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HEMOFILI FAKTOR VIII

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Topics
Coagulation Cascade Review
Pathways of coagulation, anticoagulation, and
fibrinolysis

Coagulation Factor VIII

Assay Methodology
FVIII Testing chromogenic vs. clotting assays
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Hemostasis
Hemostasis: The balance between
clotting and bleeding

Components of Hemostasis:
Vasculature
Coagulation proteins
Platelets
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Coagulation Cascade
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Coagulation Cascade
Vascular damage initiates the coagulation
cascade.
Results in the generation of thrombin at
the site of injury.
Thrombin catalyzes the conversion of
fibrinogen to an insoluble fibrin (clot)
matrix.
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Contact
Activation
Tissue Factor + VII
XIIIa
XIII
Thrombin
Fibrin
Polymer
Fibrinogen
Fibrin
Monomer
IX
XI
XIa
IXa
Xa
Va
XIIa
Prothrombin
TF-VIIa
(Prothrombinase)
PL
PL, Ca
2+
(Tenase)
VIIIa
PL, Ca
2+
X
Intrinsic Pathway
Prekallikrein
HMW
Kininogen
Extrinsic Pathway
Common Pathway
TF Pathway
Coagulation Cascade
Anticoagulation proteins:
Protein C, Protein S,
Antithrombin III
Ca
2+
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Coagulation Cascade
Critical reactions are closely checked and
localized by circlulating anticoguatlants,
such as Activated Protein C (APC), Tissue
Factor Pathway Inhibitor (TFPI), and
Antithrombin (AT).
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Coagulation Cascade
Abnormal activation of blood coagulation
and/or depressed fibrinolytic activity may
lead to the formation of a thrombus (clot).
In contrast, a defect or deficiency in the
coagulation process and/or accelerated
fibrinolysis is associated with a bleeding
tendency.
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Coagulation Cascade
The cascade scheme is organized into the
INTRINSIC and EXTRINSIC pathways,
converging into the COMMON pathway.
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Coagulation Cascade
Intrinsic Pathway:
Initiated by the activation
of FXII involving contact
factors on negatively-
charged phospholipid
surfaces (glass or kaolin
in vitro)
Factors XII, XI, IX, VIII,
prekallikrein, HMW
kininogen
Measured with aPTT
clotting assay
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Coagulation Cascade
Extrinsic Pathway:
Initiated when blood
is exposed to TF
released from
damaged
endothelium
Tissue Factor (TF),
FVII
Measured with PT
clotting assay
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Coagulation Cascade
Common
Pathway:
Factors V, X,
XIII,
Prothrombin,
Fibrinogen
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Fibrinolytic Pathway
Plasminogen
Plasmin
XL-Fibrin, fibrinogen
Tissue Plasminogen Activator (t-PA)
Urokinase (uPA)
Exogenous: streptokinase
XL- fibrin
degradation products (FDP)
Plasmin Inhibitor
PAI-1
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Coagulation Factor VIII
The role of FVIII is to accelerate the rate of
cleavage of FX by activated FIX


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FVIII in the coagulation cascade
Component Km Vmax

mol/l
mol FXa/min/mol FIXa

FIXa 299 0.0022
FIXa, Calcium 181 0.0105
FIXa, Calcium,
Phospholipids
0.0548 0.00247
FIXa, Calcium,
Phospholipids, FVIIIa
0.063 500


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FVIII in the coagulation cascade
FVIII circulates in plasma (0.1 g/ml) in non-covalent
complex with von Willebrand factor (vWF)
vWF protects FVIII from proteolysis and concentrates
it at the active sites of hemostasis
Thrombin or FXa activates FVIII
The cleavage releases FVIIIa from vWF
This enables FVIIIa to bind the phospholipid surfaces
of damaged cells
FVIIIa is inactivated by Activated Protein C (APC)
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FVIII in the coagulation cascade
XIIIa
XIII
Thrombin
Fibrin
Polymer
Fibrinogen
Fibrin
Monomer
IX
XI
XIa
IXa
Xa
Va
XIIa
Prothrombin
TF-VIIa
PL
PL, Ca
2+
(Tenase)
VIIIa
PL, Ca
2+
X
Ca
2+
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FVIII: Biochemistry
FVIII is synthesized as a single chain polypeptide

FVIII is processed by a protease and it is released as
a heterodimer

FVIII is composed of a light chain (80 kDa) joined to a
heavy chain (200 kDa)
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FVIII: Clinical Aspects


Haemophilia A (FVIII deficiency)
Thrombophilia (Elevated FVIII)
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Hemophilia A
Gene mutation (chromosome X)
Frequency: 20 per 100 000
Classification: Mild 5-25% FVIII
Moderate 1-4% FVIII
Severe <1% FVIII

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Treatment of Hemophilia
Plasma-derived Factor VIII
concentrates

Recombinant Factor VIII
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Treatment of Hemophilia
Plasma-derived Factor VIII concentrates
Two methods of purification:
1. immunoaffinity chromatography (with antibodies against FVIII
or vWF)
2. Additional step with ion-exchange, affinity or gel filtration of
intermediate purity concentrates
Concentrates contain almost exclusively FVIII and vWF
Due the nature of the preparation virucidal procedures are
applied to the final product (i.e. dry heating, solvent treatment
etc.)
Specific activity: 50-2000 IU/mg
Addition of albumin to stabilize high purified preparations
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Treatment of Hemophilia
Recombinant Factor VIII

In 1984 the FVIII gene was cloned
Next step: FVIII co-expressed with vWF to improve yields
rFVIII purified by immunoaffinity chromatography and ion-
exchange chromatography
The final product does not contain vWF
Specific activity > 5000 IU/mg
Additional safety steps: Viral inactivation, no albumin in
the final formulation
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Elevated Factor VIII
FVIII activity > 1.5 IU/mL results in 5-6-fold higher risk for DVT
than FVIII activity < 1.0 IU/mL

Confirmation of risk not associated with acute phase response

Elevated FVIII persistent over time

Familial trait observed no explanation / gene mutation
observed so far

Increase of FVIII activity is concomitant to the increase of FVIII
antigen and vWF
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What is a Chromogenic Assay?
Chromogenic substrate: peptide that
reacts with proteolytic enzymes, thus
forming color
Labeled with a chromophore that gives off
a color when hydrolyzed by specific
enzyme
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Chromogenic Assays
Pioneering chromogenic assays:
Chromogenic substrate
Peptide linked to a chromophore: p-nitroaniline (pNA)
Specific for enzymes such as FXa, Thrombin, etc.
The substrate cleavage by an enzyme releases pNA

Chromogenic method
Method for the determination of analytes by using chromogenic
substrates
The reactions cause the release of pNA which can be monitored
spectrophotometrically at 405 nm
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FVIII Units

International Unit
Factor VIII activity of a stated amount of the International Standard which
consists of a freeze-dried human blood coagulation factor VIII
concentrate. The equivalence in International Units of the International
Standard is stated by the World Health Organization.

Assay
FVIII is assayed by its biological activity as a cofactor in the activation of
FX by activated FIX in the presence of calcium ions and phospholipids.

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FVIII and the European
Pharmacopoeia recommendations
Reagents
Purified proteins derived from bovine or human origin. These include:
Factor X
Factor IXa
Factor VIII activator (thrombin)

Phospholipids from natural sources or synthetically prepared

Chromogenic substrate that is specific for FXa:
Derivatised short peptide of
between three and five amino acids
joined to a chromophore group.
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FVIII and the European
Pharmacopoeia recommendations
Assay principle
Two-stage chromogenic assay
Assay conditions during FXa generation
Components Concentration
FX 10 - 350 nmol/L
FIXa 1 - 100 nmol/L
Thrombin enough for FVIII activation
Phospholipid 1 - 50 mol/L
CaCl
2
5 - 15 mmol/L

Dilution of test and sample preparation
Buffer 1% BSA or HSA, pH 7.3 - 8.0

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Chromogenic vs. Clotting Assays
In general, chromogenic assays are more
specific, accurate, and precise; generally less
susceptible to pre-analytical variables
Clot-based assays are typically fast and less
expensive
Clot-based assays are subject to interference by
other coagulation factor levels, heparin, warfarin,
other anticoagulants, as well as the presence of
lupus anticoagulant
Both clotting and chromogenic assays can
typically be put on automated analyzers

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Measuring Factor VIII
Chromogenic assay has been
recommended as the optimal assay for
measuring elevated FVIII levels
Chromogenic assay precision is typically
better than that of one-stage clotting assay
at high FVIII levels
No interference from heparin, direct
thrombin inhibitors, or lupus anticoagulant
Assay is automatable

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Methods for Determination of
Factor VIII activity

One-stage clotting assay
Two-stage clotting assay
Chromogenic assay
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One-stage Clotting Assay
Principle:
APTT based assay

Diluted sample
+
FVIII Def. Plasma
+
PL, Ca
2+
, Surface
activator





time for clot formation

Most widely used method
Cheap, rapid and simple
but..........

Accuracy and precision influenced
by a large number of variables
Sensitive to pre-activation of the
coagulation cascade
Over estimation in assessment of
FVIII concentrates potency
Requires considerable amount of
FVIII deficient plasma


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Two-stage Clotting Assay
Principle:
Stage 1
FIXa Ca2+ Phospholipids
FX FXa
FVIII

FXa +
Ca
2+
, FV Complex
Phospholipids


Stage 2

Complex
Prothrombin Thrombin


Fibrinogen + Thrombin





Fibrin clot formation
Less variation than one-
stage assay
No need of FVIII-deficient
plasma
In the past, it was the
method of choice by the
British and European
Pharmacopoeia..........


......It has been replaced by
the chromogenic method
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FVIII Assays
One stage clotting assays give different results from two-
stage clotting and chromogenic assays

The difference between methods is more pronounced for
products of higher purity

The discrepancies, sometimes up to 25 -50%, create
problems when determining potencies and therapeutic
dosages

One-stage assays give under-estimation of FVIII levels;
therefore may treat patients with more FVIII than needed
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FVIII Assays
Chromogenic assay is useful for
measuring FVIII activity in
industry and as well as in the
clinical laboratory
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