Nigel Mackman, Ph.D.Nigel Mackman, Ph.D.

The Scripps Research InstituteThe Scripps Research Institute

La JollaLa Jolla

Molecular Events in ThrombosisMolecular Events in Thrombosis

Tissue Factor-Factor VIIaInitiated Coagulation

5/05

4_05

Tissue Factor Pathway Inhibitor (TFPI)Tissue Factor Pathway Inhibitor (TFPI)

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 MiceLessons from KO Mice

6_3/04

Phenotype of Mouse KOs Deficient in Phenotype of Mouse KOs Deficient in Clotting Factors and PARsClotting Factors and PARs

KOKOAdult Adult

survival (%)survival (%)

TFTF

VIIVII

XIXI

IXIX

VIIIVIII

Perinatal Perinatal lethality (%)lethality (%)

Embryonic Embryonic lethality (%) lethality (%)

100100

100100

00

00

00

00

00

100100

100100

100100

Extrinsic Extrinsic PathwayPathway

Intrinsic Intrinsic PathwayPathway

PAR-4/FibrinogenPAR-4/Fibrinogen

PAR-1PAR-1

FibrinogenFibrinogen

PAR-4PAR-47575

00

2525

100100

100100

00

00

5050

9090

00

00

00

00

00

00

00

5050

(MT)(MT)

VVXXCommon Common

PathwayPathway

5050

1010

100100

100100

00

00

Prothrombin 5050 100100 00

Phenotype of Clotting Factor KOs Phenotype of Clotting Factor KOs and PAR KOsand PAR KOs

Phenotype of Anticoagulant KOsPhenotype of Anticoagulant KOs

KOKOPerinatal Perinatal

lethality (%)lethality (%) Embryonic Embryonic

lethality (%) lethality (%)

100100

100100

100100

100100

00

TFPITFPI

ATAT

TMTM

EPCREPCR

PCPC

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

Adult Adult survival (%)survival (%)

00

00

00

00

00(MT)(MT)

00

00

00

00

100100

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

= Low TF Mice= Low TF Mice1% expression1% expression

Step 1:Step 1:

mTFmTF+/++/+/hTF/hTF++

Make Transgenic MiceMake Transgenic Mice

hTF minigenehTF minigene

Step 2:Step 2: Rescue mTFRescue mTF-/- -/- EmbryosEmbryos

mTFmTF+/-+/-/hTF/hTF++ mTFmTF+/-+/-

XX

mTFmTF-/--/-/hTF/hTF++

Generation of Low TF MiceGeneration of Low TF Mice

2/05

Complete deficiency of TF results in embryonic lethalityComplete deficiency of TF results in embryonic lethality

hTF promoterhTF promoter

Hemostatic BalanceHemostatic Balance

AT AT APC APC

TFPI TFPI

TFTF

ProcoagulantProcoagulant AnticoagulantAnticoagulant

Hemostatic BalanceHemostatic Balance

ProcoagulantProcoagulant AnticoagulantAnticoagulant

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

32_3/04

Genotype of Offspring at Wean Generated by Genotype of Offspring at Wean Generated by Intercrossing TFPIIntercrossing TFPI+/-+/-/mTF/mTF+/-+/-/hTF/hTF+/-+/- Mice Mice

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

ConclusionConclusion

TFPI plays an essential role in life by TFPI plays an essential role in life by

regulating TF:FVIIa activityregulating TF:FVIIa activity

2/05

Does TFPI deficiency rescue Does TFPI deficiency rescue the hemostatic defectsthe hemostatic defects

in low TF mice ? in low TF mice ?

Tissue Specific Expression of TFPI Tissue Specific Expression of TFPI in Mouse Tissuesin Mouse Tissues

HighHigh LowLow

placentaplacenta brain brain

liver liver

spleen spleen

thymus thymus

skinskin

MediumMedium

heart heart

lung lung

uterus uterus

kidney kidney

testistestis

TFPI Deficiency Prolongs the TFPI Deficiency Prolongs the Survival of Low TF MiceSurvival of Low TF Mice

4/05

TFPI Deficiency Rescues Some TFPI Deficiency Rescues Some Hemostatic Defects in Low TF MiceHemostatic Defects in Low TF Mice

HeartHeartLungLungPlacentaPlacenta

Pedersen et al. Pedersen et al. Blood Blood 20052005

Low TF / TFPI+/+

Low TF / TFPI-/-

TestisTestis

Different thresholds of TF are Different thresholds of TF are required for hemostasis in required for hemostasis in

different tissuesdifferent tissues

Role of TF in ThrombosisRole of TF in Thrombosis

AtherosclerosisAtherosclerosis

CancerCancer

SepsisSepsis

Diseases Associated with Diseases Associated with Pathological Expression of TF and Pathological Expression of TF and

Life-Threatening Thrombosis Life-Threatening Thrombosis

38_3/04

Localization of TF in Normal and Localization of TF in Normal and Atherosclerotic VesselsAtherosclerotic Vessels

40_3/04

NormalNormal AtheroscleroticAtherosclerotic

Wilcox et al., Proc.Natl.Acad.Sci.USA 86:2839-2843, 1989

Blood Borne Tissue Factor: Blood Borne Tissue Factor: Another View of ThrombosisAnother View of Thrombosis

Giesen Giesen et al., Proc.Natl.Acad.Sci.USA et al., Proc.Natl.Acad.Sci.USA 96:2311-2315, 199996:2311-2315, 1999

42_3/04

Controversy over Active Blood Controversy over Active Blood Borne TF in Healthy IndividualsBorne TF in Healthy Individuals

Against:Against: Butenas and MannButenas and Mann “... there is no reliable data “... there is no reliable data suggesting the presence of significant quantities of suggesting the presence of significant quantities of functional TF in the blood of healthy individuals.”functional TF in the blood of healthy individuals.”

For:For: Bogdanov, Hathcock and Nemerson Bogdanov, Hathcock and Nemerson “It is “It is abundantly clear that shear forces caused by laminar abundantly clear that shear forces caused by laminar flow are involved in thrombogenesis.”flow are involved in thrombogenesis.”

and and

“Whole blood clotting times are performed using non flow conditions.”

Nature MedicineNature Medicine (2004) (2004)

Contributions of Vessel Wall and Contributions of Vessel Wall and Blood Borne TF to ThrombosisBlood Borne TF to Thrombosis

Two ModelsTwo Models

Carotid artery thrombosis model Carotid artery thrombosis model (macrovasculature)(macrovasculature)

Laser injury thrombosis model Laser injury thrombosis model (microvasculature)(microvasculature)

Analyze thrombosis Analyze thrombosis in vivo in vivo in healthy in healthy mice with different levels of mice with different levels of

hematopoietic and non-hematopoietic hematopoietic and non-hematopoietic cell-derived TFcell-derived TF

50_3/04

ExperimentExperiment

Low TF Mice Have Low Levels Low TF Mice Have Low Levels of Procoagulant Activity in the of Procoagulant Activity in the

Vessel Wall Vessel Wall

Fac

tor

Xa

Gen

erat

ion

Fac

tor

Xa

Gen

erat

ion

(pM

TF

)(p

M T

F)

WTWT Low TFLow TF

Low TFLow TF

Low TF BM:Low TF BM:WTWT

WT WT

NormalNormal

WT BM:WT BM:Low TFLow TF

WTWT Low TFLow TF

NormalNormal

ThrombosisThrombosisexperimentsexperiments

6 wks6 wks

LethalLethalirradiationirradiation

Bone marrowBone marrowtransplantationtransplantation

51_3/04

Vessel wall MarrowDonor Recipient

Carotid Artery Thrombosis ModelCarotid Artery Thrombosis Model

Rose BengalRose Bengal

Time (min)Time (min)

Flo

w (

ml/

min

)F

low

(m

l/m

in)

Biarritz Slide 16_6-03

Hematopoietic Cell-derived TF Hematopoietic Cell-derived TF Does Not Contribute to Thrombosis Does Not Contribute to Thrombosis

Following Carotid Artery InjuryFollowing Carotid Artery InjuryT

ime

(min

)T

ime

(min

)

WTWT Low TFLow TF Low TFLow TF BM:BM:WTWT

WT BM:WT BM: Low TFLow TF

7/04

The vessel wall is the major source The vessel wall is the major source of TF in a carotid artery thrombosis of TF in a carotid artery thrombosis

model using healthy micemodel using healthy mice

Real-time Real-time in vivo in vivo Imaging of Platelets, Imaging of Platelets, Tissue Factor and Fibrin During Tissue Factor and Fibrin During

Thrombus Formation in the MouseThrombus Formation in the Mouse Falati et al. Falati et al. Nature MedicineNature Medicine 2002 2002

45_3/04

Bright FieldBright Field

Tissue factorTissue factor

PlateletsPlatelets

FibrinFibrin

MergeMerge

In vivoIn vivo Thrombus Formation Thrombus Formation

WT Mouse WT Mouse

LegendLegend

47_3/04

Low hTF MiceLow hTF Mice

Lack mTF geneLack mTF gene hTF mRNA and antigen ~1.0% hTF mRNA and antigen ~1.0%

of WT levelsof WT levels

Bright FieldBright Field

Tissue factorTissue factor

PlateletsPlatelets

FibrinFibrin

MergeMerge

LegendLegend

48_3/04

Donor Low TF

Donor Wild-type

Fibr

in :

Pla

tele

t (%

WT)

Fibr

in :

Pla

tele

t (%

WT)

Fibr

in :

Pla

tele

t (%

WT)

Fibr

in :

Pla

tele

t (%

WT)

Recipient Wild-type

Recipient Low TF

Low TF

Wild-type

7/04

Hematopoietic Cell-derived TF Contributes Hematopoietic Cell-derived TF Contributes to Microvascular Thrombosis to Microvascular Thrombosis

Platelet

TF

Fibrin

Hematopoietic Cell Derived TF Hematopoietic Cell Derived TF Contributes to Microvascular Contributes to Microvascular

ThrombosisThrombosis

Role of TF in Thrombosis After Rupture Role of TF in Thrombosis After Rupture of an Atherosclerotic Plaqueof an Atherosclerotic Plaque

TFTF

TF

Diseases Associated with Elevated Diseases Associated with Elevated Levels of TF-bearing MicroparticlesLevels of TF-bearing Microparticles

Acute coronary syndromesAcute coronary syndromes

SepsisSepsis

DiabetesDiabetes

Sickle cell diseaseSickle cell disease

CancerCancer

Expression of TFExpression of TF Tumor cellsTumor cells Host cells, i.e. monocytes & endothelial cellsHost cells, i.e. monocytes & endothelial cells

Microparticles containing TF released by Microparticles containing TF released by various cells various cells

TF antigen levels correlated with disease TF antigen levels correlated with disease progression, malignancy and metastasisprogression, malignancy and metastasis

TF Expression in CancerTF Expression in Cancer

Increased Procoagulant State in Increased Procoagulant State in Cancer PatientsCancer Patients

Central venous catheterCentral venous catheter

high rate of DVT high rate of DVT with catheterwith catheter

Prophylaxis not Prophylaxis not provided prior to provided prior to DVTDVT

Chemotherapy drugs

May increase TF PCAMay increase TF PCA

TF Expression

Thombosis

Tumor released factors Tumor released factors e.g. cytokines & VEGFe.g. cytokines & VEGF

Host’s inflammatory Host’s inflammatory responseresponse

SurgerySurgery

Endogenous Exogenous

+

HypothesisHypothesis

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

cancer patientscancer patients

Development of a New Functional Assay Development of a New Functional Assay to Measure Levels of Blood Borne TFto Measure Levels of Blood Borne TF

Whole blood + citrate + CTI

Platelet rich plasma (PRP)

Microparticle (MP) + platelet (P) pellet

200 x 200 x g, g, 15min15min

20,000 x 20,000 x g, g, 15min15min

5ml blood collected from healthy individuals into 5ml blood collected from healthy individuals into tubes containing sodium citrate (anti-coagulant) and tubes containing sodium citrate (anti-coagulant) and

corn trypsin inhibitor (FXII inhibitor)corn trypsin inhibitor (FXII inhibitor)

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

Stimulation Ex VivoStimulation Ex Vivo

0

5

10

15

20

0 1 2 3 4 5

TF a

ctiv

ity (m

U/m

L)

+ LPS

- LPS

Pro

coag

ula

nt

acti

vity

n = 6

– + – +TF Antibody

Levels of Blood Borne TF in Cancer Levels of Blood Borne TF in Cancer PatientsPatients

Arbitrary Threshold

0.00

0.40

0.80

1.20

1.60

2.00

0.00 1.00 2.00 3.00 4.00 5.00

TF

act

ivity

(m

U/m

L)

Healthy

Cancer patients

Pro

coag

ula

nt

Act

ivit

y

– + – +TF Antibody

Levels of blood borne TF may be a Levels of blood borne TF may be a useful marker of a pre-thrombotic useful marker of a pre-thrombotic

state in cancer patientsstate in cancer patients

Role of Monocyte TF and Role of Monocyte TF and Endothelial Cell TF in LPS Induced Endothelial Cell TF in LPS Induced

Coagulation Coagulation

Role of Monocyte and EC TF in Role of Monocyte and EC TF in LPS-induced CoagulationLPS-induced Coagulation

Bone Marrow Transplantation

Tethered anticoagulants

Cell type-specific deletion using loxP / CRE system

Experimental ApproachesExperimental Approaches

Mouse Model of Acute EndotoxemiaMouse Model of Acute Endotoxemia

LPS (LPS (E. coliE. coli serotype 0111:B4) serotype 0111:B4)10 mg/kg IP10 mg/kg IP

59_3/04

Low TF Mice have Reduced Coagulation in Low TF Mice have Reduced Coagulation in an Endotoxemia Modelan Endotoxemia Model

2/05

Vessel wall Marrow

Low TFLow TF

Low TF BM:Low TF BM:WTWT

WT WT

NormalNormal

WT BM:WT BM:WTWT

WT

NormalNormal

Donor Recipient

EndotoxemiaEndotoxemiaexperimentsexperiments

6 wks6 wks

LethalLethalirradiationirradiation

Bone marrowBone marrowtransplantationtransplantation

51_3/04

NormalNormal

WT WT

2/15/05

TF Deficiency in Hematopoietic TF Deficiency in Hematopoietic Cells Reduces Coagulation Cells Reduces Coagulation

in Endotoxemic Micein Endotoxemic Mice

WT Low TF

A539 bp

WT TF gene

717 bp Mutant TF gene

Membrane-tethered Anticoagulants Membrane-tethered Anticoagulants Reduce LPS-induced Coagulation in MiceReduce LPS-induced Coagulation in Mice

CD31 promoterhuman TFPI CD4 P-sel

hirudin

Expression of fusion proteins in secretory granules of EC, platelets and leukocytes

Chen et al. Blood 2004

Tg mice had reduced LPS-induced Tg mice had reduced LPS-induced coagulationcoagulation

BMT experiments demonstrated that BMT experiments demonstrated that expression of the fusion proteins on expression of the fusion proteins on EC was more effective in reducing EC was more effective in reducing coagulation than expression on coagulation than expression on platelets and leukocytesplatelets and leukocytes

Membrane-tethered Anticoagulants Membrane-tethered Anticoagulants Reduce LPS-induced Coagulation in MiceReduce LPS-induced Coagulation in Mice

ConclusionConclusion

Monocyte TF and EC TF contribute to Monocyte TF and EC TF contribute to

LPS-induced coagulationLPS-induced coagulation

Floxed TF Mice Floxed TF Mice

Generation of a Chimeric Mouse Generation of a Chimeric Mouse Containing a Floxed TF Allele Containing a Floxed TF Allele

Role of Monocytes and EC TF in Role of Monocytes and EC TF in LPS-induced Coagulation and LPS-induced Coagulation and

InflammationInflammation

LysM–CRELysM–CRE

Tie 2-CRETie 2-CRE

myeloidmyeloid

ECEC

Analyze the effect of cell type-specific Analyze the effect of cell type-specific deletion of the TF gene in monocytes deletion of the TF gene in monocytes

and endothelial cellsand endothelial cells

AcknowledgementsAcknowledgements

La JollaLa Jolla

Funding provided by the National Institutes of HealthFunding provided by the National Institutes of Health

R. Pawlinski R. Pawlinski

R. Tilley R. Tilley

B. Pedersen B. Pedersen

T. Holscher T. Holscher

M. Tencati M. Tencati

BostonBoston

J. Choi J. Choi

B. Furie B. Furie

B.C. B.C.

FurieFurie

MichiganMichigan

S. Day S. Day

W. W.

FayFay

What are the relative contributions What are the relative contributions of vessel wall TF and of vessel wall TF and

hematopoietic cell-derived blood hematopoietic cell-derived blood borne TF to thrombosis in borne TF to thrombosis in

healthy mice? healthy mice?