Comparison of FRETS-VWF73 to full-length VWF as a substrate for ADAMTS13 activity measurement in human plasma samples.

Thromb Haemost 2006; 95: 1049–51 Dear Sir, Thrombotic thrombocytopenic purpura (TTP) is a syndrome characterised by thrombocytopenia and microangiopathic haemolytic anaemia; it is often associated with neurological dysfunction, renal failure and fever (1). TTP is closely associated with a severe functional deficiency of ADAMTS13, the protease specifically cleaving large multimers of von Willebrand factor (VWF) in plasma (2). Both ADAMTS13 mutations (hereditary TTP) and auto-antibodies (acquired TTP) result in a severe functional ADAMTS13 deficiency in patients (3–5). As the clinical characteristics of TTP may be similar to those of other thrombotic microangiopathies (TMA) such as haemolytic uremic syndrome (HUS), an assay measuring ADAMTS13 activity would be a helpful tool for appropriate differential diagnosis and subsequent treatment. Many previous methods for the measurement of ADAMTS13 activity are not widely used at the clinical level because of technical complexity. Recently, a VWF fragment (VWF73) comprising the 73 amino acid residues (aa 1596–1668) of VWF subunit was shown to be the minimal substrate for ADAMTS13. The enzyme specifically cleaves this fragment in non-denaturing conditions and in a very short incubation period at the Tyr-Met bond. This VWF73 fragment was thus considered as an alternative substrate to full-length VWF for measurement of ADAMTS13 activity (6). Taking advantage of this substrate, a rapid and high throughput fluorescent energy transfer (FRETS) assay has been developed in which a chemically synthesized fluorogenic fragment (FRETS-VWF73) was used as a substrate to measure ADAMTS13 activity in plasma samples (7). A comparison with multimeric assay was performed and found to be highly correlated (8). The present study was designed to evaluate the FRETSVWF73 fluorescence assay when compared to our immunoradiometric assay (IRMA) using full-length VWF for the measurement of ADAMTS13 activity in plasma samples from a cohort of TMA patients and from normal subjects. Plasma from 64 TMA patients including 41 patients with acquired acute TTP, three patients with inheritedTTP and 20 patients with acute HUS were tested after obtaining appropriate consent in agreement with the institutional reviewing board of Assistance Publique – Hôpitaux de Paris and the declaration of Helsinki. Plasma of 10 healthy subjects was used as controls. ADAMTS13 activity of plasma samples was measured by a two-site IRMA using fulllength WT recombinant VWF as substrate and selected monoclonal antibodies to VWF, as previously described (9). Simultaneously, all samples were tested by FRETS-VWF73 fluorescence assay. Briefly, pooled human plasma used for calibration Letters to the Editor