Ultra-high-throughput Ca2+ assay in platelets to distinguish ITAM-linked and G-protein-coupled receptor activation
Delia I. Fernández,
Isabella Provenzale,
Hilaire Y.F. Cheung,
Jan van Groningen,
Bibian M.E. Tullemans,
Alicia Veninga,
Joanne L. Dunster,
Saman Honarnejad,
Helma van den Hurk,
Marijke J.E. Kuijpers,
Johan W.M. Heemskerk
Affiliations
Delia I. Fernández
Department of Biochemistry, Cardiovascular Research Institute Maastricht (CARIM), Maastricht University, 6229 ER Maastricht, The Netherlands; Platelet Proteomics Group, Center for Research in Molecular Medicine and Chronic Diseases (CiMUS), Universidad de Santiago de Compostela, 15782 Santiago de Compostela, Spain
Isabella Provenzale
Department of Biochemistry, Cardiovascular Research Institute Maastricht (CARIM), Maastricht University, 6229 ER Maastricht, The Netherlands; Institute for Cardiovascular and Metabolic Research, University of Reading, RG6 6AX Reading, UK
Hilaire Y.F. Cheung
Department of Biochemistry, Cardiovascular Research Institute Maastricht (CARIM), Maastricht University, 6229 ER Maastricht, The Netherlands; ISASLeibniz-Institut fur Analytische Wissenschaften-ISAS-e.V., 44227 Dortmund, Germany; Institute of Cardiovascular Sciences, Institute of Biomedical Research, College of Medical and Dental Sciences, University of Birmingham, Birmingham B15 2TT, UK
Jan van Groningen
Pivot Park Screening Centre, 5349 AB Oss, the Netherlands
Bibian M.E. Tullemans
Department of Biochemistry, Cardiovascular Research Institute Maastricht (CARIM), Maastricht University, 6229 ER Maastricht, The Netherlands
Alicia Veninga
Department of Biochemistry, Cardiovascular Research Institute Maastricht (CARIM), Maastricht University, 6229 ER Maastricht, The Netherlands
Joanne L. Dunster
Institute for Cardiovascular and Metabolic Research, University of Reading, RG6 6AX Reading, UK
Saman Honarnejad
Pivot Park Screening Centre, 5349 AB Oss, the Netherlands
Helma van den Hurk
Pivot Park Screening Centre, 5349 AB Oss, the Netherlands
Marijke J.E. Kuijpers
Department of Biochemistry, Cardiovascular Research Institute Maastricht (CARIM), Maastricht University, 6229 ER Maastricht, The Netherlands; Thrombosis Expertise Centre, Heart and Vascular Centre, Maastricht University Medical Centre, Maastricht, the Netherlands
Johan W.M. Heemskerk
Department of Biochemistry, Cardiovascular Research Institute Maastricht (CARIM), Maastricht University, 6229 ER Maastricht, The Netherlands; Synapse Research Institute, Kon. Emmaplein 7, 6214 AC, Maastricht, the Netherlands; Corresponding author
Summary: 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.
