Amino acid sequences of HNPs and the inhibitory activity of HNP1 and its mutants on proteolysis of rF-VWF73 and VWF by ADAMTS13. (A) Sequence alignment of a part of the ADAMTS13 spacer domain (G647-T669) with HNP1, HNP2, HNP3, and its mutants (HNP1-delRRY and HNP1-RRY/AAA). Highlighted in orange is the RRY motif. (B-C) The interactions between ADAMTS13-spacer and VWF-A2 fragment. Residues of Glu1660 and Asp1663 of VWF are shown as yellow sticks (PDB 3GXB). The spacer domain of ADAMTS13 is shown in silver (PDB 3GHM), with RRY shown as sticks. Arg659, Arg660, and Tyr661 appear to interact with Glu1660 and Asp1663 (dashed lines). (D-E) The interactions between HNPs1-3 and VWF73. Arg14, Arg15, and Tyr16 in HNP1-3 appear to interact with the same site (Glu1660 and Asp1663) on VWF73 where ADAMTS13 binds (dashed line). (F) The inhibitory activity of HNP1 (WT), HNP1-delRRY, HNP1-RRY/AAA, and β-defensin (0-30 µM) on proteolytic cleavage of rF-VWF73 (2 µM) by rADAMTS13 (10 nM). (G) The inhibition of HNP1, HNP1-RRY/AAA, and HNP1-delRRY or buffer controls (Con-1 and Con-2) on proteolytic cleavage of multimeric VWF (10 µg/mL) by rADAMTS13 (10 nM). Plus (+) and minus (−) signs indicate the presence and absence of the component, respectively. HMW and LMW, separated by a dashed line, represent the high and molecular weights of VWF multimers, respectively.