FRETS-VWF73, a first fluorogenic substrate for ADAMTS13 assay
FRETS-VWF73, a first fluorogenic substrate for ADAMTS13 assay
复制标题
DOI：
10.1111/j.1365-2141.2005.05420.x
复制
发表时间：
2005-04-01
影响因子：
6.5
通讯作者：
Miyata, T
中科院分区：
文献类型：
--
作者：
Kokame, K;Nobe, Y;Miyata, T
血浆金属蛋白酶ADAMTS13可切割血管性血液病因子（VWF）多聚体并下调其在血小板聚集中的活性。功能性ADAMTS13缺乏导致过度活跃的大VWF多聚体的积累，诱发危及生命的疾病，血栓性血小板减少性紫癜（TTP）。虽然测量ADAMTS13活性在TTP诊断中很重要，但现有的方法需要时间和技能。在这里，我们报告了ADAMTS13活性的荧光共振能量转移（FRET）测定。我们合成了一个73个氨基酸的肽，FRETS-VWF73。该底物在两个修饰残基之间的切割解除了完整肽中的荧光猝灭。FRETS-VWF73与正常人血浆孵育后，荧光随着时间的推移定量增加，而adamts13缺失血浆则没有影响。定量分析可以在I-h周期内实现，使用96孔格式的商业平板阅读器与普通过滤器。FRETS-VWF73检测将有助于TTP等血栓性微血管病变的表征，并可能阐明ADAMTS13活性作为各种血栓性疾病预测标志物的重要性。
A plasma metalloprotease, ADAMTS13, cleaves von Willebrand factor (VWF) multimers and downregulates their activity in platelet aggregation. Functional ADAMTS13 deficiency leads to the accumulation of hyperactive large VWF multimers, inducing a life-threatening disease, thrombotic thrombocytopenic purpura (TTP). Although measuring ADAMTS13 activity is important in TTP diagnosis, existing methods require time and skill. Here, we report a fluorescence resonance energy transfer (FRET) assay for ADAMTS13 activity. We developed a synthetic 73-amino-acid peptide, FRETS-VWF73. Cleavage of this substrate between two modified residues relieves the fluorescence quenching in the intact peptide. Incubation of FRETS-VWF73 with normal human plasma quantitatively increased fluorescence over time, while ADAMTS13-deficient plasma had no effect. Quantitative analysis could be achieved within a I-h period using a 96-well format in commercial plate readers with common filters. The FRETS-VWF73 assay will be useful for the characterization of thrombotic microangiopathies like TTP and may clarify the importance of ADAMTS13 activity as a predictive marker for various thrombotic diseases.