This study investigated the role of vascular leak-induced thrombin signaling in a mouse model of pulmonary fibrosis. The researchers found that treatment with the direct thrombin inhibitor dabigatran attenuated fibrosis by decreasing PAR1 activation, αvβ6 integrin induction, and TGF-β activation. In contrast, therapeutic anticoagulation with warfarin did not protect against fibrosis or decrease these profibrotic pathways. Blockade of αvβ6 integrin also protected against fibrosis. These findings suggest that vascular leak promotes fibrosis through a thrombin-PAR1-αvβ6-TGF-β signaling axis rather than through coagulation and fibrin deposition itself.

1. Vascular Leak-Induced Thrombin-PAR1 Signaling Drives Pulmonary
Fibrosis Following Lung Injury
Clemens K Probst1, Patricia L Brazee1, Paul H Weinreb3, Sheila M Violette3, Peter Caravan2, Andrew M
Tager1, Barry S Shea4
1Division of Pulmonary and Critical Care Medicine, Fibrosis Research Center, Massachusetts General Hospital and Harvard Medical School, Boston Massachusetts 2A. A. Martinos Center for
Biomedical Imaging, 3Biogen, Cambridge, Massachusetts, 4Division of Pulmonary, Critical Care and Sleep Medicine, Rhode Island Hospital and Warren Alpert Medical School of Brown University.
Background and Rationale: Increased vascular
permeability has been demonstrated in the lungs
of patients with idiopathic pulmonary fibrosis
(IPF), but whether this vascular leak is
mechanistically linked to the development of
fibrosis is unknown. We have previously
described a mouse model of pulmonary fibrosis
in in which disruption of endothelial barrier
integrity caused by administration of FTY720, a
functional antagonist of S1P1 signaling, shifts the
outcome of a mild lung injury, induced by low-
dose bleomycin challenge, from lung repair to
lung fibrosis. We hypothesized that increased
extravascular coagulation induced by increased
vascular permeability in this model promotes the
development of fibrosis in a manner dependent
on thrombin signaling through it major receptor
protease-activated receptor-1 (PAR1), but
independent of fibrin deposition.
Main Conclusions: Here we found dramatic
protection from fibrosis produced in our vascular
leak-dependent model with treatment with the
direct thrombin inhibitor dabigatran. Dabigatran
treatment was associated with decreased PAR1
activation, 6 intergrin induction, and TGF-
activation. Treatment with an anti-6 mAb also
protected from the development of pulmonary
fibrosis in this model. In contrast, despite
achieving therapeutic anticoagulation, treatment
with the vitamin K antagonist warfarin did not
protect from lung fibrosis in this model. Warfarin
treatment also failed to decrease PAR1
activation, 6 induction, or TGF- activation.
We conclude that vascular leak promotes
pulmonary fibrosis by promoting extravascular
coagulation, but that the pro-fibrotic effects of
extravascular coagulation are mediated by
thrombin signaling through a PAR1-6-TGF-
axis, rather than by coagulation, i.e. fibrin
generation and deposition, itself.
Therapeutic anticoagulation with
warfarin does not protect against
fibrosis
Expression of αvβ6 integrin in a
vascular leak-dependent model of
lung fibrosis
Effects of thrombin inhibition and
vitamin K antagonism on lung
αvβ6 expression and TGF-β
signaling
Figure 3. (A) Mouse plasma International Normalized Ratios
(INR) after treatment with warfarin or vehicle for 2 weeks (n
= 8-10/group). (B) Extravascular lung d-dimer content at day
14 in control mice and mice challenged with bleomycin +
FTY720 and treated with vehicle (veh) or warfarin (war) (n =
5/group). (C) Combined total lung hydroxyproline content
data from multiple experiments comparing warfarin vs.
vehicle (total n = 53-54/group) or dabigatran vs. vehicle
(total n = 56-57/group) at day 14 after bleomycin + FTY720
challenge. (D) Correlation between total lung hydroxyproline
content and total lung d- dimer content in mice challenged
with bleomycin + FTY720 and treated with warfarin vs.
vehicle. (E-G) Western blot analyses for cleaved (activated)
PAR1 in whole lung homogenates at day 14 in control mice
and mice challenged with bleomycin + FTY720 and treated
with dabigatran vs. vehicle (E, top panel; densitometry in F)
or warfarin vs. vehicle (E, bottom panel; densitometry in G).
Bar graph data are reported as mean values with SEM.*p <
0.05, **p < 0.005, ***p < 0.0005 by 2-tailed t-tests for
indicated pairwise comparisons.
Figure 4.. (A-F) Representative images of
immunohistochemical staining for the αvβ6 integrin in day
14 lung sections from control mice (A, D) and mice
challenged with bleomycin + FTY720 and treated with
vehicle (B, E) or dabigatran (C, F). N = 3/group; scale bar =
50 μm.
Figure 5. (A-C) Western blot analyses for αvβ6 (A, top
panel; densitometry in B) and pSMAD2 (A, bottom
panel; densitometry in C) in whole lung homogenates at
day 14 in control mice and mice challenged with
bleomycin + FTY720 and treated with dabigatran vs.
vehicle. (D-F) Western blot analyses for αvβ6 (D, top
panel; densitometry in E) and pSMAD2 (D, bottom panel;
densitometry in F) in whole lung homogenates at day 14
in control mice and mice challenged with bleomycin +
FTY720 and treated with warfarin vs. vehicle. Bar graph
data are reported as mean values with SEM. *p = 0.01,
**p = 0.008, ***p < 0.0005 by 2-tailed t-tests for indicated
pairwise comparisons.
Antibody blockade of αvβ6 protects
against lung fibrosis in a vascular-
leak dependent model
Figure 6. (A-C) Measurement of total lung
hydroxyproline content (A), BAL total protein
concentration (B), and BAL total leukocytes (C) at day
14 (D14) in mice challenged with i.t. PBS + i.p. sterile
water (control) or i.t. bleomycin + i.p. FTY720 (Bleo/FTY)
and treated with the αvβ6-blocking antibody (3G9) vs.
isotype control antibody (1E6) at 1 mg/kg
subcutaneously 3x/week (n = 4-5/group). Data are
reported as mean values with SEM. *p = 0.0036 by 2-
way ANOVA.
Mice were administered a single intratracheal
dose of bleomycin at 0.1 U/kg on day 0 and
administered intraperitoneal FTY720 at 1 mg/kg
three times per week, starting on day 0 and
continuing for the duration of the experiments.
For dabigatran experiments, mice were fed with
chow containing dabigatran at 10 mg/g or control
chow starting on day 0 and continued for the
duration of the experiments. For warfarin
experiments, warfarin at 1 mg/L or vehicle (0.1%
DMSO) was added to the drinking water starting
7 days prior to bleomycin administration to
achieve a steady-state of anticoagulation, and
continued for the duration of the experiments.
For anti-B6 experiments, the anti-B6 blocking
antibody (3G9) or control Ab were administered
at 1 mg/kg three times per week, starting on day
0. Mice were sacrificed at the time points
indicated for analyses of blood, bronchoalveolar
lavage (BAL) and lung tissue.
ABSTRACT
METHODS
Figure 1. (A) Plasma dilute thrombin times in mice treated
with dabigatran or vehicle for 1 week. (B-C) Representative
images of trichrome-stained mouse lung sections at day 14
after bleomycin + FTY720 challenge with vehicle (B) or
dabigatran (C) treatment (n = 3/group). (D-F) Measurement of
total lung hydroxyproline content (D), BAL total protein
concentration (E), and BAL total leukocytes (F) at day 14
(D14) in mice challenged with i.t. PBS + i.p. sterile water
(control) or i.t. bleomycin + i.p. FTY720 (Bleo/FTY) and
treated with dabigatran or vehicle (n = 4-5/group). Bar graph
data are reported as mean values with SEM. *p = 0.01 by 2-
way ANOVA; ***p < 0.0001 by 2-tailed t-test.
Thrombin inhibition attenuates fibrosis
in a vascular leak-driven model
Thrombin inhibition attenuates lung
fibrin deposition
Figure 2. (A) Extravascular lung d-dimer content at day 14 in
control mice and mice challenged with bleomycin + FTY720
and treated with vehicle or dabigatran. (B-F) Data from
ultrashort echo time lung MRI with the gadolinium-based,
fibrin-specific probe EP-2104R at day 14 in control mice and
mice challenged with bleomycin + FTY720 and treated with
vehicle or dabigatran. (B) Representative images from EP-
2104R- enhanced lung MRI. (C) Semi-quantitative analysis of
the imaging data from EP-2104R-enhanced UTE lung MRI.
(D) Quantitative assessment of lung fibrin deposition by
measurement of total lung EP-2104R content. (E) Correlation
between total lung d-dimer content and total lung EP-2104R
content of mice challenged with bleomycin + FTY720 and
treated with dabigatran vs. vehicle. (F) Correlation between
total lung OHP content and total lung EP-2104R content in
mice challenged with bleomycin + FTY720 and treated with
dabigatran vs. vehicle. Bar graph data are reported as mean
values with SEM. *p < 0.05, **p < 0.005, and ***p < 0.0005 by
2-tailed t-tests for indicated pairwise comparisons.
RESULTS
CONCLUSIONS
Schematic of the proposed mechanisms linking
vascular leak, intra-alveolar thrombin, αvβ6, and TGF-
β signaling with the development of injury-induced
lung fibrosis.
As a consequence of lung injury, there is damage to the
alveolar epithelium, denudement of the basement
membrane, and increased vascular permeability.
Extravasation of plasma constituents into the injured
alveoli causes intra-alveolar activation of the coagulation
cascade and the generation of active thrombin. Thrombin
cleaves and activates proteinase PAR1. 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). Active TGF-β can then signal through its
receptors to exert its profibrotic effects. Dabigatran, or
blockade of the αvβ6 integrin interrupts this thrombin-
PAR1-αvβ6- TGF-β axis, thereby halting the progression
from lung injury to fibrosis.
p = 0.64
p = 0.57
Figure 1
A B C
D E F
***
*
Figure 1. Thrombin inhibition attenuates fibrosis in in a vascular leak-dependent model. (A) Plasma dilute thrombin times in mice treated
with dabigatran or vehicle for 1 week. (B-C) Representative images of trichrome-stained mouse lung sections at day 14 after bleomycin +
FTY720 challenge with vehicle (B) or dabigatran (C) treatment (n = 3/group). (D-F) Measurement of total lung hydroxyproline content (D), BAL
total protein concentration (E), and BAL total leukocytes (F) at day 14 (D14) in mice challenged with i.t. PBS + i.p. sterile water (control) or i.t.
bleomycin + i.p. FTY720 (Bleo/FTY) and treated with dabigatran or vehicle (n = 4-5/group). Bar graph data are reported as mean values with
SEM. *p = 0.01 by 2-way ANOVA; ***p < 0.0001 by 2-tailed t-test.
Figure 2
r = 0.9427
p < 0.0001
**
***
A B
DC
r = 0.9275
p < 0.0001
E F
**
* *
*
Figure 2. Thrombin inhibition attenuates lung fibrin deposition. (A) Extravascular lung d-dimer content at day 14 in control mice and mice
challenged with bleomycin + FTY720 and treated with vehicle (veh) or dabigatran (dab) (n = 5/group). (B-F) Data from ultrashort echo time
(UTE) lung magnetic resonance imaging (MRI) with the gadolinium-based, fibrin-specific probe EP-2104R at day 14 in control mice and mice
challenged with bleomycin + FTY720 and treated with vehicle or dabigatran (n = 4-5/group). (B) Representative images from EP-2104R-
enhanced lung MRI. (C) Semi-quantitative analysis of the imaging data from EP-2104R-enhanced UTE lung MRI. (D) Quantitative assessment
Bleo/FTY + VehControl Bleo/FTY + Dabi
Control Bleo/FTY + Veh
*
Figure 3
A B D
p = 0.0515
r = 0.1627
p = 0.68
**
**
**
F GE
C
***
**
p = 0.35
Cleaved PAR1
Cleaved PAR1
GAPDH
GAPDH
Bleo/FTY + War
Figure 3. Therapeutic anticoagulation with warfarin does not protect against fibrosis. (A) Mouse plasma International Normalized Ratios
(INR) after treatment with warfarin or vehicle for 2 weeks (n = 8-10/group). (B) Extravascular lung d-dimer content at day 14 in control mice
and mice challenged with bleomycin + FTY720 and treated with vehicle (veh) or warfarin (war) (n = 5/group). (C) Combined total lung
hydroxyproline content data from multiple experiments comparing warfarin vs. vehicle (total n = 53-54/group) or dabigatran vs. vehicle (total
A
B
C E
F
Figure 4
Bleo/FTY + Veh D14Control Bleo/FTY + Dab D14
100X
400X
D
Figure 4. Expression of αvβ6 integrin in a vascular leak-dependent model of lung fibrosis. (A-F) Representative images of
immunohistochemical staining for the αvβ6 integrin in day 14 lung sections from control mice (A, D) and mice challenged with bleomycin +
FTY720 and treated with vehicle (B, E) or dabigatran (C, F). N = 3/group; scale bar = 50 µm.
**
Figure 5
αvβ6
β-actin
pSMAD2
GAPDH
Control Bleo/FTY + Veh Bleo/FTY + Dab
A B C
D E F
***
*
***
p = 0.86
***
***
p = 0.095
αvβ6
β-actin
pSMAD2
GAPDH
Bleo/FTY + WarBleo/FTY + VehControl
Figure 5. Effects of o o d o o αvβ6 expression and TGF-β . -C) Western blot analyses
for αvβ6 (A, top panel; densitometry in B) and pSMAD2 (A, bottom panel; densitometry in C) in whole lung homogenates at day 14 in control
mice and mice challenged with bleomycin + FTY720 and treated with dabigatran vs. vehicle. -F) Western blot analyses for αvβ6 (D, top
panel; densitometry in E) and pSMAD2 (D, bottom panel; densitometry in F) in whole lung homogenates at day 14 in control mice and mice
challenged with bleomycin + FTY720 and treated with warfarin vs. vehicle. Bar graph data are reported as mean values with SEM. *p = 0.01,
**p = 0.008, ***p < 0.0005 by 2-tailed t-tests for indicated pairwise comparisons.
Epithelial cell