von Willebrand disease (WVD)

• VWD defined as a deficiency of VWF causing impaired haemostasis.

• Synthesis and function
• von Willebrand factor (VWF) is a plasma glycoprotein which exists as large multimers 800-20,000 kDa.
• Synthesised in vascular endothelial cells and bone marrow megakaryocytes
• 2813 AA primary translation product undergoes processing in the ER to remove a signal peptide to form pro-VWF dimers (500kDa).
• Subsequently these are assembled into multimeric structures up to 20,000 kDa by N-terminal disulphide bond formation in the golgi apparatus. Extensive post translational modification then occurs (including glycosylation and sulphation).
• Then either secreted into the plasma or subendothelial matrix or stored in endothelial cell Weibel-Palade bodies and platelet alpha-granules.
• The largest multimers which are most haemostatically effective are stored in the Weibel-Palade bodies and platelet alpha-granules and are not seen in normal blood, only being released at sites of vascular injury.
• High molecular weight (HMW) multimers that are released by endothelial cells are broken down by ADAMTS 13.
• HMW multimers are important for mediating interactions between the subendothelium and platelet membrane GPIb and IIb-IIIa.
• Exact mechanism of interaction between VWF and the endothelium is uncertain but the binding and shear forces are thought to induce a conformational change in VWF which exposes the GPIb binding domain.  This slows platelet travel along the endothelium and allows the less rapid but higher affinity binding of GPIIb-IIIa to the RGD sequence on the VWF C1 domain.
• Also stabilises VIII in the circulation by binding to the D’/D3 domains – increases half life of VIII from <1h to 8-12h.

• Factors affecting the level of VWF
• Normal range 40-240 IU/dL

• Genetic defects
• Gene located at 12p13.2 comprises 52 exons spanning 178 kbp and gives rise to a 9 kbp mRNA
• Many mutations in the gene identified which alter expression, processing and function
• However some cases probably due to mutations in other genes

• ABO blood group
• Lower in blood group O (by up to 30%)

• Ethnicity
• Higher levels in African-Americans

• Age
• Neonates have higher levels falling to baseline at around 6 months
• Level increases throughout adult life by approximately 10IU /dL per decade

• OCP
• May increase levels therefore women with borderline levels should be retested when off the OCP and not pregnant

• Stress / exercise
• Mental stress for 20 min increased VWF Ag levels from 95 to 123 and FVIII levels from 125 to 217.
• Exercise can increase markedly eg to 194 after short run at maximal exertion.
• Therefore patients should rest for 15-30 min before venepuncture and avoid strenuous exercise before testing.
• Particularly problem in children

• Inflammation / malignancy / renal disease / liver disease / infection
• All increase VWF levels since it is an acute phase protein.
• Can be several times normal in malignancy

• Thyroid function
• VWF antigen is decreased in hypothyroidism / increased in hyperthyroidism

Von Willebrand Disease

• Type 1 – Partial quantitative deficiency

• Type 2 – Qualitative functional deficiency

• Type 3 – Complete quantitative deficiency

History

• Easy bruising, epistaxis, oral cavity bleeding, menorrhagia
• DH – aspirin / NSAIDs commonest cause of platelet dysfunction
• Haemostatic challenges
• Symptoms of acquired – new onset / underlying lymphoproliferative / myeloproliferative disorder
• Family history (but incomplete penetrance)

• Examination of scars of previous surgery / trauma should be examined - ? underlying collagen disorder

Investigations

1. Bleeding time
2. PFA-100
3. Platelet count
4. Coagulation screen
5. FVIII:C
6. vWF:RCo
7. vWF:Ag
8. vWF:CB
9. vWF multimer analysis
10. vWF:FVIIIB
11. RIPA
12. Platelet vWF:Ag
13. vWF inhibitor
14. Blood group

FBC

• Moderate thrombocytopenia in Type 2B
• Anaemia

APTT

• [PT, fibrinogen and TT will all be normal]

Bleeding time

• No longer has a role in screening for VWD (only 50% sensistivity) but is of value in the composite assessment of haemostasis

PFA-100

• Whole blood is aspirated through an aperture in a membrane coated with collagen impregnated with adrenaline or ADP.
• Needs adequate PCV and platelet count
• Platelets adhere, aggregate and occlude the aperture defining the closure time
• Simulates haemostasis in the high shear stress environment that occurs after small vessel injury
• 95% sensitivity for VWD
• However it cannot avoid the natural fluctuations that make diagnosing VWD difficult

Factor VIII assay (FVIII:C)

• Frequently reduced in VWD since half life is reduced but normal level doesn’t exclude

von Willebrand factor antigen (VWF:Ag)

• Plasma VWF:Ag levels measured by immunological (latex) methods (immunoradiometric IRMA – false positives with RhF) or enzyme linked immunosorbant assays ELISA – the reference method
• Limit of detection should be <1 IU/dL

von Willebrand factor ristocetin cofactor activity (VWF:RCo)

• Functional assay of GpIb binding (ristocetin is an antibiotic obtained from Nocardia lurida which promotes the interaction between GpIb and vWF)
• Washed platelets do not agglutinate in the presence of ristocetin unless there is vWF present.
• Assay performed by measuring the agglutination of normal platelets in dilutions of test plasma containing an excess of ristocetin.
• Ristocetin dimers bind to both VWF and platelet GpIb leading to the crosslinking of platelets. 
• A proposed ristocetin-binding site on VWF is the GpIb binding region.
• The result depends on the presence of HMW multimers and on an intact GpIb-binding site
• High inter-assay and inter-laboratory variability
• ELISA with antibody binding to the GpIb domain has been developed but is not as sensitive as the RCo (particularly for type 2A) – therefore not recommended

von Willebrand factor collagen binding assay (VWF:CB)

• ELISA in which the plate is coated with human type III collagen and the  amount of VWF which binds is measured
• The binding is very dependent on high molecular weight multimers and an intact collagen-binding site
• Therefore is more discriminatory than ricoff in separating 2A and 2B from 2M
• Less inter-assay variability than VWF:RCo and may be useful when there is diagnostic difficulty (particularly when trying to differentiate type 1 from 2 when the results are near the limits).
• May not detect type 2 defects due to GpIb binding region mutations (type 2M) but may detect type 2 defects due to collagen binding mutations that have a normal VWF:RCo.

vWF factor VIII binding studies (vWF:FVIIIB)

• ELISA based assay
• Serum incubated on plate incubated with vWF-Ab
• Recombinant FVIII added to the plate, incubated and washed
• Enzyme linked Ab to FVIII then added to detect the FVIII bound to the vWF

Ristocetin-induced platelet aggregation (RIPA)

• Ristocetin is added to the patient’s platelet-rich plasma at different concentrations (0.5 and 1.25mg/mL) to assess the affinity of VWF for platelets to determine the lowest concentration for aggregation.
• Aggregation at <0.5 mg/mL indicates VWF- platelet hyperactivity and is an essential diagnostic criteria for type 2B VWD and platelet type pseudo-VWD.
• RIPA is absent in severe forms of VWD but is frequently normal in those with VWF:RCo >30 U/dL

Multimeric analysis

• SDS-PAGE electrophoresis of test plasma samples using non-reducing agarose gels.
• The vWF multimers are then detected by using a peroxidise-conjugated anti-vWF antibody (or radiolabelled Ab)

• In normal plasma multimers appear as bands separated by the mass of 2 subunits
• Higher resolution gels reveal the presence of triplet bands due to proteolytic cleavage in the circulating blood.
• 2A have abnormal triplets

• VWF-FVIII binding assay
• VWF is isolated from test plasma by antibody capture onto a microtitre plate. The endogenous VIII is then washed off using a high concentration of calcium chloride.  Purified normal VIII is then added.  VWF and bound VIII are then quantified using ELISA and the chromogenic VIII assay.
• Markedly reduced binding diagnostic of type 2N.
• Large area of overlap between normal individuals and the intermediate binding profile of carriers of type 2N mutations.
• Consider in families affected by apparent mild haemophilia A without strict X-linked inheritance
• Normal range  vWF:FVIII >0.6

• Molecular analyses
• Can be helpful in some circumstances

• Plasma/Platelet mixing studies
• To distinguish type 2B from platelet-type pseudo-VWD
• Platelet type pseudo vWD, gain of function of GP1b (ie. platelet abnormality rather than vWF)
• RIPA will be heightened in both and reduced HMWM absent in both
• Addition of plasma to normal platelets will result in an enhanced RIPA in type 2B VWD (but not platelet-type pseudo-VWD)

• Platelet von Willebrand factor
• May be of value in to diagnose subtypes identified in the old classification
• Sometimes helpful when the bleeding tendency is discordant with the plasma level of VWF

Diagnosis

• To make a definitive diagnosis the patient must have:
• History of mucosal bleeding
• May be missing if no significant challenges or some form of prophylaxis has been introduced
• Reduced functional VWF levels
• Not clear that in vitro assessments of VWF function in vivo.  Thus tendency to bleeding cannot be confidently predicted when the measurements lie within a large grey area at the lower end of the normal range.
• Mutation in the VWF gene or a family history of VWD
• VWF gene is large and difficult to analyse so a genetic defect cannot be identified in many cases (particularly in type 1).  Some patients with VWD do not have defects in the gene.
• Incomplete penetrance.

• Recommendation that VWF:Ag and function is measured on at least 2 occasions with consistent results

1  

vWD vicenza

• normal production, but very rapid clearance
• Low Ag and ricoff but normal platelet vWF levels
• Normal RIPA
• Larger than normal vWF multimers
• After DDAVP, increase in FVIII and Ag levels, but virtually back to baseline by 4 hours 
  

Acquired VWD

• Presentation
• Extremely variable reflecting the diverse underlying conditions and pathological mechanisms:
• Decreased synthesis ( hypothyroidism, angiodysplasia, valproate)
• Autoantibody – will respond to IVIg
• Increased proteolytic degradation (cipro, MPD)
• Adsorption of VWF onto cells
• Mechanical degradation by high shear stress – decreased HMW multimers
• Precipitation of vWF eg HES

• Investigations also variable depending on the cause
• Autoantibodies only detected in about 10%
• Mixing studies and RIPA
• vWF Ag II assay – high levels indicative of increased clearance
• Half life of infused vWF

• Diseases associated with VWD
• Lymphoproliferative (inc MGUS)
• Myeloproliferative
• Neoplasia
• AI disease
• Cardiovascular (structural cardiac defects / angiodysplasia)
• Drugs
• Valproate, ciprofloxacin

• Treat with DDVAP, concentrates, IVIG

Management

DDAVP

• Synthetic vasopressin analogue which increases endogenous FVIII and vWF
• Iv – 0.3mcg/kg over 20 minutes
• Nasal 300mcg in adult or 150mcg in child (equivalent to 0.2mcg/kg iv)
• FVIII and VWF increase 2-5x baseline with a peak at about 60 minutes
• 2^nd dose can be given 12 hours later
• Use
• No therapaeutic use in type 3
• 2A and 2M, increases abnormal vWF and has a variable clinical effect
• 2B, controversial because may aggrevate thrombocytopenia
• 2N, increases FVIII but it has a short half life
• Side effects and contraindications
• Hypotension and facial flushing
• Fluid overload and hyponatraemia (fluid restrict to 1L after DDAVP)
• Avoid if heart failure, diuretics, atherosclerosis
• Avoid if <2 years, risk of hyponatraemia and seizures

Tranexamic acid

• Inhibits binding of plasminogen to fibrin
• Oral, iv or mouthwash
• Plasma half-life is 2 hours
• Side effects
• Nausea, vomiting, abdo pain
• Contraindicated
• Thromboembolic disorders
• Bleeding from upper urinary tract

Blood products

• All concentrates should be virally inactivated
• Manufactured from plasma from countries without CJD
• Most contain FVIII and VWF and there is concern regarding  thromboembolic risk
• Prolonged treatment, monitor factor VIII levels to avoid >100
• BPL 8Y
• Intermediate purity FVIII concentrate
• Dose should be calculated to achieve a target FVIII level
• Haemate P
• Intermediate purity FVIII concentrate
• Can be used as a continuous infusion
• Alphanate
• High purity
• Fanhdi
• Very high purity VWF concentrate
• Eg wilfactin
• May be useful if factor VIII levels high
• Platelets
• Can be considered ib bleeding persists with prolonged PFA 100 despite adequate replacement therapy
• Cryoprecipitate
• Not virally inactivated, but does contain functionally active VWF
• Should not be used unless other treatment modalities have failed

Dental treatment

• Local anaesthesia, no specific treatment required
• Inferior dental block, DDAVP or concentrate may be needed

Menorrhagia

• Consider hormonal treatments

Pregnancy

• Levels start to rise by week 6
• Many patients will achieve normal levels
• In type 2B, the increase can worsen thrombocytopenia
• Delivery plan and should be delivered in a major obstetric unit
• DDAVP is contraindicated in pre-eclampsia
• Consider antenatal diagnosis
• Type 3, levels don’t rise and concentrate will be needed to cover delivery
• Type 1 and 2, check levels at 34-36 weeks
• Vaginal delivery safe if ricof >40, >50 for CS
• Treatment if perineal tear or episiotomy
• Levels may fall rapidly after delivery and concentrate or DDAVP may be required for PPH
• Women should be warned of risk of bleeding at day 4-5
• C/S or type 3 will require treatment for 7 days
• Epidural anaesthesia not recommended in type 2/3
• If fetus at risk of type 2 or 3, avoid
• Fetal scalp monitoring
• Rotational forceps
• Ventouse
• Cord blood for testing (RIPA or FVIII (2N))
• USS to exclude ICH

Major Surgery

• Performed at an appropriate centre with appropriate laboratory
• DDAVP in responsive patients
• If unresponsive or CI, virus inactivated concentrate
• If mucosal surface involved, TA
• FVIII  and ricof levels
• 100 for major surgery and >50 post-op for about 7 days

Inhibitors to VWF

• May occur in type 3
• Little experience
• Novoseven or continuous infusion high dose rVIII
• May experience anaphylaxis to VWF