Equilibrium binding of factor VIII to plasma vWF and to recombinant vWF. (A) Binding of fluorescein-labeled factor VIII to immobilized plasma vWF. Purified vWF from human plasma was immobilized onto MoAb vW/13.7.9-coated Superose-12 beads. After 1 hour of incubation at room temperature, increasing amounts of fluorescein-labeled factor VIII were added and incubated for 15 minutes. Fluorescence per bead was monitored by flow cytometry, as described under the Materials and Methods. The best fit curve corresponds to a K_D of 0.8 nmol/L. (B) Displacement of factor VIII binding to PS-containing lipospheres by plasma vWF and recombinant vWF purified from CHO cells. (□) Plasma vWF; (○) recombinant vWF. In this assay, displacement of factor VIII from lipospheres does not reach zero, apparently because vWF binds with low affinity to lipospheres (data not shown). Therefore, the portion of the curve fitted correlates to displacement of 60% of factor VIII. The residual fluorescence, apparently not subject to displacement under these conditions, was excluded from quantitative analysis.