Kinetics of Factor VIII-von Willebrand Factor Association

The binding of factor Vlll t o von Willebrand factor (vWF) is essential for the protection of factor Vlll 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, Swe-den). Factor Vlll was incubated with vWF-RU1-Sepharose and unbound factor Vlll was separated from bound factor Vlll by centrifugation. The amount of bound factor Vlll was determined from the decrease of factor Vlll activity in the supernatant. Factor Vlll binding to vWF-RU1-Sepharose con-formed to the Langmuir model for independent binding sites with a K, of 0.46 k 0.12 nmol/L, and a stoichiometry of 1.3 factor Vlll molecules per vWF monomer at saturation