HEMOFILIA -FVIII

8/10/2019 HEMOFILIA -FVIII

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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 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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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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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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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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HEMOFILIA -FVIII