The modern concept of blood

coagulation and methods for

laboratory evaluation

I. ELALAMY & G. T. GEROTZIAFAS

Service d’Hématologie Biologique

HOPITAL TENON – ER2 UPMC PARIS

Plaque Rupture or Vascular Lesion

Hemostasis Initiation

Endothelium lesion…

Smooth muscle cells exposure… Tissue Factor expression…

Collagen exposure…

Endothelial injury, Platelet adhesionPlatelet activation

Platelet AggregationThrombus formation

Platelet Adhesion ThrombusFormation

Normal situation

Endothelial Cells

Sub-Endothelium

Plaquettes

Adapted from Ferguson JJ. Platelet physiology. In: Ferguson JJ, Chronos N, Harrington RA (Eds).

Antiplatelet Therapy in Clinical Practice. London: Martin Dunitz; 2000: pp.15–35.

PLATELET PRIORITY = VASCULAR INTEGRITY

Mediating …

Adhesion

Facilitating

Secretion

Receptors…

Allowing

Aggregation

Calvo C. www.platelets.se 2009

Endothelium

PLATELET

GpIb

Sub-endothelium (collagen)

VWF

PLATELET ADHESION

SHAPE CHANGE = CYTOSKELETON

Microtubules Microfilaments

Tubulin

Actin/Myosin

POP-CORN PLATELETS

Platelet Page : www.akh.wien.ac.at

SHAPE CHANGE = PSEUDOPODS

Platelet Page : www.akh.wien.ac.at

GRANULAR SECRETION = AMPLIFICATION

Granules

ADPATPpyrophosphateserotonincalcium +++

a GranulesPF4, βTG…PDGF, EGF, VEGF…FV, FXIII, FWPAIFibronectinTSPCD62PGPIIbIIIaCD36IgAlbuminVitronectin

Lysosomes

hydrolasesphosphataseselastasescollagenases

Platelet Page : www.akh.wien.ac.at

Thrombin

ADP

Collagen

Vasopressine

fibrinogen

GPIIb-IIIa

Resting platelets

Reversible aggregation Irreversible aggregation

TXA2 ADP

Gawaz M, Blood platelets, Thieme 2001

PLATELET AGGREGATION

…Thrombus growth...

A Cellular process leading to restoration of vessel patency

Continuous activation... recruitment

THROMBUS FORMATION

STIMULATION

PE

PI

PS

PC

PE : phosphatidyletanolaminePI : phsphatidylinositolPS : phosphatidylserinePC : phosphatidylcholine

RESTING

Elalamy EMC Angiology 2007

FLIP-FLOP = PHOSPHOLIPIDS TRANSLOCATION

FOCAL THROMBIN GENERATION

Zwall et al Blood 1997

FOCAL THROMBIN GENERATION

PLATELET RESPONSE

Adhesion and activation and aggregation …

47 kD transmembrane

glycoprotein

219 AA

21 AA

23 AA

Tissue Factor

The international nomenclature of blood coagulation factors

I

II

III

IV

V

VII

VIII

IX

X

XI

XII

XIII

Fibrinogen

Prothrombin

Thromboplastin

Ionised Calcium

Proaccelerin

Proconvertin

Antihemophilic factor A

Antihemophilic factor B

Factor Stuart

Factor Rosenthal (PTA*)

Factor Hageman

Factor of fibrin stabilisation

* Plasma Thromboplastin Antecedent

NB : the letter « a » = active (example : factor Xa)

The actors of blood coagulation and their classification

Classification 1 :

The cofactors

Va - VIIIa

The serine proteases

IIa - VIIa - IXa - Xa - XIa - XIIa

The substrate I or Fibrinogen

The zymogens of serine proteases

II

V

VII

VIII

IX

X

XI

XII

XIII

PC

Prothrombin

Proaccelerin

Proconvertin

Antihaemophilic factor A

Factor Stuart

Factor Rosenthal (PTA)

Factor Hageman

Protein CAntihaemophilic factor B

Fibrin stabilisation factor

COAGULATION FACTORS

Factor Name SynthesisHemostatic

minimal rate

Vitamine K

dépendent

I Fibrinogen Liver 0,5 to 1 g/l No

II Prothrombin Liver 40 % Yes

V Proaccelerin Liver 10 to 15 % No

VII Proconvertin Liver 5 to 10 % Yes

VIII Factor anti-haemophilic A Liver 30 to 50 % No

IX Factor anti-haemophilic B Liver 30 to 50 % Yes

X Factor Stuart Liver 10 to 20 % Yes

XI Factor Rosenthal Liver 30 % ? No

XII Factor Hageman Liver ? - No

XIIIFactor thrombin

stabilisatingLiver 2 to 3 % No

From « Introduction à l’étude de l’hémostase et de la thrombose », B. Boneu et J.P. Cazenave

COAGULATION : A SURFACE PHENOMENON

Localized Process:

Cellular Membrane Surface (catalytic surface)

Activated platelet membrane (Annexin V),

Anionic Phospholipids : phosphatidylserine

CLASSICAL COAGULATION SCHEME

Fibrinogen Fibrin

Monomers

II IIa

Ca2+

PLVa

V

PROTHROMBINASE

IXIXa

XaX

VIII

VIIIaCa2+

PL

TENASE

Polymerized

Fibrin

XIII

XIIIa

XI XIa

INTRINSIC

PATHWAY

KHPMXII

XIIa

Prekallicrein Kallicrein

- - - - - - - -EXTRINSIC

PATHWAY

VIIa VII

TF

Ca2+

X

IX

ProthrombinaseXa - Va - Phospholipids

Co

mm

on

path

way

Intrinsic pathway

• Tissue factor

• Factor VII

• Factor V

• Factor X

• Factor XII

• Factor XI

• Factor IX

• Factor VIII

Extrinsic pathwayP

roth

rom

bin

Tim

e

PT

Ac

tiv

ate

dP

art

ial

Th

rom

bo

pla

sti

nT

ime

(aP

TT

)

The old « cascade » of blood coagulation

PROTHROMBIN

Factor II

FIBRINOGEN

Factor I

THROMBIN

Factor IIa

FIBRIN

Factor Ia

TF

The TF-pathway is dominant

The modern scheme of blood coagulation In vivo

Initiation of coagulation

+ VIIa

« trigger »

3 consecutive phases

• Initiation phase• Propagation phase

• Limitation phase

Amplification forces + Inhibitory forces

Thrombin generation

(procoagulant) (anticoagulant)

FT

Tissue factor (FT)

Initial step for thrombin generation

Membrane phospholipids and

Ca++

VII / FTVIIa

Vascular lesionAtherosclerotic plaque

rupture

Triggering of coagulation

Evolution of the initiation phase

Tissue factor (TF)

Phospholipids

Ca++

Vascular lesion Atherosclerotic plaque rupture

TF

/TFVIIaVII

IX IXa

FIRST TRACES

OF THROMBIN

IXaXaX

Evolution of the initiation phase

Tissue Factor

Phospholipids

Ca++

Vascular lesion Atherosclerotic plaque rapture

FT

/ FTVIIaVII

IX IXa

FIRST TRACES

OF THROMBINE

IXaXaX

TFPI / Xa

INTRINSIC TENASE

Phospholipids

Ca++

IXaIXa

PROTHROMBINASE

Phospholipids

Ca++

Xa

From the initiation to the propagation phase

PLATELET

ACTIVATION

XI

XIa

Va

VVa

IX IXa

Tissue factor

Phospholipids

Ca++

Vascular lesion Atherosclerotic plaque rupture

FT

/ FTVIIaVII

X Xa

FIRST TRACES OF

THROMBIN

VIIIaVIII

VIIIa IXaIXa

IXa

The propagation phase

X Xa IX IXa

XIa

XI

PROTHROMBINASE

Phospholipids

Ca++

XaVa

BURST OF

THROMBIN

GENERATION

XIII XIIIa

Fibrinogen Fibrin

IXa

Xa

Xa IXa

INTRINSIC TENASE

Phospholipids

Ca++

VIIIa IXa

Limitation (inhibition) of thrombin generation

The natural

coagulation inhibitors

Major role in the

modulation of

thrombogenesis

Classified in 2 groups :

- stoechiometric inhibitors

- dynamic inhibitors

Their dysfunction

Excessive thrombin generation

The stoechiometric inhibitors

X Xa IX IXa

XIa

Phospholipids

Ca++

TF

/ FTVIIaVII

Intrinsic Tenase

Phospholipids

Ca++

IXaVIIIa

PROTHROMBINASE

Phospholipids

Ca++

XaVa

THROMBIN

TFPI / Xa

AT

The dynamic inhibitory pathway (protein C system)

THROMBIN

THROMBOMODULIN

PC

INTRINSIC TENASE

Phospholipids

Ca++

IXaVIIIa

PROTHROMBINASE

Phospholipids

Ca++

XaVa

Phospholipids

+

PS

PCa

IXaXa

Thrombin

Microparticules

x

PLATELET COAGULATION AND ENDOTHELIUM

Gawaz M, Blood platelets, Thieme 2001

Collaboration and Equilibrium= Key of Happiness!

THROMBUS FORMATION KINETICS

Platelets Fibrin Tisue Factor Platelets+Fibrin

Fibrin+Tissue Factor Platelets+Fibrin+Tissue Factor

Celi et al, Thromb Haemost 2003

Thrombin generation after TF pathway activation in platelet rich plasma

(according to C. Hemker et K. Mann)

0

50

100

150

200

thro

mb

in (

nM

)

time (min)

0 10 20

XaFree

Initiation phase

/ TFVIIa

= activation of platelets, FVIII et du FVFIRST TRACES

OF THROMBIN

Clot Formation

PROTHROMBINASE

INTRINSIC

TENASE

Activation of TAFI* to TAFIa

* Thrombin Activatable Fibrinolysis Inhibitor

The activation of TAFI to TAFIa

increases the resistance of the

clot to fibrinolysis

0 10 20time (min)

thro

mbin (nM

)

0

50

100

150

200

250

Profiles of thrombin generation

normal

hypocoagulability

Hypercoagulable state

Antithrombotic treatment

Main orientations of

a modern haemostasis laboratory

Diagnosis of Hypercoagulable states

✓ Hereditary thrombophilia

Acquired acute or chronic hypercoagulable states

Diagnosis of Bleeding disorders

✓ Hereditary

✓ Acquired

Monitoring of antithrombotic treatment

Monitoring of haemostatic treatment

Routine assays in use

• Global Clotting times

• PT extrinsic pathway

• aPTT intrinsic pathway

• Dosage of clotting factors and natural inhibitors of

coagulation (quantitative and qualitative)

• Primary haemostasis and platelet

function/phenotype

• D-Dimers marker of fibrin formation/lysis

Methods for the antithrombotic

treatment monitoring

Test Drugs Comments

PT / INR VKAStandardized

Clinically relevant

aPTT UFHStandardized

Clinically relevant

anti-Xa activityLMWHs

FXa direct inhibitors

Pharmacological test

clinically relevant?

Ecarin clotting time hirudins Pharmacological test

Thrombin generation test Universal Not standardized

Clotting test UFH LMWH* Fondaparinux VKA

PT - - - +++

aPTT ++ ± - ±

TT ++ ± - -

*clinically relevant concentration

Effect of currently used antithrombotic drugs

on routine coagulation tests

Adapted from: Hylek EM, Singer DE, Ann Int Med 1994;120:897-902

Risk of Intracranial Hemorrhage in Outpatients

Aim of the study

• To explore the effect of the association of LMWH and VKA

treatments on TF triggered thrombin generation

• In vitro study : Modelization of TG inhibition by

enoxaparin in plasma from patients with increasing INR

• Ex vivo study : thrombin generation in plasma from

patients treated with

• VKA alone or

• enoxaparin alone or

• VKA and enoxaparin

Gerotziafas GT, Dupont C, Spyropoulos AC, Hatmi M, Samama MM, Kiskinis D, Elalamy I. Differential

inhibition of thrombin generation by vitamin K antagonists alone and associated with low-molecular-

weight heparin. Thromb Haemost. 2009;102:42-8.

Materials and Methods

Parameters of thrombogram

lag time

time to peak (ttPeak)

endogenous thrombin potentiel (ETP)

peak of thrombin (Peak)

Mean rate index (MRI) was calculated by the formula peak/ (ttpeak-LT)

Pro-time and INR were determined using human thromboplastin (Thromborel® Siemens)

Anti-Xa activity was measured using a standardised chromogenic assay (Coamatic® Heparin,Chromogenix)

Patient population

VKA group : 97 patients with AF

Enoxaparin* group : 42 patients with AF

Enoxaparin*/VKA group : 41 patients with AF

Control group : 20 healthy subjects

* Therapeutic dose : 100 anti-Xa IU/kg b.i.d.

Basic characteristics of patients

VKA group

(n=97)

LMWH/VKA group

(n=41)

LMWH group

(n=42)

Age 69,5±13,4 ys

(range 39-92)

68,1±15,2 ys

(range 18-88)

65±20 ys

(range 18-70)

Sex (m/f) 44/53 14/27 35/53

INR 3,38±1,9

(0,96-12,2)

2,39±1,9*

(0,97-10)

-

<2 26 (26,8%) 22 (53,7%)* -

2 - 3 25 (25,8%) 12 (29,3%) -

>3 46 (47%) 7 (17,1%)* -

anti-Xa activity in

PPP (anti-Xa IU/m)

- 0,67 ± 0,32

(range 0,1 - 1,27)

<0,5 anti-Xa IU/ml

16 patients

>0,5 anti-Xa IU/ml

26 patients

*p<0,05 vs VKA group

0

200

400

600

800

1000

1200

1400

1600

1800

2000

0 1 2 3 4 5 6 7 8 9 10

Inhibition of thrombin generation

during VKA treatmentE

TP

(n

mxm

in)

INR

Inhibition of thrombin generation

during enoxaparin treatmentE

TP

(n

mxm

in)

anti-Xa IU/ml

0

500

1000

1500

2000

2500

3000

0 0,2 0,4 0,6 0,8 1 1,2 1,4

Thrombin generation

in sub-groups stratified according to INR

VKA

VKA + enoxaparin

*p>0,05

p<0,05 when compared VKA versus LMWH/VKA in each subgroup of INR

Representative thrombogram traces

0

100

200

300

400

500

-5 5 15 25 35

control

VKA INR 2,1

VKA INR 6

VKA/Enoxa INR 2,7; anti-Xa 0,6 IU/ml

Severe hemophilia FVIII = 2%

time (min)

ET

P (

nM

xm

in)

Routine assays in use

• Global Clotting times

• PT extrinsic pathway

• aPTT intrinsic pathway

• Dosage of clotting factors and natural inhibitors of

coagulation (quantitative and qualitative)

• Primary haemostasis and platelet

function/phenotype

• D-Dimers marker of fibrin formation/lysis

FIBRINOGEN

PROTHROMBIN

SolubleFibrin

F1+2TAT Complexes

FPA

THROMBIN

Monomer of fibrin-1

THROMBIN

FPB

Monomer of fibrin-2

1

2

3 4

DDimers Generation

Fibrin Degradation Products

High Molecular Weight of

Products of degradation

DD-E

PLASMIN

Fibrin Polymer

Monomer of fibrin-2

Factor XIIIa

Ca ++

PAP Complexes

USE OF D-DIMER TESTING

• Diagnostic algorithms are necessary in suspected DVT/PE.

• They diminish costs and increase safety :

Of value in excluding DVT (high sensitivity and NPV) +++

D-Dimer testing => Ultrasonography or CT scan needs

• Only validated tests/algorithms should be used.

• Assessment of clinical probability is the pre-requisite for a valid

algorithm.

• Validated algorithms for diagnosing DVT/PE should be implemented in all

institutions

CUS or MDCTA

D-dimer

Low or intermediate High

Prior Clinical Probability

Below cutoff Above cutoff

Negative Positive

No Rx Rx

One example of diagnostic algorithm

Righini M et al. J Thromb Haemost. 2008; 6:1059-71

D-DIMER MEASUREMENT DURING OAC

• 72 yo man with AF received VKA

• 10 days ago serious knee injury with immobilisation

• Left lower leg swollen and painful => DVT possible cause?

• Can you use D-Dimer testing even under OAC?

• small studies +++

• inhibition of thrombin generation +++

• Not safe to use D-Dimers to exclude VTE => Always compression US!

(Grade D)

Bruinstroop et al Eur J Intern Med 2009

Acute Coronary Syndromes

Gerotziafas GT et al, Br J Haemat 2003;120:611-7

0,5

1

1,5

2

2,5

3

3,5

1 3 5 7 9 11 13 15 17 19 21 23time from start (hours)

pro

thro

mb

in F

1+

2

(nM

)

0

0,1

0,2

0,3

0,4

0,5

0,6

0,7

0,8

0,9

1

0

enoxaparin enoxaparin

14 pts with UA given enoxaparin 1 mg/kg s.c. q 12h

Upper normal limit of F1+2

An

ti-Xa

(IU/m

l)

Is there any place for thromboelastometry

for antithrombotic treatment?

Modification of classic thromboelastography

Global clotting test performed in whole blood

Possibility to study plasma/blood cells interactions

Capacity to evaluate the phases of blood coagulation

Initiation phase

Propagation phase

Clot firmness

But….In-TEM and Ex-TEM

Are not sensitive to LMWHs and fondaparinux

Is there any place for thromboelastometry

for antithrombotic treatment?

CT :

Clotting Time

CFT :

Clot Formation Time

aAngle :

velocity

MCF:

Maximum Clot

Firmness

Effect of fondaparinux on min-TF WB Rotem and TGT

control

0.1 μg/ml

0.25 μg/ml

10 20 30

time(min)

20

40

60

80

100

120

140

160

180

200

thro

mb

in(n

M)

10 20 30

time(min)

20

40

60

80

100

120

140

160

180

200

thro

mb

in(n

M)

10 20 30 40

time(min)

20

40

60

80

100

120

140

160

180

200

thro

mb

in(n

M)

1.25 μg/ml

0.5 μg/ml

0.8 μg/ml

0

0

Control

0,4 μg/ml

0,8 μg/ml

1 μg/ml

PRP

Effect of enoxaparin on min-TF WB Rotem and TGT

Control

0,4 anti-Xa IU/ml

0,8 anti-Xa IU/ml

1 anti-Xa IU/ml

control

0.1 anti-Xa IU/ml

0.25 anti-Xa IU/ml

20

40

60

80

100

120

140

160

180

200

10 20 30 40

time (min)

20

40

60

80

100

120

140

160

180

200

10 20 30 40

time (min)

20

40

60

80

100

120

140

160

180

200

10 20 30 40

time (min)

1 anti-Xa IU/ml

0.5 anti-Xa IU/ml

0.8 anti-Xa IU/ml

0

0

PRP

Effect of lepirudin on min-TF WB Rotem and TGT

0

50

100

150

200

250

300

350

400

450

500

0 5 10 15 20

time (min)

thro

mbin

(nM

)

contrôle

0.25 μg/ml

0.5 μg/ml

0.75 μg/ml

1 μg/ml

1,25 μg/ml

1,5 μg/ml

2 μg/ml

Control

0,5 μg/ml

0,75 μg/ml

1 μg/ml

PRP

Control group : 20 healthy volunteers

60 patients undergoing Carotid Artery Stenting treated

with Nadroparin 5,750 antiXa IU/12h

18 patients were tested before and after LMWH

administration

Ex vivo effect of LMWH treatment on

min-TF Rotem and TGT

CT

(sec)

CFT

(sec)

α angle

(degree)

MCF

(mm)

Control

Group

(n = 20)

293±57 86±15 73±2 68±13

LMWH Group

(n = 60)380±96 123±51 67±6 64±10

Influence of LMWH treatment on min-TF WB

ROTEM parameters

11 out of 18 pts > NMaxL (61,1%)

CF

10 out of 18 pts < NMinL (55,5%)

• Values…………................ 0,61±0,15 anti-Xa IU/ml

• (0,4-0,86 anti-Xa IU/ml)

prolongation of CFT (Clot Formation Time)

was well correlated with anti-Xa activity (p=0,04 και r=0,7).

Measurement of Anti-Xa activity

Conclusions

Min-TF WB Rotem is a physiologically relevant system sensitive to detect

the antithrombotic effect of LMWHs, specific FXa inhibitors and specific FIIa

inhibitors at clinically relevant concentrations

Min-TF Rotem is sensitive to detect ex vivo the antithrombotic effect of

LMWH treatment

α-angle is the most relevant parameter for the evaluation of the

antithrombotic activity of these drugs

Modifications of thromboelastometry pattern in vitro and ex vivo induced by

antithrombotic drugs are complementary to the thrombin generation

inhibition

Reagent and lot variability influences min-TF WB Rotem

α-angle and MCF are less influenced by these preanalytical

conditions

Unmet needs and perspectives

• Which is the clinical relevance of these findings?

• Which group of patients might benefit from an optimisation

of antithrombotic therapy?

• Which are the hemostatic changes related to the

treatment and the underlying pathology or surgery that are

mandatory for bleeding or thrombotic events occurrence in

post-operative period?

CONCERNING LMWH MONITORING

WHICH ITEM IS CORRECT?

A.In every LMWH treatment, a control of platelet count twice a week is mandatory whatever the treatment duration

B. In prophylaxis use, no biological testing is required (Anti-Xa activity, platelet count)

C. In curative use, anti-Xa activity is more reliable for treatment efficacy and/or safety

D.Monitoring curative dosage may be based on ApTT prolongation

Response : C

UFH IS NOT ABLE TO INDUCE A PROLONGATION OF WHICH TEST?

A.Bleeding Time Ivy

B. Prothrombin Time

C. Activated partial Thromboplastin Time

D.Thrombin Time

Response : A

Mrs M. 85 y.o. pulmonary embolism hospitalized in ICU

UFH IV infusion and …: Day 1 Plt 215 G/L

ApTT : 80 sec /32 sec (R 2.5)

PT : 78%

Day 5 Plt: 50 G/L

ApTT : 128 sec (R 4)

PT: 35%

Which etiology is not probable?

A.Heparin-Induced Thrombocytopenia

B. DIC

C. Pre-analytical problem

D.Heparin Overdosage

Response : D