title: An autoantibody epitope comprising residues R660, Y661, and Y665 in the ADAMTS13 spacer domain identifies a binding site for the A2 domain of VWF.
abstract: In the majority of patients with acquired thrombotic thrombocytopenic purpura (TTP), antibodies are directed toward the spacer domain of ADAMTS13. We have previously shown that region Y658-Y665 is involved. We now show that replacement of R660, Y661, or Y665 with alanine in ADAMTS13 reduced/abolished the binding of 2 previously isolated human monoclonal antibodies and polyclonal antibodies derived from plasma of 6 patients with acquired TTP. We investigated whether these residues also influenced cleavage of short von Willebrand factor (VWF) fragment substrate VWF115. An ADAMTS13 variant (R660A/Y661A/Y665A, ADAMTS13-RYY) showed a 12-fold reduced catalytic efficiency (k(cat)/K(m)) arising from greatly reduced (> 25-fold) binding, demonstrated by surface plasmon resonance. The influence of these residue changes on full-length VWF was determined with denaturing and flow assays. ADAMTS13-RYY had reduced activity in both, with proteolysis of VWF unaffected by autoantibody. Binding of ADAMTS13-RYY mutant to VWF was, however, similar to normal. Our results demonstrate that residues within Y658-Y665 of the ADAMTS13 spacer domain that are targeted by autoantibodies in TTP directly interact with a complementary exosite (E1660-R1668) within the VWF A2 domain. Residues R660, Y661, and Y665 are critical for proteolysis of short VWF substrates, but wider domain interactions also make important contributions to cleavage of full-length VWF.
authors.affiliation: Department of Plasma Proteins, Sanquin-AMC Landsteiner Laboratory, Amsterdam, The Netherlands.
authors.forename: Wouter
authors.initials: W
authors.lastname: Pos
authors.forename: James T B
authors.initials: JT
authors.lastname: Crawley
authors.forename: Rob
authors.initials: R
authors.lastname: Fijnheer
authors.forename: Jan
authors.initials: J
authors.lastname: Voorberg
authors.forename: David A
authors.initials: DA
authors.lastname: Lane
authors.forename: Brenda M
authors.initials: BM
authors.lastname: Luken
mesh_terms: D051722:ADAM Proteins / Q000235:genetics / Q000378:metabolism*; D000071120:ADAMTS13 Protein; D019943:Amino Acid Substitution; D001323:Autoantibodies / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D018604:Epitope Mapping / Q000379:methods; D000939:Epitopes / Q000235:genetics / Q000378:metabolism*; D005260:Female; D006801:Humans; D008297:Male; D020125:Mutation, Missense; D011485:Protein Binding; D017434:Protein Structure, Tertiary / Q000235:genetics; D011697:Purpura, Thrombotic Thrombocytopenic / Q000235:genetics / Q000378:metabolism*; D020349:Surface Plasmon Resonance; D014841:von Willebrand Factor / Q000235:genetics / Q000378:metabolism*
chemical_list: D001323:Autoantibodies; D000939:Epitopes; D014841:von Willebrand Factor; D051722:ADAM Proteins; D000071120:ADAMTS13 Protein; C099604:ADAMTS13 protein, human