An enzyme immunoassay of ADAMTS13 distinguishes patients with thrombotic thrombocytopenic purpura from normal individuals and carriers of ADAMTS13 mutations

Summary

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 antiGST 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 antiGST coated microtiter plate wells lost its anti-6His binding activity in a timeand 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.

Keywords

von Willebrand factor - ADAMTS13 - metalloprotease - thrombotic thrombocytopenic purpura - enzyme immunoassay

• References

• 1 Bukowski RM. Thrombotic thrombocytopenic purpura. A review. Prog Hemost Thromb 1982; 06: 287-337.
• 2 Furlan M, Robles R, Solenthaler M. et al. Deficient activity of von Willebrand factorcleaving protease in chronic relapsing thrombotic thrombocytopenic purpura. Blood 1997; 89: 3097-103.
• 3 Furlan M, Robles R, Galbusera M. et al. von Willebrand factor-cleaving protease in thrombotic thrombocytopenic purpura and the hemolytic-uremic syndrome. N Engl J Med 1998; 339: 1578-84.
• 4 Tsai HM, Lian EC. Antibodies to von Willebrand factor-cleaving protease in acute thrombotic thrombocytopenic purpura. N Engl J Med 1998; 339: 1585-94.
• 5 Levy GG, Nichols WC, Lian EC. et al. Mutations in a member of the ADAMTS gene family cause thrombotic thrombocytopenic purpura. Nature 2001; 413: 488-94.
• 6 Savasan S, Lee SK, Ginsburg D. et al. ADAMTS13 gene mutation in congenital thrombotic thrombocytopenic purpura with previously reported normal VWF cleaving protease activity. Blood 2003; 101: 4449-51.
• 7 Studt JD, Bohm M, Budde U. et al. Measurement of von Willebrand factor-cleaving protease (ADAMTS-13) activity in plasma: a multicenter comparison of different assay methods. J Thromb Haemost 2003; 01: 1882-7.
• 8 Kokame K, Matsumoto M, Fujimura Y. et al. VWF73, a region from D1596 to R1668 of von Willebrand factor, provides a minimal substrate for ADAMTS-13. Blood. 2003 (September 25 ePub).
• 9 Dent JA, Berkowitz SD, Ware J. et al. Identification of a cleavage site directing the immunochemical detection of molecular abnormalities in type IIA von Willebrand factor. Proc Natl Acad Sci U S A 1990; 87: 6306-10.
• 10 Tsai HM. Advances in the Pathogenesis, Diagnosis, and Treatment of Thrombotic Thrombocytopenic Purpura. J Am Soc Nephrol 2003; 14: 1072-81.
• 11 Tsai HM. Physiologic cleavage of von Willebrand factor by a plasma protease is dependent on its conformation and requires calcium ion. Blood 1996; 87: 4235-44.
• 12 Gerritsen HE, Turecek PL, Schwarz HP. et al. Assay of von Willebrand factor (vWF)-cleaving protease based on decreased collagen binding affinity of degraded vWF: a tool for the diagnosis of thrombotic thrombocytopenic purpura (TTP). Thromb Haemost 1999; 82: 1386-9.
• 13 Mannucci PM, Canciani MT, Forza I. et al. Changes in health and disease of the metalloprotease that cleaves von Willebrand factor. Blood 2001; 98: 2730-5.