The affinity and stoichiometry of binding of human factor VIII to von Willebrand factor.
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.
Vlot
A J
AJ
Department of Haematology, University Hospital, Utrecht, The Netherlands.
Koppelman
S J
SJ
van den Berg
M H
MH
Bouma
B N
BN
Sixma
J J
JJ
D000328:Adult; D000911:Antibodies, Monoclonal / Q000276:immunology; D000937:Antigen-Antibody Reactions; D001667:Binding, Competitive; D002850:Chromatography, Gel; D005169:Factor VIII / Q000276:immunology / Q000378:metabolism*; D017979:Gold Colloid / Q000378:metabolism; D006467:Hemophilia A / Q000097:blood; D006801:Humans; D007150:Immunohistochemistry; D008297:Male; D011485:Protein Binding; D011487:Protein Conformation; D014841:von Willebrand Factor / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D017979:Gold Colloid; D014841:von Willebrand Factor; D005169:Factor VIII