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. Epub 2023 Apr 8.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Blood Platelets* / metabolism
  • Calcium / metabolism
  • Calcium Channels* / metabolism
  • Calcium Chloride / pharmacology
  • Calcium Signaling
  • Egtazic Acid / metabolism
  • Humans
  • Ion Channels / metabolism
  • ORAI1 Protein / metabolism
  • Protein-Tyrosine Kinases / metabolism
  • Protein-Tyrosine Kinases / pharmacology
  • Receptors, G-Protein-Coupled / metabolism
  • Stromal Interaction Molecule 1 / metabolism


  • Calcium Channels
  • Protein-Tyrosine Kinases
  • Calcium Chloride
  • Egtazic Acid
  • Receptors, G-Protein-Coupled
  • Calcium
  • Stromal Interaction Molecule 1
  • ORAI1 Protein
  • PIEZO1 protein, human
  • Ion Channels