The Evaluation of Factor VIII Binding Activity of von Willebrand Factor by Means of an ELISA Method:Significance and Practical Implications

Abstract

One of the functions of von Willebrand factor (vWF) is to serve as a carrier of clotting factor VIII (FVIII). Deficiency of this function results in the von Willebrand disease (vWD) variant type 2N, which resembles hemophilia A. We describe a new sandwich enzyme-linked immunosorbent assay (ELISA) to study the ability of vWF to bind exogenous recombinant FVIII (rFVIII), in which anti-vWF-coated plates are incubated with plasma vWF, followed by exogenous FVIII and a peroxidase-coupled anti-FVIII antibody. Dose-response curves obtained using normal plasma vWF and purified normal vWF revealed a hyperbolic relationship between the optical density and the vWF concentration. The assay allows the quantification of FVIII binding with values expressed in U/dL; 100 U/dL was the amount present in normal plasma. The sensitivity and specificity of the method are demonstrated by its ability to measure binding levels as low as 1 to 2 U/dL and the fact that no FVIII binding was observed using plasma known to contain less than 1 U/dL vWF. To verify the accuracy of the assay, three patients with type 2N vWD with characterized vWF gene mutations were studied using an existing chromogenic assay and our ELISA. A patient who was homozygous for the R53W mutation and had no FVIII binding capacity according to the chromogenic method showed undetectable FVIII binding by ELISA. The remaining two patients, one who was homozygous for the R91Q mutation and one with compound heterozygosity for the R91Q and R53W mutations, showed markedly decreased FVIII binding by the chromogenic method and yielded ELISA values ranging from 4 to 8 U/dL. Therefore, although the two methods produce qualitatively similar results, the ELISA method offers the advantage of allowing quantification of the FVIII binding function. FVIII binding was also analyzed in 20 patients with type 1 vWD; we found a decrease of FVIII binding that was proportionate to the decrease in vWF levels, showing a normal FVIII binding activity/vWF molecule ratio. We define the binding activity measured by this assay as vWF:FVIII binding activity and propose its use in the functional analysis of vWF.

Type 2N von Willebrand disease: Characterization and diagnostic difficulties

“…22 The VWF:FVIIIB assay was measured with a home-made ELISA method. 19 Briefly, microtitration plates were coated with anti-VWF polyclonal antibody and left overnight at +4°C (Dako, Glostrup, Denmark Genetic analysis was performed on exons 17-20 of the VWF gene as previously described. 13 Amino acid residues were numbered from the ATG initiation codon (residue 1) of the pre-pro-VWF.…”

“…19 Type 2N exhibits FVIII levels from 1 to 40 U/dL, associated with normal or reduced VWF levels, but a low FVIII/VWF:Ag ratio, 20 in a phenotype that may prompt a misdiagnosis of mild haemophilia A. 21 Here we report on our 15-year experience of diagnosing type 2N…”

Severe and moderate hemophilia A: identification of 38 new genetic alterations

“…Type 2N von Willebrand disease 4 and a combined FV-FVIII deficiency were ruled out. Assays FVIII:C and antigenic von Willebrand factor (VWF:Ag) were performed in all women in parallel with genetic studies.…”

A common ancestor more than 10,000 years old for patients with R854Q-related type 2N von Willebrand's disease in Italy

“…VWF antigen (VWF:Ag), its collagen binding capacity (VWF:CB), factor VIII activity (FVIII:C) and the VWF's FVIII binding capacity (VWF:FVIIIB) were assessed as reported elsewhere. 4,17 Briefly, for the VWF:FVIIIB assay, after coating microtiter plates with anti-VWF polyclonal antibody, plasma VWF was added and the plates were incubated for 1 h at room temperature. After washing, bound endogenous FVIII was removed with CaCl 2 ; 1 U/mL of recombinant FVIII (rFVIII) (Helixate; Aventis, Marburg, Germany) was added and the plates were incubated again for 1 h. After washing again, the amount of bound rFVIII was assessed with an anti-FVIII horseradish peroxidase-conjugated polyclonal antibody (Enzyme Research, South Bend, IN, USA).…”

“…Type 2N VWD is easy to identify by means of a test that explores the FVIII binding capacity of VWF (VWF:FVIIIB) in vitro, using exogenous recombinant FVIII. 4,5 The FVIII binding domain of VWF is located in the first 272 amino acids of the mature VWF. 6 This region is encoded by exons 18-23 of the VWF gene, but 85% of all mutations occur in exons 18-20.…”