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The affinity and stoichiometry of binding of human factor VIII to von
Willebrand factor
AJ Vlot, SJ Koppelman, MH van den Berg, BN Bouma and JJ Sixma
Department of Haematology, University Hospital, Utrecht, The Netherlands.
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.
Volume 85,
Issue 11,
pp. 3150-3157,
06/01/1995
Copyright © 1995 by The American Society of Hematology
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