Monitoring the intracellular store Ca2+ concentration in agonist‐stimulated,intact human platelets by using Fluo‐5N

Summary. Background: Most Ca²⁺ signaling research in platelets has relied solely on monitoring the cytosolic Ca²⁺ concentration ([Ca²⁺]_cyt). Changes in [Ca²⁺]_cyt constitute the net effect of Ca²⁺ fluxes into the cytosol across the plasma membrane (PM) and from intracellular stores, and Ca²⁺ sequestration into the stores and Ca²⁺ removal across the PM. This makes interpretation of the effects of pharmacologic or genetic interventions on Ca²⁺ signaling difficult and subject to error. Objectives: To validate the use of the low‐affinity Ca²⁺ indicator Fluo‐5N to monitor the concentration of Ca²⁺ in the intracellular stores ([Ca²⁺]_st) of human platelets as a first step in developing assays for a systems‐level analysis of platelet Ca²⁺ signaling. Methods: Fluo‐5N‐loaded and Fura‐2‐loaded human platelets were used to observe the effects of agonist stimulation and other manipulations on [Ca²⁺]_cyt and [Ca²⁺]_st. Results: Fluo‐5N fluorescence changed appropriately in response to compounds that induce passive depletion of intracellular Ca²⁺ stores and to physiologic agonists. Ca²⁺ reuptake inhibitors and blockers of Ca²⁺ release channels had the expected effects on Fura‐2 and Fluo‐5N fluorescence. Agonist‐evoked Ca²⁺ release was reversed by Ca²⁺ addition to the medium, and required intact Ca²⁺ reuptake mechanisms. Store refilling was observed in the presence of sarcoplasmic/endoplasmic reticulum Ca²⁺‐ATPase (SERCA) inhibitors and ionomycin, suggesting the presence of a non‐SERCA Ca²⁺ reuptake mechanism. Evidence for a role for Ca²⁺‐induced Ca²⁺ release in agonist‐evoked responses was obtained. Conclusions: Our data provide a validation of the use of Fluo‐5N as a method for monitoring changes in [Ca²⁺]_st in human platelets.