An optimized fluorogenic ADAMTS13 assay with increased sensitivity for the investigation of patients with thrombotic thrombocytopenic purpura

Abstract

Summary Background Most ADAMTS13 assays use non-physiological conditions (low ionic strength, low pH, barium chloride), are subject to interference from plasma proteins, hemoglobin and bilirubin, and have limited sensitivity, especially for inhibitors. Objectives We addressed these constraints by designing a substrate that can be used in undiluted plasma. Methods A polypeptide was expressed in E. coli that corresponds to von Willebrand factor Gln1599-Arg1668, with mutations N1610C and K1617R and an N-terminal Gly. Substrate FRETS-rVWF71 was prepared by modifying Cys1610 with DyLight 633 (abs 638 nm, em 658 nm) and the N-terminus with IRDye QC-1 (abs 500-800 nm). Assays were performed at pH 7.4 in 150 mM NaCl, 10 mM CaCl2. Results Serum and plasma anticoagulated with citrate or heparin had equivalent ADAMTS13 activity with FRETS-rVWF71. Neither bilirubin (≤20 mg/dL) nor hemoglobin (≤20 g/L) interfered with product detection. Assays with FRETS-rVWF71 and FRETS-VWF73 gave similar results (R2 = 0.95) for plasma from 80 subjects with thrombotic microangiopathy, 22 subjects with other causes of thrombocytopenia, and 20 healthy controls. The limit of detection with FRETS-rVWF71 for ADAMTS13 activity was ≤0.3%. Inhibitor assays with FRETS-rVWF71 gave titers ~2.5-fold higher than with FRETS-VWF73 and clearly distinguished patients with and without inhibitors. Conclusions FRETS-rVWF71 is suitable for ADAMTS13 assays in minimally diluted plasma or serum without interference from proteins, bilirubin or free hemoglobin in plasma. Optimized detection of ADAMTS13 inhibitors will facilitate the monitoring of antibody responses during the treatment of thrombotic thrombocytopenic purpura.

Diagnostic and prognostic values of ADAMTS13 activity measured during daily plasma exchange therapy in patients with acquired thrombotic thrombocytopenic purpura

“…It has been reported that serum and plasma anticoagulated with citrate or heparin had equivalent ADAMTS13 activity. 29 However, for patients without blood drawn for ADAMTS13 before start of PE treatment, physicians can still order an ADAMTS13 test as long as the sample is drawn before PE procedure of fourth daily PE treatment. In the majority of these cases, posttreatment samples will help to confirm the clinical suspicion of acquired TTP.…”

Allosteric activation of ADAMTS13 by von Willebrand factor

“…We (22) and others (23,24) have observed that full-length ADAMTS13 is most active at pH 6, with markedly decreased activity at pH 7.4 ( Fig. 1D and Fig.…”

“…When assayed with a fluorogenic ADAMTS13 substrate, VWF71 (Fig. 1B) (22), patient BCW49 had high-titer autoantibodies that paradoxically activated exogenous ADAMTS13 ∼threefold (Fig. 1C).…”

“…FRETS-rVWF25, FRETS-rVWF44, and FRETS-rVWF62 were prepared similarly except with C-terminal residues Asp 1622 , Arg 1641 , and Arg 1659 , respectively. ADAMTS13 was assayed with fluorogenic substrates as previously described (22). Standard assays included 50 mM Bis-Tris (pH 6) or Hepes (pH 7.4), 150 mM NaCl, 10 mM CaCl 2 , 0.05% Tween-20, 1 mg/mL BSA, 1 μM fluorogenic substrate, and PNP or recombinant ADAMTS13 variants in a total volume of 200 μL.…”

“…Fluorogenic peptide substrate FRETS-rVWF71 (VWF71) has an N-terminal Gly followed by VWF Gln 1599 -Arg 1668 with mutations N1610C and K1617R. The N terminus is modified with IRDye QC-1 Nhydroxysuccinimide ester (Li-COR), and Cys 1610 is modified with DyLight 633 maleimide (Thermo Scientific) (22). FRETS-rVWF25, FRETS-rVWF44, and FRETS-rVWF62 were prepared similarly except with C-terminal residues Asp 1622 , Arg 1641 , and Arg 1659 , respectively.…”