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ADAMTS13 cleavage efficiency is altered by mutagenic and, to a lesser extent, polymorphic sequence changes in the A1 and A2 domains of von Willebrand factor
Author(s) -
Pruss Cynthia M.,
Notley Colleen R. P.,
Hegadorn Carol A.,
O’Brien Lee A.,
Lillicrap David
Publication year - 2008
Publication title -
british journal of haematology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.907
H-Index - 186
eISSN - 1365-2141
pISSN - 0007-1048
DOI - 10.1111/j.1365-2141.2008.07266.x
Subject(s) - von willebrand factor , adamts13 , cleavage (geology) , sequence (biology) , genetics , biology , microbiology and biotechnology , chemistry , immunology , platelet , paleontology , fracture (geology)
Summary The multimeric plasma protein von Willebrand factor (VWF) is regulated in size by its protease, ADAMTS13 (a disintegrin and metalloproteinase with thrombospondin type 1 motif, member 13). Y1605‐M1606 cleavage site mutations and single nucleotide polymorphisms (SNPs) in the VWF A1 and A2 domains were examined for alteration in ADAMTS13‐mediated cleavage of VWF. Recombinant human full‐length VWF (rVWF) was digested with recombinant human ADAMTS13 (rADAMTS13) using a dialysis membrane method with 1·5 mol/l urea, and analyzed via multimer migration distance. The glutathione‐ S ‐transferase (GST) and histidine‐tagged construct, E1554‐R1668 of VWF (VWF115) was assayed via enzyme‐linked immunosorbent assay: VWF115 was bound to anti‐GST coated plates, digested with rADAMTS13, and intact VWF115 detected via horseradish peroxidase‐labelled anti‐histidine tag antibody. All alterations examined in the Y1605‐M1606 cleavage site greatly reduced the cleavability of VWF by ADAMTS13 in the rVWF assay. Greatest cleavage resistance in both assays was observed in Y1605A/M1606A. In contrast, Y1605H and M1606L show a loss of cleavability only in the rVWF assay, suggesting that an aromatic ring at 1605 is critical for ADAMTS13 recognition. Additionally, under our rVWF assay conditions, the G1643S polymorphism showed increased cleavage, suggesting a Type 2A VWD phenotype, while D1472H, Q1571H and P1601T showed slightly decreased ADAMTS13 cleavage. Our two complementary assay conditions show that A‐domain changes in VWF alter ADAMTS13‐mediated proteolysis.