ADP receptor P2Y12 is the capstone of the cross-talk between Ca2+ mobilization pathways dependent on Ca2+ ATPases sarcoplasmic/endoplasmic reticulum type 3 and type 2b in platelets

Figures

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  Figure 1. Effect of P2-class purinergic receptor type Y subtype 1 (P2Y1) and P2-class purinergic receptor type Y subtype 12 (P2Y12) antagonists on Ca2+ mobilization in WT and Serca3-/- mouse platelets. (A) Washed WT (blue tracings) or Serca3-/- (orange tracings) platelets resuspended in the presence of EGTA (100 μmol/L) to prevent Ca2+ influx were preincubated with the Ca2+ fluorophore BAPTA for 30 minutes, then stimulated, in absence of external Ca2+ (100 μmol/L EGTA), with Thr (40 mU/mL) for 5 minutes, and fluorescence induced by Ca2+ mobilization assessed by cell sorting. The ratio of fluorescence of activated vs resting platelets (set as 1) is expressed as a function of time in seconds. Then, platelets were preincubated for 10 minutes with (B) the inhibitor of P2Y1 (MRS2179 [MRS], 100 μmol/L) or (C) the inhibitor of P2Y12 (ARC69931MX [ARC], 20 μmol/L) or (D) both, or (E) the ADP scavenger apyrase (5 U/mL) before stimulation with Thr (40 mU/mL). (F) Area under curves for 1 minute was assessed in arbitrary units for WT (blue) and Serca3-/- (orange) platelets (n = 3). Data are expressed as mean ± SEM, and probability P calculated by the 2 way anova using the Tukey's multiple comparison…

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  Figure 2. Inhibition of the P2-class purinergic receptor type Y subtype 1 (P2Y1) and P2-class purinergic receptor type Y subtype 12 (P2Y12) ADP receptors and effect on secretion of WT and Serca3-/- platelets. Mouse WT (blue) or Serca3-/- (orange) platelets were washed, resuspended at 3 x 108 platelets/mL in Tyrode buffer without Ca2+, and preincubated with buffer (-) or with the antagonists (+) MRS2179 (MRS) (P2Y1 specific, 100 μmol/L), ARC69931MX (ARC) (P2Y12 specific, 20 μmol/L) or both for 10 minutes. Platelets were then stimulated with Thr (40 mU/mL) in absence of stirring, for 5 seconds (A), 10 seconds (B), 1 minute (C), or 3 minutes (D) before stopping secretion by addition of phenylalanyl-L-prolyl-L-arginine chloromethyl ketone (PPACK) (100 μmol/L) and immediately chilling at 0°C for 5 minutes, recovering supernatants by centrifugation and assessing ATP concentrations (see Section 2). Data are expressed after subtraction of ATP values obtained in absence of thrombin stimulation. In other experiments, Thr (40 mU/mL) (E), Thr (40 mU/mL), and ADP (10 μmol/L) (F) were added (+) or not (-) and incubated for 3 minutes before stopping activation. Data are means ± SEM, and probability P…

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  Figure 3. Potentiation of sarco-endoplasmic reticulum calcium ATPase type 2b (SERCA2b)-dependent secretion by sarco-endoplasmic reticulum calcium ATPase type 3 (SERCA3)-dependent ADP is altered in P2-class purinergic receptor type Y subtype 12 (P2Y12)- but not in P2-class purinergic receptor type Y subtype 1 (P2Y1)-defective mouse platelets. (A) Washed WT (blue tracings), P2Y1-/- (gray tracings), or P2Y12-/- (green tracings) platelets resuspended in the presence of EGTA (100 μmol/L) to prevent Ca2+ influx were preincubated with the Ca2+ fluorophore Oregon green 488 BAPTA for 30 minutes, then stimulated with Thr (40 mU/mL) for 5 minutes, and fluorescence induced by Ca2+ mobilization assessed by flow cytometry. The ratio of fluorescence of activated vs resting platelets (set as 1) is expressed as a function of time in seconds. (B) Area under curves for 1 minute was assessed in arbitrary units (A.U.) for WT (blue), P2Y1-/- (gray), or P2Y12-/- (green) platelets (n = 7). (C) Quantification of Ca2+ mobilization in the absence (-) or the presence of the nicotinic acid adenosine dinucleotide phosphate (NAADP) antagonist Ned-19 (10 μmol/L) is shown as area under curves for 2 minutes, assessed in…

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  Figure 4. P2-class purinergic receptor type Y subtype 12 (P2Y12), not P2-class purinergic receptor type Y subtype 1 (P2Y1), is involved in Ca2+ mobilization elicited by Thrombin and in late secretion. Washed control human platelets resuspended in the presence of EGTA (100 μmol/L) to prevent Ca2+ influx were preincubated with the Ca2+ fluorophore Oregon green 488 BAPTA for 30 minutes, then stimulated with thrombin (40 mU/mL) for 5 minutes, and fluorescence induced by Ca2+ mobilization assessed by flow cytometry. The ratio of fluorescence of activated vs resting platelets (set as 1) is expressed as a function of time in seconds. Before activation, platelets were preincubated for 10 minutes with buffer (A), the P2Y12 antagonist ARC69931MX (ARC) (20 μmol/L, B), the nucleoside phosphate scavenger apyrase (5 U/mL, C), apyrase and ARC (D), the nicotinic acid adenosine dinucleotide phosphate antagonist Ned-19 (10 μmol/L) (E), Ned-19 and ARC (F), and apyrase and Ned19 (G). Area under curves for 1 minute was assessed in arbitrary units (A.U.) after stimulation with Thrombin in the absence (-) or the presence (+) of ARC, apyrase or Ned-19 (n = 3). (H) Tracings illustrated in the figure are…

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  Figure 5. Illustration of the links between sarco-endoplasmic reticulum calcium ATPase type 3 (SERCA3)- and sarco-endoplasmic reticulum calcium ATPase type 2b (SERCA2b)-dependent pathway in platelet activation. Thrombin stimulation at low concentration activates 2 signaling pathways, the first one leading to the production of nicotinic acid adenosine dinucleotide phosphate (NAADP) promoting SERCA3 stores mobilization and the second one activating phospholipase C and production of 1,4,5, inositol trisphosphate (IP3), releasing Ca2+ from SERCA2b stores. The first pathway results in a rapid secretion of ADP (red arrow) that potentiates the second SERCA2b-dependent pathway via the ADP receptor P2-class purinergic receptor type Y subtype 12 (P2Y12) (not P2-class purinergic receptor type Y subtype 1 [P2Y1]) [18]. Note that P2Y12 cannot activate Phospholipase C directly but probably indirectly by TxA2 and its receptor TPα, which activate phospholipase C and the release of IP3, as suggested by the fact that the potentiation step is indomethacin-sensitive [18]. Note that ADP released via this pathway (blue arrow) may also activate P2Y12.

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  Supplementary Figure S1.

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  Supplementary Figure S2.