Uncoupling of the profibrotic and hemostatic effects of thrombin in lung fibrosis
Barry S. Shea, Clemens K. Probst, Patricia L. Brazee, Nicholas J. Rotile, Francesco Blasi, Paul H. Weinreb, Katharine E. Black, David E. Sosnovik, Elizabeth M. Van Cott, Shelia M. Violette, Peter Caravan, Andrew M. Tager
Barry S. Shea, Clemens K. Probst, Patricia L. Brazee, Nicholas J. Rotile, Francesco Blasi, Paul H. Weinreb, Katharine E. Black, David E. Sosnovik, Elizabeth M. Van Cott, Shelia M. Violette, Peter Caravan, Andrew M. Tager
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Research Article Inflammation Pulmonology

Uncoupling of the profibrotic and hemostatic effects of thrombin in lung fibrosis

Abstract

Fibrotic lung disease, most notably idiopathic pulmonary fibrosis (IPF), is thought to result from aberrant wound-healing responses to repetitive lung injury. Increased vascular permeability is a cardinal response to tissue injury, but whether it is mechanistically linked to lung fibrosis is unknown. We previously described a model in which exaggeration of vascular leak after lung injury shifts the outcome of wound-healing responses from normal repair to pathological fibrosis. Here we report that the fibrosis produced in this model is highly dependent on thrombin activity and its downstream signaling pathways. Direct thrombin inhibition with dabigatran significantly inhibited protease-activated receptor-1 (PAR1) activation, integrin αvβ6 induction, TGF-β activation, and the development of pulmonary fibrosis in this vascular leak–dependent model. We used a potentially novel imaging method — ultashort echo time (UTE) lung magnetic resonance imaging (MRI) with the gadolinium-based, fibrin-specific probe EP-2104R — to directly visualize fibrin accumulation in injured mouse lungs, and to correlate the antifibrotic effects of dabigatran with attenuation of fibrin deposition. We found that inhibition of the profibrotic effects of thrombin can be uncoupled from inhibition of hemostasis, as therapeutic anticoagulation with warfarin failed to downregulate the PAR1/αvβ6/TGF-β axis or significantly protect against fibrosis. These findings have direct and important clinical implications, given recent findings that warfarin treatment is not beneficial in IPF, and the clinical availability of direct thrombin inhibitors that our data suggest could benefit these patients.

Authors

Barry S. Shea, Clemens K. Probst, Patricia L. Brazee, Nicholas J. Rotile, Francesco Blasi, Paul H. Weinreb, Katharine E. Black, David E. Sosnovik, Elizabeth M. Van Cott, Shelia M. Violette, Peter Caravan, Andrew M. Tager

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Figure 7

Schematic of the proposed mechanisms linking vascular leak, intra-alveolar thrombin, αvβ6, and TGF-β signaling with the development of injury-induced lung fibrosis.

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Schematic of the proposed mechanisms linking vascular leak, intra-alveol...
As a consequence of lung injury, there is damage to the alveolar epithelium, denudement of the basement membrane, and increased vascular permeability, with extravasation of plasma constituents into the injured alveoli (orange arrows). Among these extravasated plasma contents are the clotting factors, leading to intra-alveolar activation of the coagulation cascade and the generation of active thrombin. In addition to its proteolytic cleavage of fibrinogen to generate fibrin in the airspaces, thrombin also cleaves and activates proteinase activated receptor 1 (PAR1). Based on previous work by Jenkins et al. (ref. 26) and Munger et al. (ref. 27), it has been shown that activation of PAR1 on alveolar epithelial cells consequently activates the αvβ6 integrin (in a manner dependent on RhoA and Rho kinase, a.k.a. ROCK), which results in the release of extracellular active TGF-β from the latency-associated peptide (LAP). In an autocrine fashion, active TGF-β can then signal through its receptors (TGF-βR; here shown on a neighboring fibroblast) to exert its profibrotic effects. Dabigatran, a direct thrombin inhibitor (DTI), or blockade of the αvβ6 integrin interrupts this thrombin/PAR1/αvβ6/TGF-β axis, thereby halting the progression from lung injury to fibrosis. Conversely, while warfarin is effective at reducing intra-alveolar fibrin accumulation (solid red line), it appears to have only weak effects on overall thrombin activity (dashed red line) in the injured airspaces, which may explain its inability to significantly attenuate the development of fibrosis after lung injury.