Cattle-FRETS71, a novel fluorogenic substrate with broad applicability for characterizing ADAMTS13 properties and function

Background: Current ADAMTS13 activity assays are important for diagnosing thrombotic thrombocytopenic purpura (TTP) but are unreliable to assay ADAMTS13 activity in animal models. The Cattle-FRETS71 assay is capable of detecting ADAMTS13 activity in plasma from multiple animal species, making it a potentially useful reagent at all stages of clinical research. The performance of Cattle-FRETS71 in TTP diagnosis is not yet known.Objectives: We evaluated the performance of the Cattle-FRETS71 substrate against the human FRETS-rVWF71 and the FRETS-VWF73 commercial substrates in human plasma and serum samples to validate its utility in diagnosing TTP in patients.Methods: Internal validation was performed using heparinized plasma samples (n = 81). External validation was a blinded study using serum samples from the Oklahoma TTP Registry (n = 118, collected 2004-2014) that had been initially assayed by FRETSVWF73 within 1 year of collection. Additional validation was performed with citrated plasma samples with variable ADAMTS13 activities (n = 32) that were analyzed by FRETS-VWF73. Results: There was an excellent correlation (r = 0.94) between Cattle-FRETS71 and FRETS-rVWF71 for assayed heparinized plasma samples (n = 81). Assay results between Cattle-FRETS71 and FRETS-VWF73 of Oklahoma TTP Registry serum samples (n = 118) and citrated plasma samples (n = 32) were comparably good (r = 0.81 and r = 0.85, respectively).Conclusion: The Cattle-FRETS71 assay is comparable with other assays in quantifying ADAMTS13 activity in human plasma collected from patients with documented or suspected TTP. The versatility of Cattle-FRETS71, combined with its specificity and sensitivity, makes it a useful tool for the standardization of ADAMTS13 activity across basic and clinical research paradigms.