Transcript Role of the Tissue Factor-Thrombin Pathway in Hemostasis

Molecular Events in Thrombosis
Nigel Mackman, Ph.D.
The Scripps Research Institute
La Jolla
Tissue Factor-Factor VIIa
Initiated Coagulation
5/05
Tissue Factor Pathway Inhibitor (TFPI)
4_05
Targets of New Anticoagulants
5_3/04
Recombinant Factor VIIa (Novo Seven)
as a Hemostatic Agent
 Hemophilia patients with or without
inhibitory antibodies
 Patients with diffuse bleeding that
are unresponsive to conventional
therapies
6_3/04
Lessons from KO Mice
6_3/04
Phenotype of Mouse KOs Deficient in
Clotting Factors and PARs
Extrinsic
Pathway
Intrinsic
Pathway
Common
Pathway
KO
Embryonic
lethality (%)
TF
VII
XI
IX
VIII
V
X
90
0 (MT)
0
0
0
50
10
100
100
0
0
0
100
100
0
0
100
100
100
0
0
Prothrombin
Fibrinogen
PAR-4
50
0
0
100
0
75
0
25
100
PAR-4/Fibrinogen
0
50
100
0
0
50
PAR-1
Perinatal
lethality (%)
Adult
survival (%)
Phenotype of Clotting Factor KOs
and PAR KOs
Phenotype of Anticoagulant KOs
KO
TFPI
AT
TM
EPCR
PC
Embryonic
lethality (%)
100
100
100
100
0 (MT)
Perinatal
lethality (%)
Adult
survival (%)
0
0
0
0
100
0
0
0
0
0
Each of the three anticoagulants is required for
regulation of the coagulation protease cascade
Mouse Models of TF
 Knock-out mice (mTF-/-) (1996) – embryonic lethal
 Low TF mice (mTF-/- / hTF+) (1998) – 1% hTF expression
 Cardiac specific TF mice – role of cardiomyocyte TF
 Floxed TF mice – permits cell type-specific deletion
 HCV mice (mTF-/-/HCV+)– wild-type levels of hTF
4/05
Generation of Low TF Mice
Complete deficiency of TF results in embryonic lethality
Step 1:
Make Transgenic Mice
hTF minigene hTF promoter
mTF+/+/hTF+
Step 2:
Rescue mTF-/- Embryos
X
mTF+/-
mTF+/-/hTF+
= Low TF Mice
1% expression
2/05
mTF-/-/hTF+
Hemostatic Balance
Procoagulant
TF
Anticoagulant
AT
APC
TFPI
Hemostatic Balance
Procoagulant
Anticoagulant
Do low levels of TF rescue TFPI-/- embryos ?
Genotype of Offspring at Wean Generated by
Intercrossing TFPI+/-/mTF+/-/hTF+/- Mice
Death due to no TF ( ) or no TFPI ( )
32_3/04
Conclusion
TFPI plays an essential role in life by
regulating TF:FVIIa activity
2/05
Does TFPI deficiency rescue
the hemostatic defects
in low TF mice ?
Tissue Specific Expression of TFPI
in Mouse Tissues
High
Medium
Low
placenta
heart
brain
lung
liver
uterus
spleen
kidney
thymus
testis
skin
TFPI Deficiency Prolongs the
Survival of Low TF Mice
4/05
TFPI Deficiency Rescues Some
Hemostatic Defects in Low TF Mice
Placenta
Lung
Heart
Low TF /
TFPI+/+
Low TF /
TFPI-/Pedersen et al. Blood 2005
Testis
Different thresholds of TF are
required for hemostasis in
different tissues
Role of TF in Thrombosis
Diseases Associated with
Pathological Expression of TF and
Life-Threatening Thrombosis
 Atherosclerosis
 Cancer
 Sepsis
38_3/04
Localization of TF in Normal and
Atherosclerotic Vessels
Normal
Atherosclerotic
Wilcox et al., Proc.Natl.Acad.Sci.USA 86:2839-2843, 1989
40_3/04
Blood Borne Tissue Factor:
Another View of Thrombosis
Giesen et al., Proc.Natl.Acad.Sci.USA 96:2311-2315, 1999
42_3/04
Controversy over Active Blood
Borne TF in Healthy Individuals
Nature Medicine (2004)
 Against: Butenas and Mann “... there is no reliable data
suggesting the presence of significant quantities of
functional TF in the blood of healthy individuals.”
 For: Bogdanov, Hathcock and Nemerson “It is
abundantly clear that shear forces caused by laminar
flow are involved in thrombogenesis.”
and
“Whole blood clotting times are performed using non
flow conditions.”
Contributions of Vessel Wall and Blood
Borne TF to Thrombosis
Two Models
 Carotid artery thrombosis model
(macrovasculature)
 Laser injury thrombosis model
(microvasculature)
Experiment
Analyze thrombosis in vivo in healthy
mice with different levels of
hematopoietic and non-hematopoietic
cell-derived TF
50_3/04
(pM TF)
Factor Xa Generation
Low TF Mice Have Low Levels
of Procoagulant Activity in the
Vessel Wall
WT
Low TF
6 wks
Bone marrow
transplantation
Lethal
irradiation
Donor
Recipient
Low TF
WT
Vessel wall
Low TF BM:WT
WT
Marrow
Normal
Low TF
WT BM:Low TF
51_3/04
Thrombosis
experiments
Normal
Carotid Artery Thrombosis Model
Flow (ml/min)
Rose Bengal
Time (min)
Biarritz Slide 16_6-03
Time (min)
Hematopoietic Cell-derived TF
Does Not Contribute to Thrombosis
Following Carotid Artery Injury
WT
7/04
Low TF Low TF WT BM:
BM:WT Low TF
The vessel wall is the major source
of TF in a carotid artery thrombosis
model using healthy mice
Real-time in vivo Imaging of Platelets,
Tissue Factor and Fibrin During
Thrombus Formation in the Mouse
Falati et al. Nature Medicine 2002
45_3/04
In vivo Thrombus Formation
Legend
WT Mouse
Bright Field
Tissue factor
Platelets
Fibrin
Merge
47_3/04
Low hTF Mice
Legend
Bright Field
Tissue factor
Platelets
Fibrin
Merge
48_3/04
 Lack mTF gene
 hTF mRNA and antigen ~1.0%
of WT levels
Donor
Low TF
Donor
Wild-type
7/04
Fibrin : Platelet (%WT)
Low TF
Recipient
Wild-type
Recipient
Low TF
Fibrin : Platelet (%WT)
Fibrin
Wild-type
Fibrin : Platelet (%WT)
Platelet
TF
Fibrin : Platelet (%WT)
Hematopoietic Cell-derived TF Contributes
to Microvascular Thrombosis
Hematopoietic Cell Derived TF
Contributes to Microvascular
Thrombosis
Role of TF in Thrombosis After Rupture
of an Atherosclerotic Plaque
TF
TF
TF
Diseases Associated with Elevated
Levels of TF-bearing Microparticles
 Acute coronary syndromes
 Sepsis
 Diabetes
 Sickle cell disease
 Cancer
TF Expression in Cancer
 Expression of TF
 Tumor cells
 Host cells, i.e. monocytes & endothelial cells
 Microparticles containing TF released by
various cells
 TF antigen levels correlated with disease
progression, malignancy and metastasis
Increased Procoagulant State in
Cancer Patients
Host’s inflammatory
response
Tumor released factors
e.g. cytokines & VEGF
TF Expression
+
Thombosis
Central venous catheter
Chemotherapy drugs
Surgery
 May increase TF PCA
Endogenous
Exogenous
 high rate of DVT
with catheter
 Prophylaxis not
provided prior to
DVT
Hypothesis
Blood borne TF is a marker of a prethrombotic state and may contribute
to thrombosis in cancer patients
Development of a New Functional Assay
to Measure Levels of Blood Borne TF
5ml blood collected from healthy individuals into
tubes containing sodium citrate (anti-coagulant) and
corn trypsin inhibitor (FXII inhibitor)
Whole blood + citrate + CTI
200 x g, 15min
Platelet rich
plasma (PRP)
20,000 x g, 15min
Microparticle (MP) +
platelet (P) pellet
TF activity (mU/mL)
Procoagulant activity
Levels of Blood Borne TF in Blood of
Healthy Individuals with or without LPS
Stimulation Ex Vivo
+ LPS
20
15
10
- LPS
n=6
5
0
0
TF Antibody
–1
2
+
–3
+4
5
Levels of Blood Borne TF in Cancer
Patients
TF activity (mU/mL)
Activity
Procoagulant
Cancer patients
2.00
1.60
1.20
Healthy
0.80
Arbitrary
Threshold
0.40
0.00
0.00
TF Antibody
1.00
–
2.00
+
3.00
–
4.00
+
5.00
Levels of blood borne TF may be a
useful marker of a pre-thrombotic
state in cancer patients
Role of Monocyte TF and
Endothelial Cell TF in LPS Induced
Coagulation
Role of Monocyte and EC TF in
LPS-induced Coagulation
Experimental Approaches
 Bone Marrow Transplantation
 Tethered anticoagulants
 Cell type-specific deletion
using loxP / CRE system
Mouse Model of Acute Endotoxemia
LPS (E. coli serotype 0111:B4)
10 mg/kg IP
59_3/04
Low TF Mice have Reduced Coagulation in
an Endotoxemia Model
2/05
6 wks
Bone marrow
transplantation
Lethal
irradiation
Donor
Recipient
Low TF
WT
WT
51_3/04
Endotoxemia
experiments
Vessel wall
Low TF BM:WT
Normal
WT BM:WT
Normal
Marrow
WT
Normal
TF Deficiency in Hematopoietic
Cells Reduces Coagulation
in Endotoxemic Mice
A
WT TF gene
539 bp
Mutant TF
gene 717 bp
WT
2/15/05
Low TF
Membrane-tethered Anticoagulants
Reduce LPS-induced Coagulation in Mice
human TFPI
CD4
P-sel
CD31 promoter
hirudin
Expression of fusion proteins in secretory granules
of EC, platelets and leukocytes
Chen et al. Blood 2004
Membrane-tethered Anticoagulants
Reduce LPS-induced Coagulation in Mice
 Tg mice had reduced LPS-induced
coagulation
 BMT experiments demonstrated that
expression of the fusion proteins on
EC was more effective in reducing
coagulation than expression on
platelets and leukocytes
Conclusion
Monocyte TF and EC TF contribute to
LPS-induced coagulation
Floxed TF Mice
Generation of a Chimeric Mouse
Containing a Floxed TF Allele
Role of Monocytes and EC TF in
LPS-induced Coagulation and
Inflammation
Analyze the effect of cell type-specific
deletion of the TF gene in monocytes
and endothelial cells
LysM–CRE
Tie 2-CRE
myeloid
EC
Acknowledgements
La Jolla
Boston
Michigan
R. Pawlinski
R. Tilley
B. Pedersen T.
Holscher M.
J. Choi
B. Furie
B.C. Furie
S. Day
W. Fay
Tencati
Funding provided by the National Institutes of Health
What are the relative contributions
of vessel wall TF and
hematopoietic cell-derived blood
borne TF to thrombosis in
healthy mice?