Impact of Grin1 deletion on intracellular Ca²⁺ responses in mouse platelets. (A-H [i panels]) Line graphs showing mean ± SEM relative levels of intracellular Ca²⁺ in platelets loaded with fura-2-AM (15 μM) determined from a normalized fluorescent 340/380 nm ratio (F₃₄₀/F₃₈₀) recorded over 535 seconds under the conditions indicated. Platelets were prepared and tested in the presence of platelet inhibitors (100 μM of aspirin, 0.32 U/mL of apyrase, and 50 μg/mL of Intergrilin [eptifibatide]). Line graphs start with showing the relative level of intracellular Ca²⁺ in platelets recorded in the presence of 2 mM of Ca²⁺ (30-second baseline), followed by the measurements recorded after the addition of EGTA (2 mM; dashed line) and then after the readdition of extracellular Ca²⁺ (2 mM; solid line). Downward arrow indicates a time point when a modulator was added: NMDA (100 μM) (A-B), thapsigargin (TG; 2 μM) C,E,G), or ADP (5 μM) (D,F,H). The first wave of the Ca²⁺ response, recorded after the addition of a modulator, corresponds to the release of intracellular Ca²⁺ stores, and the second wave, recorded after the readdition of extracellular Ca²⁺, corresponds to SOCE. In panels E, F, and G, “+ NMDA” refers to a 10-minute preincubation of platelets with NMDA (100 μM) performed before the commencement of the recordings; panels C and D were recorded without preincubation with NMDA; panels G and H show the knockout platelet controls for both conditions. WT platelet controls are shown in supplemental Figure 10. (A-H [ii panels]) Corresponding scatterplots showing changes in intracellular Ca²⁺ store release and SOCE quantified from an area under the curve measured using Prism software for the first and second waves of the Ca²⁺ response, respectively. Five independent experiments were performed for each condition, with 3 technical replicates. Data points are mean ± SEM. Mouse genotype is indicated. Statistical significance is shown (unpaired Student t test). ns, not significant.