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

In platelets, elevated cytosolic Ca is a crucial second messenger, involved in most functional responses, including shape change, secretion, aggregation and procoagulant activity. The platelet Ca response consists of Ca mobilization from endoplasmic reticulum stores, complemented with store-operated or receptor-operated Ca entry pathways. Several channels can contribute to the Ca 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] measurements in the presence of EGTA or CaCl. Per agonist condition, this resulted in sets of EGTA, CaCl and Ca entry ratio curves, defined by six parameters, reflecting different Ca ion fluxes. We report that threshold stimulation of GPVI or PAR, with a variable contribution of secondary mediators, induces a maximal Ca entry ratio of 3-7. Strikingly, in combination with Ca-ATPase inhibition by thapsigargin, the maximal Ca entry ratio increased to 400 (GPVI) or 40 (PAR), pointing to a strong receptor-dependent enhancement of store-operated Ca entry. By pharmacological blockage of specific Ca channels in platelets, we found that, regardless of GPVI or PAR stimulation, the Ca entry ratio was strongest affected by inhibition of ORAI1 (2-APB, Synta66) > Na/Ca exchange (NCE) > P2× (only initial). In contrast, inhibition of TRPC6, Piezo1/2 or STIM1 was without effect. Together, these data reveal ORAI1 and NCE as dominating Ca carriers regulating GPVI- and PAR-induced Ca entry in human platelets.