FRETS-VWF73 rather than CBA assay reflects ADAMTS13 proteolytic activity in acquired thrombotic thrombocytopenic purpura patients

Summary

Collagen-binding activity (CBA) and FRETS-VWF73 assays are widely adopted methods for the measurement of the plasmatic activity of ADAMTS13, the von Willebrand factor (VWF) cleaving-protease. Accurately assessing the severe deficiency of ADAMTS13 is important in the management of thrombotic thrombocytopenic purpura (TTP). However, non-concordant results between the two assays have been reported in a small but relevant percentage of TTP cases. We investigated whether CBA or FRETS-VWF73 assay reflects ADAMTS13 proteolytic activity in acquired TTP patients with non-concordant measurements. Twenty plasma samples with non-concordant ADAMTS13 activity results, <10% using FRETS-VWF73 and ≥20% using CBA, and 11 samples with concordant results, <10% using either FRETS-VWF73 and CBA assays, were analysed. FRETS-VWF73 was performed in the presence of 1.5 M urea. ADAMTS13 activities were also measured under flow conditions and the VWF multimer pattern was defined in order to verify the presence of ultra-large VWF due to ADAMTS13 deficiency. In FRETS-VWF73 assay with 1.5 M urea, ADAMTS13 activity significantly increased in roughly 50% of the samples with non-concordant results, whereas it remained undetectable in all samples with concordant measurements. Under flow conditions, all tested samples showed reduced ADAMTS13 activity. Finally, samples with non-concordant results showed a ratio of high molecular weight VWF multimers higher than normal. Our results support the use of FRETS-VWF73 over CBA assay for the assessment of ADAMTS13 severe deficiency and indicate urea as one cause of the observed differences.

Keywords

ADAMTS13 activity assay - TTP - autoantibodies - von Willebrand factor - flow assay

• References

• 1 Moake JL, Rudy CK, Troll JH. et al. Unusually large plasma factor VIII:von Willebrand factor multimers in chronic relapsing thrombotic thrombocytopenic purpura. N Engl J Med 1982; 307: 1432-1435.
• 2 Sadler JE. Von Willebrand factor, ADAMTS13, and thrombotic thrombocytopenic purpura. Blood 2008; 112: 11-18.
• 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: 1578-1584.
• 4 Tsai HM, Lian EC. Antibodies to von Willebrand factor-cleaving protease in acute thrombotic thrombocytopenic purpura. N Engl J Med 1998; 339: 1585-1594.
• 5 Peyvandi F, Palla R, Lotta LA. et al. ADAMTS-13 assays in thrombotic thrombocytopenic purpura. J Thromb Haemost 2010; 08: 631-640.
• 6 Mannucci PM, Franchini M. Advantages and limits of ADAMTS13 testing in the prognostic assessment of thrombotic thrombocytopenic purpura. Presse Med 2012; 41: e157-162.
• 7 Kremer Hovinga JA, Vesely SK, Terrell DR. et al. Survival and relapse in patients with thrombotic thrombocytopenic purpura. Blood 2010; 115: 1500-1511. quiz 1662.
• 8 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: 1386-1389.
• 9 Kokame K, Nobe Y, Kokubo Y. et al. FRETS-VWF73, a first fluorogenic substrate for ADAMTS13 assay. Br J Haematol 2005; 129: 93-100.
• 10 Palla R, Valsecchi C, Bajetta M. et al. Evaluation of assay methods to measure plasma ADAMTS13 activity in thrombotic microangiopathies. Thromb Haemost 2011; 105: 381-385.
• 11 Peyvandi F, Lavoretano S, Palla R. et al. ADAMTS13 and anti-ADAMTS13 antibodies as markers for recurrence of acquired thrombotic thrombocytopenic purpura during remission. Haematologica 2008; 93: 232-239.
• 12 Lotta LA, Mariani M, Consonni D. et al. Different clinical severity of first episodes and recurrences of thrombotic thrombocytopenic purpura. Br J Haematol 2010; 151: 488-494.
• 13 Meyer SC, Sulzer I, Lammle B. et al. Hyperbilirubinemia interferes with ADAMTS-13 activity measurement by FRETS-VWF73 assay: diagnostic relevance in patients suffering from acute thrombotic microangiopathies. J Thromb Haemost 2007; 05: 866-867.
• 14 Tripodi A, Chantarangkul V, Bohm M. et al. Measurement of von Willebrand factor cleaving protease (ADAMTS-13): results of an international collaborative study involving 11 methods testing the same set of coded plasmas. J Thromb Haemost 2004; 02: 1601-1609.
• 15 De Ceunynck K, Rocha S, Feys HB. et al. Local elongation of endothelial cell-anchored von Willebrand factor strings precedes ADAMTS13 protein-mediated proteolysis. J Biol Chem 2011; 286: 36361-36367.
• 16 Bettoni G, Palla R, Valsecchi C. et al. ADAMTS-13 activity and autoantibodies classes and subclasses as prognostic predictors in acquired thrombotic thrombocytopenic purpura. J Thromb Haemost 2012; 10: 1556-1565.
• 17 Lotta LA, Lombardi R, Mariani M. et al. Platelet reactive conformation and multimeric pattern of von Willebrand factor in acquired thrombotic thrombocytopenic purpura during acute disease and remission. J Thromb Haemost 2011; 09: 1744-1751.
• 18 Mackie I, Langley K, Chitolie A. et al. Discrepancies between ADAMTS13 activity assays in patients with thrombotic microangiopathies. Thromb Haemost 2013; 109: 488-496.
• 19 Froehlich-Zahnd R, George JN, Vesely SK. et al. Evidence for a role of anti-ADAMTS13 autoantibodies despite normal ADAMTS13 activity in recurrent thrombotic thrombocytopenic purpura. Haematologica 2012; 97: 297-303.