title: FRETS-VWF73 rather than CBA assay reflects ADAMTS13 proteolytic activity in acquired thrombotic thrombocytopenic purpura patients.
abstract: Collagen-binding activity (CBA) and FRETS-VWF73 assays are widely adopted methods for the measurement of the plasmatic activity of ADAMTS13, the von Willebrand factor (VWF) cleaving-protease. Accurately assessing the severe deficiency of ADAMTS13 is important in the management of thrombotic thrombocytopenic purpura (TTP). However, non-concordant results between the two assays have been reported in a small but relevant percentage of TTP cases. We investigated whether CBA or FRETS-VWF73 assay reflects ADAMTS13 proteolytic activity in acquired TTP patients with non-concordant measurements. Twenty plasma samples with non-concordant ADAMTS13 activity results, <10% using FRETS-VWF73 and ≥20% using CBA, and 11 samples with concordant results, <10% using either FRETS-VWF73 and CBA assays, were analysed. FRETS-VWF73 was performed in the presence of 1.5 M urea. ADAMTS13 activities were also measured under flow conditions and the VWF multimer pattern was defined in order to verify the presence of ultra-large VWF due to ADAMTS13 deficiency. In FRETS-VWF73 assay with 1.5 M urea, ADAMTS13 activity significantly increased in roughly 50% of the samples with non-concordant results, whereas it remained undetectable in all samples with concordant measurements. Under flow conditions, all tested samples showed reduced ADAMTS13 activity. Finally, samples with non-concordant results showed a ratio of high molecular weight VWF multimers higher than normal. Our results support the use of FRETS-VWF73 over CBA assay for the assessment of ADAMTS13 severe deficiency and indicate urea as one cause of the observed differences.
authors.forename: I
authors.initials: I
authors.lastname: Mancini
authors.forename: C
authors.initials: C
authors.lastname: Valsecchi
authors.forename: L A
authors.initials: LA
authors.lastname: Lotta
authors.forename: L
authors.initials: L
authors.lastname: Deforche
authors.forename: S
authors.initials: S
authors.lastname: Pontiggia
authors.forename: M
authors.initials: M
authors.lastname: Bajetta
authors.forename: R
authors.initials: R
authors.lastname: Palla
authors.forename: K
authors.initials: K
authors.lastname: Vanhoorelbeke
authors.affiliation: Prof. Flora Peyvandi, Angelo Bianchi Bonomi Hemophilia and Thrombosis Center, Fondazione IRCCS Ca' Granda - Ospedale Maggiore Policlinico, Department of Pathophysiology and Transplantation, Università degli Studi di Milano and Luigi Villa Foundation, Via Pace 9, 20122, Milan, Italy, Tel.: +39 02 5503 5414, Fax: +39 02 54 100 125, E-mail: flora.peyvandi@unimi.it.
authors.forename: F
authors.initials: F
authors.lastname: Peyvandi
mesh_terms: D051722:ADAM Proteins / Q000172:deficiency*; D000071120:ADAMTS13 Protein; D003094:Collagen / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D011237:Predictive Value of Tests; D011485:Protein Binding; D011489:Protein Denaturation; D059748:Proteolysis; D011697:Purpura, Thrombotic Thrombocytopenic / Q000097:blood / Q000175:diagnosis* / Q000201:enzymology; D012042:Registries; D015203:Reproducibility of Results; D014508:Urea / Q000737:chemistry; D014841:von Willebrand Factor / Q000378:metabolism*
chemical_list: D014841:von Willebrand Factor; D014508:Urea; D003094:Collagen; D051722:ADAM Proteins; D000071120:ADAMTS13 Protein; C099604:ADAMTS13 protein, human
keywords: ADAMTS13 activity assay; TTP; autoantibodies; flow assay; von Willebrand factor