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Introduction to coagulation and laboratory tests

Marc Jacquemin

Special Haemostasis Laboratory

Center for Molecular and Vascular Biology University of Leuven

Coagulation in a blood vessel: fibrin stabilises the hemostatic clot

Platelet immunostaining Fibrin staining

Polymerisation of fibrin stabilises the hemostatic clot = secondary hemostasis

Norledge BV, et al. Proteins. 2003;53:640-8

Expression of Tissue Factor (TF) and Factor VIIa on phospholipid surfaces in damaged tissues

Va V

IXa IX

XIa XI

X Xa

VIIIa VIII

VIIa/TF

Initiation of coagulation

Prothrombin Thrombin

Amplification Fibrinogen Fibrin

Sustained hemostasis

FXIII

FVIII is bound to vWF in plasma

A1

FVIII

vWF

2

*

Factor VIIIa Factor IXa

Phospholipids

* A3!

A2!

A1!

C2!

* *

X 100.000 by Factor VIII

Factor X

Factor Xa

Haemostasis

Activated Factor VIII as cofactor of Factor IXa

*

Hydrophobic residues

Basic residues Phospholipid binding site

Pratt et al., Nature 2000

FV and FVIII binding to phospholipids

d Ca ++

Gla Gla

Val Leu

Phe

hydrophobic

basic

Phospholipids

Gla: γ carboxy glutamic acid

FVII, FIX, FX, FII binding to phospholipids:

Gla domain

Glutamic acid oxidized vitamin K

reduced vitamin K

vitamin K

-OOC γ-carboxy glutamic acid

Cγ CH2

COO-

H Cγ CH2

COO-

γ-carboxylase

protein

VKOR

VKR

vitamin K cycle

anti-vit K

CH2

CH2

Coagulation and phospholipids Anti-phospholipid antibodies

Thrombosis

but no bleedings

3

Coagulation assays

X Xa

VIIa

Prothrombin Thrombin

Fibrinogen Fibrin

Prothrombin time (PT) + TF + phospholipids + Ca++

time between the addition of reagents to citrate plasma and detection of a clot.

PT results

•  time (seconds)

•  percent (%) by comparison to PT with dilutions of plasma 13 sec = 100% 40 sec = 10%

•  International Normalized Ratio (INR) 13 sec = 1 40 sec = 3.5

Follow up of Anti-vit K treatment Va V

IXa IX

XIa XI

X Xa

VIIIa VIII

Prothrombin Thrombin

Fibrinogen Fibrin

APTT (Activated Partial Thromboplastin Time)

XIIa XII

HK / PK Contact Activation pathway

+ phospholipids + Ca++

+ non physiological surface (glass, silica, kaolin)

Interpretation of coagulations assays

Va V

IXa IX

XIa XI

X Xa

VIIIa VIII

Prothrombin Thrombin (IIa)

Fibrinogen Fibrin

APTT nl XIIa XII

HMWK / PK

X Xa

VIIa

Prothrombin Thrombin (IIa)

Fibrinogen Fibrin

PT >

Prolonged isolated PT

4

Isolated prolonged PT (lower % value, increased INR)

acquired decreased FVII

•  early liver disease •  early anti-vit K treatment

« anti-phospholipid » antibody *

constitutive FVII defect

not bleeding disorders *

Va V

IXa IX

XIa XI

X Xa

VIIIa VIII

Prothrombin Thrombin (IIa)

Fibrinogen Fibrin

APTT > XIIa XII

HMWK / PK

X Xa

VIIa

Prothrombin Thrombin (IIa)

Fibrinogen Fibrin

PT nl

Prolonged isolated APTT

Isolated prolonged APTT

acquired •  inhibitor antibody (FVIII) •  « anti-phospholipid » antibody

constitutive •  Von Willebrand disease •  hemophilia A and B (FVIII, FIX) •  FXI •  FXII (contact activation pathway)

not bleeding disorders *

*

*

FVIII vWF

Differential diagnosis of isolated prolonged APTT

mix of patient’s plasma with normal plasma

NO correction of the APTT:

ANTIBODY

inhibitor antibody? anti-phospholipid antibody?

Correction of the APTT:

DECREASED FACTOR low titer or slow acting antibody

Factors?

Prolonged PT and APTT

Va V

IXa IX

XIa XI

X Xa

VIIIa VIII

Prothrombin Thrombin (IIa)

Fibrinogen Fibrin

APTT

XIIa XII

HMWK / K

X Xa

VIIa

Prothrombin Thrombin (IIa)

Fibrinogen Fibrin

PT

5

Acquired prolonged aPTT and PT

Clinical state Deficiency •  advanced liver disease VII, V, IX, X , II •  vit K deficiency VII, IX, X, II

•  acute DIC V •  massive blood transfusion V

•  lupus inhibitor none after dilution •  antibody to FV V

•  rare systemic diseases Gaucher’s disease V, X

Nieman Pick disease Amyloid

•  heparin •  oral anticoag treatment VII, IX, X, II