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

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.