ADAMTS13 and VWF substrates. (A) Active ADAMTS13 consists of a metalloprotease domain (M), a disintegrin-like domain (D), a thrombospondin type 1 repeat (TSP1, T), a Cys-rich domain (Cys, C), a spacer domain (Spacer, S), 7 additional TSP1 repeats (2-8), and 2 CUB domains. In addition to full-length ADAMTS13 (FL), truncated enzymes were constructed with stop codons after Gln²⁸⁹ (M), Gly³⁸⁵ (MD), Glu⁴³⁹ (MDT), Cys⁵⁵⁵ (MDTC), and Ala⁶⁸⁵ (MDTCS). All constructs contain a C-terminal V5 epitope and 6 × His tag. (B) GST fusion substrates consist of a GST moiety followed by the linker SDLEVLFQGPLGS, a segment of VWF domain A2 (VWFxxx), and 6 His residues (6 × His). Rhinovirus 3C protease cleaves the indicated Gln-Gly bond in the linker sequence to produce substrates for MALDI MS analysis. (C) The amino acid sequence of VWF from Asp¹⁵⁹⁶-Arg¹⁶⁶⁸ is annotated to show the extent of num-bered α-helices and β-strands from a homology model of the A2 domain.¹  The Tyr¹⁶⁰⁵-Met¹⁶⁰⁶ bond cleaved by ADAMTS13 is marked by arrowheads. Segments of VWF contained in GST constructs are indicated. (D) Substrates VWF73nl and VWF106nl represent N-terminal elongations of VWF64 and contain the sequence shown, annotated to indicate the extent of predicted α-helices and β-strands.¹  The asterisk (*) indicates N-glycosylated residue Asn¹⁵⁷⁴.²