High-throughput assessment identifying major platelet Ca2+ entry pathways via tyrosine kinase-linked and G protein-coupled receptors

Hilaire Yam Fung Cheung, Jinmi Zou, Chukiat Tantiwong, Delia I Fernandez, Jingnan Huang, Robert Ahrends, Mark Roest, Rachel Cavill, Jon Gibbins, Johan W M Heemskerk*

*Corresponding author for this work

Research output: Contribution to journalArticleAcademicpeer-review

Abstract

In platelets, elevated cytosolic Ca 2+ is a crucial second messenger, involved in most functional responses, including shape change, secretion, aggregation and procoagulant activity. The platelet Ca 2+ response consists of Ca 2+ mobilization from endoplasmic reticulum stores, complemented with store-operated or receptor-operated Ca 2+ entry pathways. Several channels can contribute to the Ca 2+ entry, but their relative contribution is unclear upon stimulation of ITAM-linked receptors such as glycoprotein VI (GPVI) and G-protein coupled receptors such as the protease-activated receptors (PAR) for thrombin. We employed a 96-well plate high-throughput assay with Fura-2-loaded human platelets to perform parallel [Ca 2+] i measurements in the presence of EGTA or CaCl 2. Per agonist condition, this resulted in sets of EGTA, CaCl 2 and Ca 2+ entry ratio curves, defined by six parameters, reflecting different Ca 2+ ion fluxes. We report that threshold stimulation of GPVI or PAR, with a variable contribution of secondary mediators, induces a maximal Ca 2+ entry ratio of 3-7. Strikingly, in combination with Ca 2+-ATPase inhibition by thapsigargin, the maximal Ca 2+ entry ratio increased to 400 (GPVI) or 40 (PAR), pointing to a strong receptor-dependent enhancement of store-operated Ca 2+ entry. By pharmacological blockage of specific Ca 2+ channels in platelets, we found that, regardless of GPVI or PAR stimulation, the Ca 2+ entry ratio was strongest affected by inhibition of ORAI1 (2-APB, Synta66) > Na +/Ca 2+ exchange (NCE) > P2× 1 (only initial). In contrast, inhibition of TRPC6, Piezo1/2 or STIM1 was without effect. Together, these data reveal ORAI1 and NCE as dominating Ca 2+ carriers regulating GPVI- and PAR-induced Ca 2+ entry in human platelets.

Original languageEnglish
Article number102738
Number of pages15
JournalCell Calcium
Volume112
Early online date8 Apr 2023
DOIs
Publication statusPublished - Jun 2023

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