High-throughput assessment identifying major platelet Ca 2+ entry pathways via tyrosine kinase-linked and G protein-coupled receptors.

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.
Competing Interests: Declaration of Competing Interest MR is an employee of Synapse Research Institute. JWMH is a scientific advisor of Synapse Research Institute. The other authors report no relevant conflicts of interest.
(Copyright © 2023. Published by Elsevier Ltd.)