Transcript Slide 1

Von Willebrand Disease
Ali Akalin
Resident in Pathology
UCHSC
Von Willebrand Factor
• The gene for vWF subunit is located on chromosome 12p.
• Homomultimeric glycoprotein ranging in size from 600,000
to 20 million daltons
• The carbohydrate component is estimated to add
approximately 10-15% to the molecular mass.
• Synthesized in endothelial cells and megakaryocytes and
stored in Weibel-Palade bodies and platelet alpha granules,
respectively.
• vWF is initially formed in the ER as a pre-pro VWF
molecule, which would assembly into homomultimeric
protein after glycosylation, dimerization and
multimerization in the golgi organelle and storage places in
the cells.
Biosynthesis of VWF
Biosynthesis of VWF
Structure of vWF
Function of vWF
• Serves as the carrier protein for factor VIII (probably
factor VIII and vWF are brought together in storage
granules).
• Serves as the ligand that binds to glycoptrotein Ib receptor
on platelets to initiate platelet adhesion to damaged blood
vessel walls.
• vWF needs to be activated to be able to bind to GP 1b
receptor on platelets (Ristocetin, high shear force, collagen,
etc)
Variations in vWF Activity
in Health and Disease
• There is a continium of vWF levels between normal
subjects and patients with vWD.
• In patients with borderline results, it is necessary to repeat
diagnostic testing 2 or 3 times in 4-6 weeks intervals.
• vWF level varies with blood type.
Overlap of vWF levels
Variations in vWF by blood type
Variations in vWF Activity
in Health and Disease
• In patients with type O blood and moderate reductions in
plasma vWF, the presence or absence of a bleeding history and
family studies are needed to confirm or exclude a diagnosis of
vWD.
• Thyroid hormone and estrogen promote vWF synthesis.
• Deficiency of thyroid hormone reduces vWF in both normal
subjects and patients with vWD.
• Patients with mild vWD who take birth control pills or estrogen
replacement therapy may increase their slightly low vWF levels
into normal range.
• Both factor VIII and vWF are acute phase reactants and their
levels increase 1-3 times during exercise, inflammatory
conditions, pregnancy, adrenergic stimulation, etc…
Von Willebrand Disease
• First described by Erik von Willlebrand in individuals
living on the Aland Islands, an archipeloga between
Sweden and Finland in 1926.
• Characterized by mutations that lead to an impairment in
the synthesis or function of vWF. Acquired forms are
caused by different pathophysiologic mechanisms.
• Manifests as mucocutaneuos bleeding (epistaxis,
menorrhagia, GI bleed, ecchymosis).
• Most common inherited bleeding disorder affecting up to
1% of population.
• Autosomal inheritance
Classification
A- Quantitative deficiency of VWF
Type 1: Partial quantitative deficiency of vWF
Type 3: Virtually complete deficiency of vWF
B- Qualitative deficiency of VWF
Type 2A: Qualitative variants with decreased platelet dependent function
associated with the absence of high and intermediate molecular
weight vWF multimers
Type 2B: Qualitative variants with increased affinity for platelet GPIb
Type 2M: Qualitative variants with decreased platelet dependent function
not caused by the absence of high-molecular weight vWF
multimers
Type 2N: Qualitative variants with markedly decreased affinity for
factor VIII
Type I VWD
• Accounts for ~ 70 % of patients with VWD.
• Autosomal dominant inheritance.
• Clinical presentation ranges from mild to moderately severe
bleeding.
• Some patients are asymptomatic and detected incidentally in
studies investigating a relative with type 3 disease.
• Result from increased clearance of vWF from the circulation as
well as from decreased synthesis.
• Lab findings:
– Concordant reduction in VWF antigen and activity (ristocetin
cofactor activity), and factor VIII activity.
– All vWF multimers are present, although in decreased
concentration.
– RIPA is impaired, if VWF levels are sufficiently low.
– Platelet VWF activity and antigen are usually within the normal
range, but may be reduced.
Type 3 vWD
• Very rare (estimated incidence 1/million).
• Autosomal recessive inheritance.
• Characterized by marked decrease or absence of vWF as
a result of mutations in both alleles.
• Presents with severe bleeding into soft tissue and joints, in
addition to mucocutaneous bleeding.
• Lab findings:
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vWF antigen and activity below the limits of detection
RIPA is absent
Factor VIII activity is 1-10 % of normal.
vWF multimers are not visible on gel electrophoresis.
Type 2A vWD
• Accounts for ~ 10-15 % of cases of vWD.
• Autosomal dominant inheritance.
• Affected patients typically present with moderate to
moderately severe bleeding.
• Results from mutations in A2 domain that
– cause a defect in the intracellular assembly and
transport of normal VWF multimers (group 1), or
– affect a normal cleavage site in VWF and cause
increased susceptibility to proteolysis by the VWFcleaving protease after secretion (group 2)
Type 2A vWD
• Recessive forms of type 2A are caused by mutations within
the propeptide, resulting in impaired multimer formation
(type 2C) or dimer formation (type 2D).
• Lab findings:
• Discordant reduction in vWF activity and antigen
(Activity/Ag < 0.6).
• RIPA is reduced
• vWF multimer analysis show an absence of high and
intermediate molecular weight multimers.
• Factor VIII may be normal or reduced.
Type 2B
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Accounts for ~ 5 % of cases with vWD.
Autosomal dominant inheritance.
Presents with moderate to moderately severe bleeding.
Caused by mutations in A1 domain that result in readily
binding of vWF to GP 1b on platelets (gain of function
mutations).
• The increase in binding of larger multimers to platelet GP
1b results in their loss from the circulation.
• Lab findings:
– Discordant reduction in vWF activity and antigen (Activity/Ag <
0.6).
– RIPA is increased (aggregation in low concentrations of ristocetin).
– vWF multimer analysis show an absence of high molecular weight
multimers.
– Thrombocytopenia
– Factor VIII may be normal or reduced
Type 2B
• Type 2B vWD should be differentiated from platelet type
vWD.
• A distinction can be made by modifying RIPA assay as
follow:
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Patient plasma (vWF) + exagenous fresh normal platelets (1)
Patient platelet + exageneous plasma with normal VWF (2)
Type 2B: Low dose RIPA present in test 1, absent in test 2.
Platelet type vWD: Opposite
Type 2M
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Rare
Autosomal dominant inheritance.
Significant bleeding symptoms
Mutations in A1 domain that result in reduced binding of vWF
to platelet GP 1b.
Vicenza variant show larger than normal multimers in plasma
Some variants contain the propeptide in the multimers
Lab findings:
Discordant reduction in vWF activity and antigen (Activity/Ag <
0.6)
vWF multimer analysis shows the presence of full spectrum of
multimers.
RIPA is reduced.
Factor VIII level is reduced if vWF is sufficiently low.
Differentiate Between BernardSoulier- Type1- Type 2M
vWF:RCo
Type 1 vWD
RIPA
Often
Normal
Type 2M VwD
Bernard-Soulier
Syndrome
Normal
Absent
Type 2N (Normandy) vWD
• Uncommon
• Autosomal recessive inheritance.
• Mutations affecting the N-terminus (D’ and D3) of mature
vWF monomer within the binding site for factor VIII,
causing decreased binding to factor VIII.
• Presents with bleeding into soft tissue, joints.
• Low levels of factor VIII ( 5-15 %).
• Should be suspected who presents with isolated factor VIII
deficiency and in families in which an autosomal
inheritance pattern for factor VIII is suggested.
Type 2N (Normandy) vWD
• Lab findings:
– Normal vWF activity, antigen, RIPA and vWF multimer
analysis.
– Factor VIII is usually 5-15 % of normal.
– Decreased binding of normal factor VIII to the patient’s
VWF.
Diagnostic Laboratory
Testing for vWD
A- Tests to establish the
diagnosis
1- PTT
2- VWF antigen
3- vWF:R:Co
4- Factor VIII
5- vWF multimers
B- Tests to further define
vWD
1- Low dose RIPA
2- Collagen binding
3- vWF- Factor VIII
binding (Type 2N)
4- vWF antibodies
5- Platelet vWF
6- vWF AgII
7- DNA analysis
vWF Antigen Testing
• Immunoelectrophoresis (Laurell) (In the past)
• ELISA
• Automated latex bead assay (Rheumatoid factor might
cause false elevation of vWF antigen level in this assay)
vWF Activity
• Ristocetin cafactor activity (vWF:RCo): tests the ability of
the patient’s vWF to agglutinate formalin fixed commercial
platelets by binding to platelet GP 1b receptor in the
presence of ristocetin.
• (Patient plasma(vWF)+ formalin fixed commercial
platelet+Ristocetin)
Collagen Binding Assay
(vWF:CB)
• Patient plasma (vWF) is added onto collagen-coated plates. The
amount of vWF bound is proportional to the patient’s plasma vWF
level.
Ristocetin-Induced Platelet
Aggregation (RIPA)
• Measures the affinity with which vWF binds to the platelet GP
1b receptor by limiting the concentration of ristocetin in the
assay.
• Patient’s fresh platelet rich plasma (provides both platelet and
vWF) is mixed with sequentially lower concentrations of
ristocetin, covering a range of concentrations from 0.4 to 1.2
mg/ml. The absence or presence of aggregation at each
concentration is recorded.
• Platelet-rich plasma from patients with normal vWF does not
show aggregation to ristocetin concentrations below 0.6-0.8
mg/ml.
• Platelet-rich plasma from patients with type 2B vWD show
aggregation to ristocetin concentrations 0.4-0.5 mg/ml.
Ristocetin-Induced Platelet
Aggregation (RIPA)
• RIPA assay assumes that the patient’s platelet GP 1b is
qualitatively and quantitatively normal.
• Platelets from patients with Bernard-Soulier syndrome
(decrease or abnormality in platelet GP 1b) do not aggregate in
the presence of normal vWF and ristocetin.
• Gain of funtion mutations in platelet GP 1b cause an increase in
the affinity between vWF and GP 1b, resulting in aggregation of
platelets at low concentrations of ristocetin, which mimics type
2b vWD.
• A distinction can be made by modifying RIPA assay as follow:
– Patient plasma (vWF) + exagenous fresh normal platelets
– Patient Platelet + exageneous plasma with normal VWF
RIPA
vWF Multimers
• Patient’s plasma vWF
multimers (rarely eluted vWF
from platelets) are sizeseparated by electrophoresis
in low concentration agorose
gels to look for the presence
or absence of high,
intermediate and low
molecular weight vWF
multimers.
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1: Type 3
2: Normal
3: Type 2A
4: Type 2B
5: vWF extracted from platelets
Acquired vWD
• Assocciated with a number of different disease states.
Acquired vWD
• Multimer analysis reveals a decrease in high molecular
weight multimers, suggesting preferential removal of high
molecular weight multimers.
• Pathophysiologic mechanisms include:
– Antibody formation to vWF (SLE)
• Mixing studies
• ELISA
• Immunoblotting
– Proteolysis
• Plasmin (decompensated cirrhosis, pancreatitis, DIC,
thrombolytic tx)
• Elastase-lik e enzymes (myeloproliferative disorders)
Acquired vWD
– Binding to tumor cells with increased clearance (Wilms’
tumor, multiple myeloma, Waldenstrom macroglobulinemia)
– Decreased synthesis (hypothyroidism)
– High shear force (Noncyanotic congenital heart disease, high
grade aortic stenosis)
• vWF readily binds to platelets and/or proteolysis by the vWFcleaving protease at sites of high shear force.
Treatment
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General Considerations
Treatment of Inherited vWD
Treatment of Acquired vWD
Treatment of vWD during pregnancy
General Considerations
• Accurate and complete diagnosis.
• Patient’s
– past history of bleeding,
– response to treatment,
– associated medical conditions
– current medications.
• Aspirin containing medications
• NSAID
Treatment of Inherited vWD
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Desmopressin
Replacement therapy with vWF
Antifibrinolytic therapy
Estrogen
Topical agents
Treatment of Acquired vWD
• Depends on underlying disease and pathogenetic
mechanism
– Treatment of underlying disease
– Desmopressin
– vWF replacement therapy
– IVIG
– Corticosteroids
– Plasmapheresis
– Extracorporeal immunoadsorption
– Immunosuppressive medications (cyclophosphomide for
SLE)
– Factor VIIa
Treatment of vWD
During Pregnancy
• Not needed in the majority of women with vWD because
vWF levels rise during second and third trimesters.
• If needed during pregnancy, desmopressin should not be
used.
• Low factor VIII level appears to be most important
determinant of increased bleeding during delivery.
Recommendation is >50 % factor VIII level.
• The average time of onset for postpartum hemorrhage is
between 11-23 days post-delivery.
• Desmopressin and/or vWF replacement therapy is
apropriate during the first 2-4 weeks of post-delivery, if
needed.
References
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Rick M. E. www.uptodate.online.com
Ginsburg D., Wagner D. D. Hoffman: Hematology: Basic Principles and Practice, 4th ed.,
Copyright © 2005 Elsevier
Sadler JE. New concepts in von Willebrand disease.Annu Rev Med. 2005;56:173-91.
Federici AB. Clinical diagnosis of von Willebrand disease. Haemophilia. 2004 Oct;10 Suppl
4:169-76.
Favaloro E. J. et al. von Willebrand disease: laboratory aspects of diagnosis and treatment.
Haemophilia. 2004 Oct;10 Suppl 4:164-8.
Behrman: Nelson Textbook of Pediatrics, 17th ed., Copyright © 2004 Elsevier
Colman R. W. et al (editors). Hemostasis and thrombosis : basic principles and clinical
practice. Philadelphia : Lippincott Williams & Wilkins, c2001. 4th Edition.