ADAMTS-13 plasma level determination uncovers antigen absence in acquired thrombotic thrombocytopenic purpura and ethnic differences

Background: The recently discovered plasma enzyme ADAMTS-13 cleaves the A2-domain of von Willebrand factor (VWF). A defective cleaving protease results in unusually large VWF multimers, which cause thrombotic thrombocytopenic purpura (TTP). Aim: Analysis of the ADAMTS-13 antigen levels in TTP patients compared with normal donors. Methods: An antigen ELISA test was built, based on high affinity anti-ADAMTS-13 monoclonal antibodies, which were generated using genetic immunization. Results: Specificity of the ADAMTS-13 antigen test was confirmed, as (1) plasma from a patient with acquired TTP but presenting without inhibitor did not contain antigen and (ii) the binding of recombinant ADAMTS-13 was inhibited by increasing amounts of normal plasma. The assay is sensitive as it can detect antigen levels as low as 1.6% of normal. The concentration in normal pooled human plasma was determined (1.03 +/- 0.15 mu g mL(-1)) and arbitrarily set to 1 U mL(1-). The antigen levels in congenital TTP samples (34 +/- 21 rnU mL(-1), it = 2), as well as in samples from patients with acquired TTP (231 +/- 287 mU mL(-1), n = 11), were clearly reduced when compared with normal Caucasian donors (951 +/- 206 mU mL(-1), n = 16). Remarkably, normal Chinese donors have a significantly lower antigen titer (601 +/- 129 mU mL(-1), n = 15), when compared with normal Caucasians. Conclusions: Our results show that acquired TTP patients suffer mainly from ADAMTS-13 antigen depletion, thereby indicating the importance of ADAMTS-13 antigen determination in diagnosis and patient follow-up.