Effect of SERCA3 deletion or pharmacological inhibition on Ca²⁺ mobilization, Ca²⁺ influx, aggregation, and secretion in washed platelets. (A) Ca²⁺ mobilization was assessed in unstirred control (WT, black tracings) and SERCA3^−/− (gray tracings) platelets preincubated with the cytosolic Ca²⁺ fluorescent probe Oregon Green BAPTA-AM after stimulation with 40 mU/mL thrombin (“Thr”) by flow cytometry in conditions of no external Ca²⁺ (1 mM EGTA). Ca²⁺ influx was assessed after 4 minutes by addition of 1 mM CaCl₂ (“Ca²⁺”). Global Ca²⁺ signaling was also assessed by addition of 1 mM Ca²⁺ together with thrombin (“Thr + Ca²⁺”) (right tracing). Data are expressed as nM Ca²⁺, as calculated from calibration experiments (see supplemental Methods). (B) Ca²⁺ mobilization was assessed in the same conditions as in panel A, but in the presence of apyrase (5 U/mL) (“+ Apy”) or ADP (1 µM) (“+ ADP”). (C) Ca²⁺ mobilization by 40 mU/mL thrombin was assessed after preincubation with the SERCA3-specific inhibitor tBHQ (C; 10 µM) or with Tg (D) at a concentration affecting only SERCA2b (200 nM; see supplemental Figure 5A). (E) Maximal Ca²⁺ mobilization and Ca²⁺ influx from experiments in panel A (stimulation with 40 mU/mL thrombin in the presence of 100 µM EGTA and Ca²⁺ influx after CaCl₂ [300 µM] addition) and in panel B (same as in panel A, but in the presence of 5 U/mL apyrase [Thr + Apy] or of 10 µM ADP [Thr + ADP]). Values were calculated after subtraction of unstimulated Ca²⁺ level (ΔCa²⁺ nM). Data presented are means ± SEM, n = 3, using 1-way ANOVA followed by Tukey’s multiple comparison test: ns, not significant; **P < .01; ***P < .001. (F) Washed control platelets were preincubated with dimethyl sulfoxide (control) or tBHQ 10 µM (tBHQ) or tBHQ and ADP (10 µM each) for 4 minutes prior to addition of 40 mU/mL thrombin (Thr). (G) Washed control (WT, black bars) or SERCA3^−/− (gray bars) platelets were preincubated with either buffer alone “-”, tBHQ (10 µM), or Tg (200 nM), and then either buffer (“0”), thrombin 40 mU/mL, or thrombin and 10 µM ADP (“+ ADP”) and incubated further for 3 minutes. ATP secretion was then measured in supernatants. Data presented are means ± SEM, n = 3, using 1-way ANOVA followed by Tukey’s multiple comparison test: ns, not significant; ***P < .001.