Multimerization increases the affinity of vWF subunits for factor VIII. vWF purified from human plasma or dimeric mutant ΔPro-vWF, incapable of multimerizing and purified from CHO cells, were immobilized on MoAb vW/13.7.9-Superose-12 beads. Increasing concentrations of fluorescein-conjugated factor VIII were added and incubated for 15 minutes to allow equilibrium. Fluorescence per particle was monitored by flow cytometry and shown in (A). Parameters fitted were K_D = 0.94 and 5 nmol/L for vWF and ΔPro-vWF, respectively. Binding of factor VIII to vWF in solution was measured in a competition binding assay and is shown in (B). Purified vWF or ΔPro-vWF, at various concentrations, was incubated with 2 nmol/L fluorescein-labeled factor VIII for 15 minutes at room temperature. Lipospheres were added and incubated for an additional 10 minutes and liposphere-bound fluorescein-labeled factor VIII was measured by flow cytometry. vWF-bound factor VIII was interpreted as the fraction not available for binding to lipospheres. The best-fit curves corresponds to the following parameter values: K_D= 0.68 ± 0.28 (n = 3) and 6.4 ± 0.64 (n = 3) nmol/L and a stoichiometry of 0.9 and 0.8 factor VIII molecules per vWF subunit, for plasma vWF and Δpro-vWF, respectively. (○) vWF; (▵) Δpro-vWF.