(582eb) E. Coli Derived Substrate Quantifies ADAMTS13 Protease Activity in Blood Plasma Using Flow Cytometry

The metalloprotease ADAMTS13 regulates the size of the multimeric blood protein von Willebrand factor (VWF). Reduction or inhibition of ADAMTS13 activity in humans can lead to a bleeding disorder called Thrombotic Thrombocytopenic Purpura (TTP). In this paper, we describe a flow cytometry based assay to quantify ADAMTS13 activity in human clinical samples using an E. coli derived 77-amino acid peptide substrate that is based on a mutated form of the A2-domain of VWF. This substrate was tagged using NHS (N-hydroxysuccinimide)-fluorescein at the N-terminus and Maleimide-PEG(Polyethylene glycol)_n-biotin at the C-terminus. PEG repeat unit length was 2 in the case of ‘A2-77p-short’ and it was 77 for ‘A2-77p-long’. Biotin-streptavidin bonding was used to immobilize the substrates onto streptavidin coated microspheres. In flow cytometry assays, fluorescein signal associated with A2-77p-short/A2-77p-long decreased inversely with recombinant ADAMTS13 concentration. A2-77p-long also detected plasma ADAMTS13 activity levels down to 5% of normal activity levels within 30 min. While previous substrates were limited in their ability to detect ADAMTS13 activity when clinically elevated levels of bilirubin are present, the current substrate immobilization strategy along with the specific protein design overcame this problem. As shown, A2-77p-long cleavage could be measured in the presence of bilirubin up to 400 µM. Further, enzyme catalytic activity could be tuned by varying buffer calcium concentration with lower divalent ion concentrations enhancing cleavage rate. Overall, the new peptide substrates allow rapid quantitation of ADAMTS13 activity in blood plasma and highlight a role of calcium in regulating VWF proteolysis activity.