Time-dependent cleavage of oxidized or nonoxidized VWF A2 peptide by ADAMTS13. (A) Representative SDS-PAGE of ADAMTS13 cleavage of the A2 peptide substrate. In the unoxidized substrate, on the left, cleavage products of 9 kDa and 4 kDa accumulate over time, with concomitant disappearance of the 13-kDa substrate. Cleavage is markedly reduced after oxidation of the substrate peptide with 75μM HOCl (right side of gel). (B) Rate of cleavage quantified by densitometry. (C) Schematic depiction of the method used to quantify ADAMTS13 cleavage of the substrate peptide by MS. Note that the tryptic fragment that arises from the region immediately N-terminal to the ADAMTS13 cleavage site was analyzed. This fragment is identical whether or not the methionine has been oxidized. (D) ADAMTS13 cleavage of the unoxidized or oxidized substrate peptide as determined by MS. (E) Similar cleavage rates were obtained using SDS-PAGE/densitometry and MS.