FRETS-VWF73, a first fluorogenic substrate for ADAMTS13 assay

被引:493
作者
Kokame, K
Nobe, Y
Kokubo, Y
Okayama, A
Miyata, T
机构
[1] Natl Cardiovasc Ctr, Res Inst, Osaka 5658565, Japan
[2] Natl Cardiovasc Ctr, Dept Prevent Cardiol, Osaka 5658565, Japan
关键词
ADAMTS13; von Willebrand factor; platelet; thrombotic thrombocytopenic purpura; fluorescence resonance energy transfer;
D O I
10.1111/j.1365-2141.2005.05420.x
中图分类号
R5 [内科学];
学科分类号
1002 ; 100201 ;
摘要
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.
引用
收藏
页码:93 / 100
页数:8
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