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J d D hb iJavad Dehbozorgian

von Willebrand Factor &von Willebrand Disease

First Description of vWD made by E-A von Willebrand in 1926 .H i i d i l bHeterogenetiy in symptoms and in laboratory parameter.Zi d FVIII i t d iti i 1971Zimmerman named FVIII associated anitigen in 1971Different designation of the disease:

P d h hili- Pseudohemophilia- Constitutional thrombocytopenia- Vascular hemophilia

Gene & gene productProtein encoding gene is located in chromosome 12 with

about 180 Kb in size and 52 exons (Nonprocessed d ith 97% h l f 23 44 ipseudogene with 97% homology for exons 23-44 is

located on chromosome 22.Gene product is synthesised by endothelial cells andGene product is synthesised by endothelial cells and

megakaryocytes.Mature vWF contain 2050 amino acids , cysteins are y

abundant in 2 domains including : N-terminal and C-Terminali d b h d i d fEstimated carbohydrate content is 18% and some of

carbohydrate express ABO blood group antigen.

D i ith k t t l & f ti lDomains with known structural & functional ProcessOnly the largest Multimers are fully activeOnly the largest Multimers are fully active.

Largest multimers is 10fold more active than smaller multimer and 100fold

more than nonmonomeric isolatedmore than nonmonomeric isolated. A1 Domain:

GpIbaCollagen type VICollagen type VISnake venom bothrocetinHeparin binding

Mutation in A1 if :More reactive is type 2BLess reactive is type 2A&2M

A3 domain bind to collagen type I & III.FVIII binding domain including D’domain and D3domain .

Structure of vWFCirculating vWF has complex Multomerric structure.Dimer is the smallest detectable subunit and largest molecules exceed 10000 (MW:540 kDa)Proteolytic modification in blood by disulfide reductase (Thrombospondin-I & Specific Metalloproteinase)

ll i l h b i bMetalloproteinase cleaves the subunit between Tyr842 and Met843 leading to fragment 140 & 176 Kb.

GpIb (VWF:RCo)VWF:CBGpIIb/IIIa

VWF:FVIIIB

synthesis               proteolysis                           steady state(normal)(normal)

=

clearance

Function of vWF

Platelet adhesion and aggregation inPlatelet adhesion and aggregation in vessells.

Stabillisation of FVIII

von Willebrand Factor in Action

S dl J E Bl d 2007 109 3

Copyright ©2007 American Society of Hematology.  

Sadler, J. E. Blood 2007;109:3

vWF FunctionAdhesion

Mediates the adhesion of platelets to sites of vascular injury (subendothelium)

Links exposed collagen to platelets

Mediates platelet to platelet i t tiinteraction

Binds GPIb and GPIIb-IIIa on activated plateletsStabilizes the hemostaticStabilizes the hemostatic plug against shear forces

EPIDEMIOLOGYMost frequently inherited bleeding disorder.Only small function of these suffer from a clinically hemorrhagic diathesis(125 peer million in Sweden)No difference between male & Female 35U/dl my be normal for blood O group vWF & FVIII are acute phase reactant.i l l d i i d fincrease level during aging , pregnancy and course of malignancy disease.

VWD Comes in Three VarietiesNormal individual: normal VWF

Type 1: reduced quantity, essentially normal VWF 70% of cases

Type 2: functionally deficient VWF % f

70% of cases

Type 3: virtually no VWF

25% of cases

Type 3: virtually no VWF 5% of cases

Clinical ManifestationHistory is obligatory first step in patient identification.Family history for indentified type of inheritance.In most of cases clinical symptoms of primary hemostatic defect are present.T i l fi di f WD i M h hTypical finding for vWD is Mucocutaneous hemorrhage and history of autosomal inheritance.

Clinical Manifestation(cont)Easy bruisingProlonged epistaxisBl di ft t th t ti d i th lBleeding after tooth extraction and surgery in the oral cavityMucous membrane bleedingMucous membrane bleeding Gastro intestinal blood loss.In woman menorrhagia my be predominant finding.

In mild form of type I and many patients with type 2A,2B and 2M the concentration of vWF is >40u/dl, thus secondary hemostasis is not detectablesecondary hemostasis is not detectable.

Laboratory diagnosis of vWDScreening tests or Procedure :

Family historyBleeding TimeFilter testsAPTTPlatelet Count

Confirmatory Tests:FVIII:c activityyvWF antigen(vWF-Ag)Ristocetin cofactor activity (vWF-Rco)vWF collagen binding capacity(vWF-CB)vWF collagen binding capacity(vWF-CB) Epitop specific Elisa

Tests performs specialized lab:Ri i I d d Pl l A i (RIPA)Ristocetin Induced Platelet Aggregation(RIPA)Bothrocetin Induced Platelet Aggregation(BIPA)Platelet vWFvWF propeptide (vWF:AgII)vWF Multimeric StructurevWF subunitsvWF subunitsFVIII binding capacity (vWF:FVIII B)Molecular genetic methods

Classification of vWD Severity of bleeding symptoms is the primary clinical parameter.

Quantitative defects:-Type 1 (decrease amount)T 3 (Vi l Ab )-Type 3 (Virtual Absent)

Qualitative defects: Type2M t f bt d ib d b lti l iMost of subtypes were described by multimer analysis.

V Will b d di t 1 h t it f li i l dVon Willebrand disease type 1: heterogeneity of clinical and laboratory phenotype

Inherited type 2 variants (2A, 2B, 2M)yp ( , , )

di i i l i i i bi di l idimerization  multimerization              binding  proteolysis structure

IID        IIC             IIE  N          2B         2A          2M          N 

/NP     IIC                  2N          IIE           2B   2M      IIA    CBD                          2M/1          IID

D1            D2          D‘         D3             A1         A2        A3           D4      B C1    C2  CK

SP       Propeptide mature VWF

Characterial pattern of the different type of vWD

Diff ti ti B t VWD TDifferentiation Between VWD Types

VWF:RCo / VWF:Ag ratio <0.6 (types 2A and 2M)RIPA (for type 2B)Low FVIII:C / VWF:Ag ratio (type 2N)Loss of high molecular weight multimers (types 2A and 2B)and 2B)RIPA suitable for differentiation between 2M & type1vonWillebrand AntigenII suitable for differentiationvonWillebrand AntigenII suitable for differentiation between aquired and inherited vWD.

Main diseases associated with acquired von Willebrand syndromeLymphoproliferative

Monoclonal gammopathy of undetermined significanceCardiovascular• Congenital and acquired cardiacMonoclonal gammopathy of undetermined significance

Multiple myeloma M. Waldenstrbm

MyeloproliferativeEssential thrombocythemia

• Congenital and acquired cardiac defects: • Multiple or not defined • Ventricular septal defect • Atrial septal defecty

Polycythemia veraChronic lymphoid leukemia MyelofibrosisReactive

• Atrial septal defect • Aortic stenosis • Mitral valve prolapse • Angiodisplasia • Generalized atherosclerosisNeoplasia

Wilm's tumor Carcinomas and solid tumors

Immune S t i l th t

• Generalized atherosclerosis Miscellaneous Drugs :Hepatitis C

Other systemic diseases:Systemic lupus erythematosusAutoimmune disease Mixed connective tissue disease Graft-versus-host disease Ehlers Danlos syndrome

Other systemic diseases: Hypothyroidism Diabetes Uremia HemoglobinopathiesEhlers Danlos syndrome Hemoglobinopathies

list of the pathogenic mechanisms that operate in different p g pdisorders (Cont.)

Nonspecific (plasmin) Primary hyperfibrinolysisPrimary hyperfibrinolysisSecondary- hyperfibrinolysisLysis therapy

Enhanced shear stressCongenital cardiac defeats Aortic stenosisEndocarditisMalformation 'of vessels (M. Osler, Kasabach-Memt syndrome) Severe atherosclerosis β-thalassemia

Decreased synthesisDecreased synthesis Hypothyroidism

Unknown Valproic acidValproic acid Virus disease Liver transplantation

HypothesisHypothesisypyp

Proteolysis

decreased

AbADAMTS13

enhanced

stim AbAdsorptionfunctionADAMTS13

deficiencystim.

secretionfunction structure Clearance

VWD IIA TTPVWD IIBshearET

DDAVP LPDVWD IIC, IID, IIE, 2M

IIE, 2M(V)1, LPD

list of the pathogenic mechanisms that operate in different disorders

Specific autoantibodies or nonspecific autoantibodies that form circulating I l d h th l f WFImmune complexes and enhance the clearance of vWFLymphoproltteratlve disorders Neoplastic diseases Immunologic disordersImmunologic disorders

Adsorption of vWF onto malignant cell clones or other cell surfaces Lymphoproliferative disorders y p pNeoplastic diseases Myetoproliferative disorders Enhanced shear stress

Increased proteolytic degradation of vWF Specific Myeloproliferative disorders Enhanced shear stressU iUremia Ciprofloxacin

Laboratory tests for the diagnosisLaboratory tests for the diagnosis of avWS

Orientating ProcedurePatient & Family HistoryBleeding Time Filter testsAPTTPlatelet CountConfirmatory Tests:vWF:AgvWF:RCOFVIII activityvWF:CBTests performed specialized laboratory:RIPAMultimer Structure(low resolution gel)Platelet vWFvWF:AgII

Search for antibodies against theSearch for antibodies against the FVIII/vWF-complex

Direct methods Tests according to the Bethesda method torFvlll inhibitors (vWF: RCo, vWf.: CB,

vWF: Ag). Direct binding to immobilized vWF (high background for test with anti human IgMDirect binding to immobilized vWF (high background for test with anti-human IgM

and to lesser extent IgG, because blood group antigens are part of the vWF molecule even in patients with blood group 0).

Adsorption of the immune complex on a solid phase (protein A sepharose). Protein A binds vWF directly thus spurious posltlve results are possible whenever thebinds. vWF directly, thus spurious posltlve results are possible, whenever the reaction is not verified by rigorous control mechanisms.

Acquired hemophilia A was has to be ruled out in aflcases of avWS by the absence of factor VIII auto-antibooles in the Bethesda assay.

Indirect meas resIndirect measures Response of the vWr= to DDAVP and to factor Vill/vWF concentrate infusion and to

high dose . intravenous gammaglobulin. Analysis of the underlying mechanism causing avWS. Correction. of avWS in response y y g g p

to appropriate treatment of the underlying disorder