Departments of Medicine, Washington University School of Medicine, St Louis, MO 63110, USA.
J Thromb Haemost. 2013 Aug;11(8):1511-8. doi: 10.1111/jth.12319.
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.
We addressed these constraints by designing a substrate that can be used in undiluted plasma.
A polypeptide was expressed in E. coli that corresponds to von Willebrand factor Gln(1599) -Arg(1668) , with mutations N1610C and K1617R and an N-terminal Gly. Substrate FRETS-rVWF71 was prepared by modifying Cys(1610) 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 .
Serum and plasma anticoagulated with citrate or heparin had equivalent ADAMTS13 activity with FRETS-rVWF71. Neither bilirubin (≤ 20 mg dL(-1) ) nor hemoglobin (≤ 20 g L(-1) ) interfered with product detection. Assays with FRETS-rVWF71 and FRETS-VWF73 gave similar results (R(2 ) = 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.
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.
大多数 ADAMTS13 检测方法使用非生理条件（低离子强度、低 pH 值、氯化钡），易受血浆蛋白、血红蛋白和胆红素的干扰，且灵敏度有限，尤其是对抑制剂。
通过设计可用于未稀释血浆的底物来解决这些限制。
在大肠杆菌中表达与血管性血友病因子 Gln(1599)-Arg(1668)相对应的多肽，突变 N1610C 和 K1617R 以及 N 端甘氨酸。通过用 DyLight 633 修饰 Cys(1610)（吸收 638nm，发射 658nm）和用 IRDye QC-1 修饰 N 端（吸收 500-800nm）制备底物 FRETS-rVWF71。在 pH7.4、150mmNaCl 和 10mmCaCl2 条件下进行检测。
用柠檬酸盐或肝素抗凝的血清和血浆用 FRETS-rVWF71 检测具有等效的 ADAMTS13 活性。胆红素（≤20mg/dL(-1)）和血红蛋白（≤20g/L(-1)）均不干扰产物检测。用 FRETS-rVWF71 和 FRETS-VWF73 对 80 例血栓性微血管病患者、22 例其他血小板减少症患者和 20 例健康对照者的血浆进行检测，结果具有很好的相关性（R(2)=0.95）。用 FRETS-rVWF71 检测 ADAMTS13 活性的检测限≤0.3%。用 FRETS-rVWF71 进行抑制剂检测，效价比用 FRETS-VWF73 高约 2.5 倍，且能明确区分有和无抑制剂的患者。
FRETS-rVWF71 适用于在最小稀释的血浆或血清中进行 ADAMTS13 检测，不受血浆中蛋白质、胆红素或游离血红蛋白的干扰。优化 ADAMTS13 抑制剂的检测将有助于在治疗血栓性血小板减少性紫癜期间监测抗体反应。
