PHYSIOLOGIE DE LA COAGULATIONPHYSIOLOGIE DE LA COAGULATION

Pr Yesim DARGAUD

DIU d’Hémostase Biologique et Biochimie de l’Hémostase Lyon, 2015 01 26

Timing of hemostasis

Blood Blood CoagulationCoagulation

• activation of clotting factors

Blood Blood CoagulationCoagulation

• activation of clotting factors

FibrinolysisFibrinolysis

• activation of fibrinolytic factors

FibrinolysisFibrinolysis

• activation of fibrinolytic factors

PrimaryPrimaryHemostasisHemostasis

• vasoconstriction(immediately)

PrimaryPrimaryHemostasisHemostasis

• vasoconstriction(immediately)

IIII

NNNN

JJJJ clotting factors(seconds)

• fibrin formation(minutes)

clotting factors(seconds)

• fibrin formation(minutes)

fibrinolytic factors(immediately)

• clot lysis(hours)

fibrinolytic factors(immediately)

• clot lysis(hours)

(immediately)

• platelet adhesion(seconds)

• platelet aggregation(minutes)

(immediately)

• platelet adhesion(seconds)

• platelet aggregation(minutes)

JJJJ

UUUU

RRRR

YYYY

Voie IntrinsèqueTCA

FXI FXIa

FXII, KHPM, PK

FIX FIXaFVIIIa

FVIIa + FT

1. Modèle Plasmatique en « Cascade »

FVIIIa

FX FXaFVa

FII FIIa

Thrombine

Fibrinogène Fibrine

Voie extrinsèqueTP

FX

Ce modèle n’expliquepas pourquoi saignentles hémophiles

Voie Intrinsèque

FX FXIa

FXII, KHPM, PK

FIX FIXaFVIIIa Voie extrinsèque

FVIIa + FT

TCA allongé mais pas de signe hémorragique

TCA allongé mais hémorragie d’intensité variable

TCA allongé et syndrome hémorragique sévère

FVIIIa

FX FXaFVa

FII FIIaThrombine

Fibrinogène Fibrine

Voie extrinsèque

FX

Pourquoi les hémophiles saignent ?

Tissue FactorTissue Factor

ExtracellularExtracellularExtracellularExtracellular

TransTrans--membranemembrane

IntracellularIntracellular

FVIIa

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

FIXa

FX

Surfaces Cellulaires et Génération de Thrombine

FVIIa

FT

Site actif

Phase d’initiation

FVIIa FVIIaFIXa

FXa

Cellules exprimantle facteur tissulaire

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

Thrombine

FT

Monocytes activ.C. endoth. activ.Fibroblastes FXa

FII

(IIa: 10-17 – 10-14 mol/L)

A, Numeric simulations of the earliest events leadi ng to catalyst formation with a tissue factor insult.

Mann K G Circulation 2011;124:225-235

Copyright © American Heart Association

150

250

300

thro

mbi

n (n

M)

The amount of thrombin formed is more important

than the moment of clotting

0

50

thro

mbi

n

time (min)time (min)00 1515 3030

Initiation phaseInitiation phase

First traces of IIa≤ 10nM

5% of prothrombin converted to IIa

Clot formationClot formationTF

Mann KG et al Haemostaseology 2009

Thrombine

Amplification

FVIIa

FT

FXa

CellulesExprimant le FT

FXaFII

FV *

FIXa

FXIa

VIIIa

FX

Fibrine

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

Activation plaquettaire (PAR 1)

collagène

VWF

Plaquette

collagène

VWF

Plaquette

FVa

collagène

VWF

Plaquette

T

V W FRoberts et al. 2006

105 - 106 fois plus actif

Propagation

FIXaFXFVIIIa

FIXa

FX

Plaquetteactivée

PAR

GPIIb IIIa

Tenase

50 fois plus actif

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

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

FXa

FVIIaFT

FX

GPIb IX V

PAR

Amplification Cascade Model

• This preceding model can be interpreted as follows:

If each of 100 individual Factor VIIa enzymes

catalyzed the activation of 100 Factor Xa enzymes,

and each one of these 10,000 Factor Xa enzymes

catalyzed 100 thrombin activations, and so on -

there is a resulting million-fold activation from there is a resulting million-fold activation from

the start of the pathway to fibrinogen cleavage.

• It is this amplification process that allows a clot

to form rapidly and also illustrates the magnitude

that one defect in these steps could have on the

whole process

AmplificationAmplificationThe ‘snowball’ effectThe ‘snowball’ effect

FVIIa+TFFVIIa+TF

STARTSTART

ThrombinThrombin

STARTSTART

FINISHFINISHDargaud, 2012 01 24

Thrombine

Thrombine

Thrombine

Thrombine

Thrombine

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

FVa

FIIFXa

Plaquette activée

FXaThrombine

Thrombine

ThrombineThrombine

Thrombine

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

activée

Lyon, 2013 01 22

Pathways of prothrombin activation.

Mann K G Circulation 2011;124:225-235

Copyright © American Heart Association

Butenas S et al. Arterioscler Thromb Vasc Biol 2009;29:1989-1996

Copyright © American Heart Association

A numeric simulation of consecutive resupply of ele ctronic plasma reactants to a quiescent thrombus at 790, 1200, and 1800 seconds, which resu lts in a more intense thrombin

generation and provides higher concentrations of pr othrombinase (Xa=Va).

Mann K G Circulation 2011;124:225-235

Copyright © American Heart Association

Phenotypic Hetrogeneity in the Blood Coagulation System..

FII: 50-75-100-125-150 IU/dL

Thrombin Formation *

CHEST. 2003;124(3_suppl):4S-10S.

• An example is antithrombin, which

tightly binds and inactivates

thrombin - this complex is later

cleared from circulation in the

Anti-protease system

cleared from circulation in the

liver. The clinical administration

of heparin promotes the association

between antithrombin and thrombin.

This is the basis for use of heparin

as an anti-coagulant.

Mann KG, Chest 2003

Auto-regulation of thrombin

• Besides cleaving fibrinogen, thrombin

has forms complexes with an endothelial

cell protein receptor termed

thrombomodulin. Thrombomodulin and

calcium act as co-factors of thrombin calcium act as co-factors of thrombin

activation of Protein C, a Gla

containing protease. When activated,

Protein C in conjunction with another

protein co-factor, Protein S, will

proteolyze and inactivate Factors Va and

VIIIa.

Gα

Surfaces Cellulaires et Régulation de la Génération de Thrombine

FXa

FVIIaFT

CellulesExprimant le FT

FXa FX a

AT

FVIIIactivé

AT½ vie ≤ 1 min

en milieu plasmatique

Monroe DM et al. Blood Coag Fibrinolysis 1996 Briede JJ et al. Thromb Haemost 2001Hoffman M et al. Thromb Haemost 2001 Monroe D. ATVB 2005

Thrombine

PCa

FVactivé

TM

T

EPCR

collagène

TT

T

x1000

x20

A, A hypothetical construct displaying the potentia l molar concentrations of thrombomodulin in various regions of the vasculature.

Mann K G Circulation 2011;124:225-235

Copyright © American Heart Association

PS+TFPI dep et APC+

PS

TFPI

PS+TFPI

DIAGNOSIS ofbleeding disorders

aPTT, PT and other routinecoagulation assays

Global HaemostasisAssays

150

250

300

thro

mbi

n (n

M)

The amount of thrombin formed is more important

than the moment of clotting

0

50

thro

mbi

n

time (min)time (min)00 1515 3030

Initiation phaseInitiation phase

First traces of IIa≤ 10nM

5% of prothrombin converted to IIa

Clot formationClot formationTF

Fibrinogen ActivationPeptides cleaved by thrombin contain many Asp, Glu and sulfated-Tyrresidues, thus very highly negatively charged

Eb

A A

a a

D D

a

B B

b b

Fibrinogène

Eb

A A

a a

Thrombine

Libération des sites de polymérisations A et B

D

E

D

a

B B

b b

Monomère de fibrine

D

E

D D

E

D

D

E

D D

E

D D

E

D D D D

D

E

D D

E

D

Polymérisation des monomères de fibrines

Fibrine soluble

Factor XIIIa

• The "soft clot" of fibrin monomers is stabilized and converted into the final clot by covalent cross-linkages between specific glutamine residues on one monomer and lysine residues on another monomer. This peptide (amide) bond is catalyzed by activated Factor XIIIa, a transglutaminase:

D

E

D D

E

D

D

E

D D

E

D D

E

FXIII

D

E

D D

E

D

FXIIIaLiaisons covalentes

Fibrine stable insolubleFibrine stable insoluble

Clot Dissolving Pathway

Dissolving of Blood Clots:

Fibrinolysis