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PubMed · 15801961

FRETS-VWF73, a first fluorogenic substrate for ADAMTS13 assay.

Koichi Kokame·Yuko Nobe·Yoshihiro Kokubo·Akira Okayama·Toshiyuki Miyata
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Abstract

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 1-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.

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ADAM ProteinsADAMTS13 ProteinAdultAgedAged, 80 and overAgingBiomarkersFemaleFluorescence Resonance Energy TransferFluorescent DyesHumansIndicators and ReagentsMaleMetalloendopeptidasesMiddle AgedPurpura, Thrombotic ThrombocytopenicRecombinant ProteinsReference ValuesReproducibility of ResultsSex CharacteristicsEvaluation StudyJournal ArticleResearch Support, Non-U.S. Gov't

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Koichi Kokame, Yuko Nobe, Yoshihiro Kokubo, Akira Okayama, Toshiyuki Miyata. 2005. FRETS-VWF73, a first fluorogenic substrate for ADAMTS13 assay.. https://doi.org/10.1111/j.1365-2141.2005.05420.x

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