title: Escherichia coli-derived von Willebrand factor-A2 domain fluorescence/Förster resonance energy transfer proteins that quantify ADAMTS13 activity.
abstract: The cleavage of the A2 domain of von Willebrand factor (VWF) by the metalloprotease ADAMTS13 regulates VWF size and platelet thrombosis rates. Reduction or inhibition of this enzyme activity leads to thrombotic thrombocytopenic purpura (TTP). We generated a set of novel molecules called VWF-A2 FRET (fluorescence/Förster resonance energy transfer) proteins, where variants of yellow fluorescent protein (Venus) and cyan fluorescent protein (Cerulean) flank either the entire VWF-A2 domain (175 amino acids) or truncated fragments (141, 113, and 77 amino acids) of this domain. These proteins were expressed in Escherichia coli in soluble form, and they exhibited FRET properties. Results show that the introduction of Venus/Cerulean itself did not alter the ability of VWF-A2 to undergo ADAMTS13-mediated cleavage. The smallest FRET protein, XS-VWF, detected plasma ADAMTS13 activity down to 10% of normal levels. Tests of acquired and inherited TTP could be completed within 30 min. VWF-A2 conformation changed progressively, and not abruptly, on increasing urea concentrations. Although proteins with 77 and 113 VWF-A2 residues were cleaved in the absence of denaturant, 4M urea was required for the efficient cleavage of larger constructs. Overall, VWF-A2 FRET proteins can be applied both for the rapid diagnosis of plasma ADAMTS13 activity and as a tool to study VWF-A2 conformation dynamics.
authors.affiliation: Department of Chemical and Biological Engineering, State University of New York at Buffalo, Buffalo, NY 14260, USA.
authors.forename: Kannayakanahalli M
authors.initials: KM
authors.lastname: Dayananda
authors.forename: Shobhit
authors.initials: S
authors.lastname: Gogia
authors.forename: Sriram
authors.initials: S
authors.lastname: Neelamegham
mesh_terms: D051722:ADAM Proteins / Q000097:blood* / Q000737:chemistry*; D000071120:ADAMTS13 Protein; D001774:Blood Chemical Analysis / Q000379:methods; D001777:Blood Coagulation / Q000502:physiology; D002460:Cell Line; D004926:Escherichia coli / Q000378:metabolism; D005453:Fluorescence; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D006801:Humans; D011487:Protein Conformation; D011697:Purpura, Thrombotic Thrombocytopenic / Q000097:blood / Q000175:diagnosis; D014842:von Willebrand Diseases / Q000097:blood / Q000175:diagnosis; D014841:von Willebrand Factor / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics
chemical_list: D014841:von Willebrand Factor; D051722:ADAM Proteins; D000071120:ADAMTS13 Protein; C099604:ADAMTS13 protein, human