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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.

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molecular events in thrombosis

Molecular Events in Thrombosis

Nigel Mackman, Ph.D.

The Scripps Research Institute

La Jolla

recombinant factor viia novo seven as a hemostatic agent
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

phenotype of mouse kos deficient in clotting factors and pars
Phenotype of Mouse KOs Deficient in Clotting Factors and PARs

Embryonic lethality (%)

Perinatal lethality (%)

Adult survival (%)

KO

90

0

0

0

0

TF

VII

XI

IX

VIII

100

100

0

0

0

0

0

100

100

100

Extrinsic Pathway

(MT)

Intrinsic Pathway

50

10

100

100

0

0

V

X

Common Pathway

Prothrombin

50

100

0

Fibrinogen

PAR-4

0

0

75

0

25

100

0

50

PAR-4/Fibrinogen

PAR-1

100

0

0

50

phenotype of anticoagulant kos
Phenotype of Anticoagulant KOs

Embryonic lethality (%)

Perinatal lethality (%)

Adult survival (%)

KO

TFPI

AT

TM

EPCR

PC

100

100

100

100

0

0

0

0

0

0

0

0

0

0

100

(MT)

Each of the three anticoagulants is required for regulation of the coagulation protease cascade

mouse models of tf
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

slide13

Generation of Low TF Mice

Complete deficiency of TF results in embryonic lethality

Step 1:

Make Transgenic Mice

hTF promoter

hTF minigene

mTF+/+/hTF+

Step 2:

Rescue mTF-/- Embryos

X

mTF+/-

mTF+/-/hTF+

= Low TF Mice

1% expression

mTF-/-/hTF+

2/05

hemostatic balance

Procoagulant

Anticoagulant

AT APC TFPI

TF

Hemostatic Balance
hemostatic balance1
Hemostatic Balance

Procoagulant

Anticoagulant

Do low levels of TF rescue TFPI-/- embryos ?

genotype of offspring at wean generated by intercrossing tfpi mtf htf mice
Genotype of Offspring at Wean Generated by Intercrossing TFPI+/-/mTF+/-/hTF+/- Mice

Death due to no TF ( ) or no TFPI ( )

32_3/04

conclusion
Conclusion

TFPI plays an essential role in life by regulating TF:FVIIa activity

2/05

tissue specific expression of tfpi in mouse tissues
Tissue Specific Expression of TFPI in Mouse Tissues

High

Medium

Low

placenta

heart lung uterus kidney testis

brain liver spleen thymus skin

tfpi deficiency rescues some hemostatic defects in low tf mice
TFPI Deficiency Rescues Some Hemostatic Defects in Low TF Mice

Placenta

Lung

Heart

Testis

Low TF / TFPI+/+

Low TF / TFPI-/-

Pedersen et al. Blood 2005

slide24

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
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
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
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.”

two models
Two Models
  • Carotid artery thrombosis model (macrovasculature)
  • Laser injury thrombosis model (microvasculature)
slide30

Experiment

Analyze thrombosis in vivo in healthy mice with different levels of hematopoietic and non-hematopoietic cell-derived TF

50_3/04

low tf mice have low levels of procoagulant activity in the vessel wall
Low TF Mice Have Low Levels of Procoagulant Activity in the Vessel Wall

(pM TF)

Factor Xa Generation

WT

Low TF

slide32

6 wks

Thrombosis

experiments

Bone marrow

transplantation

Lethal

irradiation

Donor

Recipient

Vessel wall

Marrow

Low TF

WT

Low TF BM:WT

Normal

Low TF

WT

WT BM:Low TF

Normal

51_3/04

slide33

Carotid Artery Thrombosis Model

Rose Bengal

Flow (ml/min)

Time (min)

Biarritz Slide 16_6-03

hematopoietic cell derived tf does not contribute to thrombosis following carotid artery injury
Hematopoietic Cell-derived TF Does Not Contribute to Thrombosis Following Carotid Artery Injury

Time (min)

WT

Low TF

Low TFBM:WT

WT BM:Low TF

7/04

slide35

The vessel wall is the major source of TF in a carotid artery thrombosis model using healthy mice

slide36
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

slide37

Bright Field

Tissue factor

Platelets

Fibrin

Merge

In vivo Thrombus Formation

Legend

WT Mouse

47_3/04

low htf mice

Bright Field

Tissue factor

Platelets

Fibrin

Merge

Low hTF Mice

Legend

  • Lack mTF gene
  • hTF mRNA and antigen ~1.0% of WT levels

48_3/04

hematopoietic cell derived tf contributes to microvascular thrombosis
Hematopoietic Cell-derived TF Contributes to Microvascular Thrombosis

Wild-type

Fibrin : Platelet (%WT)

Platelet

TF

Fibrin

Low TF

Fibrin : Platelet (%WT)

Donor Low TF

Recipient Wild-type

Fibrin : Platelet (%WT)

Donor Wild-type

Recipient Low TF

Fibrin : Platelet (%WT)

7/04

slide41

TF

TF

TF

Role of TF in Thrombosis After Rupture of an Atherosclerotic Plaque

diseases associated with elevated levels of tf bearing microparticles
Diseases Associated with Elevated Levels of TF-bearing Microparticles
  • Acute coronary syndromes
  • Sepsis
  • Diabetes
  • Sickle cell disease
  • Cancer
tf expression in 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

high rate of DVT with catheter

  • Prophylaxis not provided prior to DVT
  • May increase TF PCA
Increased Procoagulant State in Cancer Patients

Tumor released factors e.g. cytokines & VEGF

Host’s inflammatory response

TF Expression

Thombosis

+

Endogenous

Exogenous

Chemotherapy drugs

Central venous catheter

Surgery

hypothesis
Hypothesis

Blood borne TF is a marker of a pre-thrombotic state and may contribute to thrombosis in cancer patients

development of a new functional assay to measure levels of blood borne tf
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

levels of blood borne tf in blood of healthy individuals with or without lps stimulation ex vivo

+ LPS

- LPS

Levels of Blood Borne TF in Blood of Healthy Individuals with or without LPS Stimulation Ex Vivo

Procoagulant activity

n = 6

TF Antibody

+

+

levels of blood borne tf in cancer patients

Cancer patients

Healthy

Procoagulant Activity

Arbitrary Threshold

+

+

TF Antibody

Levels of Blood Borne TF in Cancer Patients
slide49

Levels of blood borne TF may be a useful marker of a pre-thrombotic state in cancer patients

role of monocyte and ec tf in lps induced coagulation
Role of Monocyte and EC TF in LPS-induced Coagulation
  • Bone Marrow Transplantation
  • Tethered anticoagulants
  • Cell type-specific deletion using loxP / CRE system

Experimental Approaches

mouse model of acute endotoxemia
Mouse Model of Acute Endotoxemia

LPS (E. coli serotype 0111:B4)

10 mg/kg IP

59_3/04

slide55

6 wks

Endotoxemia

experiments

Bone marrow

transplantation

Lethal

irradiation

Donor

Recipient

Vessel wall

Marrow

Low TF

WT

Low TF BM:WT

Normal

WT

WT

WT BM:WT

Normal

Normal

51_3/04

tf deficiency in hematopoietic cells reduces coagulation in endotoxemic mice
TF Deficiency in Hematopoietic Cells Reduces Coagulation in Endotoxemic Mice

A

WT TF gene

539 bp

Mutant TF gene

717 bp

WT

Low TF

2/15/05

membrane tethered anticoagulants reduce lps induced coagulation in mice
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 mice1
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
conclusion1

Conclusion

Monocyte TF and EC TF contribute to LPS-induced coagulation

role of monocytes and ec tf in lps induced coagulation and inflammation
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

myeloid

Tie 2-CRE

EC

slide63

Acknowledgements

Michigan

La Jolla

Boston

S. Day W. Fay

R. Pawlinski R. Tilley B. Pedersen T. Holscher M. Tencati

J. Choi B. Furie B.C. Furie

Funding provided by the National Institutes of Health

slide64

What are the relative contributions of vessel wall TF and hematopoietic cell-derived blood borne TF to thrombosis in healthy mice?

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