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

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

  • Cell Calcium. 2023 Jun:112:102738. doi: 10.1016/j.ceca.2023.102738.
Hilaire Yam Fung Cheung 1,  Jinmi Zou 2,  Chukiat Tantiwong 3,  Delia I Fernandez 4,  Jingnan Huang 5,  Robert Ahrends 6,  Mark Roest 7,  Rachel Cavill 8,  Jon Gibbins 9,  Johan W M Heemskerk 10
Affiliations

Affiliations

  • 1 Department of Biochemistry, Cardiovascular Research Institute Maastricht (CARIM), Maastricht University, Maastricht, The Netherlands; Leibniz-Institut für Analytische Wissenschaften-ISAS-e.V, Dortmund, Germany; Institute of Cardiovascular Sciences, College of Medical and Dental Sciences, University of Birmingham, Birmingham, United Kingdom.
  • 2 Department of Biochemistry, Cardiovascular Research Institute Maastricht (CARIM), Maastricht University, Maastricht, The Netherlands; Synapse Research Institute Maastricht, 6217 KD Maastricht, The Netherlands.
  • 3 Department of Biochemistry, Cardiovascular Research Institute Maastricht (CARIM), Maastricht University, Maastricht, The Netherlands; Institute for Cardiovascular and Metabolic Research (ICMR), School of Biological Sciences, University of Reading, Reading, United Kingdom.
  • 4 Department of Biochemistry, Cardiovascular Research Institute Maastricht (CARIM), Maastricht University, Maastricht, The Netherlands; Center for Research in Molecular Medicine and Chronic Diseases (CIMUS), Universidade de Santiago de Compostela, Santiago de Compostela, 15706, Spain.
  • 5 Department of Biochemistry, Cardiovascular Research Institute Maastricht (CARIM), Maastricht University, Maastricht, The Netherlands; Leibniz-Institut für Analytische Wissenschaften-ISAS-e.V, Dortmund, Germany; Center for Research in Molecular Medicine and Chronic Diseases (CIMUS), Universidade de Santiago de Compostela, Santiago de Compostela, 15706, Spain.
  • 6 Leibniz-Institut für Analytische Wissenschaften-ISAS-e.V, Dortmund, Germany; Dept. of Analytical Chemistry, University of Vienna, Vienna, Austria.
  • 7 Synapse Research Institute Maastricht, 6217 KD Maastricht, The Netherlands.
  • 8 Department of Advanced Computing Sciences, Maastricht University, 6229 ER Maastricht, The Netherlands.
  • 9 Institute for Cardiovascular and Metabolic Research (ICMR), School of Biological Sciences, University of Reading, Reading, United Kingdom.
  • 10 Department of Biochemistry, Cardiovascular Research Institute Maastricht (CARIM), Maastricht University, Maastricht, The Netherlands; Synapse Research Institute Maastricht, 6217 KD Maastricht, The Netherlands. Electronic address: [email protected].
PMID: 37060673 DOI: 10.1016/j.ceca.2023.102738
Abstract

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.

Keywords

ORAI1; Platelet; STIM1; Sodium-calcium exchange; Store-regulated calcium entry.

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