ADAMTS13-mediated proteolysis modulates binding of VWF to KB-VWF-D3.1 and KB-VWF-F1.1. (A) Purified pdVWF was incubated with recombinant ADAMTS13 and exposed to vortex-induced shear. Samples taken at indicated time (T)-points (0-3 hours) were analyzed via SDS-agarose electrophoresis. As a control, pdVWF was exposed to ADAMTS13 and shear for 3 hours in the presence of EDTA, a chelating agent that blocks ADAMTS13 activity. (B) Samples were analyzed for total VWF-antigen using polyclonal antibodies, for the presence of intact-VWF using KB-VWF-D3.1 and for the presence of degraded-VWF using KB-VWF-F1.1. Presented is the ratio of intact VWF to total VWF antigen (red circles; left y-axis) and the ratio of degraded VWF to total VWF antigen (blue squares; right y-axis) vs exposure time to ADAMTS13. Normal pooled plasma was used as calibrator for KB-VWF-D3.1, whereas a degraded-VWF preparation was used as calibrator for KB-VWF-F1.1. Data represent mean ± SD of 3 independent experiments.