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

Fernández, D. I. ORCID: https://orcid.org/0000-0002-5055-9019, Provenzale, I., Cheung, H. Y. F., van Groningen, J., Tullemans, B. M. E., Veninga, A., Dunster, J. L. ORCID: https://orcid.org/0000-0001-8986-4902, Honarnejad, S. ORCID: https://orcid.org/0000-0002-4558-501X, van den Hurk, H., Kuijpers, M. J. E. and Heemskerk, J. W. M. (2021) Ultra-high throughput Ca2+ assay in platelets to distinguish ITAM-linked and G-protein coupled receptor activation. iScience, 25 (1). 103718. ISSN 2589-0042 doi: 10.1016/j.isci.2021.103718

Abstract/Summary

Antiplatelet drugs targeting G-protein coupled receptors (GPCR), 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 rises to differentiate GPVI and GPCR effects on platelets. In 96-, 384- or 1536-well formats, Calcium-6-loaded human platelets displayed a slow-prolonged or fast-transient [Ca2+]i rise when stimulated with the GPVI agonist collagen-related peptide (CRP), or with thrombin and other GPCR agonists, respectively. Semi-automated curve fitting revealed five early and late 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 provide proof-of-principle of distinct receptor-type dependent Ca2+ signaling curves platelets, which allow identification of new inhibitors in a UHT way.

Item Type	Article
URI	https://reading-clone.eprints-hosting.org/id/eprint/102124
Identification Number/DOI	10.1016/j.isci.2021.103718
Refereed	Yes
Divisions	Interdisciplinary centres and themes > Institute for Cardiovascular and Metabolic Research (ICMR) Life Sciences > School of Biological Sciences > Biomedical Sciences
Publisher	Cell Press
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