Novel recombinant glycosylphosphatidylinositol (GPI)-anchored ADAMTS13 and variants for assessment of anti-ADAMTS13 autoantibodies in patients with thrombotic thrombocytopenic purpura

Summary

Immunoglobulin Gs (IgGs) against ADAMTS13 are major causes of acquired (idiopathic) thrombotic thrombocytopenic purpura (TTP). We report here a novel cell-based assay using glycosylphosphatidylinositol (GPI)-anchored ADAMTS13 or variants expressed on cell membrane for assessment of autoantibodies in patients with TTP. We showed that IgGs from all 26 patients with acquired TTP bound to cells expressing a GPI anchored full-length ADAMTS13 (gFL) and a variant truncated after the spacer domain (gS). Also, IgGs from 25/26 (96.7%) of these TTP patients bound to cells expressing a GPI-anchored C-terminal fragment, TSP1 2–8 plus CUB (gT2C). In contrast, none of the 20 healthy blood donors showed detectable binding of their IgGs to the cells expressing gFL, gS, and gT2C. A moderate, but statistically significant correlation was observed between plasma concentrations of anti-ADAMTS13 IgG and positive cells expressing gFL (r=0.65), gS (r=0.67), and gT2C (r=0.42). These results suggest that the microtiter-plate assay and the cell-based assay may detect differential antigenic epitopes. Moreover, antigens clustered on cell membranes may enhance antibody binding affinity, thereby increasing analytical sensitivity. Finally, our assay was able to determine kinetic changes of plasma levels of anti-ADAMTS13 IgGs in TTP patients during plasma therapy. Together, our findings suggest that the novel cell-based assay may be applicable for rapid identification and mapping of anti-ADAMTS13 autoantibodies in patients with acquired TTP.

Keywords

von Willebrand factor cleaving protease - thrombotic microangio-pathies - diagnostic test - autoantibody

• References

• 1 Tsai HM. Current concepts in thrombotic thrombocytopenic purpura. Annu Rev Med 2006; 57: 419-436.
• 2 Levy GG, Nichols WC, Lian EC. et al. Mutations in a member of the ADAMTS gene family cause thrombotic thrombocytopenic purpura. Nature 2001; 413: 488-494.
• 3 Klaus C, Plaimauer B, Studt JD. et al. Epitope mapping of ADAMTS13 autoantibodies in acquired thrombotic thrombocytopenic purpura. Blood 2004; 103: 4514-4519.
• 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 Esplin MS, Branch DW. Diagnosis and management of thrombotic microangiopathies during pregnancy. Clin Obstet Gynecol 1999; 42: 360-367.
• 6 Elliott MA, Nichols Jr. WL, Plumhoff EA. et al. Posttransplantation thrombotic thrombocytopenic purpura: a single-center experience and a contemporary review. Mayo Clin Proc 2003; 78: 421-430.
• 7 Bar Meir E, Amital H, Levy Y. et al. Mycoplasma-pneumoniae-induced thrombotic thrombocytopenic purpura. Acta Haematol 2000; 103: 112-115.
• 8 Fontana S, Gerritsen HE, Kremer Hovinga J. et al. Microangiopathic haemolytic anaemia in metastasizing malignant tumours is not associated with a severe deficiency of the von Willebrand factor-cleaving protease. Br J Haematol 2001; 113: 100-102.
• 9 Bennett CL, Kim B, Zakarija A. et al. Two mechanistic pathways for thienopyridine-associated thrombotic thrombocytopenic purpura: a report from the SERF-TTP Research Group and the RADAR Project. J Am Coll Cardiol 2007; 50: 1138-1143.
• 10 Tsai HM. Is severe deficiency of ADAMTS-13 specific for thrombotic thrombocytopenic purpura? Yes. J Thromb Haemost 2003; 1: 625-631.
• 11 Veyradier A, Obert B, Houllier A. et al. Specific von Willebrand factor-cleaving protease in thrombotic microangiopathies: a study of 111 cases. Blood 2001; 98: 1765-1772.
• 12 Zheng XL. ADAMTS13 testing: why bother?. Blood 2010; 115: 1475-1476.
• 13 Coppo P, Wolf M, Veyradier A. et al. Prognostic value of inhibitory anti-ADAMTS13 antibodies in adult-acquired thrombotic thrombocytopenic purpura. Br J Haematol 2006; 132: 66-74.
• 14 Hovinga JA, Vesely SK, Terrell DR. et al. Survival and relapse in patients with thrombotic thrombocytopenic purpura. Blood 2010; 115: 1500-1511.
• 15 Zheng XL, Richard KM, Goodnough LT. et al. Effect of plasma exchange on plasma ADAMTS13 metalloprotease activity, inhibitor level, and clinical outcome in patients with idiopathic and non-idiopathic thrombotic thrombocytopenic purpura. Blood 2004; 103: 4043-4049.
• 16 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.
• 17 Cataland SR, Jin M, Lin S. et al. Cyclosporin and plasma exchange in thrombotic thrombocytopenic purpura: long-term follow-up with serial analysis of ADAMTS13 activity. Br J Haematol 2007; 139: 486-493.
• 18 Zheng XL, Pallera AM, Goodnough LT. et al. Remission of chronic thrombotic thrombocytopenic purpura after treatment with cyclophosphamide and rituximab. Ann Intern Med 2003; 138: 105-108.
• 19 Fakhouri F, Teixeira L, Delarue R. et al. Responsiveness of thrombotic thrombocytopenic purpura to rituximab and cyclophosphamide. Ann Intern Med 2004; 140: 314-315.
• 20 Bresin E, Gastoldi S, Daina E. et al. Rituximab as pre-emptive treatment in patients with thrombotic thrombocytopenic purpura and evidence of anti-ADAMTS13 autoantibodies. Thromb Haemost 2009; 101: 233-238.
• 21 Scully M, Cohen H, Cavenagh J. et al. Remission in acute refractory and relapsing thrombotic thrombocytopenic purpura following rituximab is associated with a reduction in IgG antibodies to ADAMTS-13. Br J Haematol 2007; 136: 451-461.
• 22 Furlan M, Robles R, Solenthaler M. et al. Acquired deficiency of von Willebrand factor-cleaving protease in a patient with thrombotic thrombocytopenic purpura. Blood 1998; 91: 2839-2846.
• 23 Shelat SG, Smith AG, Ai J. et al. Inhibitory autoantibodies against ADAMTS-13 in patients with thrombotic thrombocytopenic purpura bind ADAMTS-13 protease and may accelerate its clearance in vivo. J Thromb Haemost 2006; 4: 1707-1717.
• 24 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.
• 25 Ferrari S, Scheiflinger F, Rieger M. et al. Prognostic value of anti-ADAMTS 13 antibody features (Ig isotype, titer, and inhibitory effect) in a cohort of 35 adult French patients undergoing a first episode of thrombotic microangiopathy with undetectable ADAMTS 13 activity. Blood 2007; 109: 2815-2822.
• 26 Ferrari S, Mudde GC, Rieger M. et al. IgG subclass distribution of anti-ADAMTS13 antibodies in patients with acquired thrombotic thrombocytopenic purpura. J Thromb Haemost 2009; 7: 1703-1710.
• 27 Zheng XL, Wu HM, Shang D. et al. Multiple domains of ADAMTS13 are targeted by autoantibodies against ADAMTS13 in patients with acquired idiopathic thrombotic thrombocytopenic purpura. Haematologica 2010; 95: 1555-1562.
• 28 Vesely SK, George JN, Lammle B. et al. ADAMTS13 activity in thrombotic thrombocytopenic purpura-hemolytic uremic syndrome: relation to presenting features and clinical outcomes in a prospective cohort of 142 patients. Blood 2003; 102: 60-68.
• 29 Han Y, Xiao J, Falls E. et al. A shear-based assay for assessing plasma ADAMTS13 activity and inhibitors in patients with thrombotic thrombocytopenic purpura. Transfusion 2011; 51: 1580-1591.
• 30 Tsai HM, Raoufi M, Zhou W. et al. ADAMTS13-binding IgG are present in patients with thrombotic thrombocytopenic purpura. Thromb Haemost 2006; 95: 886-892.
• 31 Rieger M, Mannucci PM, Kremer Hovinga JA. et al. ADAMTS13 autoantibodies in patients with thrombotic microangiopathies and other immunomediated diseases. Blood 2005; 106: 1262-1267.
• 32 Lublin DM, Atkinson JP. Decay-accelerating factor: biochemistry, molecular biology, and function. Annu Rev Immunol 1989; 7: 35-58.
• 33 Ai J, Smith P, Wang S. et al. The proximal carboxyl-terminal domains of ADAMTS13 determine substrate specificity and are all required for cleavage of von Willebrand factor. J Biol Chem 2005; 280: 29428-29434.
• 34 Zheng XL, Nishio K, Majerus EM. et al. Cleavage of von Willebrand factor requires the spacer domain of the metalloprotease ADAMTS13. J Biol Chem 2003; 278: 30136-30141.
• 35 Kokame K, Nobe Y, Kokubo Y. et al. FRETS-VWF73, a first fluorogenic substrate for ADAMTS13 assay. Br J Haematol 2005; 129: 93-100.
• 36 Luken BM, Turenhout EA, Hulstein JJ. et al. The spacer domain of ADAMTS13 contains a major binding site for antibodies in patients with thrombotic thrombocytopenic purpura. Thromb Haemost 2005; 93: 267-274.
• 37 Muniz M, Riezman H. Intracellular transport of GPI-anchored proteins. EMBO J 2000; 19: 10-15.
• 38 Allford SL, Hunt BJ, Rose P. et al. Guidelines on the diagnosis and management of the thrombotic microangiopathic haemolytic anaemias. Br J Haematol 2003; 120: 556-573.
• 39 Fakhouri F, Vernant JP, Veyradier A. et al. Efficiency of curative and prophylactic treatment with rituximab in ADAMTS13 deficient-thrombotic thrombocytopenic purpura: a study of 11 cases. Blood 2005; 106: 1932-1937.