Department of Pathology and Laboratory Medicine, The Children's Hospital of Philadelphia and The University of Pennsylvania Medical Center, Philadelphia, Pennsylvania 19104, USA.
Transfusion. 2011 Jul;51(7):1580-91. doi: 10.1111/j.1537-2995.2010.03020.x. Epub 2011 Jan 20.
Severe deficiency of plasma ADAMTS13 activity is a frequent finding in patients with hereditary and acquired thrombotic thrombocytopenic purpura (TTP). To date, plasma ADAMTS13 activity is determined by cleavage of either predenatured von Willebrand factor (VWF) or small peptides derived from the VWF-A2 domain. The physiologic relevance of the assay results is uncertain.
We sought to develop a novel shear-based assay to assess plasma ADAMTS13 activity and inhibitors. We also compared this assay with a fluorogenic peptide assay.
We found that an incubation of purified plasma VWF with 0.5 to 1.0 µL of citrated plasma under constant vortexing at 2500 rpm for 60 minutes in the presence of 5 mmol/L CaCl(2) and 1.7 µmol/L ZnCl(2) and low concentration of NaCl resulted in the maximal cleavage of VWF. The cleavage product could be separated by a 2.5% agarose gel and detected by Western blotting. The assay revealed that plasma and recombinant ADAMTS13 are highly sensitive to inhibition by zinc and chloride ions. Under the optimal conditions, the shear-based assay appeared to be more sensitive than the guanidine-denaturization assay for determining plasma ADAMTS13 activity.
Our fluid shear-based assay may be useful for investigating basic biologic function and regulation of ADAMTS13 metalloprotease. It may also be applicable for assessing plasma ADAMTS13 activity and inhibitors in TTP patients.
严重缺乏血浆 ADAMTS13 活性是遗传性和获得性血栓性血小板减少性紫癜（TTP）患者的常见现象。迄今为止，血浆 ADAMTS13 活性是通过切割预先变性的血管性血友病因子（VWF）或 VWF-A2 结构域衍生的小肽来确定的。该测定结果的生理相关性尚不确定。
我们试图开发一种新的基于剪切的测定法来评估血浆 ADAMTS13 活性和抑制剂。我们还将该测定法与荧光肽测定法进行了比较。
我们发现，在 2500rpm 下以 0.5 至 1.0μL 的柠檬酸血浆孵育纯化的血浆 VWF，在 5mmol/L CaCl2 和 1.7μmol/L ZnCl2 以及低浓度 NaCl 的存在下持续涡旋 60 分钟，可导致 VWF 最大程度的切割。通过 2.5%琼脂糖凝胶分离切割产物，并通过 Western blot 检测。该测定法表明，血浆和重组 ADAMTS13 对锌离子和氯离子的抑制高度敏感。在最佳条件下，与胍变性测定法相比，基于剪切的测定法似乎更能灵敏地测定血浆 ADAMTS13 活性。
我们的流体剪切测定法可能有助于研究 ADAMTS13 金属蛋白酶的基本生物学功能和调节。它也可能适用于评估 TTP 患者的血浆 ADAMTS13 活性和抑制剂。
