Whitelock J L, Nolasco L, Bernardo A, Moake J, Dong J-F, Cruz M A
Department of Medicine, Thrombosis Research Section, Baylor College of Medicine, Houston, TX, USA.
J Thromb Haemost. 2004 Mar;2(3):485-91. doi: 10.1111/j.1538-7836.2004.00601.x.
The metalloprotease ADAMTS-13 cleaves von Willebrand factor (VWF) at the Y842/M843 peptide bond located in the A2 domain. Measurement of ADAMTS-13 activity is a clinical utility for thrombotic diseases, but the current assays used for diagnostic and clinical research are non-physiological and time consuming. We have expressed in bacteria a recombinant VWF-A2 peptide (aa 718-905) that contains both a 6xHis tag at the N-terminal end and a Tag-100 epitope at the C-terminal end. Diluted plasma was mixed with the VWF-A2 peptide and digestion was allowed to proceed in a Ni2+-coated microtiter well plate for 2 h. The immobilized Ni2+ captures the VWF-A2 peptide by its 6xHis tag and cleavage of the A2 peptide is measured by the removal of the C-terminus fragment of the A2 peptide that contains the Tag-100. The cleavage activity for this assay was defined by the low detection of A2 peptide containing the Tag-100 epitope by the antiTag-100 monoclonal antibody. The assay was completed in <5 h. We then used the assay to analyze ADAMTS-13 activity in plasma from 39 healthy donors and 16 samples from patients diagnosed as thrombotic thrombocytopenic purpura. The average of enzyme activity +/- SEM for normal plasmas diluted 1 : 50 was 40 +/- 4.2% while the value obtained for the patients was 2.4 +/- 0.7%. These results were validated by a traditional long incubation assay (24 h). Our assay provides significant advantages over currently used assays because it is quicker, reproducible, cost effective and measures ADAMTS-13 activity under physiological and non-denaturing conditions. This assay is clinically useful and significant in measuring ADAMTS-13 activity in plasma.
金属蛋白酶ADAMTS-13在位于A2结构域的Y842/M843肽键处切割血管性血友病因子（VWF）。ADAMTS-13活性的测定对血栓性疾病具有临床应用价值，但目前用于诊断和临床研究的检测方法不符合生理情况且耗时。我们在细菌中表达了一种重组VWF-A2肽（氨基酸718 - 905），该肽在N末端含有一个6xHis标签，在C末端含有一个Tag-100表位。将稀释的血浆与VWF-A2肽混合，并在涂有Ni2+的微量滴定板中进行消化2小时。固定化的Ni2+通过其6xHis标签捕获VWF-A2肽，并通过去除含有Tag-100的A2肽的C末端片段来测量A2肽的切割情况。该检测方法的切割活性通过抗Tag-100单克隆抗体对含有Tag-100表位的A2肽的低检测来定义。该检测在<5小时内完成。然后我们使用该检测方法分析了39名健康供体血浆和16份被诊断为血栓性血小板减少性紫癜患者样本中的ADAMTS-13活性。稀释1:50的正常血浆的酶活性平均值±SEM为40±4.2%，而患者样本的值为2.4±0.7%。这些结果通过传统的长时间孵育检测方法（24小时）得到了验证。我们的检测方法比目前使用的检测方法具有显著优势，因为它更快、可重复、成本效益高，并且能在生理和非变性条件下测量ADAMTS-13活性。该检测方法在测量血浆中ADAMTS-13活性方面具有临床实用性和重要意义。
