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Published on: May 13, 2020
FRETS-VWF73, a first fluorogenic substrate for ADAMTS13 assay
Koichi Kokame1, Yuko Nobe, Yoshihiro Kokubo
1National Cardiovascular Centre Research Institute, Suita, Osaka, Japan. kame@ri.ncvc.go.jp
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.
Insights
A new assay rapidly measures ADAMTS13 enzyme activity, crucial for diagnosing thrombotic thrombocytopenic purpura (TTP). This fluorescence-based method offers a faster, more accessible diagnostic tool for TTP and related thrombotic diseases.
Area of Science:
- Biochemistry
- Hematology
- Enzymology
Background:
- ADAMTS13 is a plasma metalloprotease that regulates von Willebrand factor (VWF) activity.
- Deficiency in ADAMTS13 activity causes thrombotic thrombocytopenic purpura (TTP) due to VWF multimer accumulation.
- Current methods for measuring ADAMTS13 activity are time-consuming and require specialized expertise.
Purpose of the Study:
- To develop a rapid and efficient assay for quantifying ADAMTS13 activity.
- To establish a fluorescence resonance energy transfer (FRET) based assay using a synthetic substrate.
- To enable faster diagnosis and characterization of thrombotic microangiopathies.
Main Methods:
- Development of a synthetic peptide substrate, FRETS-VWF73, containing modified residues.
- Utilizing FRET technology where cleavage of the substrate by ADAMTS13 relieves fluorescence quenching.
- Incubation of the substrate with human plasma samples and measurement of fluorescence in a 96-well plate format.
Main Results:
- The FRETS-VWF73 assay demonstrated quantitative measurement of ADAMTS13 activity within one hour.
- Normal human plasma showed increased fluorescence over time, indicating substrate cleavage.
- ADAMTS13-deficient plasma did not result in increased fluorescence, confirming assay specificity.
- The assay is compatible with standard laboratory equipment, including commercial plate readers.
Conclusions:
- The developed FRET assay provides a rapid, quantitative, and accessible method for measuring ADAMTS13 activity.
- This assay can aid in the diagnosis and management of thrombotic thrombocytopenic purpura (TTP).
- The assay holds potential for predicting thrombotic disease risk by assessing ADAMTS13 activity levels.
