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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 Testingchromogenic vs. clotting assays
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Hemostasis
Hemostasis:The balance betweenclotting and bleeding
Components of Hemostasis:
Vasculature
Coagulation proteinsPlatelets
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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
ActivationTissue Factor + VII
XIIIa
XIII
Thrombin
Fibrin
Polymer
Fibrinogen Fibrin
Monomer
IX
XI
XIa
IXa
Xa
Va
XIIa
Prothrombin
TF-VIIa
(Prothrombinase)
PL
PL, Ca2+(Tenase)
VIIIa
PL, Ca2+
X
Intrinsic Pathway
Prekallikrein
HMW
Kininogen
Extrinsic Pathway
Common Pathway
TF Pathway
Coagulation Cascade
Anticoagulation proteins:
Protein C, Protein S,
Antithrombin III
Ca2+
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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), andAntithrombin (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 phospholipidsurfaces (glass or kaolin
in vitro)
Factors XII, XI, IX, VIII,
prekallikrein, HMWkininogen
Measured with aPTT
clotting assay
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Coagulation Cascade
Extrinsic Pathway:
Initiated when blood
is exposed to TF
released fromdamaged
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- fibrindegradation 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
Compon ent Km Vmax
mol/l mol FXa/min/mol FIXa
FIXa 299 0.0022
FIXa, Calcium 181 0.0105FIXa, 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
FibrinPolymer
Fibrinogen FibrinMonomer
IX
XI
XIa
IXa
XaVa
XIIa
Prothrombin
TF-VIIa
PL
PL, Ca2+(Tenase)
VIIIaPL, Ca2+
XCa2+
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FVIII: Biochemistry
FVIII is synthesized as a single chain polypeptide
FVIII is processed by a protease and it is released asa 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% FVIIIModerate 1-4% FVIII
Severe
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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 ofintermediate 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 inthe final formulation
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Elevated Factor VIII
FVIII activity > 1.5 IU/mL results in 5-6-fold higher risk for DVTthan 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 mutationobserved 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 monitoredspectrophotometrically at 405 nm
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FVIII Units
International Unit
Factor VIII activity of a stated amount of the International Standard whichconsists of a freeze-dried human blood coagulation factor VIIIconcentrate. 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 ofFX 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 IXaFactor 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 Concentrat ion
FX 10 - 350 nmol/LFIXa 1 - 100 nmol/L
Thrombin enough for FVIII activation
Phospholipid 1 - 50 mol/L
CaCl2 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 morespecific, accurate, and precise; generally lesssusceptible 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 cantypically be put on automated analyzers
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Measuring Factor VIII
Chromogenic assay has beenrecommended as the optimal assay formeasuring elevated FVIII levels
Chromogenic assay precision is typicallybetter than that of one-stage clotting assayat high FVIII levels
No interference from heparin, directthrombin 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, Ca2+, Surfaceactivator
time for clot formation
Most widely used method
Cheap, rapid and simple
but..........
Accuracy and precision influencedby a large number of variables
Sensitive to pre-activation of the
coagulation cascade
Over estimation in assessment of
FVIII concentrates potency
Requires considerable amount ofFVIII deficient plasma
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Two-stage Clotting Assay
Principle:
Stage 1FIXa Ca2+ Phospholipids
FX FXa
FVIIIFXa +Ca2+, FV ComplexPhospholipids
Stage 2
ComplexProthrombin Thrombin
Fibrinogen + Thrombin
Fibrin clot formation
Less variation than one-stage assay
No need of FVIII-deficientplasma
In the past, it was themethod of choice by theBritish and EuropeanPharmacopoeia..........
......It has been replaced bythe 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%, createproblems 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 theclinical laboratory
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