The affinity and stoichiometry of binding of human factor VIII to von Willebrand factor

Abstract

To study the interaction between factor VIII and von Willebrand factor (vWF), binding experiments were performed using immobilized plasma vWF. Plasma was obtained from healthy donors and from patients with severe hemophilia A. For normal and hemophilic vWF, the dissociation constants (kd) for binding of factor VIII to vWF were 0.21 +/- 0.04 and 0.22 +/- 0.05 nmol/L, respectively. At saturation, the stoichiometry was one factor VIII molecule per 50 vWF monomers. In gel-filtration experiments, vWF was saturated by 23 times more factor VIII. However, when this FVIII-vWF complex was immobilized on microtiter plates, the ratio of factor VIII/vWF decreased to the same ratio as in the solid-phase binding assay. To exclude any effect of antibody binding, colloidal gold particles with a diameter of 15 nm were coupled to purified vWF. This vWF-gold complex remained immunoreactive toward polyclonal and monoclonal antibodies, and was able to bind factor VIII, specifically, saturably, and reversibly. After incubation of vWF-gold with factor VIII, unbound and bound factor VIII were separated by centrifugation. Binding isotherms of these fluid-phase binding experiments indicated a kd of 0.32 +/- 0.09 nmol/L and a stoichiometry of approximately 0.5 factor VIII molecule per vWF monomer. We conclude that vWF-binding to a surface, with or without an antibody, may induce a conformational change causing a dissociation of bound factor VIII from vWF.

Parameters influencing FVIII pharmacokinetics in patients with severe and moderate haemophilia A

“…Parameters, such as LRP1 or PC levels also had no influence on FVIII PK. Our findings of a major influence of VWF on FVIII half-life are in accordance with data from Fisher et al [11], Fijnvandraat et al [28] and Vlot AJ et al [29]. VWF is known to act as a protector against FVIII degradation [13], e.g.…”

Functional characteristics of N8, a new recombinant FVIII

“…This was confirmed in this study where K d values of 0.2 nm for VWF binding were obtained by ELISA and SPR. The high affinity of N8 for VWF is in good agreement with published data [5,16]. In plasma, the majority of FVIII is bound to VWF, whereas a small pool (2-5%) at any given time exists in the non-bound state [14].…”

A qualitative and quantitative analysis of von Willebrand factor contained in a very high‐purity plasma‐derived FVIII concentrate

“…3(a) (1 unit of VWF for roughly 10 units of FVIII) is not representative because it was obtained under specific experimental conditions. According to the literature, the VWF/FVIII ratio is highly variable and depends on experimental conditions such as whether the assay was performed in solid‐phase or in solution [5, 42].…”