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

doi:10.1016/j.isci.2021.103718

Ultra-high-throughput Ca²⁺ assay in platelets to distinguish ITAM-linked and G-protein-coupled receptor activation

iScience. 2021 Dec 31;25(1):103718. doi: 10.1016/j.isci.2021.103718. eCollection 2022 Jan 21.

Authors

Delia I Fernández^{1

2}, Isabella Provenzale^{1

3}, Hilaire Y F Cheung^{1

4

5}, Jan van Groningen⁶, Bibian M E Tullemans¹, Alicia Veninga¹, Joanne L Dunster³, Saman Honarnejad⁶, Helma van den Hurk⁶, Marijke J E Kuijpers^{1

7}, Johan W M Heemskerk^{1

8}

Affiliations

¹ Department of Biochemistry, Cardiovascular Research Institute Maastricht (CARIM), Maastricht University, 6229 ER Maastricht, The Netherlands.
² Platelet Proteomics Group, Center for Research in Molecular Medicine and Chronic Diseases (CiMUS), Universidad de Santiago de Compostela, 15782 Santiago de Compostela, Spain.
³ Institute for Cardiovascular and Metabolic Research, University of Reading, RG6 6AX Reading, UK.
⁴ ISASLeibniz-Institut fur Analytische Wissenschaften-ISAS-e.V., 44227 Dortmund, Germany.
⁵ Institute of Cardiovascular Sciences, Institute of Biomedical Research, College of Medical and Dental Sciences, University of Birmingham, Birmingham B15 2TT, UK.
⁶ Pivot Park Screening Centre, 5349 AB Oss, the Netherlands.
⁷ Thrombosis Expertise Centre, Heart and Vascular Centre, Maastricht University Medical Centre, Maastricht, the Netherlands.
⁸ Synapse Research Institute, Kon. Emmaplein 7, 6214 AC, Maastricht, the Netherlands.

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: Cell biology; Functional aspects of cell biology; Methodology in biological sciences.

Abstract

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