FRETS-VWF73, a first fluorogenic substrate for ADAMTS13 assay.
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.
Kokame
Koichi
K
National Cardiovascular Centre Research Institute, Suita, Osaka, Japan. kame@ri.ncvc.go.jp
Nobe
Yuko
Y
Kokubo
Yoshihiro
Y
Okayama
Akira
A
Miyata
Toshiyuki
T
D051722:ADAM Proteins; D000071120:ADAMTS13 Protein; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D000375:Aging / Q000097:blood; D015415:Biomarkers / Q000097:blood; D005260:Female; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry; D006801:Humans; D007202:Indicators and Reagents; D008297:Male; D008666:Metalloendopeptidases / Q000097:blood*; D008875:Middle Aged; D011697:Purpura, Thrombotic Thrombocytopenic / Q000175:diagnosis*; D011994:Recombinant Proteins; D012016:Reference Values; D015203:Reproducibility of Results; D012727:Sex Characteristics
D015415:Biomarkers; D005456:Fluorescent Dyes; D007202:Indicators and Reagents; D011994:Recombinant Proteins; D051722:ADAM Proteins; D008666:Metalloendopeptidases; D000071120:ADAMTS13 Protein; C099604:ADAMTS13 protein, human