Shear-induced cleavage of VWF multimers by ADAMTS13. Human plasma VWF (40 nM) was sheared by vortexing for the indicated times with ADAMTS13 variants or human recombinant ADAMTS13 (rADAMTS13, 50 nM). Reactions were stopped by adding 10 mM EDTA. Cleavage products were analyzed by gel electrophoresis and western blotting with rabbit anti-VWF and goat anti-rabbit IRDye 680RD. The major 350-kDa dimeric VWF cleavage product was quantitated by fluorescence imaging. Cleavage rates for ADAMTS13 mutants were normalized to the cleavage rate for concurrent controls with human rADAMTS13. Data from duplicates are summarized in Table 3.