[33] Measurement of platelet cytoplasmic ionized calcium concentration with aequorin and fluorescent indicators

Publisher Summary Two technical discoveries have enabled the direct measurement of [Ca 2+ ]i in intact platelets using the Ca 2+ -sensitive fluorophore Quin 2 and the Ca 2+ -sensitive photoprotein aequorin. In addition, a second generation of Ca 2+ -sensitive fluorophores—Fura 2 and Indo 1—has been developed in which signaling is much improved. This chapter compares the features of aequorin and Quin 2 as prototypes of their respective indicator classes and describes the newer fluorophores, their calibrations, and potential utilities. Quin 2 can be loaded into intact cells by reversibly masking its hydrophilic carboxylic acid functional groups with an acetoxymethyl group. Intracellular accumulation of Quin 2 depends upon the extracellular concentration of the lipophilic form to which the cell is exposed. The fact that aequorin has a biologic source suggests that its Ca 2+ -response characteristics are ideal for intracellular applications.