A Rapid Sensitive Assay for Measuring ADAMTS13 Activity in Plasma Utilizing a Novel Recombinant Alexa488-vWF86 (Q1599\P1611C) FRET Substrate.

ADAMTS13 (von Willebrand Factor cleaving protease) is a zinc metalloprotease that cleaves the Tyr(1605)/Met(1606) bond within the A2 domain of von Willebrand Factor (vWF). Low levels of ADAMTS13 activity (<5%) are strongly associated with the life threatening hemostatic condition thrombotic thrombocytopenic purpura (TTP). A rapid clinical assay for ADAMTS13 is necessary to provide critical information for the early diagnosis of TTP and to guide and monitor the treatment of TTP.

Present methods for measuring ADAMTS13 activity (e.g. collagen binding, vWF multimer analysis and ELISA) are cumbersome, take a long time to complete and lack sensitivity. A fluorescence resonance energy transfer (FRET) assay for ADAMTS13 has been described, which uses the costly and difficult to synthesize truncated A2 domain VWF73aa peptide as substrate (1). However, the lower limit of detection of ADAMTS13 assays using VWF73 FRET substrate is only about 5–10% of normal ADAMTS13 activity levels (1,2). Recently, Zhang et al (3) reported a novel recombinant VWF A2 FRET substrate of 86 amino acids in which cysteine residues were substituted for residues Q1599 and P1611, respectively. The cysteine residues were chemically labeled with fluorescein maleimide to produce a FRET substrate whereby the two fluorescein dyes flanking the ADAMTS13 Tyr/Met cleavage site interact sufficiently such that auto-quenching of fluorescence occurs.

In this study, we developed a rapid more sensitive fluorescent assay for ADAMTS13 using a novel recombinant VWF86(Q1599C/P1611C) FRET substrate. VWF86(Q1599C/P1611C) peptide was chemically coupled via the cysteine residues to the brighter, more photostable Alexa488 fluorochrome. Assay conditions were optimized to obtain higher analytical sensitivity < 5% normal ADAMTS13 activity (0.5 ng/ml) and shorter assay times (20–30 minutes). ADAMTS13 activity in 50 normal individuals as well as subjects with TTP, thrombosis and lupus anticoagulant will be presented. The rapid FRET assay using the novel recombinant Alexa488-vWF86 (Q1599\P1611C) substrate should prove clinically useful for quantitation of ADAMTS13 activity and the rapid diagnosis of TTP.