Proteolysis of recombinant A2 domain fragments. (A) Purified recombinant A2 domain was analyzed by 12% SDS-PAGE and Coomassie staining. VWF A2 domain was visible as a single band of approximately 29 kDa corresponding to its predicted mass (lane 1). A2-N1574Q (lane 2) and PNGase F–digested A2 (lane 3) were also analyzed on the same gel for comparison. (B) wtA2, A2-N1574Q, and A2-PNG (5 μM) were incubated with 10 nM ADAMTS13 in 5 mM CaCl₂, 50 mM NaCl, and 20 mM Tris-HCl (pH 7.8), and 1.5 M urea. At various time points subsamples were taken and the reaction was stopped with EDTA, and proteolysis was assessed by SDS-PAGE in 16% Tris-tricine gels and Coomassie staining.