Identification of hydrophobic VWF A2 domain residues that contribute to ADAMTS13 binding. (A) A total of 6 µM VWF106 (Glu1554-Arg1659) was incubated with WT ADAMTS13 or ADAMTS13 Cys5 (17 nM) in conditioned medium, and aliquots taken at the indicated time points were analyzed by SDS-PAGE and Coomassie staining. (B) VWF115(3S) and VWF115(5S) containing composite mutations of hydrophobic residues to serine were used in functional analyses as in panel A, except that 1 nM WT ADAMTS13 was used. (C) Binding of WT ADAMTS13 to VWF115(3S) and (5S). VWF115 or variant (100 nM) was immobilized on microtiter wells, and binding was assessed as in Figure 4B. Mean values ± SD are shown (n = 3). (D-E) Proteolysis of VWF115(3S) (D) and VWf115(5S) (E) by 24 nM WT ADAMTS13 and ADAMTS13 Cys5. Cleavage was analyzed as in panel A-B.