Von Willebrand Disease Alison Street Malaysia April 2010 OUTLINE - - PowerPoint PPT Presentation

Von Willebrand Disease

Alison Street Malaysia April 2010

OUTLINE

• Physiology of VWF
• Clinical presentation of VWD
• Classification of VWD with emphases on

Type 1, 2B and 2N disease

• Testing for VWD
• Treatment

Pedigree of the original family described by Erik von Willebrand in 1926

VWF Gene

• Located at chromosome 12p13.2
• 52 exons spanning 178 kb
• 9kb mRNA
• Partial pseudogene at chromosome 22

– VWF and pseudogene diverge by 3.1% in sequence – Probable relatively recent origin of pseudogene by partial gene duplication

5’ 3’ N C

• -S--

N N C C C C C C C C C C

• -S--
• -S--
• -S--
• -S--
• -S--
• -S--

VWF mRNA Pro-VWF Pro-VWF dimer Pro-VWF multimers Propeptide dimer VWF multimers

RER Golgi

VWF biosynthesis and processing

VWF synthesis and processing

• VWF synthesised in endothelial cells and

megakaryocytes

• Primary translation product processed in ER

to form pro-VWF dimers

• In Golgi apparatus VWF propolypeptide

mediates assembly of dimers into multimers

• f molecular wt. up to 20 x 106
• Mature VWF secreted directly into plasma or

subendothelium, or stored in endothelial cell Weibel-Palade bodies and platelet alpha granules

VWF FUNCTION

• Platelet-dependent function in primary

haemostasis

– High shear stress – High molecular weight multimers

• Carrier for FVIII
• VWF plasma half-life ~12 h

VWF gene and protein – structure / function relationships

VWF gene (chromosome 12) VWF primary translation product Mature secreted VWF protomer - functional sites

Region duplicated in partial VWF pseudogene (chromosome 22)

VWF binds via A3 domain to collagen inducing a conformational change Which allows GP1b to bind to VWF A1 domain This slows the platelet travel and allows activation of FVIII Activation of platelets leads to the binding of GPIIb/IIIa to VWF C2 domain which is slower but has higher affinity

VWF functionality - high molecular weight multimers

• Essential to promote platelet-vessel wall

and platelet-platelet interactions at high shear

• Circulating VWF multimer size is

controlled by proteolytic cleavage by ADAMTS13

ADAMTS 13

• A specific plasma protease which

proteolyses the bond between Tyr 1605 and Met 1606 (Tyr 842 and Met 843 of mature sub-unit)

• Generates a spectrum of circulating vWF

species (single – twenty dimer multimers)

• f which larger ones have most affinity for

platelet Gp1b and Gp11b/111a receptors

VWF multimer analysis

Normal High MW Crucial for normal function Low MW

Von Willebrand Disease

• Inherited deficiency or dysfunction of VWF
• Bleeding results due to impaired platelet

adhesion and lower levels of FVIII

• VWD prevalence haemostasis centres :

0.0023-0.01%

• Abnormal VWF prevalence (screening):

0.6-1.3%

Clinical presentations

• Bleeding: mucous membrane and skin sites
• Personal history of bleeding
• Family history of bleeding
• Bleeding: severity, site, duration, type of

injury or insult, ease of stopping, concurrent medications e.g. aspirin, clopidogrel, warfarin, heparin

• Liver,kidney, bone marrow disorder
• Examination: bruising/bleeding & exclude
• ther diagnoses

Bleeding symptoms are common in people with normal levels of VWF!

• 23%of healthy controls, replying to a

bleeding questionnaire, report at least one symptom of bleeding compared with 88%

• f patients with a diagnosis of VWD Type

1.

• Standardised bleeding scores do not

predict VWF gene or plasma levels within families but do predict for post-operative bleeding

1994 Classification of VWD

• Type 1 VWD (~ 70% of cases??)

– Partial quantitative deficiency of VWF

• Type 2 VWD

– Qualitative deficiency of VWF – Sub-types 2A, 2B, 2M, 2N

• Type 3 VWD

– Virtual complete deficiency of VWF

Sadler, Thromb Haemost 1994, 71, 520-5

Clinical assessment

• Bleeding: mucous membrane and skin sites
• Personal history of bleeding
• Family history of bleeding
• Bleeding: severity, site, duration, type of

injury or insult, ease of stopping, concurrent medications e.g. aspirin, clopidogrel, warfarin, heparin

• Exclusion of liver, kidney or bone marrow

disorders

• Examination: bruising/bleeding & exclusion of
• ther diagnoses

Classification

type description inheritance prevalence bleeding 1* partial quantitative deficiency AD up to 1% Mild-mod 2A VWF-dep platelet adhesion Loss high & int MW multimers AD or AR uncommon variable- usually moderate 2B affinity for platelet GPIb Loss high MW multimers AD 2M VWF-dep platelet adhesion without selective loss high MW multimers AD or AR 2N binding affinity for FVIII AR 3* almost complete deficiency AR rare high * Quantitative deficiency vs qualitative deficiency

Laboratory testing

• Skin Bleeding Time
• Platelet Function Analyser/ Aggregation
• Plasma testing for VWF antigenic and

activity levels

• Activity measured by Ristocetin Co Factor

and Collagen Binding assays

• Full Blood Examination
• F VIII levels/ F VIII binding

Summary of criteria for diagnosis and classification of VWD

Type 1 Type 3 Type 2A Type 2B Type 2M Type 2N VWF:Ag Decreased < 5% Decreased (Normal) Decreased (Normal) Variable Normal VWF activity Decreased Absent Markedly decreased Decreased (Normal) Decreased relative to VWF:Ag Normal RIPA Reduced (Normal) Absent Markedly reduced Increased Reduced (Normal) Normal Multimers Essentially normal Absent HMW absent HMW usually absent Normal Normal VWF: FVIIIB Normal NA Normal Normal Normal Reduced

Approach to classification of VWD

Quantitative defect Qualitative defect Type 1 or Type 3 VWD Type 2 VWD Normal platelet-dependent Defective platelet- VWF function & dependent VWF function defective FVIII binding Type 2N VWD Gain in function Reduced function Type 2B VWD Loss of HMW multimers HMW multimers present Type 2A VWD Type 2M VWD

Laboratory features of Type I VWD

• Partial quantitative deficiency of VWF
• Concordant VWF:Ag & CBA/RistoCoF
• Normal multimer pattern
• When VWF <20 IU/dL may identify mutations

which interfere with intracellular transport of dimeric proVWF or promote rapid clearance

• f VWF from circulation
• Lower VWF levels, more likely to have VWF

gene mutations, significant bleeding history + strong Family History*

*Goodeve et al. Blood 2007;109:112-121, James et al. Blood 2007;109:145-154

Many diagnoses of Type 1 VWD are false positives

• Past bleeding history is a better guide to

risk assessment for future bleeding particularly when the VWF is between 30 and 50 IU/dl (RR 50-200%)

• Neither symptoms nor VWF level are

predicted by VWF genetic testing at this level of deficiency

50% 0% VWF Level

• eg. Dominant Negative

VWF Mutations VWF Mutations

missense splicing transcriptional

+

ABO Blood Group

+

Other Genetic Modifiers ~35% of cases Incomplete Penetrance Highly Penetrant

Is it just a low VWF level or VWD?

• Genetic factors account for minority of

heritable variation in VWF levels

• No linkage to VWF locus when VWF >30
• Other inheritable and environmental factors

influence plasma VWF

*Mannucci et. al. Blood 1989;74:2433-2436

ISTH VWF mutation database

(www.vwf.group.shef.ac.uk)

• Pre-Canadian, EU, and UK studies - 14

different VWF gene mutations reported in association with type 1 VWD

• By 31 July 2007 - 117 different mutations
• r candidate mutations reported

D1 D2

D’

D3 D4

A2 A3 A1

NH2 COOH 1 2813

B1-3 preproVWF VWF Monomer

22aa 741aa 2050aa

CK

C1 C2

5’ Regulatory Sequence Mutations

• 2731C>T
• 2714G>A
• 2639G>A
• 2615A>G
• 2533G>A
• 2522C>T
• 2487G>A
• 2328T>G
• 1896C>T
• 1886A>C
• 1873A>G
• 1665G>C
• 1522del13

Propeptide Sequence Mutations G19R L129M D141G G160W N166I c.1109+2T>C (splice) c.1534-3C>A (splice) M576I A641V W642X D’-D3 Mutations K762E c.3072delC M771I c.3108+5G>A (splice) c.2435delC I1094T R816W C1111Y R854Q c.3379+1G>A (splice) c.2685+2T>C (splice) Y1146C c.3537+1G>A (splice) C1190R c.2686-1G>C (splice) R1205H R924Q R924W C996E D3, A1-A3 Mutations 1546_1548del3 P1413L V1229G N1421K N1231T Q1475X P1266L R1583W V1279I Y1584C c.3839_3845dup7 c.4944delT R1315C R1668S L1361S R1379C K1405del A3-D4 Mutations c.5180insTT E2233G V1760I c.6798+1G>T (splice) L1774S R2287W K1794E N1818S V1850M P2063S R2185Q c.6599-20A>T (splice) T2104I B1-B3,C1,C2 Mutations C2304Y T2647M R2313H C2693Y C2340R P2722A G2343V c.8412insTCCC R2379C R2464C c.7437+1G>A (splice) S2497P G2518S Q2544X

Candidate VWF mutations in type 1 VWD – Canadian, EU and UK studies

Genetics of type 1 VWD (2010)

• Molecular mechanisms more clearly

understood

• But functional studies assessed in only

about 10% of reported candidate mutations

• A proportion of type 1 VWD likely to be

due to defects away from VWF locus

• Definition of type 1 VWD not restricted

to VWF mutations

Type 2 VWD

• Qualitative variants of VWF
• The proportion of large VWF multimers is

reduced in Types 2A and B with consequent loss of function in 2A and gain in 2B

• Mutations lead to interference with assembly
• r secretion of large multimers or increased

susceptibility to proteolytic degradation

• Type 2N results in impaired binding of FVIII

and may be confused with mild haemophilia

Proposed model of VWF synthesis and catabolism

Sadler et al, JTH 2006, 4, 2103-14

Normal VWD variants

Summary of criteria for diagnosis and classification of VWD

Type 1 Type 3 Type 2A Type 2B Type 2M Type 2N VWF:Ag Decreased < 5% Decreased (Normal) Decreased (Normal) Variable Normal VWF activity Decreased Absent Markedly decreased Decreased (Normal) Decreased relative to VWF:Ag Normal RIPA Reduced (Normal) Absent Markedly reduced Increased Reduced (Normal) Normal Multimers Essentially normal Absent HMW absent HMW usually absent Normal Normal VWF: FVIIIB Normal NA Normal Normal Normal Reduced

Genetic diagnosis in type 2 VWD

• Generally well-characterised genetic disorders
• Screening for type 2N VWD in non X-linked mild or

moderate FVIII deficiency

• Differentiation of type 2B VWD and platelet-type

pseudo VWD

• Differential diagnosis of type 1 and type 2M VWD

Type 2B VWD

• Increased affinity of mutant VWF for

platelet glycoprotein 1b

• Circulating platelets coated with mutant

VWF - may prevent platelet adhesion at sites of injury

• Variable thrombocytopenia - reversible

sequestration of VWF-platelet aggregates in microcirculation

Type 2B VWD

• VWF bound to platelets susceptible to

proteolysis by ADAMTS13 → high molecular weight multimers usually absent in plasma

• VWF activity usually reduced
• VWF:Ag reduced or normal
• Increased Ristocetin Induced Platelet

Aggregation.

• May be confused with platelet type or

“pseudo” VWD

Genetics of Type 2B VWD

• Autosomal dominant inheritance
• “Gain of function” missense mutations

confined to VWF A1 domain (containing platelet GP1b binding site)

• 4 VWF gene mutations account for

~ 90% of type 2B VWD

Type 2N VWD (VWD Normandy)

• Reduced affinity of VWF for factor VIII
• Phenotype similar to mild or moderate

haemophilia A or haemophilia A carriers.

• Should be considered and tested in

all cases of spontaneous mild haemophilia

VWF-FVIII binding assay (VWF:FVIIIB)

Normal Patient Heterozygous control Homozygous control

Functional domains and mutations reported in VWF up to 2006

Type 2N VWD

• Recognition and differentiation of type 2N

VWD from haemophilia A important for:

– Precise genetic counselling – Accurate carrier or prenatal diagnosis – Appropriate treatment of bleeding episodes

• Consider type 2N VWD as a possible

diagnosis in patients with FVIII deficiency which is not clearly X-chromosome linked

Treatment of VWD

Selected according to

• Past bleeding experiences
• Level of VWF activity
• Type of procedure
• Product availabilty (need to use VWF

containing concentrates, not recombinant Factor VIII)

DDAVP

• Stimulates endothelial production and

secretion of VWF from endothelial cells. Can give for 4-5 days

• Of particular value in Type 1 disease

where baseline level >10%

• Should give a trial dose to assess effect

and tolerability (need to monitor Na+)

Special considerations

• Beware all cases of “mild” Haemophilia, it

might be VWD Type 2N

• It is important to develop and

communicate care plans for delivery and post partum management in women with VWD, secondary bleeding is common.

• Acquired VWD may complicate

lymphoproliferative disorders (Type 2A like)

THANK YOU

Plasma VWF levels rise with

• Increased age
• Non-O blood group

(mean VWF O=75U/dL, 95% VWF levels for O blood donors = 36-157 U/dL* due to reduced vWFsurvival)

• Lewis blood group (secretor)
• Adrenaline, Thrombin (DDAVP)
• Inflammatory mediators
• Endocrine hormones

(periods/pregnancy/OCP)

*Gill et. al. Blood 1987;69:1691-1695

Von Willebrand Factor cleavage

Chromosome 12, 178kb, 52 exons

Sadler et. al. J Thromb & Haemost 2006, 4: 2103-2114

ADAMTS13 cleavage site

Platelet GPIb binding site THBS-1 binding site Probable ADAMTS13 binding site

Normal physiology of ADAMTS13

Adapted from Moake JL. NEJM 2002;347:589-600

Weibel Palade body

Endothelial cell Secretion of multimers

Binding site

Unusually large von Willebrand multimers

ADAMTS13

ADAMTS13 processing & VWF: a delicate balance between thrombosis and bleeding

ULvWF Monomers vWF

ADAMTS13

 ULvWF: Congenital or acquired deficiency

• f ADAMTS13

TTP Monomers vWF: Mutations in vWF  cleavage by ADAMTS13 Type IIA vWD

Normal conditions

• FVIII-VWF circulates but doesn’t strongly interact

with platelets or endothelial cells

NHLBI Guideline VWD 2007

FVIII VWF multimers Resting platelets

Vascular injury

• VWF adheres to vessel subendothelial matrix
• With shear, VWF multimers uncoil, platelets adhere

and become activated

NHLBI Guideline VWD 2007

VWF multimers activated platelets

Platelet-fibrin plug

• Activated platelets expose phosphatidyl serine and

bind and activate FVIII  clotting

• Platelet-fibrin plug  thrombolysis  tissue repair

NHLBI Guideline VWD 2007

Platelet type pseudo-VWD (pVWD)

• Rare autosomal dominant disorder
• Mutations in platelet GpIb/IX receptor

cause increased platelet-VWF binding

• Clinical presentation variable
• Laboratory presentation very similar to

type 2B VWD (mild thrombocytopenia, increased RIPA, decreased HMW multimers)

• Potential for misdiagnosis

Differentiation of type 2B VWD and platelet-type pseudo-VWD (pVWD)

• Genotyping

– Targeted screening of VWF gene exon 28 and platelet GpIb/IX receptor gene

• Plasma / platelet mixing studies

– Patient platelets (PRP) + normal VWF (cryoprecipitate)

• Spontaneous aggregation in pVWD
• Absence of platelet aggregation in type 2B VWD

(reliability?) – Patient VWF (PPP) + normal platelets (PRP)

• Enhanced RIPA in type 2B VWD
• RIPA not enhanced in pVWD

A Disintegrin and Metalloprotease with ThromboSpondin motifs 13

Levy,G.G. et al. Blood 2005;106:11-17