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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
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