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

Cheung, H. Y. F., Zou, J., Tantiwong, C., Fernandez, D. I., Huang, J., Ahrends, R., Roest, M., Cavill, R., Gibbins, J. ORCID: and Heemskerk, J. W. M. (2023) High-throughput assessment identifying major platelet Ca2+ entry pathways via tyrosine kinase-linked and G protein-coupled receptors. Cell Calcium, 112. 102738. ISSN 0143-4160

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To link to this item DOI: 10.1016/j.ceca.2023.102738


In platelets, elevated cytosolic Ca2+ is a crucial second messenger, involved in most functional responses, including shape change, secretion, aggregation and procoagulant activity. The platelet Ca2+ response consists of Ca2+ mobilization from endoplasmic reticulum stores, complemented with store-operated or receptor-operated Ca2+ entry pathways. Several channels can contribute to the Ca2+ 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 [Ca2+]i measurements in the presence of EGTA or CaCl2. Per agonist condition, this resulted in sets of EGTA, CaCl2 and Ca2+ entry ratio curves, defined by six parameters, reflecting different Ca2+ ion fluxes. We report that threshold stimulation of GPVI or PAR, with a variable contribution of secondary mediators, induces a maximal Ca2+ entry ratio of 3-7. Strikingly, in combination with Ca2+-ATPase inhibition by thapsigargin, the maximal Ca2+ entry ratio increased to 400 (GPVI) or 40 (PAR), pointing to a strong receptor-dependent enhancement of store-operated Ca2+ entry. By pharmacological blockage of specific Ca2+ channels in platelets, we found that, regardless of GPVI or PAR stimulation, the Ca2+ entry ratio was strongest affected by inhibition of ORAI1 (2-APB, Synta66) > Na+/Ca2+ exchange (NCE) > P2X1 (only initial). In contrast, inhibition of TRPC6, Piezo1/2 or STIM1 was without effect. Together, these data reveal ORAI1 and NCE as dominating Ca2+ carriers regulating GPVI- and PAR-induced Ca2+ entry in human platelets.

Item Type:Article
Divisions:Interdisciplinary centres and themes > Institute for Cardiovascular and Metabolic Research (ICMR)
Life Sciences > School of Biological Sciences > Biomedical Sciences
ID Code:111544
Uncontrolled Keywords:ORAI1, platelet, sodium-calcium exchange, STIM1, store-regulated calcium entry

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