Fibrosis in the Acute Respiratory Distress Syndrome

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D.C.J. Howell, R.C. Chambers, and G.J. Laurent

Introduction

Sepsis often leads to severe pulmonary dysfunction and a large proportion of patients will develop acute lung injury/acute respiratory distress syndrome (ALI/ARDS) [1]. Although sepsis is frequently an initiating factor in the development of ALI/ARDS, the etiology of ALI/ARDS is diverse and the disorders associated with the condition can broadly be divided into those which cause direct or indirect lung injury (Table 1). The current American/European definition of the condition has been designed to reflect the underlying severity of lung injury in ALI/ARDS (Table 2). Although not specifically part of the diagnostic criteria, it is well documented that a proportion of patients with ALI/ARDS develop aggressive pulmonary fibrosis that ultimately leads to their demise.

Table 1. Etiology of ALI/ARDS

Direct Lung Injury Indirect Lung Injury

Bronchopneumonia Sepsis

Gastric aspiration Multiple trauma with shock

Pulmonary contusion Drug overdose

Inhalational injury Acute pancreatitis

Near-drowning Transfusion-associated acute

Reperfusion injury lung injury (TRALI)

Fat emboli Cardiopulmonary bypass

Pathogenesis of ALI/ARDS

ALI/ARDS is classically thought to exhibit three phases: i) exudative/inﬂammatory;

ii) proliferative; and iii) fibrotic (reviewed in [2]). Briefly, the exudative phase is characterized histologically by diffuse alveolar damage as the microvascular endothelial and alveolar epithelium, which form the alveolar-capillary barrier, are disrupted. Intense neutrophil infiltration is also a major feature of this phase of

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Table 2. Diagnostic criteria in ALI/ARDS

Acute lung injury Acute respiratory distress syndrome

Chest Xray Bilateral inﬁltrates Bilateral inﬁltrates

Clinical scenario Acute onset Acute onset

Pulmonary artery wedge pressure

< 18 mmHg < 18 mmHg

Oxygenation PaO₂/FiO₂ratio

< 300 mmHg

PaO₂/FiO₂ratio

< 200 mmHg

ALI/ARDS. Once injured, the endothelial barrier becomes increasingly permeable resulting in highly proteinaceous, hemorrhagic pulmonary edema fluid flooding into alveoli, with resultant formation of fibrinous hyaline membranes. Epithelial integrity is also breached, as a result of damage to type I and II pneumocytes, which leads to exacerbation of alveolar edema as permeability of the epithelium increases and its resorptive function ceases. In addition, as type II cells are also injured, surfactant production is reduced. Lack of efficient endothelial and epithelial repair is thought to be critical in the progression of ALI/ARDS as the endothelium plays a vital role in remodeling of the alveolar capillary barrier [3], and an intact epithelial layer plays an important role in suppressing fibroblast proliferation and matrix production.

During the proliferative phase of ALI/ARDS, damage to the delicate capillary network of the lung is a major feature with intimal proliferation in small blood vessels. Following necrosis of type I pneumocytes, the epithelial basement mem- brane is exposed and type II cells proliferate in an attempt to repair the damaged epithelium. Fibroblasts/myofibroblasts emerge in the interstitial space and alveolar lumen. As fibrinous exudates become organized, they are replaced by collagen fibrils. The fibrotic phase is characterized by extensive alveolar septal and intra- alveolar fibrosis, as well as myointimal thickening and mural fibrosis of vessels, which contribute to the degree of pulmonary hypertension observed in this condition. There is a progressive increase in lung collagen with the duration of the condition, the severity of which correlates with increase in mortality.

A concept that has been challenged over recent years concerns the sequential relationship of the three phases of ALI/ARDS. Whereas it was once thought that these were distinct and develop as the condition progresses, there is now increasing evidence that there is much overlap between the three phases. In particular, a number of studies have shown that the fibrotic/fibroproliferative response occurs much earlier than previously thought. For example, N-terminal procollagen peptide III (N-PCP-III), which is a marker of collagen turnover, is elevated in bronchoalveolar lavage (BAL) fluid and tracheal aspirates from patients with ALI/ARDS within 24 hours of diagnosis [4–6]. In addition, fibroproliferation has been shown to occur early in ALI/ARDS and also predicts a poor outcome [7]. Another more recent study has further shown that extensive thin-section computed tomography (CT)

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changes, indicative of ﬁbroproliferation, are independently predictive of a poor prognosis in patients with clinical early-stage ALI/ARDS [8].

What Drives the Fibrotic Response in ALI/ARDS?

A number of factors, including genetic influences, oxidant stress, anti-apoptotic agents, and excessive mechanical ventilation, leading to shear-stress of alveoli, are likely to play critical roles in orchestrating the fibrotic response to lung injury in ALI/ARDS. In addition, pro-inflammatory and pro-fibrotic cytokines, chemokines, and growth factors are released from resident and recruited inflammatory cells that influence the progression of this condition. Although many potential fibrotic mediators have been proposed to play a role in chronic forms of pulmonary fibrosis, such as usual interstitial pneumonia [9, 10], less is currently known about specific factors that directly affect fibroproliferation and the resultant fibrotic response in ALI/ARDS. However, a number of candidates have been identified from human and animal studies. For example, levels of the potent pro-fibrotic mediators, transforming growth factor-

α

^(TGF-

α

) and platelet derived growth factor (PDGF), are increased in BAL ﬂuid obtained from patients with ALI/ARDS [11, 12]. Further- more, expression of a tumor necrosis factor-

α

^(TNF-

α

) transgene in murine lung leads to an alveolitis that steadily progresses to ﬁbrosis, suggesting the possible importance of this cytokine in ALI/ARDS [13]. In addition, we have recently obtained evidence that angiotensin II, possibly generated locally within the lung, may play an important role in the ﬁbrotic response to experimentally-induced lung injury, at least in part via the action of TGF-

β

[14]. Th-2 cytokines, including IL-4 and IL-13, have also been implicated in the pathogenesis of ﬁbroproliferative lung disorders [15]. More recently, BAL ﬂuid from patients with ALI/ARDS was shown to contain active TGF-

β

1 which was capable of inducing procollagen I pro- moter activity in human lung ﬁbroblasts in vitro [16]. Finally, there is increasing evidence that a prevailing procoagulant microenvironment with generation of coagulation proteinases such as thrombin and factor Xa, may also play a crucial role in regulating the ﬁbrotic response in this condition.

Evidence for the Role of the Coagulation Cascade in ALI/ARDS

Consistent with the concept that the coagulation cascade is activated in ALI/ARDS, extravascular and intra-alveolar accumulation of ﬁbrin is a characteristic feature of this condition [17, 18]. The excessive procoagulant activity observed in the lung in ALI/ARDS is thought to arise from an imbalance between pro- and anti-coagulant factors. For example, BAL ﬂuid from patients with ALI/ARDS has been shown to contain tissue factor/factor VII/VIIa complexes [18], which can activate factor X and trigger activation of the extrinsic pathway of coagulation.

The prevailing balance between the pro- and anti-coagulant state in the lung following injury is also affected by regulatory mechanisms, which control the

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clearance of deposited fibrin (fibrinolysis). This process, which occurs at all sites of wound healing, is initiated when plasminogen is converted to plasmin by the proteinases, urokinase-type plasminogen activator (u-PA) or tissue-type plasminogen activator (t-PA). Plasmin subsequently cleaves fibrin into a range of fibrin degradation products (FDPs). Fibrinolytic activity in the vasculature is largely under the control of t-PA; whereas extravascular fibrinolysis in the lung is controlled by u-PA. The conversion of plasminogen to plasmin by t-PA and u-PA is reg- ulated by the endogenous inhibitor, plasminogen activator inhibitor-1 (PAI-1).

PAI-1 activity is increased in ALI/ARDS, particularly in the alveolar compartment, thus favoring fibrin persistence [19]. The fibrinolytic system is also influenced by the plasma glycoprotein thrombin-activatable fibrinolysis inhibitor (TAFI). Dur- ing fibrin degradation, plasmin exposes C-terminal lysine residues on the fibrin molecule to potentiate its clearance. TAFI cleaves these residues, which, therefore, favors fibrin persistence. Although it has not been shown in patients with ALI/ARDS, it is noteworthy that levels of TAFI are increased in BAL fluid from patients with interstitial lung disease [20].

In terms of a deﬁciency of anticoagulant factors, levels of antithrombin are reduced in patients with ALI/ARDS [21]. In addition, it has been shown that levels of protein C in the intra-alveolar compartment from patients with ALI/ARDS are reduced compared with plasma levels and correlate with a poor clinical outcome [22,23]. Levels of the major endogenous inhibitor of the extrinsic coagulation cascade, tissue factor pathway inhibitor (TFPI), are markedly increased following experimental lung injury [24]. However, studies by Gando and colleagues [25]

suggest that systemic activation of the tissue factor-dependent pathway is not adequately balanced by TFPI in patients with ARDS.

A number of studies performed in experimental animal models have examined the effects of modulating the coagulation cascade in ALI/ARDS. For example, exogenous delivery of the highly specific direct thrombin inhibitor, hirudin, or of antithrombin, have been shown to be protective in animal models of ALI/ARDS [26–28]. In addition, administration of heparin, which inhibits coagulation proteinases by potentiating the formation of antithrombin/serine proteinase complexes, but also has anti-inflammatory properties, leads to improved gas exchange in an animal model of ALI/ARDS [29]. Heparin has also been shown to attenuate bleomycin-induced pulmonary fibrosis in mice [30], although in this study, it was uncertain whether heparin was delivered at an anticoagulant dose and whether the protective effects were due to its direct anti-proliferative effects, or due to blocking proteinase activity. The animal model of bleomycin-induced fibrosis, based on intratracheal delivery of this agent, is a well-established model of ALI/ARDS. Characteristic pathogenetic features of ALI/ARDS are observed in the lung following bleomycin instillation, including the rapid influx of inflammatory cells, an increase in microvascular permeability, and aggressive fibroproliferation, culminating in established interstitial fibrosis. Of note, intratracheal administration of activated protein C (APC) and intratracheal gene transfer of TFPI both attenuate bleomycin-induced fibrosis in rodent studies [31, 32]. BAL fluid levels

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of the coagulation proteinase, thrombin, are increased in this animal model of ALI/ARDS [33].

Thrombin and Proteinase Activated Receptors (PARs)

In addition to its critical role in blood coagulation, thrombin exerts potent cellular responses via its ability to activate the family of proteinase activated receptors (PARs). A number of these cellular effects are likely to play important roles in inﬂammatory and tissue repair processes in ALI/ARDS and will, therefore, be discussed in greater detail.

The PARs belong to the family of seven transmembrane G-protein coupled receptors, which exhibit a unique mechanism of activation that involves the un- masking of a tethered ligand by limited proteolysis of specific amino acid sequences from the N-terminus of the receptor [34]. Following proteolytic cleavage, the newly generated tethered ligand binds intramolecularly to the second extracellular loop of the receptor, inducing a conformational shape change that allows it to interact with heterotrimeric G-proteins and initiate downstream signaling responses. To date, four PARs have been characterized, of which three, PAR-1, -3, and -4, are activated by thrombin. Synthetic peptides corresponding to the tethered ligands of PAR-1, -2 and -4 are capable of mimicking a number of cellular responses elicited by their respective endogenous activators. The first PAR to be cloned and characterized was PAR-1 [34], which has subsequently been shown to be the major receptor involved in mediating thrombin’s cellular effects, in particular in terms of fibroblast responses [35–37]. PAR-1 has a wide tissue distribution and is present on a number of cell types including platelets, endothelial cells, epithelial cells, fibroblasts, smooth muscle cells, monocytes, lymphocytes, mast cells, and certain tumor cell lines (reviewed in [38]). PAR-2 and PAR-4 are similarly expressed on numerous cell types in the airways, blood, and cardiovascular system, whereas PAR-3 appears to have a more restricted expression pattern.

PAR-Mediated Cellular Effects of Thrombin Pertinent to Fibrosis in ALI/ARDS

PAR-1 is the major high-affinity thrombin signaling receptor and is abundantly expressed in the injured lung. PAR-1 mediated cellular responses elicited by thrombin that are likely to be important in the pathogenesis of ALI/ARDS include the ability of thrombin to promote platelet aggregation, influence vascular tone and permeability, stimulate angiogenesis and vascular repair, and promote inflammatory cell trafficking. Of particular importance to the fibrotic response in ALI/ARDS, thrombin is a fibroblast mitogen and chemoattractant [39–41]. In addition, thrombin stimulates lung fibroblast differentiation to the myofibroblast phenotype [42, 43]

and mesenchymal cell procollagen production and gene expression [36,44]. These

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effects can be mimicked with PAR-1 agonists; whereas ﬁbroblasts derived from PAR-1 deﬁcient mice are unresponsive to thrombin in terms of MAP kinase signaling, proliferation [35], and procollagen

α

1(I) gene promoter activity [45]. Throm- bin has been shown to be a major fibroblast mitogen in BAL fluid from patients with pulmonary fibrosis associated with systemic sclerosis [46, 47]. To our knowledge, similar studies in patients with ARDS/ALI have not yet been reported.

There is good evidence that most of the cellular effects of thrombin are mediated via the induction and release of secondary mediators [38]. For example, PAR-1 activation by thrombin induces the production and release of PDGF, connective tissue growth factor (CTGF), TGF-

β

and pro-inflammatory mediators, such as IL- 6, IL-8 and monocyte chemotactic protein-1 (MCP-1/CCL2). These mediators are, in turn, responsible for thrombin’s mitogenic, pro-fibrotic, and pro-inflammatory effects via both autocrine and paracrine mechanisms.

We have specifically examined the procoagulant and downstream cellular ef- fects of thrombin and PAR-1 activation in the bleomycin model of ALI in vivo using the direct thrombin inhibitor, UK-156406, in rats [48] and comparing responses in wild type and PAR-1 knockout (PAR-1 -/-) mice [49]. These studies revealed that thrombin and PAR-1 immunoreactivity in the lung were markedly increased following bleomycin instillation and were predominantly associated with fibroblasts and infiltrating macrophages. This is, to our knowledge, the first demonstration that expression of thrombin and PAR-1 is increased in a model of ALI/ARDS.

In animals given bleomycin, lung collagen content characteristically doubled and was preceded by signiﬁcant elevations in

α

1(I) procollagen and CTGF mRNA levels. However, in bleomycin-treated animals receiving an anticoagulant dose of UK-156046, lung collagen accumulation was signiﬁcantly attenuated, a feature that was also preceded by a signiﬁcant reduction in

α

1(I) procollagen and CTGF gene expression.

The protective effect of direct thrombin inhibition in this model may have been due to blocking thrombin’s procoagulant (fibrin generation) or PAR-mediated cellular effects. In order to specifically dissect the potential contribution of PAR-1 activation in this model, we examined the response of PAR-1 -/- mice. Total lung collagen accumulation following bleomycin injury was dramatically reduced in PAR-1 -/- mice compared with that found in correspondingly injured wild type animals, as was BAL fluid inflammatory cell recruitment and microvascular permeability. This protection was associated with attenuation in lung levels of the potent PAR-1 inducible pro-inflammatory and pro-fibrotic growth factors, MCP-1, CTGF, and TGF-

β

[49]. Taken together, these data provide evidence that thrombin and PAR-1 play a critical role in inﬂammation, microvascular leak, and ﬁbrotic responses in this model of ALI and may, therefore, also contribute to the pathogenesis of ALI/ARDS in humans.

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Emerging Concepts Regarding PARs and Fibrosis

A number of important studies have been published that have challenged conven- tional dogma on coagulation cascade proteinases and PAR activation (Fig. 1). It was previously thought that thrombin was the only major activator of PAR-1, -3 and -4 and that trypsin and mast cell tryptase activated PAR-2 [50]. However, it is now known that thrombin is not the only coagulation proteinase that is capable of exerting functional responses via proteolytic cleavage of PAR-1. Limited proteolysis of PAR-1 by factor Xa initiates downstream functional effects, such as ﬁbroblast proliferation and procollagen production [45, 51]. Furthermore, plasmin has been shown to activate PAR-1 to induce the expression of Cyr 61, a member of the CCN family of proteins which includes CTGF [52]. Riewald and Ruf have also shown that nascent factor Xa, in the procoagulant transient tissue factor-factor VIIa-factor Xa ternary complex generated following activation of the extrinsic coagulation cascade, signals via both PAR-1 and PAR-2 in endothelial cells [53]. This study raises the possibility that tissue factor dependent initiation of the coagulation cascade is mechanistically coupled to PAR-dependent cellular signaling.

There is good evidence that PAR-2 can be transactivated by cleaved PAR-1 [54]

and that the tissue factor-factor VIIa complex can also signal via PAR-2 in endothelial cells [55]. Non-coagulation proteinases, such as trypsin, elastase, and the neutrophil proteinase, cathepsin G, have also been shown to cleave PAR-1. This was previously thought to occur at non-activating sites producing no functional effects [38, 56]. However, neutrophil elastase has recently been shown to induce apoptosis in human lung epithelial cells via a PAR-1 dependent mechanism. Since epithelial cell apoptosis is a central process in ALI/ARDS [57, 58], this observation may be particularly relevant in the context of this condition.

Fig. 1. Potential activators of proteinase activated receptors (PARs) in ALI/ARDS

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Of particular pertinence to the pathogenesis of sepsis, the anticoagulant, APC, has recently also been shown to be capable of activating PAR-1 on endothelial cells, via a process that utilizes the endothelial protein C receptor (EPCR) as a co-receptor [59]. These studies suggest that rather than producing deleterious effects, activation of PAR-1 by APC on the endothelium is cytoprotective and anti- inflammatory. A model has been proposed based on the existence of different threshold concentrations of thrombin within the vasculature, exerting opposing effects. This model suggests that at low concentrations of thrombin, below the procoagulant threshold, thrombin binds to thrombomodulin. Formation of the resultant stoichiometric complex inhibits the enzymatic activity of thrombin and blocks direct PAR-1 activation. Thrombin bound to thrombomodulin can then fa- vorably cleave zymogen protein C to its product, APC. Both substrate and product of this reaction bind to the EPCR. Endogenous production of APC by thrombin is dependent on EPCR binding. EPCR-bound APC subsequently cleaves PAR-1 and induces anti-inflammatory events. When thrombin is generated at higher concentrations that exceed the procoagulant threshold, PAR-1 is activated via the transient tissue factor-factor VIIa-factor Xa complex when coagulation is initiated and in the propagation phase of thrombin generation, which is required for the conversion of fibrinogen to fibrin. In terms of the relevance of these events to excessive intravascular coagulation, such as in sepsis, once the procoagulant threshold is exceeded, disease progression is rapid and this may negate the protective effects of EPCR-bound APC activation of PAR-1 [60]. This may be a plausible mechanism by which APC exerts the favorable effects observed in the PROWESS trial in humans [61]. However, this theory is not universally accepted and is currently at the center of a very interesting debate [62, 63]. In contrast to a clear role for PAR-1 in the bleomycin model of ALI, two murine studies have shown that PAR-1 deficiency is not protective in models of endotoxemia [64, 65]. However, the former study showed that a combination of PAR-2 deficiency and thrombin inhibition was associated with a favorable outcome [64], suggesting that blockade of all PAR-mediated cellular effects may be necessary for protection in endotoxemia. The contribution of PAR-1 (and other PARs) may, therefore, be dependent on both the nature and the initiating site of lung injury.

Clinical Implications and Conclusion

Despite intense research efforts, there are still no pharmacological agents which have been shown to improve mortality rates in ALI/ARDS. The recent North Ameri- can Late Steroid Rescue Study (LaSRS), conducted by the ARDSNet group, assessed the role of methylprednisolone based on previous favorable results in a smaller study [5]. Patients receiving steroids had early physiologic and clinical beneﬁt, displaying improved oxygenation and lung compliance, and earlier withdrawal of mechanical ventilation. However, there was no difference in mortality at 60 and 180 days compared with the control group. Subgroup analysis showed that if BAL ﬂuid procollagen III peptide levels were high at enrolment into the study, there was

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a survival benefit with steroid therapy, suggesting that identification of patients with an early fibrotic phenotype may be vital to aid the development of successful pharmacological strategies in the future (presented at the American Thoracic Society Conference, San Diego, 2005).

A number of ﬁbrotic mediators have been identiﬁed in ALI/ARDS, including TGF-

α

^{, TGF-}

β

^{and TNF-}

α

, which, if successfully targeted, may lead to a therapeutic breakthrough for the treatment of this condition. We further propose that modu- lation of the coagulation cascade, and more speciﬁcally, PAR-1 mediated cellular effects of coagulation proteinases, may also warrant further evaluation as potential therapeutic targets in this condition (Fig. 2). As described above, a number of anticoagulant agents, such as TFPI, site inactivated factor VIIa, heparin, and APC, have shown promise in animal models of ALI/ARDS, but successful clinical trials using these agents have yet to be described. Furthermore, the potential risk of bleeding complications observed in the recent PROWESS trial of APC in sepsis [61] suggests that the use of direct thrombin inhibitors or other antico- agulants in ALI/ARDS may prove problematic. PAR-1 antagonists and blocking antibodies have been developed as potential anti-thrombotic agents [66, 67] and PAR-1 antagonist peptides have been shown to be anti-thrombotic and successful in preventing restenosis in an animal model of vascular thrombosis in non-human primates [68, 69], suggesting that a suitable agent for use in humans is a realistic

Fig. 2. Potential mechanism for the interaction of the coagulation cascade in the ﬁbrotic pathway in ALI/ARDS. TGF: transforming growth factor

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possibility. Strategies aimed at blocking PAR-1 may provide a unique opportu- nity for the treatment of ALI/ARDS by selectively interfering with the pro-ﬁbrotic and pro-inﬂammatory effects of excessive proteinase signaling, whilst avoiding potential hemostatic complications associated with direct proteolytic inhibitors.

References

1. Ware LB, Matthay MA (2000) The acute respiratory distress syndrome. N Engl J Med 342:1334–1349

2. Bellingan GJ (2002) The pulmonary physician in critical care * 6: The pathogenesis of ALI/ARDS. Thorax 57:540–546

3. Orfanos SE, Mavrommati I, Korovesi I, et al (2004) Pulmonary endothelium in acute lung injury: from basic science to the critically ill. Intensive Care Med 30:1702–1714

4. Clark JG, Milberg JA, Steinberg KP, et al (1995) Type III procollagen peptide in the adult respiratory distress syndrome. Association of increased peptide levels in bronchoalveolar lavage ﬂuid with increased risk for death. Ann Intern Med 122:17–23

5. Meduri GU, Headley AS, Golden E, et al (1998) Effect of prolonged methylprednisolone therapy in unresolving acute respiratory distress syndrome: a randomized controlled trial.

JAMA 280:159–165

6. Chesnutt AN, Matthay MA, Tibayan FA, et al (1997) Early detection of type III procollagen peptide in acute lung injury. Pathogenetic and prognostic signiﬁcance.

Am J Respir Crit Care Med 156:840–845

7. Marshall RP, Bellingan G, Webb S, et al (2000) Fibroproliferation occurs early in the acute respiratory distress syndrome and impacts on outcome. Am J Respir Crit Care Med 162:1783–

1788

8. Ichikado K, Suga M, Muranaka H, et al (2006) Prediction of prognosis for acute respiratory distress syndrome with thin-section CT: validation in 44 cases. Radiology 238:321–329 9. McAnulty RJ, Laurent GJ (1995) Pathogenesis of lung ﬁbrosis and potential new therapeutic

strategies. Exp Nephrol 3:96–107

10. Chapman HA (2004) Disorders of lung matrix remodeling. J Clin Invest 113:148–157 11. Madtes DK, Rubenfeld G, Klima LD, et al (1998) Elevated transforming growth factor-alpha

levels in bronchoalveolar lavage ﬂuid of patients with acute respiratory distress syndrome.

Am J Respir Crit Care Med 158:424–430

12. Snyder LS, Hertz MI, Peterson MS, et al (1991) Acute lung injury. Pathogenesis of intraalveolar ﬁbrosis. J Clin Invest 88:663–673

13. Miyazaki Y, Araki K, Vesin C, et al (1995) Expression of a tumor necrosis factor-alpha transgene in murine lung causes lymphocytic and ﬁbrosing alveolitis. A mouse model of progressive pulmonary ﬁbrosis. J Clin Invest 96:250–259

14. Marshall RP, Gohlke P, Chambers RC, et al (2004) Angiotensin II and the ﬁbroproliferative response to acute lung injury. Am J Physiol Lung Cell Mol Physiol 286:L156–L164

15. Jakubzick C, Choi ES, Joshi BH, et al (2003) Therapeutic attenuation of pulmonary ﬁbrosis via targeting of IL-4- and IL-13-responsive cells. J Immunol 171:2684–2693

16. Budinger GR, Chandel NS, Donnelly HK, et al (2005) Active transforming growth factor- beta1 activates the procollagen I promoter in patients with acute lung injury. Intensive Care Med 31:121–128

17. Bachofen M, Weibel ER (1982) Structural alterations of lung parenchyma in the adult respiratory distress syndrome. Clin Chest Med 3:35–56

18. Idell S, Gonzalez K, Bradford H, et al (1987) Procoagulant activity in bronchoalveolar lavage in the adult respiratory distress syndrome. Contribution of tissue factor associated with factor VII. Am Rev Respir Dis 136:1466–1474

(11)

19. Prabhakaran P, Ware LB, White KE, Cross MT, Matthay MA, Ohnan MA (2003) Elevated levels of plasminogen activator inhibitor-1 in pulmonary edema ﬂuid are associated with mortality in acute lung injury. Am J Physiol Lung Cell Mol Physiol 285:L20–L28

20. Fujimoto H, Gabazza EC, Hataji O, et al (2003) Thrombin-activatable ﬁbrinolysis inhibitor and protein C inhibitor in interstitial lung disease. Am J Respir Crit Care Med 167:1687–1694 21. Kirschstein W, Heene DL (1985) Fibrinolysis inhibition in acute respiratory distress syn-

drome. Scand J Clin Lab Invest Suppl 178:87–94

22. Ware LB, Fang X, Matthay MA (2003) Protein C and thrombomodulin in human acute lung injury. Am J Physiol Lung Cell Mol Physiol 285:L514–L521

23. Matthay MA, Ware LB (2004) Plasma protein C levels in patients with acute lung injury:

prognostic signiﬁcance. Crit Care Med 32 (Suppl 5):S229–S232

24. Sabharwal AK, Bajaj SP, Ameri A, et al (1995) Tissue factor pathway inhibitor and von Willebrand factor antigen levels in adult respiratory distress syndrome and in a primate model of sepsis. Am J Respir Crit Care Med 151:758–767

25. Gando S, Kameue T, Matsuda N, et al (2003) Imbalances between the levels of tissue factor and tissue factor pathway inhibitor in ARDS patients. Thromb Res 109:119–124

26. Hoffmann H, Siebeck M, Spannagl M, et al (1990) Effect of recombinant hirudin, a speciﬁc inhibitor of thrombin, on endotoxin-induced intravascular coagulation and acute lung injury in pigs. Am Rev Respir Dis 142:782–788

27. Schmidt B, Davis P, La-Pointe H, et al (1996) Thrombin inhibitors reduce intrapulmonary accumulation of ﬁbrinogen and procoagulant activity of bronchoalveolar lavage ﬂuid during acute lung injury induced by pulmonary overdistention in newborn piglets. Pediatr Res 39:798–804

28. Uchiba M, Okajima K (1997) Antithrombin III (AT III) prevents LPS-induced pulmonary vascular injury: novel biological activity of AT III. Semin Thromb Hemost 23:583–590 29. Abubakar K, Schmidt B, Monkman S, et al (1998) Heparin improves gas exchange during

experimental acute lung injury in newborn piglets. Am J Respir Crit Care Med 158:1620–1625 30. Piguet PF, Van GY, Guo J (1996) Heparin attenuates bleomycin but not silica-induced pulmonary fibrosis in mice: possible relationship with involvement of myofibroblasts in bleomycin, and fibroblasts in silica-induced fibrosis. Int J Exp Pathol 77:155–116

31. Yasui H, Gabazza EC, Tamaki S, 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

32. Kijiyama N, Ueno H, Sugimoto I, et al (2006) Intratracheal gene transfer of tissue factor pathway inhibitor attenuates pulmonary ﬁbrosis. Biochem Biophys Res Commun 339:1113–

1119

33. Tani K, Yasuoka S, Ogushi F, et al (1991) Thrombin enhances lung ﬁbroblast proliferation in bleomycin-induced pulmonary ﬁbrosis. Am J Respir Cell Mol Biol 5:34–40

34. 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 35. 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

36. Chambers RC, Dabbagh K, McAnulty RJ, Gray AJ, Blanc-Brude O, Laurent GJ (1998) Thrombin stimulates ﬁbroblast pro-collagen production via proteolytic activation of protease-activated receptor 1. Biochem J 333:121–127

37. Chambers RC, Leoni P, Blanc-Brude O, 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

38. Dery O, Corvera C, Steinhoff M, Bunnett NW (1998) Proteinase-activated receptors:novel mechanisms of signalling by serine proteases. Am J Physiol 274:C1429–C1452

(12)

39. Dawes KE, Gray AJ, Laurent GJ (1993) Thrombin stimulates ﬁbroblast chemotaxis and repli- cation. Eur J Cell Sci 61:126–130

40. Chen LB, Buchanan JM (1975) Mitogenic activity of blood components. I. Thrombin and prothrombin. Proc Natl Acad Sci USA 72:131–135

41. Carney DH, Cunningham DD (1978) Cell surface action of thrombin is sufﬁcient to initiate division of chick cells. Cell 14:811–823

42. Bogatkevich GS, Tourkina E, Silver RM, et al (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

43. Bogatkevich GS, Tourkina E, Abrams CS, et al (2003) Contractile activity and smooth muscle {alpha}-actin organization in thrombin-induced human lung myoﬁbroblasts. Am J Physiol Lung Cell Mol Physiol 285:L334–L343

44. Dabbagh K, Laurent GJ, McAnulty RJ, et al (1998) Thrombin stimulates smooth muscle cell procollagen synthesis and mRNA levels via a PAR-1 mediated mechanism. Thromb Haemost 79:405–409

45. Blanc-Brude OP, Archer F, Leoni P, et al (2005) Factor Xa stimulates ﬁbroblast procollagen production, proliferation, and calcium signaling via PAR1 activation. Exp Cell Res 304:16–27 46. Hernandez-Rodriguez NA, Cambrey AD, Harrison NK, et al (1995) Role of thrombin in

pulmonary ﬁbrosis. Lancet 346:1071–1073

47. Ohba T, McDonald JK, Silver RM (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

48. Howell DC, Goldsack NR, Marshall RP, 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

49. Howell DC, Johns RH, Lasky JA, 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

50. Macfarlane SR, Seatter MJ, Kanke T, et al (2001) Proteinase-activated receptors. Pharmacol Rev 53:245–282

51. 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

52. Pendurthi UR, Ngyuen M, Andrade-Gordon P (2002) Plasmin induces Cyr61 gene expression in ﬁbroblasts via protease-activated receptor-1 and p44/42 mitogen-activated protein kinase- dependent signalling pathway. Arterioscler Thromb Vasc Biol 22:1421–1426

53. Riewald M, Ruf W (2001) Mechanistic coupling of protease signalling and initiation of coagulation by tissue factor. Proc Natl Acad Sci USA 98:7742–7747

54. O’Brien PJ, Molino M, Kahn M, et al (2001) Protease activated receptors: theme and variations.

Oncogene 20:1570–1581

55. Camerer E, Huang W, Coughlin SR (2000) Tissue factor- and factor X-dependent activation of protease-activated receptor 2 by factor VIIa. Proc Natl Acad Sci USA 97:5255–5260 56. Nakayama T, Hirano K, Shintani Y, et al (2003) Unproductive cleavage and the inactivation

of protease-activated receptor-1 by trypsin in vascular endothelial cells. Br J Pharmacol 138:121–130

57. Suzuki T, Moraes TJ, Vachon E, 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 58. Laurent GJ (2005) No bit PARt for PAR-1. Am J Respir Cell Mol Biol 33:213–215

59. Riewald M, Petrovan RJ, Donner A, et al (2002) Activation of endothelial cell protease activated receptor 1 by the protein C pathway. Science 296:1880–1882

60. Ruf W, Riewald M (2003) Tissue factor-dependent coagulation protease signalling in acute lung injury. Crit Care Med 31 (Suppl 4): S231–S237

(13)

61. Bernard GR, Vincent JL, Laterre PF, et al (2001) Recombinant human protein C Worldwide Evaluation in Severe Sepsis (PROWESS) study group. Efﬁcacy and safety of recombinant human activated protein C for severe sepsis. N Engl J Med 344:699–709

62. Ruf W (2005) Is APC activation of endothelial cell PAR1 important in severe sepsis?: Yes.

J Thromb Haemost 3:1912–1914

63. Esmon CT (2005) Is APC activation of endothelial cell PAR1 important in severe sepsis?: No.

J Thromb Haemost 3:1910–1911

64. Pawlinski R, Pedersen B, Schabbauer G, et al (2004) Role of tissue factor and protease- activated receptors in a mouse model of endotoxemia. Blood 103:1342–1347

65. Camerer E, Cornelissen I, Kataoka H, et al (2006) Roles of protease-activated receptors in a mouse model of endotoxemia. Blood 107:3912–3921

66. Brass LF (1997) Thrombin receptor antagonists: a work in progress. Coron Artery Dis 8:49–58 67. Bernatowicz MS, Klimas CE, Hartl KS, Peluso M, Allegretto NJ, Seiler SM (1996) Development

of potent thrombin receptor antagonist peptides. J Med Chem 39:4879–4887

68. Andrade-Gordon P, Derian CK, Maryanoff BE, et al (2001) Administration of a potent antagonist of protease-activated receptor-1 (PAR-1) attenuates vascular restenosis following balloon angioplasty in rats. J Pharmacol Exp Ther 298:34–42

69. Derian CK, Damiano BP, Addo MF (2003) Blockade of the thrombin receptor protease- activated receptor-1 with a small-molecule antagonist prevents thrombus formation and vascular occlusion in nonhuman primates. J Pharmacol Exp Ther 304:855–861