollagen production and proliferation, and focused on identifying its signaling receptor. We show for the first time that factor Xa stimulates procollagen production by both human and mouse fibroblasts. We also demonstrate that the
Acknowledgments
The authors gratefully acknowledge generous support from Johnson and Johnson Medical-UK and the Wellcome Trust (Programme Grant No 051154). Calcium measurements were performed in the laboratory of Stephen Bolsover, Department of Physiology, University College London (UK), and supported by grants from the Wellcome Trust, Action Research, the Biotechnology and Biological Sciences Research Council and the Medical Research Council.
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2018, Journal of Molecular and Cellular CardiologyCitation Excerpt :Factor Xa and thrombin increase the proliferation, collagen production, and myofibroblast differentiation capability of lung fibroblasts through PAR1 signaling [34,35]. TFLLR-NH2, a PAR1 agonist peptide, has been proven to increase collagen production, and Ca2+ signaling of fibroblasts [35,36]. In the present study, we found that rivaroxaban attenuated the increased migratory capability, pro-collagen type I production, and proliferation capability of thrombin or TFLLR-NH2-treated atrial fibroblasts.
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