The specific aims of this presentation are: 1) to review the evidence that activation of the coagulation cascade is a common feature of fibrotic lung disease and other respiratory conditions associated with excessive deposition of extracellular matrix; 2) to describe the profibrotic effects of coagulation proteases and the signalling receptors involved; and 3) to provide evidence that coagulation proteases and their signalling receptors contribute to experimentally induced lung fibrosis.
Introduction
The fragile architecture of the lung is constantly under threat from both external and internal insults. When these insults lead to damage of the extensive vascular network of the lung, activation of the coagulation cascade (fig. 1⇓) ensures that there is minimal blood loss by temporarily plugging damaged vessels with a stable clot consisting of aggregated platelets enmeshed in fibrin. However, excessive or smouldering activation of the coagulation cascade has been implicated in promoting lung inflammation and subsequent interstitial and alveolar fibrosis 1, 2. Intra-alveolar accumulation of fibrin occurs in the lungs of patients with pulmonary fibrosis 3, in acute lung injury and in the acute respiratory distress syndrome (ARDS) 4, in which rapid fibroproliferation and matrix synthesis can lead to the development of extensive fibrotic lesions. Bronchoalveolar lavage fluid from patients with ARDS has also been reported to contain tissue factor-factor VII/VIIa complexes that can trigger activation of the extrinsic coagulation cascade 4. Levels of active thrombin have been shown to be increased in the lungs of patients with pulmonary fibrosis associated with systemic sclerosis 5, in pulmonary fibrosis associated with chronic lung disease of prematurity 6 and in asthma 7. Several procoagulant factors (fibrinogen, factors VII and X) have also been identified in patients with intra-alveolar fibrosis associated with idiopathic bronchiolitis obliterans-organising pneumonia 8.
The coagulation cascade. The figure shows the stepwise activation of coagulation proteases leading to the generation of thrombin via both the extrinsic and intrinsic coagulation pathways. Thrombin converts soluble fibrinogen to insoluble fibrin and exerts numerous cellular effects, including promoting platelet aggregation, via activation of protease-activated receptors.
The importance of excessive and persistent fibrin deposition in lung repair and fibrosis has been demonstrated by extensive studies performed in experimental models using genetically modified mice in which fibrinolytic capacity in the lung was either up- or downregulated. Bleomycin-induced pulmonary fibrosis is increased in mice overexpressing plasminogen activator inhibitor (PAI)‐1 (favouring fibrin persistence) and is decreased in PAI‐1‐knockout mice (favouring fibrin clearance) 9. Fibrin is thought to influence the fibrotic response by acting as a provisional matrix and reservoir of growth factors for fibroblasts and inflammatory cells. However, fibrinogen-knockout mice were not protected in this model 10, 11, so that fibrin would seem to promote, but is not required for fibrosis.
Protease-activated receptors: sensors for lung injury and initiators of repair and fibrosis
In addition to their critical role in blood coagulation, thrombin and immediate upstream coagulation proteases of the extrinsic coagulation cascade exert a number of cellular effects by activating a novel family of seven transmembrane G‐protein coupled receptors, termed protease-activated receptors (PARs) 12. These receptors display a unique mechanism of activation involving the unmasking of a tethered ligand by limited proteolysis (fig. 2⇓). Conformational changes induced following interaction of the tethered ligand with the second extracellular loop initiates cell signalling via heterotrimeric G‐proteins. To date, four such receptors have been characterised, of which three (PAR‐1, ‐3 and ‐4) are activated by the main coagulation protease, thrombin, whereas factor Xa and tissue factor-factor VIIa‐factor Xa complexes activate both PAR‐1 and PAR‐2 2. There is increasing evidence that coagulation protease signalling is mechanistically coupled and thus an integrated part of the tissue factor-VIIa-initiated coagulation pathway. This has led to the suggestion that PARs may act as cellular sensors of tissue injury. In addition, the cellular responses initiated upon activation of PARs are consistent with the notion that these receptors play a critical role in orchestrating subsequent inflammatory and repair responses, as part of the normal response to tissue injury.
Activation of protease-activated receptors (PARs). PARs are activated by limited proteolysis and the unmasking of a tethered ligand. Interaction of this ligand with the second extracellular loop of the receptor initiates cell signalling via interaction with heterotrimeric G‐proteins.
Profibrotic effects of coagulation proteases in vitro
Research performed in the author's laboratory has concentrated on elucidating the effects of thrombin and its major cellular receptor PAR‐1 on fibroblast function in vitro and lung fibrosis in vivo. It has been shown that thrombin is a potent promoter of fibroblast proliferation 13 and extracellular matrix production 14. Thrombin has also been shown to promote the transformation of fibroblasts into smooth muscle α‐actin-positive contractile myofibroblasts 15. This is the predominant fibroblast phenotype present in active fibrotic lesions and responsible for excessive deposition of matrix proteins. Although lung fibroblasts express all four PARs, current evidence suggests that PAR‐1 is the main receptor responsible for mediating thrombin's effects on fibroblast function 2. More recent unpublished data from the author's laboratory has also shown that the profibrotic effects of factor Xa, including its effects on fibroblast proliferation and procollagen production, are mediated via activation of PAR‐1 rather than PAR‐2. Current in vitro evidence has further shown that these coagulation proteases do not influence fibroblast function following PAR‐1 activation directly but act via the induction of a host of secondary mediators, including platelet-derived growth factor 16 and possibly connective tissue growth factor (CTGF) 17.
Role of coagulation proteases and protease-activated receptor‐1 in lung fibrosis
Further work has confirmed a major role for thrombin and PAR‐1 in lung injury and fibrosis. PAR‐1 and thrombin expression is increased in inflammatory and fibroproliferative foci following bleomycin-induced lung injury in rats, and direct thrombin inhibition has been shown to attenuate lung collagen deposition and CTGF messenger ribonucleic acid levels in this model 18. Similar findings have also been reported with the anticoagulant, activated protein C, in mice 19. Recent preliminary data obtained using PAR‐1‐deficient mice further indicate that these mice are protected from bleomycin-induced lung injury. Studies to unravel the key pathways by which PAR‐1 contributes to fibrosis are currently ongoing.
Clinical implications
There have, to date, been no studies of anticoagulants in fibrotic lung disease. However, anticoagulants, including tissue factor pathway inhibitor and antithrombin III, have been trialled in humans with sepsis, a major risk factor for the development of ARDS, but the results have been largely disappointing. The results of a recent phase‐III, randomised, double-blind, placebo-controlled, multicentre Protein C Worldwide Evaluation of Severe Sepsis (PROWESS) trial of intravenous infusion of activated protein C in severe sepsis (that included patients with ARDS) and subsequent reduction in mortality, resulted in Food and Drug Administration approval for the use of this drug in severe sepsis, despite greater risk of serious bleeding 20. ARDS subgroup analysis was not presented, so the ability of this agent to protect the lung in ARDS remains to be determined.
Conclusion
To conclude, there is increasing evidence that modulation of the coagulation cascade, and more specifically the pro-fibrotic effects of coagulation proteases, warrant further investigation as potential therapeutic strategies in a number of respiratory conditions associated with excessive deposition of matrix proteins. Targeting the signalling receptors rather than the enzymes involved in their activation may allow selective interference with the cellular responses of coagulation proteases without the bleeding complications associated with the use of traditional anticoagulants. Since protease-activated receptor‐1 antagonists are currently being developed and are showing promise as antithrombotic agents in preclinical studies in primates 21, they may be available for use in a variety of disease settings, including fibrotic lung disease, in the not too distant future.
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