An enzyme immunoassay of ADAMTS13 distinguishes patients with thrombotic thrombocytopenic purpura from normal individuals and carriers of ADAMTS13 mutations.
Recent studies demonstrate that assay of ADAMTS13, a circulating zinc metalloprotease that cleaves von Willebrand factor (VWF) at the Y1605-M1606 bond, is an important tool in the diagnosis of thrombotic thrombocytopenic purpura (TTP). In order to develop a method that could be adapted for general use, we describe an enzyme immunoassay (EIA)-based method for measuring the activity of ADAMTS13 in patient plasma samples. A monomeric peptide consisting of the amino acid residues D1596-R1668 of VWF was produced that spanned the ADAMTS13 cleavage site and was franked by glutathione s-transferase (GST) and a 6His sequences at the amino and carboxyl termini respectively. When probed with either anti-GST or anti-6His, the VWF fragment appeared as a 38.1-kDa band. After incubation with normal plasma, the VWF fragment was replaced by a 30.4-kDa band, which was recognized by anti-GST but not by anti-6His, consistent with the expected cleavage at the Y1605-M1606 bond. EDTA or plasma samples from patients with TTP inhibited this cleavage. After incubation with normal plasma, the VWF fusion protein immobilized onto anti-GST coated microtiter plate wells lost its anti-6His binding activity in a time- and plasma concentration-dependent manner. By using this EIA, the ADAMTS13 activity level was less than 0.12 U/mL in patients with acquired or hereditary TTP, distinguishing these patients from normal individuals or carriers of one copy of mutant ADAMTS13 allele. These results suggest the EIA method based on the VWF fusion protein is a simple but promising alternative for measuring ADAMTS13 activity.
Zhou
Wenhua
W
Division of Hematology, Montefiore Medical Center, 111 East 210th Street, Bronx, New York, 10467, USA.
Tsai
Han-Mou
HM
D051722:ADAM Proteins; D000071120:ADAMTS13 Protein; D000595:Amino Acid Sequence; D016022:Case-Control Studies; D005982:Glutathione Transferase; D006579:Heterozygote; D006639:Histidine; D006720:Homozygote; D006801:Humans; D007124:Immunoenzyme Techniques / Q000379:methods* / Q000592:standards; D008666:Metalloendopeptidases / Q000235:genetics* / Q000378:metabolism; D009154:Mutation / Q000502:physiology*; D010446:Peptide Fragments / Q000378:metabolism; D011697:Purpura, Thrombotic Thrombocytopenic / Q000175:diagnosis* / Q000201:enzymology / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012680:Sensitivity and Specificity; D014841:von Willebrand Factor / Q000378:metabolism
D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D014841:von Willebrand Factor; D006639:Histidine; D005982:Glutathione Transferase; D051722:ADAM Proteins; D008666:Metalloendopeptidases; D000071120:ADAMTS13 Protein; C099604:ADAMTS13 protein, human