The Coagulation Cascade

1
The Coagulation Cascade Mechanisms of Lung
Fibrosis

• Rachel C Chambers
• University College London

2
Fibroproliferative Response to Lung Injury
Epithelial Injury
Epithelial cell death

• Activation of
• Coagulation cascade
• Inflammation
• AEC II cell proliferation

TF/FVIIa/FXa
Thrombin
Profibrotic mediator release
TGFb PDGF
Fibroblast recruitment proliferation
differentiation
Capillary
ECM deposition
NORMAL ALVEOLUS
INJURED ALVEOLUS
3
Pulmonary Fibrosis
Epithelial Injury
TF/FVIIa/FXa
Thrombin
TGFb PDGF
Capillary
NORMAL ALVEOLUS
INJURED ALVEOLUS
4
Myofibroblast central effector cell in
pulmonary fibrosis
Myofibroblast

• a-SMA positive contractile
• Highly synthetic
• ECM collagens I III, ED-A fibronectin,
  proteoglycans
• Pro-inflammatory mediators IL-6, IL-8, CCL2 etc
• Pro-fibrotic mediators TGFb, CTGF, PDGF
• MMPs capable of degrading the ECM
• Resistant to apoptosis

Reviewed in Scotton Chambers, Chest 132, 2007
5
Activation of the coagulation cascade in
fibroproliferative lung disease
6
Blocking the coagulation cascade attenuates
experimental lung fibrosis

• Direct thrombin inhibition
• Howell et al, Am J Pathol 2001
• Activated protein C
• Yasui et al, Am J Respir Crit Care Med 2001
• Heparin
• Gunther et al, Am J Respir Crit Care Med 2003
• Tissue factor pathway inhibitor
• Kijiyama et al, Biochem Biophys Res Commun 2006

plt0.01
Fibrinogen knockout mice are not protected from
lung fibrosis Hattori et al, J Clin Invest 2000
Ploplis et al, Am J Pathol 2000
7
Anticoagulant Therapy for Idiopathic Pulmonary
Fibrosis?

• Small non-blinded trial of prednisolone alone
  versus prednisolone plus anticoagulant therapy
• Mortality associated with acute exacerbation
  reduced in anticoagulant group

Kubo et al., Chest 20051281475-1482
8
Proteinase-activated receptors sensors of tissue
injury
9
Proteinase-activated receptors sensors of tissue
injury
Reviewed in Chambers RC. Br J Pharmacol
2008153S367-S378
10
PAR1 mediates pluripotent cellular responses
Endothelial barrier function Platelet aggregation
Low thrombin protective High thrombin
disruptive
Pro-inflammatory responses Production of IL-1,
IL2, IL-6 Production of chemokines (CCL2
etc) Promotion of adhesion molecule expression
?
Mitogenic responses Fibroblasts, endothelial
cells, smooth muscle cells Indirect mediated via
autocrine production of PDGF/PDGFaR
Fibrogenic responses Myofibroblast
differentiation ECM production Induction and
activation of fibrogenic mediators CTGF, TGFb
etc
Reviewed in Chambers RC. Br J Pharmacol
2008153S367-S378
11
PAR1 signaling pathways
PAR1
Ga12/13 bg
Gaq/11 bg
Gai/o bg
Rho GEFs
PLCb
Adenylyl Cyclase
Downstream effectors
Downstream effectors
Downstream effectors
Cell shape, secretion, metabolic response,
proliferation, differentiation, motility etc
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PAR1 is highly expressed in IPF
Control
IPF
IPF
Laser capture of alveolar septae

• Macrophages
• Alveolar epithelial cells
• Fibroblasts
• Endothelial cells
• Smooth muscle cells

Fold increase in PAR1 mRNA levels
plt0.01
Mercer, Johns, Scotton et al. (in revision)
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PAR1-/- mice are protected from bleomycin-induced
lung inflammation
Global transcriptional profiling
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plt0.01
10
8
BALF Total Protein (mg/ml)
6

4
2
0
Sal
Bleo
Sal
Bleo
plt0.01
BALF Inflammatory Cell No

(x 10-5)
Sal
Bleo
Sal
Bleo
Howell, Johns et al., Am J Pathol 1661353-65,
2005 Scotton et al. unpublished Deng et al. Mol
Biol Cell 19 2520-2533, 2008
WT
PAR1-/-
14
PAR1-/- mice are protected from bleomycin-induced
fibrosis
TGFb1 Immunohistochemistry
WT
PAR1-/-
Plt0.01
12
10
8
TGF?1 immunoreactivity ( field of view ve)
6
4
2
0
Sal Bleo
Sal Bleo
WT
PAR1-/-
Howell, Johns et al., Am J Pathol 1661353-65,
2005
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Ligation of PAR1 leads to the activation of
latent TGFb
Thrombin
TGFb
avb6
Gaq
Rho
Rho kinases
LIM
Cofilin
Epithelial cell
Jenkins et al. J Clin Invest. 20061161606-14
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What are the endogenous activators of PAR1 in
lung injury?
BALF
40
plt0.01
?
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Thrombin Levels (nmol/L)
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Hernandez-Rodriguez et al. Lancet 1995
Also reported
by Ohba et al. Am J Respir Cell Mol Biol 1994
0
Volunteer SSc
17
Transcriptional profiling reveals local FX gene
induction after bleomycin lung injury
? Factor X
Scotton, Krupiczojc et al. (in revision)
18
Factor X is locally upregulated in IPF
Laser capture microdissected alveloar septae
Scotton, Krupiczojc et al. (in revision)
19
Assembly of the extrinsic coagulation pathway in
IPF
Scotton, Krupiczojc et al. (in revision)
20
FXa induces fibroblast differentiation in primary
human adult lung fibroblasts
a-sma
Western blot
a-SMA
ERK total
FXa
Control
a-SMA protein expression (fold increase relative
to control)
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a-SMA protein expression (fold increase relative
to control)
12.5
plt0.01
20
10
15
7.5
both plt0.01
10
5
5
2.5
0
0
FXa
Control
FXa RWJ-58259 SD-208
-
---
--
-
Scotton, Krupiczojc et al. (in revision)
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Role of FXa in bleomycin-induced fibrosis
FX/Xa Immunohistochemistry
Saline
Bleomycin
FXa activity in BALF
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Conclusions

• Local coagulation and coagulation signalling
  system present in the lung
• PAR1 plays a key role in mediating the interplay
  between coagulation, inflammation and tissue
  repair
• Uncontrolled coagulation and PAR1 signalling may
  lead to lung inflammation and fibrosis
• Locally-derived FXa may represent a major PAR1
  activator in lung fibrosis
• PAR1 antagonists may offer promise as potential
  novel therapeutic agents

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Acknowledgements
Post-doctoral Fellows Chris Scotton Paul
Mercer PhD Students and Clinical Training
Fellows David Howell Robin Johns Malvina
Krupizojc Xiaoling Deng Research
Assistants Danielle Copeman Steve Bottoms UCL
Collaborators Robin McAnulty Geoff
Laurent Funding bodies
External Collaborators Shaun Coughlin (UCSF,
USA) Peter Tipping (Monash, Aus) James Moffatt
(St Georges Hospital, UK) Andrew Nicholson
(Royal Brompton, UK) Oliver Eickelberg (Giessen,
Germany) Naftali Kaminski (Pittsburg, USA) Gisli
Jenkins (Nottingham, UK) Giuseppe Lungarella
(Siena, Italy) Claudia Derian (JJ, PRD,
USA) Linda Higgins (Scios, USA) Annette Gilchrist
(Caden Biosciences, USA) John Morser (Berlex,
USA)
Rosetrees Trust