Factor VIII binding to vWF. Based on ELISA, equal amounts of vWF protein in the conditioned medium from transfected cells (100 ng/mL) were incubated with increasing concentrations of recombinant-derived factor VIII. The complexes were immobilized by capture using microtiter plates coated with anti-vWF polyclonal antibody. Bound proteins were quantified by factor VIII activity as described in the Materials and Methods. (○) Wild-type vWF; (▵) ΔPro-vWF; (▴) ΔPro+A-vWF; (•) conditioned medium from mock-transfected cells. Although these results represent single values from a single experiment in which protein was analyzed in parallel by Western blot analysis (Fig 1), these binding curves were performed at least 5 times from different transfection experiments and yielded data qualitatively similar to that reported here.