In vitro cleavage of pro-vWF by PACE/furinincreases binding affinity to factor VIII. COS-1 cells were transfected with wild-type vWF, ΔPro-vWF, or cleavage site mutant R760K/K762D vWF and conditioned medium was harvested. vWF from the conditioned medium was immobilized on anti-vWF polyclonal antibody-coated microtiter plates. The immobilized vWF was then treated with concentrated conditioned medium from PACE-Sol–transfected or mock-transfected COS-1 cells as described in the Materials and Methods. In (A), proteins were recovered from the wells and analyzed by SDS-PAGE under reducing conditions and Western blot analysis using polyclonal anti-vWF antibody. (○) Pro-vWF; (•) mature vWF. (B) Factor VIII binding (1.25 nmol/L) to vWF preparations (wild-type, ΔPro-vWF and R760K/K762D vWF) that were immobilized on anti-vWF polyclonal antibody-coated microtiter plates and then treated with conditioned medium from mock-transfected or PACE-Sol-transfected (+ PACE) COS-1 cells. Although the results depicted here are from a single experiment with a single concentration of factor VIII in which Western blot analysis was performed in parallel (A), this experiment was performed several times with varying concentrations of factor VIII and gave consistent results.