Binding of wild-type ADAMTS13 and ADAMTS13-RYY to VWF studied with SPR. (A) Equal amounts of the ADAMTS13 preparations, as determined by ELISA, were used in binding experiments; see silver-stained SDS-PAGE gel (left), and Western blot with an anti-metalloprotease domain antibody (right). ADAMTS13-WT (B) or ADAMTS13-RYY (C), 12.5 to 300nM, was perfused over VWF115 immobilized to a CM5 sensor chip at a rate of 20 μL/minute for 240 seconds. Dissociation in the absence of ADAMTS13 was then studied for a further 240 seconds. (D-E) Experiments were conducted as in panels B and C but with immobilized VWF106. (F-G) Experiments were conducted as in panels B and C but with immobilized full-length VWF. In all graphs representative binding curves obtained with the use of 12.5, 25, 50, 100, 200, or 300nM wild-type ADAMTS13 or ADAMTS13-RYY are shown. Binding is represented as mol ADAMTS13/mol immobilized VWF (fragment).