COAGULATION SANGUINEMECANISMES D’ACTION DU rFVIIaMECANISMES D’ACTION DU FEIBA

Yesim DARGAUD, MD, PhD

DIU d’Hémostase Clinique & Thrombose Lyon, 2015 01 22

Lyon, 2013 01 22

Timing of hemostasis

Blood Coagulation

• activation of clotting factors(seconds)

• fibrin formation(minutes)

Blood Coagulation

• activation of clotting factors(seconds)

• fibrin formation(minutes)

Fibrinolysis

• activation of fibrinolytic factors(immediately)

• clot lysis(hours)

Fibrinolysis

• activation of fibrinolytic factors(immediately)

• clot lysis(hours)

PrimaryHemostasis

• vasoconstriction(immediately)

• platelet adhesion(seconds)

• platelet aggregation(minutes)

PrimaryHemostasis

• vasoconstriction(immediately)

• platelet adhesion(seconds)

• platelet aggregation(minutes)

IIIIIIII

NNNNNNNN

JJJJJJJJ

UUUUUUUU

RRRRRRRR

YYYYYYYY

Lyon, 2013 01 22

Voie Intrinsèque

FX FXIa

FXII, KHPM, PK

FIX FIXaFVIIIa

FX FXaFVa

FII FIIaThrombine

Fibrinogène Fibrine

TCA

Voie extrinsèque

TP

FVIIa + FT

FX

TCAFVIII(VWF)

FIXFXIFXII

SAPLFg

TPFIIFVFVIIFXFg

Lyon, 2013 01 22

Voie Intrinsèque

FX FXIa

FXII, KHPM, PK

FIX FIXaFVIIIa

FX FXaFVa

FII FIIaThrombine

Fibrinogène Fibrine

Voie extrinsèqueFVIIa + FT

FX

TCA allongé mais pas de signe hémorragique

TCA allongé mais hémorragie d’intensité variable

TCA allongé et syndrome hémorragique sévère

Pourquoi les hémophiles saignent ?

Lyon, 2013 01 22

Tissue Factor

Extracellular

Trans-membrane

Intracellular Lyon, 2013 01 22

FVIIa FVIIa

[FVII] = 10nM[FVIIa] = 1-2% [FVII] ~0.1nM FIX

FIXa

FX

FXa

Cellules exprimantle facteur tissulaire

Surfaces Cellulaires et Génération de Thrombine

Monroe DM et al. Blood Coag Fibrinolysis 1996 Allen GA et al. Blood Coag Fibrinolysis 2001 Monkovic et al. Biochemistry 1990

FVIIa

FT

Site actif

Phase d’initiation

FVa

Thrombine

FT

Monocytes activ.C. endoth. activ.Fibroblastes FXa

FII

Lyon, 2013 01 22

Thrombine

Amplification

Oliver JA et al. Blood 2002 Hultin B. Blood 19 85 Hoffman et al. Thromb Haemost 2005

FVIIa

FT

FXa

CellulesExprimant le FT

FXaFII

Activation plaquettaire (PAR 1)

collagène

VWF

Plaquette

FV *

collagène

VWF

Plaquette

FVa

collagène

VWF

Plaquette

T

V W F

FIXa

FXIa

VIIIa

FX

Roberts et al. 2006

Fibrine

Lyon, 2013 01 22

105 - 106 fois plus actif

50 fois plus actif

>90% du FXa est produit par le complexe FVIIIa-FIXa sur les plaquettes activées

Propagation

Mann KG et al. Thromb Haemost 2003 Hockin MF et al J Clin Biochem 2002

FIXaFX

FXa

FVIIaFT

FX

FVIIIa

FIXa

FX

Plaquetteactivée

GPIb IX V

PAR

GPIIb IIIa

Tenase

Lyon, 2013 01 22

FXaThrombine

Thrombine

Thrombine

Thrombine

Thrombine

ThrombineThrombine

Thrombine

Thrombine

Propagation

>96% de la thrombine est synthétisée pendant la phasede PROPAGATION sur des PLAQUETTES activées

Prothrombinase 300 000 foisplus active que le FXa seul pour transformer le FII en Thrombine

FVa

FIIFXa

Plaquette activée

Lyon, 2013 01 22

AmplificationThe ‘snowball’ effect

FVIIa+TF

Thrombin

START

FINISHDargaud, 2012 01 24

Mechanisms of Action of rFVIIa

Lyon, 2013 01 22Yesim DARGAUD

MOA #1 Burst of the initiation phase of the coagulation systemPlatelet independent MOA

Monroe D. ATVB 2005;25:2463-9

Kd=50pM/L

Les doses cliniques utilisées supposent une autre hypothèse de méc. d’actionLyon, 2013 01 22

Kjalke M, et al. J Thromb Haemost 2007; 5: 774–780 Lisman T, et al. Blood 2003; 101: 1864–1870

+ FVIIa

no FVIIa

0.00.20.40.60.81.01.21.41.61.8

100 200 300 400 500

Bou

nd F

VIIa

FVIIa (nM)

Monroe D. Brit J Haematol 1997;99:542-7

Platelets

+ PAR1 peptide

+ FVIIa

MOA #2 : Platelet dependent MOAs of rFVIIa

Lyon, 2013 01 22

MOA #2 Activation of FX on the surface of activated platelets

30 nM

50 nM

20 nM

40 nM

10 nMNo VIIa

FX

a (n

M)

Time (minutes)

0

1

2

3

4

0 10 20 30 40

–FVIIa +FVIIa

FX

FXa

Platelets

+ FVIIa

+ FX

Monroe D. Brit J Haematol 1997;99:542-7

Lyon, 2013 01 22

MOA #2 Activation of FIX on the surface of activated platelets

Rel

ativ

e F

IXa

Time (minutes)

0.0

0.2

0.4

0.6

0 10 20 30 40

40 nM

30 nM

20 nM

10 nM

No rFVIIa

Gabriel DA. J Thromb Haemost 2004;2:1816-22

Lyon, 2013 01 22

rFVIIa promotes thrombin generation

Model system

+ FVIIa

Thr

ombi

n (n

M)

Time (minutes)

0

10

20

30

40

50

60

70

0 20 40 60 80

Normal+FVIIa

D. Monroe & Allen - 2004 Lyon, 2013 01 22

MOA: rFVIIa Enhances Thrombin Generation

0

10

20

30

40

50

60

70

80

90

0 10 20 30 40 50 60 70

time (min)

thro

mbi

n (n

M)

T0 T 30min

Lyon, 2013 01 22

Thrombine20nM

Thrombine0.5nM

Wolberg A et al. Blood reviews 2007

Fibrinogène 2mg/ml

L’IMPACT DE LA THROMBINE SUR LA STRUCTURE DU CAILLO T DE FIBRINE

Système PurifiéIn vitro

Lyon, 2013 01 22

MOA: rFVIIa Improves Fibrin Clot Structure

NC FVIII<1IU/dl rFVIIa 90µg/kg r FVIIa 270µg/kg

Dargaud Y et al. Semin Hematol 2008;45: S72-3

Lyon, 2013 01 22

MOA #3 rFVIIa and platelet adhesion to collagen type III (1600 s-1)

Control + 60 U/mL rFVIIa/FX/FII

Lyon, 2013 01 22

MOA #3 rFVIIa and platelet adhesion to collagen type III at low platelet count(25,000/µL)

Control + rFVIIa/FX/FII

Lyon, 2013 01 22

Platelet aggregation

EndotheliumvWF

Site of injury – exposed collagen

GpIb

Activatedplatelet

Platelet

GpIIb/IIIaFibrinogenGranule

contents

MOA #4 rFVIIa and platelet aggregation in patients with Glanzmann’s thrombasthenia

Lisman T, et al. Blood 2004;103:1720–7

Control 100 U/mL rFVIIa

Lyon, 2013 01 22

NN1731

• V158D/E296V/M298Q

SC

EGF2

Gla

Hoffman JTH 2011;9:759

Activité biochimique6x plus élevée querFVIIa

EGF1

Lyon, 2013 01 22

Lyon, 2013 01 22

Lyon, 2013 01 22

MECANISMES D’ACTION DU FEIBA

Turecek et al.haemophilia 2004

Varadi et al. J Thromb Haemost 2004

Mann KG, Chest 2003

WHY DO WE NEED GLOBAL HAEMOSTASIS ASSAYS ?

DIAGNOSIS ofbleeding disorders

aPTT, PT and other routinecoagulation assays

Tests correlated to the clinical outcome of patient s

Global HaemostasisAssays

1 - Thromboelastography

4,75°

detection�

software�

Young et al. Haemophilia 2006;12:598-604 KaolinDehmel et al. Haemophilia 2008; 1-7

Evaluation of thromboelastography for monitoring recombinant activated factor VII ex vivo in haemophilia A and B patients with inhibitors: a multicentre trial

Results: a clear concentration-response relationship was only detected for one patient

Conclusions: 1- thromboelastography may potentially be a clinically useful tool for monitoring rFVIIa but only when the baseline curve is significantly abnormal

2- test conditions may need to be optimized before TEG can be utilised for all inhibitor patients

Young et al. Blood Coagul Fibrinolysis 2008;19:276-82

Multicentre, open-label trial

Aim:to explore the dose-response relationship between rFVIIa concentration and thromboelastography parameters

2 - Thrombin Generation

Thrombin Generating Capacity and Clinical Bleeding Phenotype

0102030405060708090

100

0 10 20 30 40 50

Time (min)

Thro

mbi

n (n

M)

FVIII<1 IU/dl FVIII<1 IU/dl

Dargaud Y et al. Thromb Haemost 2005;93:475-80Beltran-Miranda CP et al. Haemophilia 2005;11:326-34Trossaert M et al. J Thromb Haemost 2008; 6:486-93

TF 1pMPL 4µM

CAT method

Dargaud Y 19.10.2010

Varadi K et al J Thromb Haemost 2003;1:2374-80 Negrier C et al Haemophilia 2006;12:48-53Van Veen JJ et al. Int J Lab Hem 2009 ; 31:189-98 Livnat T et al. Haemophilia 2008;14:282-6

Normal range (ETP)

Dargaud Y 19.10.2010

Thrombin Generation Assay

* A three-step protocol for individually tailoring of bypassing therapy

# Patient Elective Surgery and other invasive procedur es

12345678910

DEBSCAGASDUPDUPDUPDUPROYROYLAM

Lower limb amputationBilateral total knee arthroplastyTotal knee arthroplastyAnkle arthroplastyTotal left knee arthroplastyElbow synovectomyTotal right knee arthroplastyLaser cataract surgeryPartial colectomyElbow (radioactive) synovectomy

# Patient Elective Surgery and other invasive procedur es

1234567891011121314151617

DEBSCAGASDUPDUPDUPDUPROYROYLAMSLAJHAKFSENFSENDUPDUPDUP

Lower limb amputationBilateral total knee arthroplastyTotal knee arthroplastyAnkle arthroplastyTotal left knee arthroplastyElbow synovectomyTotal right knee arthroplastyLaser cataract surgeryPartial colectomyElbow (radioactive) synovectomyImplantable venous access port insertionTotal hip arthroplastyUreteroscopic lithotripsyHemorrhoidectomyExplantation of infected total knee arthroplastyTotal knee arthroplastyEmbolization of the hepatic artery aneurysm

Ankle Arthroplasty

In vitro

FVIII < 1 IU/dlAb = 18 BU/ml

TF 1pMPL 4µMCTI 1.45µMCAT method

PRP: Platelet-rich plasmaPPP: Platelet-poor plasma

0

200

400

600

800

1000

1200

1400

1600

0 2 4 6 8 10

temps (heure)

ETP

(nM

.min

)

Ex vivo

Dargaud et al. Blood 2010

0

500

1000

1500

2000

2500

3000

0 10 20 30 40 50 60

time (hour)

ETP

(nM

.min

)

0

20

40

60

80

100

120

140

0 10 20 30 40 50 60

TIME (H)

Hb

(g/d

l)

D0 D1D2

0

20

40

60

80

100

120

140

0 10 20 30 40 50 60 70 80 90

time (min)

thro

mbi

n (n

M)

T0 T30min T 6H 2ème inj Feiba T 8h (residuel)

Ankle Arthroplasty

TF 1pMPL 4µMCTI 1.45µMCAT method

Dargaud et al. Blood 2010

Patient 1

Patient born in 1965Severe Hemophilia A with inhibitors anti-FVIII Ab + since 1973Multiple hemophilic arthropathies (elbows, knees, ankles)On demand treatment with FEIBA Tooth extraction and anal fistula surgery with Feiba in 1996In 1996, rFVIIa : clinically no responder

In 2001: bilateral ankle arthrodesis with FEIBA 75 U/kg/8hHb before surgery 123 g/LHb post-operative D1 54 g/L → Transfusion 3 packs of RCC

2004: Bilateral Total Knee Arthroplasty

In vitro

0

50

100

150

200

250

300

0 5 10 15 20 25 30 35 40 45 50

Time (min)

Thr

ombi

n (n

M)

T0 T30min T 1h T 3h T 6h T 8h T 12h Control

Ex vivoaPCC

Dargaud Y. et al. Haemophilia, 2005;11:552-8

FVIII< 1 IU/dlAb= 75 BU/ml

0200400600800

100012001400160018002000

0 100 200 300

rFVIIa (µg/kg)

ETP

(nM

.min

)

rFVIIa

0200400600800

100012001400160018002000

0 20 40 60 80 100 120

Feiba (U/ml)

ET

P (

nM.m

in)

Feiba

TF 1pMPL 4µMCTI 1.45µMCAT method

Dargaud Y. et al. Haemophilia, 2005;11:552-8

77,9

9,6

36

6

23

5,2

17,7

3,5

20

3,7 1,8

11,8

0

10

20

30

40

50

60

70

80

90

0

preoperative period from day-12 to day 0 (days)

FV

III in

hibi

tor le

vels

(BU

)

d-12 d- 11 d-8 d-6 d-3 d-1 d0

Surgery

FVIIIconcentrate

Perioperative Monitoring of FVIII & aPCC

Patient 2

Patient born in 1967Severe Hemophilia A with inhibitors anti-FVIII Ab + since 1975Severe hemophilic arthropathy of the left knee

on demand treatment with rFVIIa 90µg/kgFEIBA: partial clinical efficacy on hemarthroses

In 2001: 3 teeth extraction with rFVIIa 90µg/kgHb before surgery 153 g/LHb post-operative D2 70 g/L

In 2001: Total knee arthroplasty with rFVIIa 120µg/kgBlood loss during surgery : 1200 ccBlood loss on post-operative D2 : 2760 ccMassive hematoma with dehiscence, skin necrosis and infectionProsthetic joint infection

2007: Lower Limb Amputation in a Severe Haemophilia A Patient With Inhibitors

0200400600800

100012001400160018002000

0 50 100 150 200 250 300

rFVIIa (µg/kg)

ET

P (

nM.m

in)

PRP

PPP

In vitro

0

500

1000

1500

2000

0 50 100 150 200 250 300

rFVIIa (µg/kg)

ET

P (

nM.m

in) PRP

PPP

0

200

400

600

800

1000

1200

1400

0 50 100 150

time (minutes)E

TP

(nM

.min

)

PRP

PPP

Ex vivo rFVIIa 200µg/kg

FVIII < 1 IU/dlAb = 21 BU/ml

Dargaud Y. Haemophilia 2008;14 s4:20-7

PRP: Platelet-rich plasmaPPP: Platelet-poor plasma

TF 1pMPL 4µMCTI 1.45µMCAT method

Dargaud Y. Haemophilia 2008;14 s4:20-7

Perioperative Monitoring of rFVIIa Therapy

0

200

400

600

800

1000

1200

1400

1600

0 60 120 180 240 300 360

time (min)

ET

P (

nM.m

in)

PRP

PPP

Control

rFVIIa200µg/kg

rFVIIa200µg/kg

rFVIIa90µg/kg

rFVIIa 90µg/kg

Surgery

TF 1pMPL 4µMCTI 1.45µMCAT method

Dargaud Y 19.10.2010

0

200

400

600

800

1000

1200

1400

0 1 2 3 4 5 6 7 8 9 10 11

time (days)

ET

P (

nM.m

in II

a)

PRP PPP Control

/2H/3H

/4H rFVIIa 20µg/kg/h

0

20

40

60

80

100

120

140

160

0 1 2 3 4 5 6 7 8 9 10 11

time (days)

Hb

(g/d

l)

0

2000

4000

6000

8000

0 1 2 3 4 5 6 7 8 9

Time (days)

FV

II:C

(IU

/dl)

Dargaud Y. Haemophilia 2008;14 s4:20-7

TF 1pMPL 4µMCTI 1.45µMCAT method

Dargaud Y 19.10.2010

0

50

100

150

200

250

300

0 5 10 15 20 25 30 35 40 45 50

time (min)

thro

mbi

n (n

M)

PPP rFVIIa PPP Feiba

FVIII <1 UI/dLAc anti-FVIII = 24 UB/mL

Cataract Surgery and Partial Colectomy

Dargaud et al. Blood 2010;116:5734-7