@article{c71c566a8e694fc487ebf67104270cda,
title = "Ultra-high-throughput Ca2+ assay in platelets to distinguish ITAM-linked and G-protein-coupled receptor activation",
abstract = "Antiplatelet drugs targeting G-protein-coupled receptors (GPCRs), used for the secondary prevention of arterial thrombosis, coincide with an increased bleeding risk. Targeting ITAM- linked receptors, such as the collagen receptor glycoprotein VI (GPVI), is expected to provide a better antithrombotic-hemostatic profile. Here, we developed and characterized an ultra- high-throughput (UHT) method based on intracellular [Ca2+](i) increases to differentiate GPVI and GPCR effects on platelets. In 96-, 384-, or 1,536-well formats, Calcium-6- loaded human platelets displayed a slow-prolonged or fast-transient [Ca2+](i) increase when stimulated with the GPVI agonist collagen-related peptide or with thrombin and other GPCR agonists, respectively. Semi-automated curve fitting revealed five parameters describing the Ca2+ responses. Verification of the UHT assay was done with a robustness compound library and clinically relevant platelet inhibitors. Taken together, these results present proof of principle of distinct receptor-type-dependent Ca2+ signaling curves in platelets, which allow identification of new inhibitors in a UHT way.",
keywords = "GLYCOPROTEIN-VI, ANTIPLATELET THERAPIES, CALCIUM, INHIBITION, THROMBIN, COLLAGEN, ADP, IDENTIFICATION, RESPONSES, KINETICS",
author = "D.I. Fernandez and I. Provenzale and H.Y.F. Cheung and {van Groningen}, J. and B.M.E. Tullemans and A. Veninga and J.L. Dunster and S. Honarnejad and {van den Hurk}, H. and M.J.E. Kuijpers and J.W.M. Heemskerk",
note = "Funding Information: Support came from ZonMw project No. 40-43500-98-4006 (The Hague). This work received funding from the European Union's Horizon 2020 research and innovation program under Marie Sklodowska-Curie grant agreement TAPAS 766118. D.I.F. is registered in a joined PhD program of the Universities of Maastricht and Santiago de Compostela, I.P. is registered in a joined PhD program of the Universities of Maastricht and Reading, H.Y.F.C. is registered in a joined PhD program of the Universities of Maastricht and Birmingham. J.L.D. thanks the British Heart Foundation for funding (RG/20/7/34866 and RG/15/2/31224). D.I.F. M.J.E.K. S.H. H.v.d.H. and J.W.M.H. designed research; D.I.F, I.P. H.Y.F.C. B.M.E.T. J.v.G. and A.V. performed experiments; D.I.F. I.P. J.L.D. and S.H. analyzed data; D.I.F. M.J.E.K. and J.W.M.H. wrote the manuscript. J.W.M.H. is a cofounder and shareholder of FlowChamber B.V. The other authors declare no relevant conflicts of interest. Funding Information: Support came from ZonMw project No. 40-43500-98-4006 (The Hague). This work received funding from the European Union's Horizon 2020 research and innovation program under Marie Sklodowska-Curie grant agreement TAPAS 766118 . D.I.F. is registered in a joined PhD program of the Universities of Maastricht and Santiago de Compostela, I.P. is registered in a joined PhD program of the Universities of Maastricht and Reading, H.Y.F.C. is registered in a joined PhD program of the Universities of Maastricht and Birmingham. J.L.D. thanks the British Heart Foundation for funding ( RG/20/7/34866 and RG/15/2/31224 ). Publisher Copyright: {\textcopyright} 2021 The Author(s)",
year = "2022",
month = jan,
day = "21",
doi = "10.1016/j.isci.2021.103718",
language = "English",
volume = "25",
journal = "iScience",
issn = "2589-0042",
publisher = "Elsevier Inc.",
number = "1",
}