von Willebrand Factor Multimers

The von Willebrand factor (vWF) multimer analysis is primarily a qualitative test to identify variants of type II von Willebrand disease (vWD); therefore, there are no predefined reference ranges for its analysis. ^[1]

The plasma von Willebrand factor (vWF) multimer analysis provides a qualitative visual assessment of the size spectrum and the banding pattern of VWF multimers present in plasma compared with a normal control.

This test allows the identification of qualitatively abnormal variants of the vWF, also known as type II von Willebrand disease (vWD). vWF multimer analysis using low- and medium-resolution gels clearly distinguishes abnormal banding patterns that result from atypical vWF structures and allows their classification as vWD type IIA, IIB, IIM, or IIN, and their different subtypes (eg, IIA, IIB, IIC, IIC, IIE, and IID). ^[2]

(A study by Boender et al indicated that clinically, densitometric analysis using the medium-large visual index for vWF multimers is as accurate as visual observation for distinguishing type IIA and IIB vWD from the type IIM and IIN forms of the disorder. ^[3] )

Specifics for collection and panels associated with von Willebrand factor (vWF) multimers are as follows:

• Specimen type: Platelet-poor plasma (double-centrifuged specimen)

• Container/tube: Light-blue top (citrate)

• Collection method: Venipuncture

• Specimen volume: 1 mL

Other instructions are as follows:

• Draw specimen before coagulation factor replacement therapy.

• Spin down, remove plasma, and spin again. Double centrifugation is essential for accurate results as platelet contamination may affect the assay.

• Preferably, freeze specimen immediately to 40°C or less. Frozen specimens remain stable for 42 days. ^[4]

• vWF antigen, vWF activity, and factor VIII activity are required before performing this test. Submit separate specimens for the previously mentioned assays following their specific specimen requirements.

• Reject the specimen only if gross lipemia, hemolysis, or bilirubinemia is present.

Related tests include the following:

• vWF antigen

• Factor VIII activity

• Platelet count

• Platelet function tests

• Prothrombin time (PT)

• Activated partial thromboplastin time (aPTT)

• Bleeding time

• Factor VIII coagulant activity

• vWF ristocetin cofactor

• vWF collagen binding

• vWF antigen

• vWF propeptide

• Ristocetin-induced platelet aggregation (RIPA)

Multimeric analysis of VWF is carried out by electrophoresis of plasma samples using nonreducing agarose gels in the presence of sodium dodecyl sulphate. ^[5]

Following electrophoresis, the following steps are required:

1. The gels are fixed, washed, and dried and are then reacted with an antihuman vWF iodine I 125–labelled antibody.

2. The specimens are Western blotted and then probed with enzyme-labeled antibodies for subsequent visualization of vWF multimers using enzyme substrates.

3. Alternatively, enhanced chemiluminescence followed by autoradiography can be used to visualize the multimers.

The plasma vWF multimers analysis is a highly specialized, complex, and time consuming assay that requires specialized equipment and technical expertise but lacks standardization. ^[2]

Description

von Willebrand factor (vWF) is a large multimeric glycoprotein synthesized as a 2050 amino acid monomer in endothelium, megakaryocytes, and the subendothelial connective tissue. Every monomer contains particular binding domains that provide them the capacity to interact and bind other proteins specifically, factor VIII, collagen, and some platelet receptors. ^[6]

Monomers undergo dimerization in the endoplasmic reticulum by N -glycosylation of their C-terminal ends and then move to the Golgi apparatus where multimers are assembled by cross-linking of cysteine residues at their N-terminal ends to form vWF multimers of increasing molecular weight. Multimers of high molecular weight have the greatest hemostatic capacity.

vWF plays a major role in blood hemostasis. In response to various stimuli, vWF is released from storage granules in platelets and endothelial cells to control bleeding. Its deficiency or qualitative defect leads to a bleeding tendency known as von Willebrand disease (vWD); however, given the hemostatic capacity of its largest multimers, defects in its catabolism can predispose to thrombogenic disorders, including thrombotic thrombocytopenic purpura and hemolytic-uremic syndrome.

vWF also binds circulating factor VIII and prevents it peripheral degradation. Factor VIII is released from vWF by the action of thrombin.

The catabolism of vWF is mediated primarily by ADAMTS13, a metalloproteinase that cleaves vWF between tyrosine 842 and methionine 843 in the vWF A2 domain, generating a series of smaller multimers. It is this multimeric pattern that is analyzed in the laboratory.

In the absence of ADAMTS13, the cleavage of vWF does not occur and ultra-large vWF multimers appear in the plasma. The absence of ADAMTS13 and the generation of ultra-large vWF multimers are associated with thrombotic thrombocytopenic purpura. ^[7]

In a study of patients with acquired autoimmune thrombotic thrombocytopenic purpura, Béranger et al reported that assessment of the collagen-binding capacity of vWF (vWF:CB) can offer a more easily performed substitute for electrophoresis in determining the disease-associated loss of vWF high–molecular-weight multimers (vWF-HMWM). According to the investigators, enzyme-linked immunosorbent assay (ELISA), used to measure vWF:CB, revealed abnormally low levels of vWF-HMWM in 47.4% of the study’s patients, while electrophoresis found low values in 50% of patients. ^[8]

Indications/Applications

vWD is divided into 3 major categories: partial quantitative deficiency (type I), qualitative deficiency (type II), and total deficiency (type III).

Multimeric analysis of vWF is particularly important in the diagnosis of vWD type II, which is further divided into 4 variants (IIA, IIB, IIM, and IIN), based on the molecular characteristics of the dysfunctional vWF. The analysis shows the multimeric distribution of vWF, thus allowing classification based on the specific absence of large multimers (type IIB) or both intermediate and large (type IIA) multimers. The different subtypes have distinct clinical features and therapeutic requirements.

Manifestations of vWD include the following:

• Easy bruising

• Prolonged bleeding after minor trauma to skin or mucous membranes

• Severe hemorrhage after major surgery or delayed bleeding up to several weeks after surgery

• Heavy bleeding after tooth extraction or other oral surgery (eg, tonsillectomy and adenoidectomy)

• Menorrhagia

• Bleeding symptoms exacerbated by ingestion of aspirin and ameliorated by oral contraceptives ^[9]