Free Access

Procoagulant signalling mechanisms in lung inflammation and fibrosis: novel opportunities for pharmacological intervention?

Corresponding Author

R C Chambers

Department of Medicine, Centre for Respiratory Research, Rayne Institute, University College London, London, UK

Centre for Respiratory Research, Rayne Institute, University College London, 5 University Street, London WC1E 6JJ, UK. E-mail: [email protected]Search for more papers by this author

R C Chambers,

Corresponding Author

R C Chambers

Department of Medicine, Centre for Respiratory Research, Rayne Institute, University College London, London, UK

Centre for Respiratory Research, Rayne Institute, University College London, 5 University Street, London WC1E 6JJ, UK. E-mail: [email protected]Search for more papers by this author

First published: 29 January 2009

https://doi.org/10.1038/sj.bjp.0707603

Citations: 129

Share a link

Email
Facebook
Twitter
LinkedIn
Reddit
Wechat

Abstract

There is compelling evidence that uncontrolled activation of the coagulation cascade following lung injury contributes to the development of lung inflammation and fibrosis in acute lung injury/acute respiratory distress syndrome (ALI/ARDS) and fibrotic lung disease. This article reviews our current understanding of the mechanisms leading to the activation of the coagulation cascade in response to lung injury and the evidence that excessive procoagulant activity is of pathophysiological significance in these disease settings. Current evidence suggests that the tissue factor-dependent extrinsic pathway is the predominant mechanism by which the coagulation cascade is locally activated in the lungs of patients with ALI/ARDS and pulmonary fibrosis. Whilst, fibrin deposition might contribute to the pathophysiology of ALI/ARDS following systemic insult; current evidence suggests that the cellular effects mediated via activation of proteinase-activated receptors (PARs) may be of particular importance in influencing inflammatory and fibroproliferative responses in experimental models involving direct injury to the lung. In this regard, studies in PAR₁ knockout mice have shown that this receptor plays a major role in orchestrating the interplay between coagulation, inflammation and lung fibrosis. This review will focus on our current understanding of excessive procoagulant signalling in acute and chronic lung injury and will highlight the novel opportunities that this may present for therapeutic intervention.

British Journal of Pharmacology (2008) 153, S367–S378; doi:10.1038/sj.bjp.0707603; published online 28 January 2008

Abbreviations:

ALI: acute lung injury
APC: activated protein C
ARDS: acute respiratory distress syndrome
CCL2: chemokine (C–C motif) ligand 2
CTGF: connective tissue growth factor
FVIIa: factor VII (activated)
FXa: factor X (activated)
IPF: idiopathic pulmonary fibrosis
KO: knockout
PAI-1: plasminogen activator inhibitor-1
PAR: proteinase-activated receptor
PDGF: platelet-derived growth factor
TF: tissue factor
TFPI: tissue factor pathway inhibitor
TGFβ: transforming growth factor-beta

Overview: coagulation and fibrinolysis

The activation of the coagulation cascade is one of the earliest events initiated following tissue injury. The prime function of this complex and highly regulated proteolytic system is to generate insoluble, crosslinked fibrin strands, which bind and stabilize weak platelet haemostatic plugs, formed at sites of tissue injury. The formation of this provisional clot is critically dependent on the action of thrombin, and is generated following the stepwise activation of coagulation proteinases via the extrinsic and intrinsic systems (reviewed in ^{Mann et al., 2003}). In vivo, the activation of the coagulation cascade is initiated via the extrinsic pathway. Under normal circumstances, blood is not exposed to tissue factor (TF). However, upon tissue injury, exposure of plasma to TF expressed on non-vascular cells or on activated endothelial cells results in the formation of the TF-activated factor VII (FVIIa) complex. The TF–FVIIa complex subsequently catalyses the initial activation of FX to activated factor X (FXa) and FIX to activated factor IX. FXa in association with activated factor V catalyses the conversion of prothrombin to thrombin, which in turn converts fibrinogen to fibrin, the main constituent of a clot. This mechanism is felt to generate only limited amounts of thrombin. Sustained coagulation is achieved when thrombin synthesized through the initial TF–FVIIa–FXa complex catalyses the activation of FXI, FIX, FVIII and FX. In this manner, the intrinsic pathway is activated, leading to the sustained generation of thrombin and blood coagulation. The extrinsic pathway is therefore paramount in initiating coagulation via the activation of limited amounts of thrombin, whereas the intrinsic pathway maintains coagulation by the dramatic amplification the initial signal.

The coagulation cascade is tightly controlled by both negative feedback mechanisms, as well as by circulating and locally produced endogenous anticoagulants. The extrinsic pathway is mainly controlled by TF pathway inhibitor (TFPI), which inactivates TF–FVIIa complexes after binding to FXa. The intrinsic pathway is controlled by antithrombin, which inhibits thrombin and other serine proteases of the coagulation cascade in the presence of heparin. Other important physiological inhibitors include heparin cofactor II and protease nexin-1, which inhibit thrombin; α₂-macroglobulin and α₁-antitrypsin (also known as α₁-proteinase inhibitor), which inhibit thrombin and factors IXa, Xa and XIa; and protein Z-dependent protease inhibitor, member of the serpin superfamily of proteinase inhibitors that produces rapid inhibition of factor Xa in the presence of protein Z, procoagulant phospholipids and Ca⁺⁺. Finally, when thrombin binds to the endothelial cell surface receptor, thrombomodulin, it is converted from a procoagulant into an anticoagulant by activating protein C. Protein C activation is further enhanced by the endothelial cell surface receptor, endothelial cell protein C receptor. Activated protein C (APC), in conjunction with protein S, inactivates factors Va and VIIIa and thereby suppresses further thrombin generation.

Fibrinolysis is initiated when plasminogen is converted to plasmin by the proteinases, urokinase-type or tissue-type plasminogen activator. Plasmin subsequently cleaves fibrin into a range of fibrin degradation products. Fibrinolytic activity in the vasculature is largely under the control of tissue-type plasminogen activator, whereas extravascular fibrinolysis in the lung is controlled by urokinase-type plasminogen activator. The conversion of plasminogen to plasmin by tissue-type and urokinase-type plasminogen activators is regulated by the endogenous inhibitor, plasminogen activator inhibitor-1 (PAI-1). The fibrinolytic system is also influenced by the plasma glycoprotein, thrombin-activatable fibrinolysis inhibitor and protein C inhibitor (PCI). During fibrin degradation, plasmin exposes C-terminal lysine residues on the fibrin molecule to potentiate its clearance. Thrombin-activatable fibrinolysis inhibitor cleaves these residues, which therefore favours fibrin persistence. PCI on the other hand suppresses plasminogen activation and also blocks the activity of APC.

Activation of the coagulation cascade in acute lung injury

In the normal uninjured lung, the alveolar haemostatic balance is generally antithrombotic and pro-fibrinolytic. However, in both acute lung injury (ALI) and chronic lung diseases such as pulmonary fibrosis, this balance appears to be greatly shifted in favour of procoagulant and antifibrinolytic activity. This section will review this evidence, the underlying causes for this unbalance and its pathological significance. For ALI/acute respiratory distress syndrome (ARDS), this evidence has recently been reviewed (^{Ware et al., 2006}) and will therefore be only touched upon briefly here.

Acute lung injury and ARDS are common, life-threatening conditions leading to acute respiratory failure. These conditions arise from a variety of local and systemic insults, of which sepsis, pneumonia and trauma are the most common causes. ALI/ARDS is characterized by diffuse alveolar damage leading to disruption of the alveolar capillary barrier, pulmonary oedema and neutrophilic inflammation. Extravascular intra-alveolar accumulation of fibrin, often evident as hyaline membranes lining the denuded alveolar surface, has long been recognized as a pathological hallmark of ALI/ARDS and it is well recognized that the coagulation cascade and the subsequent fibrinolytic pathway, responsible for clearing the fibrin clot, are disregulated in these patients (reviewed in ^{Idell, 2003}). The rapid development of interstitial and intra-alveolar fibrosis can lead to the obliteration of airspaces and accounts for respiratory death in up to 40% of patients with ARDS (^{Marshall et al., 1998}).

There is good evidence that the TF-dependent extrinsic pathway is the predominant mechanism by which the coagulation cascade is locally activated in the lungs of patients with ALI/ARDS. TF–FVII procoagulant activity is increased in bronchoalveolar lavage fluid (BALF) from patients with ARDS (^{Idell et al., 1989}) and a recent study employing immunohistochemical staining for TF in human lung tissue from patients with ARDS reported prominent TF staining on alveolar epithelial cells as well as intra-alveolar macrophages and hyaline membranes (^{Bastarache et al., 2007}). Locally activated coagulation zymogens, in combination with leakage of plasma proteins (including fibrinogen) into the alveolar space, as a consequence of microvascular injury are thought to be responsible for the extensive deposition of intra-alveolar and interstitial fibrin (^{Günther et al., 2003}). In terms of anticoagulant factors, levels of antithrombin are reduced in patients with ARDS, and increased TF levels are not matched by a similar increase in TFPI levels (^{Gando et al., 2003}). Plasma and intra-alveolar protein C levels are also decreased in patients with ALI/ARDS (^{Ware et al., 2007}). The alveolar epithelium has also recently been shown to express thrombomodulin and endothelial cell protein C receptor and is capable of activating protein C (^{Wang et al., 2007}). The alveolar epithelium may therefore play an important role in modulating intra-alveolar coagulation. Inflammation or injury to the epithelium may further play a significant role in shifting this balance to a procoagulant state.

Anticoagulant drug intervention in patients with ALI/ARDS

Drug intervention studies with anticoagulants in animal models have provided strong support that excessive coagulation activity may be of pathological significance in ALI/ARDS. Extrapolating the importance of these findings to human disease pathogenesis remains a major challenge, as does the development of effective therapies aimed at interfering with uncontrolled procoagulant activity in these disease settings. Despite the overwhelming success of anti-coagulant strategies in experimental models of ALI, clinical trials of anticoagulants (for example, antithrombin III, TFPI) in patients have been largely disappointing. In contrast, the results of a phase III, randomized, double blind, placebo-controlled, multicentre PROWESS trial of intravenous infusion of the anticoagulant, APC in severe sepsis (which included patients with ARDS) and subsequent reduction in mortality resulted in Food and Drug Administration approval for the use of recombinant human APC (drotrecogin alfa (activated), Xigris) in patients with severe sepsis (sepsis associated with acute organ dysfunction) who have a high risk of death. ARDS subgroup analysis was not presented, and so the effectiveness of this agent to protect the lung in ARDS remains to be determined. Risk of catastrophic bleeding complications is the most common serious concern associated with recombinant human APC therapy, and so each patient being considered for therapy has to be carefully evaluated and anticipated benefits weighed against potential risks associated with therapy. The continued use of this agent is currently the subject of an active debate. There are several ongoing trials to determine whether anticoagulant therapy will be beneficial in ALI; one study will compare TFPI with placebo in patients with pneumonia. There are also currently two studies comparing recombinant human APC with placebo in ALI/ARDS patients and patients with infectious ALI, respectively. The results of these trials are eagerly awaited (reviewed in ^{Ware et al., 2006}).

Activation of the coagulation cascade in pulmonary fibrosis

There is also increasing evidence that therapies targeting the coagulation cascade may prove useful for respiratory conditions associated with chronic or repetitive lung injury, including pulmonary fibrosis. Pulmonary fibrosis represents the end stage of a heterogeneous group of disorders, of known and unknown cause, in which excessive deposition of collagen and other extracellular matrix proteins within the pulmonary interstitium leads to obliteration of airspaces and progressive loss of lung function (Figure 1). There is good evidence that the coagulation cascade is activated in several fibrotic lung diseases, including systemic sclerosis (^{Hernandez-Rodriguez et al., 1995}), idiopathic pulmonary fibrosis (IPF) (^{Imokawa et al., 1997}), sarcoidosis and hypersensitivity pneumonitis (^{Günther et al., 2000}). As is the case for ALI, current evidence suggests a major role for the TF-dependent extrinsic pathway as the main activator of the coagulation cascade in these conditions. TF expression is highly upregulated on type II pneumocytes and to some extent on alveolar macrophages, in close association with fibrin deposits in the lungs of patients with IPF and systemic sclerosis (^{Imokawa et al., 1997}). Levels of active thrombin are increased in bronchoalveolar lavage fluid from patients with pulmonary fibrosis associated with systemic sclerosis (^{Hernandez-Rodriguez et al., 1995}) and in pulmonary fibrosis associated with chronic lung disease of prematurity (^{Dik et al., 2003}). Several other procoagulant factors (fibrinogen, factors VII and X) have also been identified in patients with intra-alveolar fibrosis associated with bronchiolitis obliterans organizing pneumonia/cryptogenic organizing pneumonia (^{Peyrol et al., 1990}). There is also evidence that the protein C pathway is deficient in the lungs of patients with IPF and sarcoidosis as well as collagen vascular disease-associated interstitial lung disease (^{Kobayashi et al., 1998}). Moreover, decreased protein C activation was found to be associated with abnormal collagen turnover in the intra-alveolar space of patients with these conditions (^{Yasui et al., 2000}). This study, together with the observations that thrombin in bronchoalveolar lavage fluid from patients with systemic sclerosis, contributes to the mitogenic activity of this fluid for cultured lung fibroblasts; ^{Ohba et al. (1994)} and ^{Hernandez-Rodriguez et al. (1995)} provided some of the earliest evidence that the coagulation cascade might influence the development of fibrosis in these patients. Reviewing the current evidence that the coagulation cascade plays a pathophysiological role in patients with acute and chronic lung injury and the opportunities this provides for therapeutic intervention will be a focus of much of the remainder of this article.

Details are in the caption following the image — **Figure 1**
Open in figure viewer PowerPoint

Alveolar architecture of normal and fibrotic lung. Images show the stark contrast in alveolar architecture in the normal and fibrotic lung. In the fibrotic lung, the open alveolar architecture is obliterated and replaced with dense fibrotic tissue. (Histology slides courtesy of Dr Robin McAnulty, Centre for Respiratory Research, University College London.)

Anticoagulants are effective in blocking experimentally induced lung fibrosis

Strategies targeting the coagulation cascade with direct and indirect anticoagulants have provided strong support for a causative role of the coagulation cascade in experimentally induced fibrosis. For example, our laboratory has shown that direct thrombin inhibition was highly effective at attenuating lung collagen deposition in bleomycin-induced lung fibrosis in rats (^{Howell et al., 2001}). Similar findings have also been reported with the anticoagulant, APC instilled intratracheally in mice (^{Yasui et al., 2001}) and with nebulized unfractionated heparin in rabbits (^{Günther et al., 2003}). Although APC and heparin also exert anti-inflammatory effects, it seems likely that they may be acting, at least in part, via their anticoagulant properties in this model. More recently, intratracheal gene transfer of TFPI was also reported to decrease bleomycin-induced thrombin generation and pulmonary fibrosis in rats (^{Kijiyama et al., 2006}). Taken together, these data support the notion that TF-mediated coagulation in the extravascular intra-alveolar space is of paramount importance and that anticoagulant therapy could be beneficial.

Anticoagulant therapy for patients with pulmonary fibrosis?

There have been few successful trials in pulmonary fibrosis, and so this condition remains largely untreatable (reviewed in ^{Scotton and Chambers, 2007}). However, the results of a recent non-blinded, randomized trial of 56 patients with IPF given prednisolone alone or prednisolone plus anticoagulation (^{Kubo et al., 2005}) provides some support that targeting the coagulation cascade may improve outcome. In this study, the anticoagulant group had reduced mortality from acute exacerbations, with an overall significant increase in survival (63% survival at 3 years in the anticoagulant group versus 35% in the non-anticoagulant group). Although this was a small non-blinded study, and the exact mechanism of this beneficial effect is not known, it is one of the few studies describing a beneficial outcome on survival in an IPF clinical study reported on to date.

Fibrinolysis following lung injury

The prevailing balance between the pro- and anticoagulant states in the lung following injury is also affected by regulatory mechanisms that control the clearance of deposited fibrin (reviewed by ^{Idell, 2003}). There is compelling evidence that fibrinolysis is impaired in patients with both ALI/ARDS and pulmonary fibrosis. A number of human and animal studies have shown that levels of PAI-1 are increased in these conditions, thus favouring fibrin persistence (^{Olman et al., 1995}). A recent study has further shown that levels of thrombin-activatable fibrinolysis inhibitor and PCI are increased in bronchoalveolar lavage fluid from patients with interstitial lung disease and may thereby contribute to intra-alveolar hypofibrinolysis associated with these conditions (^{Fujimoto et al., 2003}).

The contribution of fibrin deposition to the development of experimental lung fibrosis has received considerable attention but remains a somewhat unresolved issue. Fibrin is thought to influence the fibrotic response in several ways. First, fibrin inhibits surfactant function and may thereby cause atelectasis (alveolar collapse). Second, according to the concept of ‘collapse induration’, by acting as a provisional matrix and reservoir of growth factors for fibroblasts and inflammatory cells, the fibrin matrix contributes to alveolar collapse and traction of remaining airspaces (honeycombing). Studies performed in experimental models using genetically modified mice in which the fibrinolytic capacity in the lung was either up- or downregulated support the notion that fibrin persistence may contribute to the development of bleomycin-induced fibrosis. Lung collagen accumulation is increased in mice overexpressing PAI-1 (favouring fibrin persistence) and is decreased in PAI-1 knockout (KO) mice (favouring fibrin clearance) (^{Eitzman et al., 1996}). In further support for a role for fibrin in experimental fibrosis, aerosolization of urokinase-type plasminogen activator was highly effective in preventing bleomycin-induced lung fibrosis in rabbits (^{Günther et al., 2003}). Similarly, the recent report that thrombin-activatable fibrinolysis inhibitor deficiency is associated with an attenuated response to bleomycin-induced lung fibrosis (^{Fujimoto et al., 2006}) also supports the notion that fibrin persistence influences the subsequent fibrotic response. In contrast, the finding that fibrinogen KO mice are not protected from bleomycin-induced fibrosis (^{Hattori et al., 2000}) suggests that fibrin per se may not be required for progression to fibrosis in bleomycin-induced lung fibrosis.

Proteinase-activated receptors: signalling receptors for coagulation proteinases

If fibrin is not required for experimental lung fibrosis, this begs the question as to how the coagulation cascade is causally involved in driving the fibrotic response. This problem was solved, at least in part, by the discovery of the proteinase-activated receptors (PARs) in the early 1990s (^{Vu et al., 1991}).

The PARs belong to a subfamily of the seven transmembrane domain G-protein-coupled receptors and derive their name from their unique mechanism of activation involving the unmasking of a tethered ligand by limited proteolysis (Figure 2). Conformational changes induced following interaction of the tethered ligand with the second extracellular loop of these receptors initiate cell signalling via heterotrimeric G proteins. The PAR family comprises four members (PAR₁–PAR₄) and collectively, the proteinases of the coagulation cascade can target all four family members. For a summary of our current understanding on the major activators, expression patterns and pharmacology of the PARs, please see Table 1. All four PARs are expressed in the lung on a variety of resident cell types, as well as on cells that are recruited to the lung following injury. Thrombin is considered to be a major activator of PAR₁, PAR₃ and PAR₄; whereas FXa, either on its own or as part of the more potent TF–FVIIa–FXa ternary complex, activates either PAR₁ or PAR₂, depending on cell type and cofactor expression (reviewed in ^{Coughlin 2005}). Synthetic peptides corresponding to the tethered ligand sequence of PAR₁, PAR₂ and PAR₄ are capable of mimicking a number of cellular responses of endogenous proteinase activators. Whereas PAR₁, PAR₂ and PAR₄ act as signalling receptors, current evidence suggests that PAR₃ acts as a thrombin-docking receptor for efficient presentation of the proteinase to PAR₄ at low thrombin concentrations. Although there is little doubt that the PARs act as major signalling receptors for coagulation proteinases, it is important to point out that the PARs can also be activated by non-coagulation proteinases. In this regard, PAR₂ is a major substrate for trypsin as well as mast cell tryptase and has received considerable attention in the setting of both asthma and airway inflammation (reviewed in ^{Moffatt et al., 2004}).

Table 1. Human proteinase-activated receptor expression and pharmacology

	No. of amino acids	High-affinity activating proteinases	Other activating proteinases	Tethered ligand sequence	Activating peptides	Antagonists	Inactivating proteinases	Expression in the lung	Cell types
PAR1	425	Thrombin	TF/FVIIa/FXa complex, FXa, granzyme A, plasmin, trypsin IV, MMP-1, tissue kallikreins	R⁴¹↑SFLLRN	SFLLRN-NH₂, TFLLR-NH₂	RWJ-5611, RWJ-58259, SCH 530348	Cathepsin G, neutrophil proteinase-3, elastase, chymase, Der p1	Airways, blood vessels, lung parenchyma	Endothelial cells, epithelial cells, fibroblasts, macrophages, mast cells, natural killer cells, neuronal cells, platelets, smooth muscle cells (airway and vascular), T cells
PAR2	397	Trypsin, tryptase, trypsin II, trypsin IV	TF/FVIIa/FXa complex, TF-FVIIa, FXa, matriptase/MT-SP1, proteinase-3Der p1, Der p3, Der p9, tissue kallikreins	R³⁴↑SLIGKV	SLIGKV-NH₂, SFLLRN-NH₂	None to date	Elastase, chymase	Airways, blood vessels, bronchial glands, lung parenchyma	Endothelial cells, epithelial cells, eosinophils, fibroblasts, mast cells, macrophages, monocytes, neuronal cells, neutrophils, platelets, smooth muscle cells (airway and vascular), T cells
PAR3	374	Thrombin	Trypsin, factor Xa	K³⁸↑TFRGAP	None known	––	Cathepsin G	Airways	Epithelial cells, fibroblasts, platelets, airway smooth muscle cells, T cells
PAR4	385	Thrombin, trypsin	Cathepsin G, tissue kallikreins	R⁴⁷↑GYPGQV	GYPGQV-NH₂, AYPGKF-NH₂	YD-3	Unknown	Airway, blood vessels, cardiovascular system	Endothelial cells, epithelial cells, smooth airway muscle cells, platelets, fibroblasts

Letters denote amino-acid sequences in one letter code; arrows denote cleavage site.
Abbreviations: Der p1, 3 and 9, house dust mite Dermatophagoides pteronyssinus proteinase 1, 3 and 9; FVIIa, activated factor VII; FXa, activated factor X; MMP-1, matrix metalloproteinase-1; MT-SP1, membrane-type serine protease 1; NH₂, amide; TF, tissue factor.

PAR₁, the high-affinity thrombin receptor, was the first PAR to be cloned and fully characterized and has subsequently been shown to mediate thrombin's pluripotent cellular effects on numerous cell types. The clearest physiological role for PAR₁ is in the activation of platelets by thrombin, one of the key events involved in blood clotting. In addition, PAR₁ plays a central role in influencing a number of cellular responses that are central to the subsequent inflammatory and tissue repair programmes initiated following tissue injury (reviewed in ^{Chambers, 2003}). This receptor is currently a major drug target in the setting of thrombosis and cardiovascular disease (reviewed in ^{Chackalamannil, 2006}). The remainder of this article will discuss the evidence that PAR₁ may represent an attractive novel target for therapeutic intervention in the settings of both acute and chronic lung injury.

PAR activation and pro-inflammatory signalling

The role of PARs in promoting inflammation has been the subject of several excellent recent reviews (^{Coughlin 2005}; ^{Bunnett 2006}) and will therefore only briefly be mentioned here. Extensive in vitro studies have revealed that activation of PAR₁ on numerous cell types, including among others fibroblasts, epithelial cells, monocytes/macrophages and vascular endothelial cells, leads to the induction and release of potent pro-inflammatory mediators and chemokines (Table 2). Similar potent pro-inflammatory effects have also been reported for factor Xa and TF–FVIIa–FXa complexes via both PAR₁- and PAR₂-dependent mechanisms and there is increasing evidence that these PAR-mediated pro-inflammatory responses may play significant roles in the context of a number of inflammatory conditions (reviewed in ^{Bunnett, 2006}). Activation of PAR₄, as well as PAR₂, with synthetic activating peptides has similarly been reported to lead to the release of interleukin-6 (IL-6), IL-8 and prostaglandin E₂ (PGE₂) by cultured bronchial epithelial cells (^{Asokananthan et al., 2002}). A number of these mediators are potent inducers of TF expression, and so PARs may play a central role in perpetuating the interplay between coagulation and inflammation (Figure 3). Moreover, thrombin has also been reported to induce the expression of endothelial cell adhesion molecules, including P-selectin and intercellular adhesion molecule-1 (ICAM-1) in vitro and may therefore facilitate the recruitment of inflammatory cells via the production of chemokine networks and upregulation of adhesion molecule expression.

Table 2. Pro-inflammatory mediators released following PAR₁ activation

Cytokine/chemokine	Cell type	Reference
IL-1β	Monocytes/macrophages	^{Naldini et al. (1998}, ²⁰⁰²⁾
IL-2	T lymphocytes	^{Mari et al. (1994)}
IL-6	Fibroblasts, epithelial cells, monocytes/macrophages, mast cells, smooth muscle cells	^{Sower et al. (1995)}, ^{Cirino et al. (1996)}, ^{Kranzhofer et al. (1996)} and ^{Naldini et al. (1998)}
IL-8	Fibroblasts, epithelial cells, monocytes/macrophages	^{Ueno et al. (1996)}, ^{Suk and Cha (1999)}, ^{Ludwicka-Bradley et al. (2000)} and ^{Asokananthan et al. (2002)}
PGE₂	Epithelial cells	^{Asokananthan et al. (2002)}
CCL2/ MCP-1	Fibroblasts, endothelial cells, monocytes/macrophages	^{Riewald et al. (2002)}, ^{Bachli et al. (2003)} and ^{Colognato et al. (2003)}
TNF-α	Monoctyes/macrophages	^{Naldini et al. (1998)}

Table lists the major cytokines/chemokines released by cell types present in the lung.
Abbreviations: CCL2, chemokine (C–C motif) ligand 2; IL, interleukin; MCP-1, monocyte chemotactic protein 1; TNFα, tumour necrosis factor-alpha.

PAR₁ is also the major thrombin receptor expressed on microvascular endothelial cells, and activation of PAR₁ by thrombin promotes endothelial cell permeability and contraction in vitro. Direct intravenous infusion of thrombin increases pulmonary vascular permeability in experimental models (reviewed in ^{Siflinger-Birnboim and Johnson 2003}). An important role for PAR₁ in mediating these effects was provided by studies showing that thrombin-induced pulmonary microvascular permeability is abrogated in lung organ cultures from PAR₁ KO mice (^{Vogel et al., 2000}). Widespread microvascular injury and leak are common features of ALI/ARDS and chronic fibrotic lung diseases and are thought to be a major mechanism leading to the extravasation of coagulation zymogens and intra-alveolar fibrin deposition (reviewed in ^{Idell, 2003}). Therapies targeting thrombin-dependent PAR₁ activation on the microvascular endothelium may therefore be of therapeutic value, although it is worth pointing out that activation of PAR₁ on the endothelium may also be cytoprotective in certain circumstances (^{Feistritzer et al., 2006}).

PAR₁ is a major pro-fibrotic signalling receptor

The myofibroblast is the key effector cell in pulmonary fibrosis responsible for the production of the bulk of extracellular matrix proteins deposited within the pulmonary interstitium. This cell type is characterized by the de novo expression of contractile α-smooth muscle actin fibres and is thought to originate from three possible sources: expansion of the resident fibroblast pool; epithelial–mesenchymal transition or recruitment of circulating mesenchymal progenitor cells (fibrocytes) to sites of lung injury (reviewed in ^{Scotton and Chambers, 2007}). Thrombin exerts potent pro-fibrotic effects in vitro by influencing fibroblast function and extensive in vitro studies involving selective PAR₁-activating peptides and cells derived from PAR₁-deficient mice have revealed that PAR₁ is the major signalling receptor involved in mediating the potent stimulatory effects of thrombin on lung fibroblast proliferation (^{Trejo et al., 1996}), extracellular matrix production (^{Chambers et al., 1998}) and fibroblast to myofibroblast differentiation (^{Bogatkevich et al., 2001}). We have further shown that PAR₁ is also the major receptor by which FXa stimulates lung fibroblast mitogenesis (^{Blanc-Brude et al., 2005}).

There is good evidence that the pro-fibrotic effects elicited following PAR₁ activation by thrombin and factor Xa are not mediated following PAR₁ activation directly but via the induction of a host of potent pro-fibrotic mediators. In terms of fibroblast mitogenic responses following PAR₁ activation, this response is critically dependent on both the induction of platelet-derived growth factor-AA and upregulation of the platelet-derived growth factor α-receptor (^{Ohba et al., 1994}; ^{Blanc-Brude et al., 2001}). Thrombin also induces the expression of platelet-derived growth factor-AA by other cell types, including macrophages (^{Tani et al., 1997}) and blocking platelet-derived growth factor signalling has been successful in attenuating experimentally induced lung fibrosis (^{Rice et al., 1999}; ^{Yoshida et al., 1999}). We and others have shown that PAR₁ activation also leads to the rapid and dramatic induction of connective tissue growth factor (CTGF) by cultured lung fibroblasts (^{Chambers et al., 2000}) and epithelial cells (CTGF) (^{Riewald et al., 2001}). CTGF has been shown to influence two discrete pro-fibrotic effects via two distinct domains: the N-terminal domain of CTGF mediates myofibroblast differentiation and collagen synthesis, whereas the C-terminal domain mediates fibroblast proliferation (^{Grotendorst and Duncan 2005}).

More recently, another major mechanism by which activation of PAR₁ may promote fibrosis was uncovered by the observation that PAR₁ ligation can lead to the activation of latent transforming growth factor-beta (TGFβ) (^{Jenkins et al., 2006}). TGFβ is one of the most potent pro-fibrotic mediators characterized to date and a major target in the context of developing novel therapeutic strategies for pulmonary fibrosis and other fibrotic conditions (reviewed in ^{Scotton and Chambers 2007}). The activation of latent TGFβ is a major rate-limiting step in the regulation of TGFβ bioavailability and involves the conversion of the latent precursor to its biologically active form through dissociation from the latency-associated peptide. In collaborative studies performed with our centre, ^{Jenkins et al. (2006)} have recently reported that PAR₁ ligation on epithelial cells leads to the activation of TGFβ via a αvβ6 integrin-dependent mechanism in vitro and that this mechanism contributes to TGFβ activity following bleomycin-induced lung injury. The significance of this finding and the potential role of CTGF downstream of PAR₁ activation in experimental lung fibrosis will be discussed in more detail in the next section. Finally, in the context of PAR₁ activation on epithelial cells, it has also been recently reported that activation of PAR₁ induces alveolar epithelial cell apoptosis in vitro (^{Suzuki et al., 2005}). The importance of this finding in the context of lung injury and fibrosis remains to be established, but this may represent another potential mechanism by which excessive procoagulant signalling may exert deleterious effects.

Lessons from PAR KO mouse, PAR agonist and antagonist studies in experimental models of lung injury

Recent studies conducted in our laboratory using PAR₁ KO mice support a major role for PAR₁ in influencing inflammatory cell recruitment, microvascular leak, lung oedema and fibrosis in response to bleomycin-induced lung injury (^{Howell et al., 2005}; ^{Jenkins et al., 2006}). The protection from bleomycin-induced lung inflammation and fibrosis in PAR₁ KO mice is associated with a reduction in the upregulation of the PAR₁-inducible mediators, chemokine (C-C motif) ligand 2 (CCL2)/monocyte chemotactic protein 1/JE and CTGF. TGFβ lung levels are also reduced compared with correspondingly injured wild-type mice, but current in vitro data obtained using cultured lung epithelial cells indicate that thrombin/PAR₁ does not upregulate the expression of TGFβ directly, but as mentioned above acts at the level of activation of the latent TGFβ complex. The significance of this finding in experimental lung injury is supported by the observation that TGFβ signalling, as evidenced by nuclear phosphorylated Smad 2 immunostaining, is attenuated in PAR₁ KO mice compared with correspondingly injured wild-type mice (^{Jenkins et al., 2006}). Once activated, TGFβ is a potent inducer of its own production in vitro and in vivo (^{Sime et al., 1997}), and so the attenuated response in terms of TGFβ expression in PAR₁ KO mice following bleomycin-induced lung injury may be explained by a diminished ability of these mice to activate latent TGFβ. More recently, we have also obtained unpublished evidence that PAR₁ may promote TGFβ activation by lung fibroblasts via a non-integrin-mediated but thrombospondin-1-dependent mechanism, suggesting that PAR₁ may play an important role in controlling TGFβ bioavailability via several activation mechanisms.

The finding that CTGF expression is blunted in PAR₁ KO mice in response to bleomycin-induced lung injury is also of particular interest in light of our previous report that protection from bleomycin-induced lung fibrosis by direct thrombin inhibition is also accompanied by a blunted CTGF response (^{Howell et al., 2001}). CTGF levels are increased in patients with fibrotic lung disease (^{Allen et al., 1999}), but the mechanisms by which CTGF contributes to the development of lung fibrosis is currently poorly understood. Although CTGF exerts pro-fibrotic effects in vitro (^{Grotendorst and Duncan, 2005}), adenoviral gene transfer of CTGF to the lung was shown to induce only a mild and transient fibrotic response, suggesting that CTGF is not a direct fibrogenic factor in this organ (^{Bonniaud et al., 2003}). However, CTGF is capable of inducing fibrosis when co-administered with bleomycin in ‘fibrosis-resistant’ BALB/c mice, potentially by promoting a non-degradative environment (^{Bonniaud et al., 2004}). The upregulation of CTGF following lung injury may therefore represent another potential mechanism by which PAR₁ may influence the subsequent fibrotic response. Finally, the diminished expression of CCL2/monocyte chemotactic protein 1/JE in PAR₁ KO mice following bleomycin injury is also of particular interest, in view of recent reports that CCL2 blockade and chemokine (C–C motif) receptor 2 deficiency (the main CCL2 signalling receptor) also lead to a blunted fibrotic response to both bleomycin and fluorescein isothiocyanate-conjugated-induced lung injury, potentially by influencing the recruitment of circulating fibrocytes (^{Moore et al., 2005}). Interestingly, PAR₁ antagonism is also protective in experimental liver fibrosis, and stellate cells were found to upregulate CCL2 following PAR₁ activation in vitro (^{Fiorucci et al., 2004}), so that the PAR1/CCL2 axis may be significant in a number of fibrotic conditions.

In other models of ALI such as during high-tidal-volume ventilation, intratracheal instillation of PAR₁-activating peptides (TFLLRN) increases lung oedema via the same αvβ6-dependent latent TGFβ activation mechanism described above (^{Jenkins et al., 2006}). In contrast to these findings, in spontaneously breathing mice, intratracheal instillation of PAR₂ (SLIGRL)-, but not PAR₁-, activating peptides was sufficient to induce acute lung inflammation via a neuropeptide-dependent mechanism (^{Su et al., 2005}). Moreover, PAR₁ KO mice are not protected in the mouse model of systemic endotoxin-induced inflammation (endotoxaemia) (^{Pawlinski et al., 2004}). Current evidence suggests that multiple PARs (PAR₁–PAR₄) and fibrin formation together contribute to systemic endotoxin-induced inflammation (^{Pawlinski et al., 2004}; ^{Camerer et al., 2006}). Taken together, these findings suggest that the contribution of PAR₁ to lung inflammation is likely to be highly dependent on the nature of the insult (bleomycin versus endotoxin) and also on whether the primary insult originates in the lung or in the systemic circulation.

Clinical implications for patients with pulmonary fibrosis: PAR₁ antagonists as novel agents for therapeutic intervention?

The protection of PAR₁ KO mice from lung oedema, inflammatory cell recruitment and the development of fibrosis in the bleomycin model of lung injury and fibrosis demonstrates that this receptor plays a central role in orchestrating the tissue response to lung injury. The finding from our and other laboratories that this receptor is highly upregulated on fibroblasts and macrophages within fibrotic foci in the lungs of patients with IPF (^{Howell et al., 2001}) and pulmonary fibrosis associated with systemic sclerosis (^{Bogatkevich et al., 2005}) is consistent with the notion that this receptor may also play a central role in the pathogenesis of human fibrotic lung disease. PAR₁ expression is upregulated in response to a number of pro-inflammatory and pro-fibrotic mediators (reviewed in ^{Sokolova and Reiser, 2007}), but the mediators responsible for controlling PAR₁ expression in the fibrotic lung are currently unknown. A recent report that autocrine production of COX-2-derived PGE₂ is responsible for downregulating the expression of PARs (PAR₁–PAR₃) following PAR₁ activation in cultured human lung fibroblasts (^{Sokolova and Reiser, 2007}) is of particular interest in view of the compelling evidence that fibroblasts from patients with IPF are unable to upregulate COX-2 gene expression in response to various pro-inflammatory and pro-fibrotic stimuli (^{Keerthisingam et al., 2001}). This may provide a plausible explanation for the high levels of PAR₁ expression on these cells in IPF and further supports the notion that uncontrolled PAR₁ signalling may be of pathological significance in this disease setting. Recent unpublished data from our laboratory suggest that this receptor is also highly expressed on infiltrating macrophages and numerous fibroblasts present in fibrotic areas in lung tissue from patients with ALI/ARDS (^{Howell et al., 2007}). Finally, there is also accumulating evidence that procoagulant signalling may contribute to other respiratory conditions associated with remodelling, including airway remodelling in asthma (^{Terada et al., 2004}) and more recently also in chronic obstructive pulmonary disease (COPD) (^{Demeo et al., 2006}), as well as fibrosis in other organs such as the liver (^{Fiorucci et al., 2004}).

Progress in the development of PAR₁ antagonists

There is now increasing pre-clinical evidence that PAR₁-blocking strategies might prove useful for the treatment of fibroproliferative lung disease. It has been argued for some time now that such an approach may be safer in terms of bleeding complications compared with strategies aimed at directly interfering with the coagulation cascade as PAR₁ antagonists would allow selective inhibition of the potentially deleterious receptor-mediated cellular effects of coagulation proteinases, while preserving their essential role in fibrin formation. The development of PAR₁-specific receptor antagonists has been fuelled by the potential for such antagonists as antithrombotic/antiplatelet agents. The development of PAR₁ antagonists has been partially successful, but the clinical utility of these agents remains to be established. This section will briefly summarize recent progress in this field. For a recent review and further details, please see ^{Chackalamannil (2006)}.

The early peptidomimetic PAR₁ antagonists were designed on the basis of the SFLLRN motif of the PAR₁-tethered ligand. Optimization of these antagonists led to the identification of the heterocycle-based peptide-mimetic of PAR₁ antagonists, indole-based RWJ-56110 and indazole-based RWJ-58259 (^{Zhang et al., 2001}; ^{Damiano et al., 2003}). Both compounds inhibit thrombin-induced platelet aggregation with IC₅₀ values of 340 and 370 nM. In the guinea pig model of ex vivo platelet aggregation, RWJ-58259 showed improved efficacy over RWJ-56110. RWJ-58259 completely inhibits thrombin-induced platelet aggregation at doses as low as 0.3 mg kg⁻¹ and this antagonist further displayed antithrombotic activity in a cynomolgus monkey arterial injury model. Studies with another related indole-based PAR₁ antagonist exerted protective effects in a rat model of liver fibrosis at a dose of 1.5 mg kg⁻¹ day⁻¹ (^{Fiorucci et al., 2004}) and pre-clinical studies with RWJ-58259 are currently ongoing in our centre in both acute and chronic lung injury models.

Good PAR₁ affinity and promising activity in functional assays have also been obtained for non-peptide PAR₁ antagonists such as the pyrroloquinazoline analogues, such as SCH 79797. Promising data have been reported in thrombin-induced platelet aggregation assays (IC₅₀=3 μM) and other cell-based assays, and SCH 79797 was reported to limit myocardial ischaemia/reperfusion injury in rat hearts at an optimal dose of 25 μg kg⁻¹ i.v. (^{Strande et al., 2007}). However, this compound has been reported to be toxic for lung fibroblasts (Sokolova and Reiser, unpublished observations in ^{Sokolova and Reiser, 2007}). Potential off-target effects have also recently been reported (^{Di Serio et al., 2007}).

PAR₁ antagonists based on the core structure of the tetracyclic piperidine alkaloid, himbacine, from Australian magnolia trees have also recently been developed (^{Chackalamannil, 2006}). The most potent PAR₁ antagonist in this series demonstrates excellent affinity (K_i of 4.3 nM), good oral bioavailability (∼62%) and blocks platelet aggregation in the cynomolgus monkey model up to 70% at a dose of 3 mg kg⁻¹ (^{Chelliah et al., 2007}).

There are currently two pharmaceutical companies developing orally active PAR₁ antagonists in clinical trials in the setting of cardiovascular disease. A phase II trial of SCH 530348 in subjects undergoing non-emergent percutaneous coronary intervention (Study P03573AM1) has recently been completed (http://clinicaltrials.gov/show/NCT00132912), and two phase III trials to assess the effects of SCH 530348 in preventing heart attack and stroke in patients with atherosclerosis (TRA 2°P—TIMI 50) (Study P04737: http://clinicaltrials.gov/ct/show/NCT00526474) and in patients with acute coronary syndrome (TRA·CER) (Study P04736) have recently been announced (http://clinicaltrials.gov/ct/show/NCT00527943). The results of these trials are eagerly awaited and may hold promise for therapeutic intervention in the setting of inflammation and fibrosis in several disease settings, including fibroproliferative lung disease.

Conclusions

There is compelling evidence that the TF-dependent extrinsic coagulation pathway is locally activated in the lungs of patients with ALI/ARDS and fibrotic lung disease and further that PAR₁, the high-affinity thrombin signalling receptor, plays a central role in orchestrating the interplay between coagulation, inflammation and fibroproliferation in experimental models of lung injury via its ability to release and activate a host of pro-inflammatory and pro-fibrotic mediators (Figure 4). Although these responses might be part of the normal programme leading to tissue repair, excessive procoagulant signalling in response to lung injury is highly deleterious. Therapeutic agents aimed at blunting excessive procoagulant activity and signalling may therefore offer promise as novel therapeutic agents in these disease settings. Targeting the lung epithelium directly with novel inhaled therapies (for example, TFPI, APC) may offer advantage over traditional anticoagulant therapy in terms of avoidance of potential bleeding complications. Moreover, therapeutic strategies aimed at targeting PAR₁ directly may offer similar benefits. PAR₁ antagonists are currently being developed as antithrombotic agents and such agents may hold promise in the setting of a number of conditions associated with excessive procoagulant signalling.

Acknowledgments

I acknowledge the contribution of past and present researchers who have contributed to the research programme on PARs in the Centre for Respiratory Research at University College London (UCL), including Dr David Howell, Dr Robin Johns, Dr Chris Scotton, Ms Malvina Krupiczojc, Dr Paul Mercer, Ms Xiaoling Deng and Mr Steven Bottoms. I am grateful to the following funding bodies who have supported this research programme, The Wellcome Trust (Programme Grant no. GR071124MA), the Medical Research Council, the British Lung Foundation (Project Grant no. P03/8), the Rockefeller Fund and the Rosetrees Trust. I am also grateful to Dr Robin McAnulty (Centre for Respiratory Research, UCL) for providing histology slides in Figure 1.

Conflict of interest

The authors state no conflict of interest.

References

Allen JT, Knight RA, Bloor CA, Spiteri MA (1999). Enhanced insulin-like growth factor binding protein-related protein 2 (connective tissue growth factor) expression in patients with idiopathic pulmonary fibrosis and pulmonary sarcoidosis. Am J Respir Cell Mol Biol 21: 693–700.
10.1165/ajrcmb.21.6.3719
CASPubMedWeb of Science®Google Scholar
Asokananthan N, Graham PT, Fink J, Knight DA, Bakker AJ, McWilliam AS et al. (2002). Activation of protease-activated receptor (PAR)-1, PAR-2, and PAR-4 stimulates IL-6, IL-8, and prostaglandin E2 release from human respiratory epithelial cells. J Immunol 168: 3577–3585.
10.4049/jimmunol.168.7.3577
CASPubMedWeb of Science®Google Scholar
Bachli EB, Pech CM, Johnson KM, Johnson DJ, Tuddenham EG, McVey JH (2003). Factor Xa and thrombin, but not factor VIIa, elicit specific cellular responses in dermal fibroblasts. J Thromb Haemost 1: 1935–1944.
10.1046/j.1538-7836.2003.00363.x
CASPubMedWeb of Science®Google Scholar
Barry GD, Le GT, Fairlie DP (2006). Agonists and antagonists of protease activated receptors (PARs). Curr Med Chem 13: 243–265.
10.2174/092986706775476070
CASPubMedWeb of Science®Google Scholar
Bastarache JA, Wang L, Geiser T, Wang Z, Albertine KH, Matthay MA et al. (2007). The alveolar epithelium can initiate the extrinsic coagulation cascade through expression of tissue factor. Thorax 62: 608–616.
10.1136/thx.2006.063305
PubMedWeb of Science®Google Scholar
Blanc-Brude OP, Archer F, Leoni P, Derian C, Bolsover S, Laurent GJ et al. (2005). Factor Xa stimulates fibroblast procollagen production, proliferation, and calcium signaling via PAR1 activation. Exp Cell Res 304: 16–27.
10.1016/j.yexcr.2004.10.021
CASPubMedWeb of Science®Google Scholar
Blanc-Brude OP, Chambers RC, Leoni P, Dik WA, Laurent GJ (2001). Factor Xa is a fibroblast mitogen via binding to effector-cell protease receptor-1 and autocrine release of PDGF. Am J Physiol Cell Physiol 281: C681–C689.
10.1152/ajpcell.2001.281.2.C681
CASPubMedWeb of Science®Google Scholar
Bogatkevich GS, Gustilo E, Oates JC, Feghali-Bostwick C, Harley RA, Silver RM et al. (2005). Distinct PKC isoforms mediate cell survival and DNA synthesis in thrombin-induced myofibroblasts. Am J Physiol Lung Cell Mol Physiol 288: L190–L201.
10.1152/ajplung.00448.2003
CASPubMedWeb of Science®Google Scholar
Bogatkevich GS, Tourkina E, Silver RM, Ludwicka-Bradley A (2001). Thrombin differentiates normal lung fibroblasts to a myofibroblast phenotype via the proteolytically activated receptor-1 and a protein kinase C-dependent pathway. J Biol Chem 276: 45184–45192.
10.1074/jbc.M106441200
CASPubMedWeb of Science®Google Scholar
Bonniaud P, Margetts PJ, Kolb M, Haberberger T, Kelly M, Robertson J et al. (2003). Adenoviral gene transfer of connective tissue growth factor in the lung induces transient fibrosis. Am J Respir Crit Care Med 168: 770–778.
10.1164/rccm.200210-1254OC
PubMedWeb of Science®Google Scholar
Bonniaud P, Martin G, Margetts PJ, Ask K, Robertson J, Gauldie J et al. (2004). Connective tissue growth factor is crucial to inducing a profibrotic environment in ‘fibrosis-resistant’ BALB/c mouse lungs. Am J Respir Cell Mol Biol 31: 510–516.
10.1165/rcmb.2004-0158OC
CASPubMedWeb of Science®Google Scholar
Bunnett NW (2006). Protease-activated receptors: how proteases signal to cells to cause inflammation and pain. Semin Thromb Hemost 32 (Suppl 1): 39–48.
10.1055/s-2006-939553
CASPubMedWeb of Science®Google Scholar
Camerer E, Cornelissen I, Kataoka H, Duong DN, Zheng YW, Coughlin SR. (2006). Roles of protease-activated receptors in a mouse model of endotoxemia. Blood 107: 3912–3921.
10.1182/blood-2005-08-3130
CASPubMedWeb of Science®Google Scholar
Chackalamannil S (2006). Thrombin receptor (protease activated receptor-1) antagonists as potent antithrombotic agents with strong antiplatelet effects. J Med Chem 49: 5390–5403.
10.1021/jm0603670
CASWeb of Science®Google Scholar
Chambers RC (2003). Proteinase-activated receptors and the pathophysiology of pulmonary fibrosis. Drug Dev Res 60: 29–35.
10.1002/ddr.10317
CASWeb of Science®Google Scholar
Chambers RC, Dabbagh K, McAnulty RJ, Gray AJ, Blanc-Brude OP, Laurent GJ (1998). Thrombin stimulates fibroblast procollagen production via proteolytic activation of protease-activated receptor 1. Biochem J 333: 121–127.
10.1042/bj3330121
CASPubMedWeb of Science®Google Scholar
Chambers RC, Leoni P, Blanc-Brude OP, Wembridge DE, Laurent GJ (2000). Thrombin is a potent inducer of connective tissue growth factor production via proteolytic activation of protease-activated receptor-1. J Biol Chem 275: 35584–35591.
10.1074/jbc.M003188200
CASPubMedWeb of Science®Google Scholar
Chelliah MV, Chackalamannil S, Xia Y, Eagen K, Clasby MC, Gao X et al. (2007). Heterotricyclic himbacine analogs as potent, orally active thrombin receptor (protease activated receptor-1) antagonists. J Med Chem 50: 5147–5160.
10.1021/jm070704k
CASPubMedWeb of Science®Google Scholar
Cirino G, Cicala C, Bucci MR, Sorrentino L, Maraganore JM, Stone SR. (1996). Thrombin functions as an inflammatory mediator through activation of its receptor. J Exp Med 183: 821–827.
10.1084/jem.183.3.821
CASPubMedWeb of Science®Google Scholar
Colognato R, Slupsky JR, Jendrach M, Burysek L, Syrovets T, Simmet T (2003). Differential expression and regulation of protease-activated receptors in human peripheral monocytes and monocyte-derived antigen-presenting cells. Blood 102: 2645–2652.
10.1182/blood-2002-08-2497
CASPubMedWeb of Science®Google Scholar
Coughlin SR (2005). Protease-activated receptors in hemostasis, thrombosis and vascular biology. J Thromb Haemost 3: 1800–1814.
10.1111/j.1538-7836.2005.01377.x
CASPubMedWeb of Science®Google Scholar
Damiano BP, Derian CK, Maryanoff BE, Zhang HC, Gordon PA. (2003). RWJ-58259: a selective antagonist of protease activated receptor-1. Cardiovasc Drug Rev 21: 313–326.
10.1111/j.1527-3466.2003.tb00124.x
CASPubMedWeb of Science®Google Scholar
Demeo DL, Mariani TJ, Lange C, Srisuma S, Litonjua AA, Celedon JC et al. (2006). The SERPINE2 gene is associated with chronic obstructive pulmonary disease. Am J Hum Genet 78: 253–264.
10.1086/499828
CASPubMedWeb of Science®Google Scholar
Di Serio C, Pellerito S, Duarte M, Massi D, Naldini A, Cirino G et al. (2007). Protease-activated receptor 1-selective antagonist SCH79797 inhibits cell proliferation and induces apoptosis by a protease-activated receptor 1-independent mechanism. Basic Clin Pharmacol Toxicol 101: 63–69.
10.1111/j.1742-7843.2007.00078.x
CASPubMedWeb of Science®Google Scholar
Dik WA, Zimmermann LJ, Naber BA, Janssen DJ, Van Kaam AH, Versnel MA (2003). Thrombin contributes to bronchoalveolar lavage fluid mitogenicity in lung disease of the premature infant. Pediatr Pulmonol 35: 34–41.
10.1002/ppul.10219
PubMedWeb of Science®Google Scholar
Eitzman DT, McCoy RD, Zheng X, Fay WP, Shen T, Ginsburg D et al. (1996). Bleomycin-induced pulmonary fibrosis in transgenic mice that either lack or overexpress the murine plasminogen activator inhibitor-1 gene. J CIin Invest 97: 232–237.
10.1172/JCI118396
CASPubMedWeb of Science®Google Scholar
Feistritzer C, Schuepbach RA, Mosnier LO, Bush LA, Di Cera E, Griffin JH et al. (2006). Protective signaling by activated protein C is mechanistically linked to protein C activation on endothelial cells. J Biol Chem 281: 20077–20084.
10.1074/jbc.M600506200
CASPubMedWeb of Science®Google Scholar
Fiorucci S, Antonelli E, Distrutti E, Severino B, Fiorentina R, Baldoni M et al. (2004). PAR1 antagonism protects against experimental liver fibrosis. Role of proteinase receptors in stellate cell activation. Hepatology 39: 365–375.
10.1002/hep.20054
CASPubMedWeb of Science®Google Scholar
Fujimoto H, Gabazza EC, Hataji O, Yuda H, D'Alessandro-Gabazza CN, Nakano M et al. (2003). Thrombin-activatable fibrinolysis inhibitor and protein C inhibitor in interstitial lung disease. Am J Respir Crit Care Med 167: 1687–1694.
10.1164/rccm.200208-905OC
PubMedWeb of Science®Google Scholar
Fujimoto H, Gabazza EC, Taguchi O, Nishii Y, Nakahara H, Bruno NE et al. (2006). Thrombin-activatable fibrinolysis inhibitor deficiency attenuates bleomycin-induced lung fibrosis. Am J Pathol 168: 1086–1096.
10.2353/ajpath.2006.050610
CASPubMedWeb of Science®Google Scholar
Gando S, Kameue T, Matsuda N, Hayakawa M, Morimoto Y, Ishitani T et al. (2003). Imbalances between the levels of tissue factor and tissue factor pathway inhibitor in ARDS patients. Thromb Res 109 (2–3): 119–124.
Google Scholar
Grotendorst GR, Duncan MR (2005). Individual domains of connective tissue growth factor regulate fibroblast proliferation and myofibroblast differentiation. FASEB J 19: 729–738.
10.1096/fj.04-3217com
CASPubMedWeb of Science®Google Scholar
Günther A, Lubke N, Ermert M, Schermuly RT, Weissmann N, Breithecker A et al. (2003). Prevention of bleomycin-induced lung fibrosis by aerosolization of heparin or urokinase in rabbits. Am J Respir Crit Care Med 168: 1358–1365.
10.1164/rccm.2201082
PubMedWeb of Science®Google Scholar
Günther A, Mosavi P, Ruppert C, Heinemann S, Temmesfeld B, Velcovsky HG et al. (2000). Enhanced tissue factor pathway activity and fibrin turnover in the alveolar compartment of patients with interstitial lung disease. Thromb Haemost 83: 853–860.
10.1055/s-0037-1613933
CASPubMedWeb of Science®Google Scholar
Hattori N, Degen JL, Sisson TH, Liu H, Moore BB, Pandrangi RG et al. (2000). Bleomycin-induced pulmonary fibrosis in fibrinogen-null mice. J Clin Invest 106: 1341–1350.
10.1172/JCI10531
CASPubMedWeb of Science®Google Scholar
Hernandez Rodriguez NA, Cambrey AD, Harrison NK, Chambers RC, Gray AJ, Southcott AM et al. (1995). Role of thrombin in pulmonary fibrosis. Lancet 346: 1071–1073.
10.1016/S0140-6736(95)91744-6
CASPubMedWeb of Science®Google Scholar
Howell DC, Falzon M, Bilbe N, Bottoms SE, Laurent GJ, Bellingan GJ et al. (2007). Pulmonary fibrosis occurs early in acute lung injury/acute respiratory distress syndrome (ALI/ARDS) and is associated with expression of protease activated receptor-1 (PAR1). Am J Respir Crit Care Med 175: A950.
Google Scholar
Howell DC, Goldsack NR, Marshall RP, McAnulty RJ, Starke R, Purdy G et al. (2001). Direct thrombin inhibition reduces lung collagen, accumulation, and connective tissue growth factor mRNA levels in bleomycin-induced pulmonary fibrosis. Am J Pathol 159: 1383–1395.
10.1016/S0002-9440(10)62525-4
CASPubMedWeb of Science®Google Scholar
Howell DC, Johns RH, Lasky JA, Shan B, Scotton CJ, Laurent GJ et al. (2005). Absence of proteinase-activated receptor-1 signaling affords protection from bleomycin-induced lung inflammation and fibrosis. Am J Pathol 166: 1353–1365.
10.1016/S0002-9440(10)62354-1
CASPubMedWeb of Science®Google Scholar
Idell S (2003). Coagulation, fibrinolysis, and fibrin deposition in acute lung injury. Crit Care Med 31: S213–S220.
10.1097/01.CCM.0000057846.21303.AB
CASPubMedWeb of Science®Google Scholar
Idell S, James KK, Levin EG, Schwartz BS, Manchanda N, Maunder RJ et al. (1989). Local abnormalities in coagulation and fibrinolytic pathways predispose to alveolar fibrin deposition in the adult respiratory distress syndrome. J Clin Invest 84AB: 695–705.
10.1172/JCI114217
Web of Science®Google Scholar
Imokawa S, Sato A, Hayakawa H, Kotani M, Urano T, Takada A (1997). Tissue factor expression and fibrin deposition in the lungs of patients with idiopathic pulmonary fibrosis and systemic sclerosis. Am J Respir Crit Care Med 156: 631–636.
10.1164/ajrccm.156.2.9608094
CASPubMedWeb of Science®Google Scholar
Jenkins RG, Su X, Su G, Scotton CJ, Camerer E, Laurent GJ et al. (2006). Ligation of protease-activated receptor 1 enhances alpha(v)beta6 integrin-dependent TGF-beta activation and promotes acute lung injury. J Clin Invest 116: 1606–1614.
10.1172/JCI27183
CASPubMedWeb of Science®Google Scholar
Keerthisingam CB, Jenkins RG, Harrison NK, Hernandez-Rodriguez NA, Booth H, Laurent GJ et al. (2001). Cyclooxygenase-2 deficiency results in a loss of the anti-proliferative response to transforming growth factor-beta in human fibrotic lung fibroblasts and promotes bleomycin-induced pulmonary fibrosis in mice. Am J Pathol 158: 1411–1422.
10.1016/S0002-9440(10)64092-8
CASPubMedWeb of Science®Google Scholar
Kijiyama N, Ueno H, Sugimoto I, Sasaguri Y, Yatera K, Kido M et al. (2006). Intratracheal gene transfer of tissue factor pathway inhibitor attenuates pulmonary fibrosis. Biochem Biophys Res Commun 339: 1113–1119.
10.1016/j.bbrc.2005.11.127
CASPubMedWeb of Science®Google Scholar
Kobayashi H, Gabazza EC, Taguchi O, Wada H, Takeya H, Nishioka J et al. (1998). Protein C anticoagulant system in patients with interstitial lung disease. Am J Respir Crit Care Med 157: 1850–1854.
10.1164/ajrccm.157.6.9709078
CASPubMedWeb of Science®Google Scholar
Kranzhofer R, Clinton SK, Ishii K, Coughlin SR, Fenton II JW, Libby P (1996). Thrombin potently stimulates cytokine production in human vascular smooth muscle cells but not in mononuclear phagocytes. Circ Res 79: 286–294.
10.1161/01.RES.79.2.286
CASPubMedWeb of Science®Google Scholar
Kubo H, Nakayama K, Yanai M, Suzuki T, Yamaya M, Watanabe M et al. (2005). Anticoagulant therapy for idiopathic pulmonary fibrosis. Chest 128: 1475–1482.
10.1378/chest.128.3.1475
CASPubMedWeb of Science®Google Scholar
Ludwicka-Bradley A, Tourkina E, Suzuki S, Tyson E, Bonner M, Fenton JW et al. (2000). Thrombin upregulates interleukin-8 in lung fibroblasts via cleavage of proteolytically activated receptor-I and protein kinase C-gamma activation. Am J Respir Cell Mol Biol 22: 235–243.
10.1165/ajrcmb.22.2.3642
CASPubMedWeb of Science®Google Scholar
Mann KG, Brummel K, Butenas S (2003). What is all that thrombin for J Thromb Haemost 1: 1504–1514.
10.1046/j.1538-7836.2003.00298.x
CASPubMedWeb of Science®Google Scholar
Mari B, Imbert V, Belhacene N, Far DF, Peyron JF, Pouyssegur J et al. (1994). Thrombin and thrombin receptor agonist peptide induce early events of T cell activation and synergize with TCR cross-linking for CD69 expression and interleukin 2 production. J Biol Chem 18: 8517–8523.
Google Scholar
Marshall R, Bellingan G, Laurent G (1998). The acute respiratory distress syndrome: fibrosis in the fast lane. Thorax 53: 815–817.
10.1136/thx.53.10.815
CASPubMedWeb of Science®Google Scholar
Moffatt JD, Page CP, Laurent GJ (2004). Shooting for PARs in lung diseases. Curr Opin Pharmacol 4: 221–229.
10.1016/j.coph.2004.01.012
CASPubMedWeb of Science®Google Scholar
Moore BB, Kolodsick JE, Thannickal VJ, Cooke K, Moore TA, Hogaboam C et al. (2005). CCR2-mediated recruitment of fibrocytes to the alveolar space after fibrotic injury. Am J Pathol 166: 675–684.
10.1016/S0002-9440(10)62289-4
CASPubMedWeb of Science®Google Scholar
Naldini A, Pucci A, Carney DH, Fanetti G, Carraro F (2002). Thrombin enhancement of interleukin-1 expression in mononuclear cells: involvement of proteinase-activated receptor-1. Cytokine 20: 191–199.
10.1006/cyto.2002.2001
CASPubMedWeb of Science®Google Scholar
Naldini A, Sower L, Bocci V, Meyers B, Carney DH (1998). Thrombin receptor expression and responsiveness of human monocytic cells to thrombin is linked to interferon-induced cellular differentiation. J Cell Physiol 177: 76–84.
10.1002/(SICI)1097-4652(199810)177:1<76::AID-JCP8>3.0.CO;2-B
CASPubMedWeb of Science®Google Scholar
Ohba T, McDonald JK, Silver RM, Strange C, LeRoy EC, Ludwicka A (1994). Scleroderma bronchoalveolar lavage fluid contains thrombin, a mediator of human lung fibroblast proliferation via induction of platelet-derived growth factor alpha-receptor. Am J Respir Cell Mol Biol 10: 405–412.
10.1165/ajrcmb.10.4.7510986
CASPubMedWeb of Science®Google Scholar
Olman MA, Mackman N, Gladson CL, Moser KM, Loskutoff DJ (1995). Changes in procoagulant and fibrinolytic gene expression during bleomycin-induced lung injury in the mouse. J Clin Invest 96: 1621–1630.
10.1172/JCI118201
CASPubMedWeb of Science®Google Scholar
Pawlinski R, Pedersen B, Schabbauer G, Tencati M, Holscher T, Boisvert W et al. (2004). Role of tissue factor and protease-activated receptors in a mouse model of endotoxemia. Blood 103: 1342–1347.
10.1182/blood-2003-09-3051
CASPubMedWeb of Science®Google Scholar
Peyrol S, Cordier JF, Grimaud JA (1990). Intra-alveolar fibrosis of idiopathic bronchiolitis obliterans-organizing pneumonia. Cell-matrix patterns. Am J Pathol 137: 155–170.
CASPubMedWeb of Science®Google Scholar
Rice AB, Moomaw CR, Morgan DL, Bonner JC (1999). Specific inhibitors of platelet-derived growth factor or epidermal growth factor receptor tyrosine kinase reduce pulmonary fibrosis in rats. Am J Pathol 55: 213–221.
10.1016/S0002-9440(10)65115-2
Web of Science®Google Scholar
Riewald M, Kravchenko VV, Petrovan RJ, O'Brien PJ, Brass LF, Ulevitch RJ et al. (2001). Gene induction by coagulation factor Xa is mediated by activation of protease-activated receptor 1. Blood 97: 3109–3116.
10.1182/blood.V97.10.3109
CASPubMedWeb of Science®Google Scholar
Riewald M, Petrovan RJ, Donner A, Mueller BM, Ruf W (2002). Activation of endothelial cell protease activated receptor 1 by the protein C pathway. Science 296: 1880–1882.
10.1126/science.1071699
CASPubMedWeb of Science®Google Scholar
Scotton CJ, Chambers RC (2007). Molecular targets in pulmonary fibrosis: the myofibroblast in focus. Chest 132: 1311–1321.
10.1378/chest.06-2568
PubMedWeb of Science®Google Scholar
Siflinger-Birnboim A, Johnson A (2003). Protein kinase C modulates pulmonary endothelial permeability: a paradigm for acute lung injury. Am J Physiol Lung Cell Mol Physiol 284: L435–L451.
10.1152/ajplung.00106.2002
CASPubMedWeb of Science®Google Scholar
Sime PJ, Xing Z, Graham FL, Csaky KG, Gauldie J (1997). Adenovector-mediated gene transfer of active transforming growth factor-beta1 induces prolonged severe fibrosis in rat lung. J Clin Invest 100: 768–776.
10.1172/JCI119590
CASPubMedWeb of Science®Google Scholar
Sokolova E, Reiser G (2007). A novel therapeutic target in various lung diseases: airway proteases and protease-activated receptors. Pharmacol Ther 115: 70–83.
10.1016/j.pharmthera.2007.04.002
CASPubMedWeb of Science®Google Scholar
Sower LE, Froelich CJ, Carney DH, Fenton JW, Klimpel GR (1995). Thrombin induces IL-6 production in fibroblasts and epithelial cells. Evidence for the involvement of the seven-transmembrane domain (STD) receptor for alpha-thrombin. J Immunol 155: 895–901.
10.4049/jimmunol.155.2.895
CASPubMedWeb of Science®Google Scholar
Strande JL, Hsu A, Su J, Fu X, Gross GJ, Baker JE (2007). SCH 79797, a selective PAR1 antagonist, limits myocardial ischemia/reperfusion injury in rat hearts. Basic Res Cardiol 102: 350–358.
10.1007/s00395-007-0653-4
CASPubMedWeb of Science®Google Scholar
Su X, Camerer E, Hamilton JR, Coughlin SR, Matthay MA (2005). Protease-activated receptor-2 activation induces acute lung inflammation by neuropeptide-dependent mechanisms. J Immunol 175: 2598–2605.
10.4049/jimmunol.175.4.2598
CASPubMedWeb of Science®Google Scholar
Suk K, Cha S (1999). Thrombin-induced interleukin-8 production and its regulation by interferon-gamma and prostaglandin E2 in human monocytic U937 cells. Immunol Lett 67: 223–227.
10.1016/S0165-2478(99)00015-2
CASPubMedWeb of Science®Google Scholar
Suzuki T, Moraes TJ, Vachon E, Ginzberg HH, Huang TT, Matthay MA et al. (2005). Proteinase-activated receptor-1 mediates elastase-induced apoptosis of human lung epithelial cells. Am J Respir Cell Mol Biol 33: 231–247.
10.1165/rcmb.2005-0109OC
CASPubMedWeb of Science®Google Scholar
Tani K, Ogushi F, Takahashi H, Kawano T, Endo T, Sone S (1997). Thrombin stimulates platelet-derived growth factor release by alveolar macrophages in rats––significance in bleomycin-induced pulmonary fibrosis. J Med Invest 44: 59–65.
CASPubMedGoogle Scholar
Terada M, Kelly EA, Jarjour NN (2004). Increased thrombin activity after allergen challenge: a potential link to airway remodeling Am J Respir Crit Care Med 169: 373–377.
10.1164/rccm.200308-1156OC
PubMedWeb of Science®Google Scholar
Trejo J, Connolly AJ, Coughlin SR (1996). The cloned thrombin receptor is necessary and sufficient for activation of mitogen-activated protein kinase and mitogenesis in mouse lung fibroblasts. Loss of responses in fibroblasts from receptor knockout mice. J Biol Chem 271: 21536–21541.
10.1074/jbc.271.35.21536
CASPubMedWeb of Science®Google Scholar
Ueno A, Murakami K, Yamanouchi K, Watanabe M, Kondo T (1996). Thrombin stimulates production of interleukin-8 in human umbilical vein endothelial cells. Immunology 88: 76–81.
10.1046/j.1365-2567.1996.d01-635.x
CASPubMedWeb of Science®Google Scholar
Vogel SM, Gao X, Mehta D, Ye RD, John TA, Andrade-Gordon P et al. (2000). Abrogation of thrombin-induced increase in pulmonary microvascular permeability in PAR-1 knockout mice. Physiol Genomics 4: 137–145.
10.1152/physiolgenomics.2000.4.2.137
CASPubMedWeb of Science®Google Scholar
Vu TK, Hung DT, Wheaton VI, Coughlin SR (1991). Molecular cloning of a functional thrombin receptor reveals a novel proteolytic mechanism of receptor activation. Cell 64: 1057–1068.
10.1016/0092-8674(91)90261-V
CASPubMedWeb of Science®Google Scholar
Wang L, Bastarache JA, Wickersham N, Fang X, Matthay MA, Ware LB (2007). Novel role of the human alveolar epithelium in regulating intra-alveolar coagulation. Am J Respir Cell Mol Biol 36: 497–503.
10.1165/rcmb.2005-0425OC
CASPubMedWeb of Science®Google Scholar
Ware LB, Camerer E, Welty-Wolf K, Schultz MJ, Matthay MA (2006). Bench to bedside: targeting coagulation and fibrinolysis in acute lung injury. Am J Physiol Lung Cell Mol Physiol 291: L307–L311.
10.1152/ajplung.00157.2006
CASPubMedWeb of Science®Google Scholar
Ware LB, Matthay MA, Parsons PE, Thompson BT, Januzzi JL, Eisner MD (2007). The National Heart, Lung, and Blood Institute Acute Respiratory Distress Syndrome Clinical Trials Network. Pathogenetic and prognostic significance of altered coagulation and fibrinolysis in acute lung injury/acute respiratory distress syndrome. Crit Care Med 35: 1821–1828.
10.1097/01.CCM.0000221922.08878.49
PubMedGoogle Scholar
Yasui H, Gabazza EC, Taguchi O, Risteli J, Risteli L, Wada H et al. (2000). Decreased protein C activation is associated with abnormal collagen turnover in the intraalveolar space of patients with interstitial lung disease. Clin Appl Thromb Hemost 6: 202–205.
10.1177/107602960000600404
CASPubMedWeb of Science®Google Scholar
Yasui H, Gabazza EC, Tamaki S, Kobayashi T, Hataji O, Yuda H et al. (2001). Intratracheal administration of activated protein C inhibits bleomycin-induced lung fibrosis in the mouse. Am J Respir Crit Care Med 163: 1660–1668.
10.1164/ajrccm.163.7.9911068
CASPubMedWeb of Science®Google Scholar
Yoshida M, Sakuma-Mochizuki J, Abe K, Arai T, Mori M, Goya S et al. (1999). In vivo gene transfer of an extracellular domain of platelet-derived growth factor beta receptor by the HVJ-liposome method ameliorates bleomycin-induced pulmonary fibrosis. Biochem Biophys Res Commun 265: 503–508.
10.1006/bbrc.1999.1647
CASPubMedWeb of Science®Google Scholar
Zhang HC, Derian CK, Andrade-Gordon P, Hoekstra WJ, McComsey DF, White KB et al. (2001). Discovery and optimization of a novel series of thrombin receptor (par-1) antagonists: potent, selective peptide mimetics based on indole and indazole templates. J Med Chem 44: 1021–1024.
10.1021/jm000506s
CASPubMedWeb of Science®Google Scholar

Citing Literature

Volume153, IssueS1

Special Issue: Life Sciences

March 2008

Pages S367-S378

Procoagulant signalling mechanisms in lung inflammation and fibrosis: novel opportunities for pharmacological intervention?

Abstract

Abbreviations:

Overview: coagulation and fibrinolysis

Activation of the coagulation cascade in acute lung injury

Anticoagulant drug intervention in patients with ALI/ARDS

Activation of the coagulation cascade in pulmonary fibrosis

Anticoagulants are effective in blocking experimentally induced lung fibrosis

Anticoagulant therapy for patients with pulmonary fibrosis?

Fibrinolysis following lung injury

Proteinase-activated receptors: signalling receptors for coagulation proteinases

PAR activation and pro-inflammatory signalling

PAR₁ is a major pro-fibrotic signalling receptor

Lessons from PAR KO mouse, PAR agonist and antagonist studies in experimental models of lung injury

Clinical implications for patients with pulmonary fibrosis: PAR₁ antagonists as novel agents for therapeutic intervention?

Progress in the development of PAR₁ antagonists

Conclusions

Acknowledgments

Conflict of interest

References

Citing Literature

Figures

References

Information

About Wiley Online Library

Help & Support

Opportunities

Connect with Wiley

Procoagulant signalling mechanisms in lung inflammation and fibrosis: novel opportunities for pharmacological intervention?

Abstract

Abbreviations:

Overview: coagulation and fibrinolysis

Activation of the coagulation cascade in acute lung injury

Anticoagulant drug intervention in patients with ALI/ARDS

Activation of the coagulation cascade in pulmonary fibrosis

Anticoagulants are effective in blocking experimentally induced lung fibrosis

Anticoagulant therapy for patients with pulmonary fibrosis?

Fibrinolysis following lung injury

Proteinase-activated receptors: signalling receptors for coagulation proteinases

PAR activation and pro-inflammatory signalling

PAR1 is a major pro-fibrotic signalling receptor

Lessons from PAR KO mouse, PAR agonist and antagonist studies in experimental models of lung injury

Clinical implications for patients with pulmonary fibrosis: PAR1 antagonists as novel agents for therapeutic intervention?

Progress in the development of PAR1 antagonists

Conclusions

Acknowledgments

Conflict of interest

References

Citing Literature

Figures

References

Related

Information

PAR₁ is a major pro-fibrotic signalling receptor

Clinical implications for patients with pulmonary fibrosis: PAR₁ antagonists as novel agents for therapeutic intervention?

Progress in the development of PAR₁ antagonists