von Willebrand factor assays

Von Willebrand factor (VWF) facilitates platelet adhesion via the glycoprotein Ib/IX/V (GPIb/IX/V) complex on the platelet surface, and the glycoprotein IIb/IIIa (GPIIb/IIIa; integrin α_IIb/β₃) complex mediates adherence to fibrin or fibronectin associated with the vascular wall. VWF activity assays measure the ability of VWF to bind platelets or GPIb. The VWF/factor VIII-binding assay is used to distinguish the 2N subtype from other von Willebrand disease (VWD) type 2 subtypes, and VWF multimer analysis is typically performed after initial VWF assays to help define the VWD variant subtype.

The VWF ristocetin cofactor activity (VWF:RCo) assay measures the ability of VWF to bind to platelets using an instrument (typically an aggregometer) to measure platelet aggregation in the presence of the antibiotic ristocetin. This assay is typically performed in conjunction with a VWF antigen assays to calculate a ratio of VWF level vs VWF function in the classification of VWD type 1, 2 or 3. Intra- and inter-laboratory variability associated with the VWF:RCo, however, can increase the potential for misdiagnosis.^1-5

The VWF:glycoprotein-IbR (VWF:GPIbR) assay uses ristocetin to trigger VWF binding to wild-type, plasma-derived or recombinant GPIb. This assay is available for automated applications and can be used as a precise and sensitive alternative to the VWF:RCo for measuring VWF activity.⁵

The VWF:glycoprotein-IbM (VWF:GPIbM) assay can also be used as an alternative to the VWF:RCo assay during differential diagnosis and classification of VWD. Similar to the VWF:RCo and VWF:GPIbR assay, the VWF:GPIbM measures VWF function. However, the VWF:GPIbM measures VWF binding to a modified gain-of-function version of a GPIb protein independent of ristocetin, which can reduce intra- and inter-laboratory variability. The VWF:GPIbM assay is used in conjunction with the VWF antigen assay to build a VWF quantity-to-function ratio for the initial classification of VWD.^2-5

The ristocetin-induced platelet aggregation (RIPA) assay is similar to the VWF:RCo assay. However, it is used to distinguish VWD type 2B and platelet-type VWD from VWD type 2A. In RIPA, a platelet rich plasma sample from the patient is exposed to low concentrations of ristocetin using an instrument (typically an aggregometer) to measure aggregation. Samples from patients with VWD type 2B and platelet-type VWD will aggregate under these conditions, whereas samples from normal patients and patients with all other types of VWD will express little to no aggregation.^1,4

von Willebrand factor platelet-binding (VWF:PB) is used to distinguish patients with VWD type 2B from those with VWD type 2A, platelet-type VWD and pseudo-VWD. The VWF:PB assay uses paraformaldehyde-fixed normal platelets in the presence of low-concentration ristocetin. The VWF binding capability is measured using a labelled antibody. Increases in VWF:PB are observed in samples from patients with VWD type 2B. Samples from patients with VWD types 1, 2A, 2M, 2N, 3 and platelet-type VWD typically do not bind platelets in this assay.^1,4

The von Willebrand factor/factor VIII-binding (VWF-FVIII-binding) assay is used to distinguish the 2N subtype from other VWD type 2 subtypes. This assay uses a modified enzyme-linked immunosorbent assay (ELISA) technique. Anti-VWF antibodies are seeded on a plate and capture VWF when exposed to a patient sample. The plate is then washed to remove endogenous FVIII bound to VWF in the sample. The plate, with the captured VWF is exposed to a known concentration of recombinant FVIII. Finally, an enzyme-linked anti-FVIII antibody is used to measure the concentration of FVIII bound to the patient’s VWF. Patients with VWD type 2N typically have decreased FVIII using this assay.^6,7

von Willebrand factor (VWF) multimer analysis is typically performed after initial VWD assays to help define the VWD variant subtype. In this qualitative assay, VWF multimers are separated by size using sodium dodecyl sulphate (SDS)-agarose gel electrophoresis. The multimer distribution is visualised using a labelled antibody that targets VWF. This analysis can also be performed through transfer of the protein from the SDS-agarose gel to a membrane using a Western blot technique. With this method, VWF multimers are visualised using immunofluorescence.^6,7

Low-resolution gels can be used to separate large molecular weight VWF multimers and often are used to differentiate VWD types 2A and 2B from VWD types 1, 2M, 2N and 3. Samples from patients with VWD types 2A and 2B have fewer high molecular weight VWF multimers, whereas samples from patients with VWD types 1, 2M and 2N typically have a normal distribution. No multimers are detected in samples from patients with VWD type 3.⁶

Typical VWD multimer analysis results.⁶

1. Nichols WL, Hultin MB, James AH, et al. von Willebrand disease (VWD): evidence-based diagnosis and management guidelines, the National Heart, Lung, and Blood Institute (NHLBI) Expert Panel report (USA). Haemophilia 2008;14:171-232.
2. Keesler DA, Flood VH. Current issues in diagnosis and treatment of von Willebrand disease. Res Pract Thromb Haemost 2018;2:34-41.
3. Sharma R, Flood VH. Advances in the diagnosis and treatment of Von Willebrand disease. Hematology Am Soc Hematol Educ Program 2017;2017:379-84.
4. James AH, Eikenboom J, Federici AB. State of the art: von Willebrand disease. Haemophilia 2016;22 Suppl 5:54-9.
5. Bodo I, Eikenboom J, Montgomery R, et al. Platelet-dependent von Willebrand factor activity. Nomenclature and methodology: communication from the SSC of the ISTH. J Thromb Haemost 2015;13:1345-50.
6. Nichols WL, Hultin MB, James AH, et al. von Willebrand disease (VWD): evidence-based diagnosis and management guidelines, the National Heart, Lung, and Blood Institute (NHLBI) Expert Panel report (USA). Haemophilia 2008;14:171-232.
7. James AH, Eikenboom J, Federici AB. State of the art: von Willebrand disease. Haemophilia 2016;22 Suppl 5:54-9.