VWF73, a region from D1596 to R1668 of von Willebrand factor, provides a minimal substrate for ADAMTS-13.
ADAMTS-13 was recently identified as a new hemostatic factor, von Willebrand factor (VWF)-cleaving protease. Either congenital or acquired defects of the enzymatic activity lead to thrombotic thrombocytopenic purpura (TTP). ADAMTS-13 specifically cleaves a peptidyl bond between Y1605 and M1606 in the A2 domain of VWF. Here, we determined the minimal region recognized as a specific substrate by ADAMTS-13. A series of partial deletions in the A2 domain flanked with N- and C-terminal tags were expressed in Escherichia coli and affinity-purified. These purified proteins were incubated with human plasma, subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and analyzed by Western blot. Judging from mobility shifts, all constructs except one were cleaved at the expected site. Data suggested that a minimal region as a functional substrate consisted of 73 amino acid residues from D1596 to R1668 of VWF, designated VWF73, and that further deletion of the E1660-R1668 region led to the loss of cleavage by ADAMTS-13. VWF73 was not cleaved by plasma from patients with congenital or acquired TTP, but cleaved by plasma from patients with hemolytic uremic syndrome, suggesting that VWF73 is a specific substrate forADAMTS-13. Thus, VWF73 will be a useful seed to develop a new rapid assay to determine ADAMTS-13 activity.
D051722:ADAM Proteins; D000071120:ADAMTS13 Protein; D019943:Amino Acid Substitution; D001483:Base Sequence; D002478:Cells, Cultured; D003001:Cloning, Molecular; D017931:DNA Primers; D004730:Endothelium, Vascular / Q000502:physiology; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D007700:Kinetics; D008666:Metalloendopeptidases / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism*; D011697:Purpura, Thrombotic Thrombocytopenic / Q000235:genetics; D012313:RNA / Q000235:genetics / Q000302:isolation & purification; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013379:Substrate Specificity; D014471:Umbilical Veins; D014841:von Willebrand Factor / Q000737:chemistry / Q000378:metabolism*
D017931:DNA Primers; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D014841:von Willebrand Factor; D012313:RNA; D051722:ADAM Proteins; D008666:Metalloendopeptidases; D000071120:ADAMTS13 Protein; C099604:ADAMTS13 protein, human