Week 5 Coagulation Lecture 2 - Von Willebrand Disease

Question 1

Von Willebrand Factor (VWF)

Answer 1

VWF is a protein that is assembled from identical subunits into linear ‘strings’ of varying size referred to as multimers
The complex cellular processing consists of:
- dimerisation in the endoplasmic reticulum (ER)
- glycosylation in the ER and Golgi
- multimerisation in the Golgi
- packaging into storage granules
When vascular injury occurs, VWF becomes tethered to the exposed sub-endothelium
The high fluid shear rates that occur in the microcirculation induce a conformational change in multimeric VWF that causes platelets to adhere, become activated, and then aggregate
Platelet and endothelial cell VWF are released locally following cellular activation where this VWF participates in the developing haemostatic plug

Question 2

ADAMTS13

Answer 2

A Disintegrin-like And Metalloprotease domain [reprolysin type] with Thrombospondin type I motifs
VWF multimers that are subjected to physiologic degradation by ADAMTS13
Deficiency of ADAMTS13 is associated with thrombotic thrombocytopenic purpura (TTP)

Question 3

Von Willebrand Disease (VWD)

Answer 3

Von Willebrand disease (VWD) is an inherited disorder of coagulation characterised by a quantitative or qualitative abnormality of von Willebrand factor (vWf)
Patients with an appropriate bleeding history and VWF
activity 0.30-0.50 iu/ml should be regarded as having primary haemostatic bleeding with reduced VWF as a risk factor rather than VWD ( ‘Low VWF’)
A VWF activity <0.30 iu/ml is usually associated with bleeding and is more likely to be associated with a mutation in VWF

Question 4

Clinical Aspects of VWD

Answer 4

Males and females both inherit mutant alleles with equal frequency, however, clinical signs more common in females, ~2:1
Mode of inheritance varies with the type and
subtype of VWD
In some patients the disorder results from the double inheritance of a recessive gene
- these cases tend to be clinically severe
Some cases may occur sporadically with no family history reflecting a de novo mutation
Clinical signs are usually distinct from the haemophilias and rare coagulation disorders (may be similar to platelet disorders)

Question 5

VWD - Symptoms

Answer 5

Bleeding is characteristically mucocutaneous
Epistaxis
Menorrhagia
Easy bruising
Many cases investigated after excessive bleeding after a dental extraction etc.

Question 6

VWD Diagnosis

Answer 6

Very important to have a COMPLETE CLINICAL HISTORY as VWF levels can be altered by a number of physiological & pathological conditions
For example:
- oestrogens may raise VWF levels: physiological variation, pregnancy, oral contraceptives
- ABO Blood Groups: VWF levels are approximately 25% lower in blood group O individuals than in ‘non-O’
- systemic disease/inflammation: VWF is increased in a number of acute and chronic systemic disorders

Question 7

Investigation of VWD

Answer 7

Initial investigation for VWD should measure:
- FVIII
- VWF:Ag
- VWF activity (function)
VWF activity should be assessed by its ability to bind both:
- GPIb
- collagen
Secondary classification of VWD should measure:
- multimer analysis
- ristocetin induced platelet agglutination
- (VWF-FVIII binding assay)

Question 8

Classification of VWD

Answer 8

Type 1
- partial quantitative deficiency of VWF
Type 2
- qualitative deficiency of VWF
Type 3
- virtually complete deficiency of VWF

Question 9

Secondary Classification of Type 2 VWD

Answer 9

Type 2A
- qualitative variants with decreased platelet dependent function that is associated with the absence of HMW VWF multimers
Type 2B
- qualitative variants with increased affinity for platelet glycoprotein 1b
Type 2M
- qualitative variants with decreased platelet dependent function that is NOT caused by the absence of HMW VWF multimers
Type 2N
- qualitative variants with markedly decreased binding affinity for FVIII

Question 10

VWD FBC

Answer 10

May be normal
Provides an assessment of platelet concentration
- platelet disorders have similar clinical signs (=> differential diagnoses)
- thrombocytopenia specifically in type 2B VWD
Additional
- e.g. microcytic hypochromic anaemia with blood loss & iron deficiency

Question 11

VWD Coagulation Studies

Answer 11

PT is normal
aPTT often normal
- but may be prolonged if the FVIII level is reduced to below 30–40 IU/dL
PFA-100 Platelet Function Analyzer may be affected
in VWD
- overall sensitivity is 90% (close to 100% in type 2(excluding 2N) and type 3 VWD, less in type 1)

Question 12

VWD - VWF:Ag Testing

Answer 12

Quantity of VWF protein (antigen) in the plasma
Measured using enzyme-linked immunosorbent assay

Question 13

VWD - VWF:RCo

Answer 13

The ristocetin cofactor activity assay determines the ability of VWF to agglutinate platelets
Ristocetin dimers bind to VWF and induce a conformational change facilitating VWF binding to platelet GPIb and thus cross-linking of platelets
The agglutination is dependent on the presence of high molecular weight (HMW) multimers and an intact GPIb binding site

Question 14

VWD - VWF Multimer Analysis

Answer 14

SDS-agarose electrophoresis is used to determine the
complement of VWF oligomers in the plasma
Normal plasma contains up to ‘40mers’ of VWF
Multimers are classified as high, intermediate, or low molecular weight by counting bands 1-5 as low molecular weight, 6-10 as intermediate molecular weight, and 10 as high-molecular-weight (HMW) forms

Question 15

VWD - Platelet Aggregometry

Answer 15

Measures platelet aggregation by the change in optical signal after addition of an agonist (ADP, collagen, arachidonic acid)
Specific use of Ristocetin as an agonist ‘Ristocetin induced platelet aggregation’ (RIPA)

Question 16

Type 1 VWD

Answer 16

Partial quantitative deficiency of VWF
Type 1 VWD accounts for ~75 percent of symptomatic cases of VWD
May be due to the following mechanisms:
- decreased production of VWF
- rapid VWF clearance (e.g. VWF Vicenza)
Function:antigen ratio >0.6
Decreases in VWF protein concentration (VWF:Ag) and assays of VWF function (VWF:RCo).
Levels of FVIII usually parallel VWF and may be reduced secondary to reduced VWF
VWF multimer gels show no significant decrease in large VWF multimers

Question 17

Type 2 VWD

Answer 17

There are 4 sub-types of type 2 vWF
- 2A, 2B, 2M, 2N
Bleeding in type 2 VWD is often thought to be more severe than in type 1 VWD
A function:antigen ratio of <0.6 should be used to identify patients with type 2 VWD

Question 18

Type 2A VWD

Answer 18

Type 2A has decreased VWF-dependent platelet adhesion with selective deficiency of high-molecular-weight multimers
The deficit of large multimers predisposes persons to bleed
May be caused by
- mutations that interfere with the assembly or secretion of large multimers
- mutations that increase the susceptibility of VWF multimers to proteolytic degradation in the circulation
Levels of VWF:Ag may be normal or modestly decreased
VWF function is abnormal as shown by markedly decreased VWF:Rco activity

Question 19

Type 2B VWD

Answer 19

Type 2B VWD is caused by mutations that increase platelet-VWF binding, which leads to the degradation and consequently depletion of large, functional VWF multimers
Large multimers are assembled in a normal fashion but after secretion they bind spontaneously to platelets and are subsequently cleaved by ADAMTS13
This results in predominantly relatively small multimers which are not able to mediate platelets adhesion effectively
In contrast to type 2A or 2M, patients typically have thrombocytopenia that is exacerbated by surgery, pregnancy, or other ‘stress’
Platelet function may also be decreased as circulating platelets are coated with mutant VWF

Question 20

Type 2M VWD

Answer 20

Type 2M VWD mutations reduce the interaction of VWF with platelet GPIb or with connective tissue but do not substantially impair multimer assembly
Also includes defects of VWF collagen binding

Question 21

Type 2N VWD

Answer 21

Most mutations that cause type 2N VWD occur within the FVIII binding site of VWF
Decreases FVIII levels so that type 2N VWD appears as an autosomal recessive form of haemophilia A
In typical cases, the FVIII level is less than 10 percent, with a normal VWF:Ag and VWF:RCo.

Question 22

Type 3 VWD

Answer 22

Type 3 VWF has a virtually complete deficiency of VWF
Characterised by ‘undetectable’ VWF:Ag
Bleeding signs also occur in 26-48% of carriers
FVIII levels usually are also very low

Question 23

VWF Gene

Answer 23

Located on the short arm chromosome 12
VWF pseudogene (VWFP) on chromosome 22 has 97% similarity to coding gene (VWF)

Question 24

Platelet-Type VWD (PT-VWD)

Answer 24

Results from gain-of-function mutations within the platelet GP1BA gene coding for the glycoprotein (GP)Ibα protein
These mutations cause excessive binding between GPIbα and von Willebrand factor
- => removal of both VWF protein and platelets from the circulation => clinical bleeding phenotype
Clinically, patients typically present with mild to moderate mucocutaneous bleeding
However, they can have life-threatening bleeding during conditions of haemostatic challenge such as surgery & childbirth

Question 25

PT-VWD - Laboratory Features

Answer 25

Varying levels of thrombocytopenia
VWF:RCo/VWF:Ag ratio < 0.6
Enhanced ristocetin-induced platelet aggregation (RIPA), typically in response to doses < 0.6 mg/dL of ristocetin
Loss of high molecular weight VWF multimers

Question 26

Acquried VWD

Answer 26

Mild to moderate bleeding disorder due to a decreased concentration of VWF
Is not caused by a mutation in the VWF gene
Is most often seen in persons older than 40 years with no previous bleeding history
Causes variety of features:
- lymphoproliferative disorders
- SLE
- severe thrombocytosis
- removal of VWF from circulation by binding to tumour cells