Recombinant VWF glycan mutants. (A) The cDNA encoding full-length VWF was cloned into pcDNA3.1 as described and transfected into HEK293T cells using PEI. Recombinant VWF was analyzed by reducing 7% SDS-PAGE followed by Western blotting and probing with anti–VWF-HRP. wtVWF (lane 1), N1515Q (lane 2), N1574Q (lane 3), and N1515/1574Q (lane 4). (B) wt and mutated recombinant VWF (5 μg/mL) was cleaved by 10 nM ADAMTS13 in the presence of 1.5 M urea and cleavage assessed by multimer analysis in 1% gels. wtVWF (lanes 1,5), N1515Q (lanes 2,6), N1574Q (lanes 3,7), and N1515/1574Q (lanes 4,8). RP indicates reference plasma; vertical line indicates a repositioned gel lane.