KINETICS OF FACTOR-VIII VON-WILLEBRAND-FACTOR ASSOCIATION

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 VII I with vWF using a centrifugation binding assay. Purified or plasma vW F was immobilized with a monoclonal antibody (MoAb RU1) covalently lin ked to Sepharose (Pharmacia LKB Biotechnology, Uppsala, Sweden). Facto r VIII was incubated with vWF-RU1-Sepharose and unbound factor VIII wa s separated from bound factor VIII by centrifugation. The amount of bo und factor VIII was determined from the decrease of factor VIII activi ty in the supernatant. Factor VIII binding to vWF-RU1-Sepharose confor med to the Langmuir model for independent binding sites with a K-d 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 conta ins a binding site for factor VIII. Competition experiments were perfo rmed with a recombinant vWF (Delta A2-rvWF), lacking residues 730 to 9 10 which contain the epitope for MoAb RU1. Delta A2-rvWF effectively d isplaced previously bound factor VIII, confirming that factor VIII bin ding to vWF-RU1-Sepharose was reversible. To determine the association rate constant (k(on)) and the dissociation rate constant (k(off)), fa ctor VIII was incubated with vWF-RU1-Sepharose for various time interv als. The observed association kinetics conformed to a simple bimolecul ar association reaction with k(on) = 5.9 +/- 1.9 x 10(6) M(-1) s(-1) a nd k(off) = 1.6 +/- 1.2 x 10(-3) s(-1) (mean +/- SD). Similar values w ere obtained from the dissociation kinetics measured after dilution of preformed factor VIII-vWF-RU1-Sepharose complexes. Identical rate con stants were obtained for factor VIII binding to vWF from normal pooled plasma and to vWF from plasma of patients with hemophilia A. The kine tic 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 VII I associates with endogenous vWF. Using the plasma concentration of vW F (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 approximate to 2 secon ds. (C) 1996 by The American Society of Hematology.