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

• Published in: Cell calcium (Edinburgh) 2023-06, Vol.112, p.102738-102738, Article 102738
• Main Authors: Cheung, Hilaire Yam Fung, Zou, Jinmi, Tantiwong, Chukiat, Fernandez, Delia I., Huang, Jingnan, Ahrends, Robert, Roest, Mark, Cavill, Rachel, Gibbins, Jon, Heemskerk, Johan W.M.
• Format: Article
• Language: English
• Subjects: ORAI1; Platelet; Sodium-calcium exchange; STIM1; Store-regulated calcium entry
• ISSN: 0143-4160; 1532-1991
• DOI: 10.1016/j.ceca.2023.102738

•In a well-plate based assay, a systematic comparison was made of cytosolic Ca2+ traces in platelets in the presence or absence of extracellular CaCl2, allowing the construction of Ca2+ entry ratio curves.•The Ca2+ entry ratios depended on the agonist strength and type, and were enforced by secondary mediators.•Upon blockage of SERCAs, Ca2+ entry ratios increased to up to 300, i.e. more with collagen-receptor than with thrombin-receptor stimulation.•Pharmacological evidence pointed to ORAI1 and sodium-calcium exchange as dominate Ca2+ entry carriers, independent of agonist type, with an initial role of P2X1 channels. 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) > 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 Ca2+ carriers regulating GPVI- and PAR-induced Ca2+ entry in human platelets. [Display omitted]