Jin M, Cataland S, Bissell M, Wu H M
Department of Pathology, Ohio State University College of Medicine and Public Health, Columbus, OH 43210, USA.
J Thromb Haemost. 2006 Feb;4(2):333-8. doi: 10.1111/j.1538-7836.2006.01758.x.
Thrombotic thrombocytopenic purpura (TTP), a life-threatening thrombotic microangiopathy, requires immediate diagnosis and plasma exchange therapy. Development of TTP is related to functional deficiency of ADAMTS-13 protease that leads to the accumulation of ultra large von Willebrand factor (VWF) and subsequent platelet thrombosis. Currently no clinical test is available for the rapid detection of ADAMTS-13 activity.
The goal is to devise a novel method to rapidly detect functional activity of ADAMTS-13 and improve clinical outcome.
A recombinant VWF substrate containing the ADAMTS-13 cleavage site and a 6X Histidine tag was cleaved by ADAMTS-13 in a dose-dependent manner, generating approximately 7739 Da peptide containing a 6X Histidine tag. This cleaved peptide, bound to an IMAC/Nickel ProteinChip, was quantified using Surface Enhanced Laser Desorption/Ionization Time-of-flight Mass Spectrometry (SELDI-TOF-MS). The assay is capable of quantifying ADAMTS-13 activity as low as 2.5% in plasma within 4 h. When the cleaved peptide was quantified as a ratio of an internal control peptide, the test displayed good reproducibility, with an average inter-assay coefficient of variation (CV) of < 33%. Further validation revealed a mean ADAMTS-13 activity of 92.5% +/- 16.6% in 39 healthy donors. Sixteen patients with idiopathic TTP displayed mean ADAMTS-13 activity of 1.73% +/- 3.62%. Further utility of this novel method includes determining the inhibitory titer of ADAMTS-13 antibody in cases of acquired TTP.
We have devised a novel SELDI-TOF-MS assay that offers a rapid, cost-effective, and functionally relevant test for timely diagnosis and management of TTP.
血栓性血小板减少性紫癜（TTP）是一种危及生命的血栓性微血管病，需要立即诊断并进行血浆置换治疗。TTP的发生与ADAMTS-13蛋白酶功能缺陷有关，该缺陷导致超大血管性血友病因子（VWF）积聚，随后引发血小板血栓形成。目前尚无用于快速检测ADAMTS-13活性的临床检测方法。
目标是设计一种新方法来快速检测ADAMTS-13的功能活性并改善临床结果。
一种含有ADAMTS-13切割位点和6X组氨酸标签的重组VWF底物被ADAMTS-13以剂量依赖方式切割，产生了一个约7739 Da的含6X组氨酸标签的肽段。该切割后的肽段与IMAC/镍蛋白芯片结合，使用表面增强激光解吸/电离飞行时间质谱（SELDI-TOF-MS）进行定量。该检测方法能够在4小时内定量血浆中低至2.5%的ADAMTS-13活性。当将切割后的肽段作为内部对照肽段的比例进行定量时，该检测显示出良好的重现性，平均批间变异系数（CV）<33%。进一步验证显示，39名健康供体的ADAMTS-13平均活性为92.5%±16.6%。16例特发性TTP患者的ADAMTS-13平均活性为1.73%±3.62%。这种新方法的进一步应用包括在获得性TTP病例中确定ADAMTS-13抗体的抑制效价。
我们设计了一种新的SELDI-TOF-MS检测方法，该方法为TTP的及时诊断和管理提供了一种快速、经济高效且功能相关的检测方法。
