The presence of the vWF propeptide inhibits factor VIII binding. COS-1 cells were transfected with wild-type and propeptide cleavage site mutant (R760K and R760K/K762D) vWF, as well as with the expression vectors encoding wild-type vWF in the absence or presence of PACE-Sol or a catalytically inactive mutant PACE-SA. Conditioned medium was harvested for factor VIII binding assays and Western immunoblot analysis of vWF (Fig 1). The reduced amount of vWF detected upon PACE-SA cotransfection was reproducible and was corrected for in the factor VIII binding assay by using increased amounts of conditioned medium for this sample. The concentration of vWF in the conditioned medium was determined by ELISA and the binding assays were performed using equal amounts of vWF and were measured by factor VIII activity assay as described in the Materials and Methods. (○) Wild-type vWF; (□) R760K vWF; (◊) R760K/K762D vWF; (▵) vWF+PACE-Sol; (▴) vWF+PACE-SA; (•) mock.