A NEW ADAMTS13 ACTIVITY ASSAY USED WITH ANTIGEN ASSAY PROVIDES DIAGNOSTIC INFORMATION IN TTP, BLEEDING OR THROMBOSIS
Abstract number: P-S-329
Guinto1 E., Grafos1 N., Fryer1 H., Owen2 J., Lawson2 H., Wu3 H.M., Greenfield1 R.
11R and D, American Diagnostica Inc, Stamford 22Hem-Onc, Wake Forest U Sch Med, Winston-Salem 33Pathology, Ohio State U, Columbus, United States
How-to-cite Guinto E, Grafos N, Fryer H, Owen J, Lawson H, Wu HM, Greenfield R. A NEW ADAMTS13 ACTIVITY ASSAY USED WITH ANTIGEN ASSAY PROVIDES DIAGNOSTIC INFORMATION IN TTP, BLEEDING OR THROMBOSIS. J Thromb Haemost 2007; 5 Supplement 2: P-S-329
Abstract
Introduction: Rapid, sensitive, and specific ADAMTS13 assays with predictive value are desired in diagnosis and treatment of thrombotic thrombocytopenic purpura (TTP) and hemolytic uremic syndrome, inherited or acquired (1).
Methods: We developed a new fluorescence energy transfer (FRET) ADAMTS13 activity assay [Actifluor ADAMTS13 assay] for rapid measurement of ADAMTS13 activity in plasma using recombinant VWF84 (P16C/Q28C) peptide(2). Two Cys flanking the Tyr-Met bond cleaved by ADAMTS13 were labelled with stable Alexa fluor 488. The FRET activity, quantified using recombinant ADAMTS13 (rADAMTS13) was measured from the rate of fluorescence change. The relationship between activity and antigen levels [Imubind ADAMTS13 ELISA] was determined on normal, TTP, pregnant, bleeding and various thrombotic conditions.
Results: Kinetics of rADAMTS13 have Km 2.7 uM, kcat 5 s-1, kcat/Km 1.9 uM-1 s-1 and inhibition by unlabelled peptide with Ki similar to Km. The activity and antigen levels in 50 normal plasmas (mean 738108 ng/mL and 586154 ng/mL) result in activity/antigen ratios of 0.73-2.1 (mean 1.25). In 29 patients with TTP associated conditions (mean 213174 ng/ml, 24 have ratios 0.0 – 0.63, 2 are within and 3 are above the normal range. Most plasmas of patients (n ~60) with thrombophilia or veinous thromboembolism have ratios below normal while patients (n ~55) with stroke, pregnancy or myocardial infarction have near normal values.
Conclusions: The Actifluor ADAMTS13 assay is suitable for routine clinical use. Low ADAMTS13 activity was seen in TTP and underlying disease. The determination of ADAMTS13 activity/antigen ratio may be a useful diagnostic indicator of bleeding or thrombosis.
References: 1. Reiger M et al, Thromb Haemost 2006; 95:212-220
2.Zhang L et al, Anal. Biochem. 358(2006) 298-300.
