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Kinetics of factor VIII-von Willebrand factor association
AJ Vlot, SJ Koppelman, JC Meijers, C Dama, HM van den Berg, BN Bouma, JJ Sixma and GM Willems
Department of Haematology, University Hospital Utrecht, The Netherlands.
The binding of factor VIII to von Willebrand factor (vWF) is essential for
the protection of factor VIII against proteolytic degradation in plasma. We
have characterized the binding kinetics of human factor VIII with vWF using
a centrifugation binding assay. Purified or plasma vWF was immobilized with
a monoclonal antibody (MoAb RU1) covalently linked to Sepharose (Pharmacia
LKB Biotechnology, Uppsala, Sweden). Factor VIII was incubated with
vWF-RU1-Sepharose and unbound factor VIII was separated from bound factor
VIII by centrifugation. The amount of bound factor VIII was determined from
the decrease of factor VIII activity in the supernatant. Factor VIII
binding to vWF-RU1-Sepharose conformed to the Langmuir model for
independent binding sites with a Kd of 0.46 +/- 0.12 nmol/L, and a
stoichiometry of 1.3 factor VIII molecules per vWF monomer at saturation,
suggesting that each vWF subunit contains a binding site for factor VIII.
Competition experiments were performed with a recombinant vWF
(deltaA2-rvWF), lacking residues 730 to 910 which contain the epitope for
MoAB RU1. DeltaA2-rvWF effectively displaced previously bound factor VIII,
confirming that factor VIII binding to vWF-RU1-Sepharose was reversible. To
determine the association rate constant (k(on)) and the dissociation rate
constant (k(off)), factor VIII was incubated with vWF-RU1-Sepharose for
various time intervals. The observed association kinetics conformed to a
simple bimolecular association reaction with k(on) = 5.9 +/- 1.9 x 10(6)
M(-1) s(-1) and k(off) = 1.6 +/- 1.2 x 10(-3) s(-1) (mean +/- SD). Similar
values were obtained from the dissociation kinetics measured after dilution
of preformed factor VIII-vWF-RU1-Sepharose complexes. Identical rate
constants were obtained for factor VIII binding to vWF from normal pooled
plasma and to vWF from plasma of patients with hemophilia A. The kinetic
parameters in this report allow estimation of the time needed for complex
formation in vivo in healthy individuals and in patients with hemophilia A,
in which monoclonally purified or recombinant factor VIII associates with
endogenous vWF. Using the plasma concentration of vWF (50 nmol/L in
monomers) and the obtained values for K(on) and K(off), the time needed to
bind 50% of factor VIII is approximately 2 seconds.
Volume 87,
Issue 5,
pp. 1809-1816,
03/01/1996
Copyright © 1996 by The American Society of Hematology
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