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Updated: Dec 20, 2025
Fluorescence-based Monitoring of PAD4 Activity via a Pro-fluorescence Substrate Analog
Published on: November 5, 2014
Recombinant expression of ecto-nucleotide pyrophosphatase/phosphodiesterase 4 (NPP4) and development of a
Vittoria Lopez1, Sang-Yong Lee1, Holger Stephan2
1Pharmaceutical Institute, Pharmaceutical & Medicinal Chemistry, University of Bonn, An der Immenburg 4, 53121, Bonn, Germany; PharmaCenter Bonn, University of Bonn, An der Immenburg 4, 53121, Bonn, Germany.
Abstract:
Nucleotide pyrophosphatase/phosphodiesterase 4 (NPP4) is a membrane-bound enzyme that hydrolyzes extracellular diadenosine polyphosphates such as diadenosine triphosphate (Ap3A) and diadenosine tetraphosphate (Ap4A) yielding mononucleotides. NPP4 on the surface of endothelial cells was reported to promote platelet aggregation by hydrolyzing Ap3A to ADP, which activates pro-thrombotic G protein-coupled P2Y1 and P2Y12 receptors. Thus, NPP4 inhibitors have potential as novel antithrombotic drugs. In the present study we expressed soluble human NPP4 in Sf9 insect cells and established an enzyme assay using diadenosine tetraphosphate (Ap4A) as a substrate. The reaction product ATP was quantified by luciferin-luciferase reaction in a 96-well plate format. The sensitive method displayed a limit of detection (LOD) of 14.6 nM, and a Z'-factor of 0.68 indicating its suitability for high-throughput screening. The new assay was applied for studying enzyme kinetics and led to the identification of the first NPP4 inhibitors.
