Introduction to coagulation and laboratory tests · Acquired prolonged aPTT and PT Clinical state...

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

VIIa/TF

Initiation of coagulation

IXa IX

X Xa

VIIa/TF

Initiation of coagulation

IXa IX

X Xa

VIIa/TF

Initiation of coagulation

Prothrombin Thrombin

Va V

IXa IX

XIa XI

X Xa

VIIIa VIII

VIIa/TF

Initiation of coagulation

Prothrombin Thrombin

Amplification

Va V

IXa IX

XIa XI

X Xa

VIIIa VIII

VIIa/TF

Initiation of coagulation

Prothrombin Thrombin

Amplification

Va V

IXa IX

XIa XI

X Xa

VIIIa VIII

VIIa/TF

Initiation of coagulation

Prothrombin Thrombin

Amplification

Va V

IXa IX

XIa XI

X Xa

VIIIa VIII

VIIa/TF

Initiation of coagulation

Prothrombin Thrombin

Amplification

Va V

IXa IX

XIa XI

X Xa

VIIIa VIII

VIIa/TF

Initiation of coagulation

Prothrombin Thrombin

Amplification Fibrinogen Fibrin

Sustained hemostasis

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

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

Hydrophobic residues

Basic residues Phospholipid

binding site

Pratt et al., Nature 2000

FV and FVIII binding to phospholipids

Hydrophobic residues

Basic residues Phospholipid

binding site

Pratt et al., Nature 2000

FV and FVIII binding to phospholipids

hydrophobic

basic

Phospholipids

FVII, FIX, FX, FII binding to phospholipids:

Gla domain

d Ca ++

Gla Gla Val

Leu Phe

hydrophobic

basic

Phospholipids

Gla:�J�carboxy glutamic acid

FVII, FIX, FX, FII binding to phospholipids:

Gla domain

Glutamic acid

H CJ

CH�

COO-

J-carboxylase

protein CH�

Glutamic acid

-OOC J-carboxy glutamic acid

CJ

CH�

COO-

H CJ

CH�

COO-

J-carboxylase

protein CH�

CH�

Glutamic acid oxidized

vitamin K

reduced vitamin K

vitamin K

-OOC J-carboxy glutamic acid

CJ

CH�

COO-

H CJ

CH�

COO-

J-carboxylase

protein

VKOR

VKR

vitamin K

cycle

CH�

CH�

Glutamic acid oxidized

vitamin K

reduced vitamin K

vitamin K

-OOC J-carboxy glutamic acid

CJ

CH�

COO-

H CJ

CH�

COO-

J-carboxylase

protein

VKOR

VKR

vitamin K

cycle

anti-vit K

CH�

CH�

Coagulation and phospholipids

Coagulation and phospholipids

Modern times, 1936

Anti-phospholipid antibodies

Anti-phospholipid antibodies

Anti-phospholipid antibodies

Thrombosis

but no bleedings

Coagulation assays

Va V

IXa IX

XIa XI

X Xa

VIIIa VIII

VIIa/TF

Initiation of coagulation

Prothrombin Thrombin

Amplification Fibrinogen Fibrin

Sustained hemostasis

FXIII

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

X Xa

VIIa

Prothrombin Thrombin

Fibrinogen Fibrin

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

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)

PT results

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

PT results

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

Anti-vit K treatment

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

APTT (Activated Partial Thromboplastin Time)

+ phospholipids + Ca++

+ non physiological surface (glass, silica, kaolin)

APTT (Activated Partial Thromboplastin Time)

XIIa XII

HK / PK Contact Activation

pathway

+ phospholipids + Ca++

+ non physiological surface (glass, silica, kaolin)

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

X Xa

VIIa

Prothrombin Thrombin (IIa)

Fibrinogen Fibrin

PT

Prolonged isolated PT

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

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

Differential diagnosis of isolated prolonged APTT

mix of patient’s plasma with normal plasma

Correction of the APTT:

DECREASED FACTOR low titer or slow acting antibody

Factors XII, FXI, FIX, FVIII?

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

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

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