Von Willebrand factor

Gene: ch 12.

Structure
- Glycoprotein (500 – 20000 kDa) composed of multimers (> 2 dimers). Mmonomer is 278 kDa
(22 aa) Signal peptide
ER
(741 aa) Propeptide → Dimer
(2050 aa) Mature subunit removal of the signal peptide∧dimerization viathe C−terminal cysteine

NH2
- Domains: – D1-D2-D/-D3-A1-A2-A3-D4-B1-B2-B3-C1-C2-CK – COOH

- (22 CHO side chains): 10-19% of the MW. 13% of them end in the ABO Ags.

- Binding sites to: Collagen – Fibrinogen - GP Ib - GP IIbIIIa.

- Ag/function ratio: > 0.7.

- VWF-F VIII complex:

1. F VIII C: the functional part.
2. VWF.
3. F VIII Ag: the antigenic part.

Origin
1- ECs:
- Constitutive pathway: Secreted directly into the plasma
- Regulated pathway: Weibel Palade bodies:
Thrombin ,− Adrenaline−Histamine plasma metalloproteinase
−Vasopressin−C ytokines Secretion ADAMTS−13 Degradation.
¿ ¿ →
❑ ❑
2- Megakaryocytes and PLTs: stored in α granules and not secreted.

Release from stores

Stress – Exercise – Occlusion – Hormones – Pregnancy – Thrombin –Adrenalin - Histamine - IL-1.

Function
1- PLTs adhesion under high shear: binding to GPIb and collagen.
 2- Carriage, stabilization and protection of F VIII from rapid degradation.
Von Willebrand disease (The commonest inherited bleeding disorder)
Type % Abnormality Inheritance VIII VWF- RiCoF Multimer analysis
Ag
1 75 Quantitative partial deficiency AD N L L Normal
in combination with other
modifying genes, in particular
the ABO locus.
2A  Mutation in A2 domain AD but
some
N N L Absent large
intermediate size. Some
and
→ ↑ susceptibility to
recessive have abnormal triplets
proteolysis by
forms may
ADAMTS-13 →
occur
Absent large and
15
intermediate size
 Mutation in
thrpropeptide, D/ - D3
or the cystien knot.
2B Mutation in A1 domain → AD N L N Absent large. Normal
Excessive affinity of VWF to triplets.
GPIb without activation →
consumption of PLTs
(thrombocytopenia) and large
multimer
2M Mutation in A1 domain → AD N N L Normal. Some have ultra-
Decreased affinity of VWF to large.
GPIb
2N Mutation in F VIII carrying site AR N N Normal
autosomal
hemophilia 10 (D/ domain) 15-35%
A

3 Quantitative complete AR L L L Virtually absent

deficiency due to inactivating or (severe)
null mutations in both alleles or
large deletion of VWF gene.

Clinical picture and complications

nd rd
- Age: 2 or 3 decades - Family history
- Bleeding: from small vessels where the high shear requires VWF for PLT adhesion:
o Muco-Cutaneous: Bruising - After minor injury (not life threatening) - No Hemarthrosis.
o Mild to Moderate
o Spontaneous (defective PLT adhesion) or post-traumatic (↓ F VIII).
- Type 2B: Thrombocytopenia during pregnancy (acute phase).
- Type 2N and type 3: resembles severe hemophilia A.
- Type 3: anaphylactic shock after VWF infusion.

Treatment
 - Severe bleeding: cryoppt.
- Less severe: dDAVP.
- For surgery: blood transfusion.
Pseudo VWD (PLT type)
- (AD) missense mutation in GPIb → Excessive binding of GPIb to VWF→ ↓
VWF.

- Washed PLT of the patient + cryoppt → spontaneous aggregation.

- Washed donor PLT + patient plasma → no spontaneous aggregation.
- PRP of the patient + low dose of ristocetin (0.5 mg/ml) → ↑ response.

- Treatment: PLT concentrate.

Acquired VWD
- Abs→ ↑ clearance or inhibition: LPD – Paraproteinaemia - MPN - Auto immune D.
- ↓ Synthesis: Hypothyroidism and diabetes.
- Shear → destruction of VWF: Severe Aortic stenosis or leaking
- Some plasma expanders as hydroxyethyl starches.
- Adsorption of VWF to tumor cells or activated PLTs: Malignancy

- ↓VWF Ag - RiCoF assay - collagen binding assay.

- ↓ VWF propeptide: ↑ clearance.
- Mixing studies.

- Treatment: dDAVP, VWF concentrates and IV immunoglobulins.

 Investigations
 Problems:
- Broad normal range.
- VWF is an acute phase protein - ↓ during menstruation - ↑ in neonates.
- 2.5 % of populations with ↓ VWF Ag have normal hemostasis.
- Differ according to the blood group (group O: 30% lower): must be 2 SD
< mean of the same blood group population.
- Family history is not helpful: weak genetic penetrance.
- History of minor bleeding episodes is very common.

VWF-Ag (N: 50 – 200 iu/dl)

a- ELISA.
b- Immunoturbidimetric (latex agglutination): False positive with +ve RhF and acquired VWD.

VWF function assay (N: 50 – 200 iu/dl)

a- Ristocetin co-factor assay (of poor reducibility): Washed normal PLTs + Patient
PPP + ristocetin → agglutination depending on patient VWF → comparing with a
standard curve.

b- Collagen binding assay (complementary to RiCoF - more precise - sensitive to

large mutlimers): ELISA coated with collagen + patient’s plasma + anti-VWF Abs.

c- F VIII binding assay: Assessment of F VIII C (clotting or chromogenic) → ↓ →

ELISA wells coated by anti VWF Abs + plasma of the patient → + recombinant F
VIII → assessment of F VIII (chromogenic or immunoassays).

Multimeric analysis (when Ag/function < 0.7)

a- Autoradiographic method (the gold standard): using radioactive iodine
b- Non-radioactive method: electrophoresis on Na dodecyle sulphate agarose gel →
fixation → conjugated VWF Ab + coloring substrate → scanning of the gel.
Normal Electrophoresis: triplet of a dark central band sandwiched between two lighter
bands.
 Genetic analysis of VWF gene
 Sequencing - PCR – RFLP.