Assays for Plasma ADAMTS13 Activity and Inhibitors Using Recombinant FRETS-VWF73 Peptide

Thrombotic thrombocytopenic purpura (TTP) is a life-threatening thrombotic microangiopathy caused by severe deficiency of plasma ADAMTS13 activity. ADAMTS13 cleaves ultra-large von Willebrand factor (ULVWF) multimers, thus preventing the pathological accumulation of platelet-VWF aggregates at sites of vascular injury. The deficiency of ADAMTS13 can be congenital or more commonly immune-mediated, involving the formation of autoantibodies that bind and inactivate plasma ADAMTS13 activity. A simple, rapid, and cost-effective assay is crucial for the diagnosis of TTP and for the conduction of translational research relevant to ADAMTS13 function. Here, we describe a FRETS-rVWF73 assay for ADAMTS13 activity and inhibition, starting from the peptide substrate expression, purification, labeling, detailed assay protocol, and potential pitfalls. This assay can be used to quantify ADAMTS13 enzymatic activity and inhibition in various biological conditions for translational research.