A G(i) -independent mechanism mediating Akt phosphorylation in platelets

Abstract

Background: The serine-threonine kinase Akt plays an important role in regulating platelet activation. Stimulation of platelets with various agonists results in Akt activation as indicated by Akt phosphorylation. However, the mechanisms of Akt phosphorylation in platelets are not completely understood.

Objectives and methods: We used P2Y₁ knockout mice to address the role of P2Y₁₂ in Akt phosphorylation in response to thrombin receptors in platelets.

Results: Thrombin or the PAR4 thrombin receptor peptide AYPGKF at high concentrations stimulated substantial phosphorylation of Akt residues Thr³⁰⁸ and Ser⁴⁷³ in P2Y₁₂-deficient platelets. AYPGKF-induced Akt phosphorylation is enhanced by expression of recombinant human PAR4 cDNA in Chinese hamster ovary (CHO) cells. P2Y₁₂ -independent Akt phosphorylation was not inhibited by integrin inhibitor peptide RGDS or integrin β₃ deficiency. Akt phosphorylation induced by thrombin or AYPGKF in P2Y₁₂-deficient platelets was inhibited by the calcium chelator dimethyl-BAPTA, the Src family kinase inhibitor PP2, and PI3K inhibitors, respectively.

Conclusions: Our results reveal a novel P2Y₁₂-independent signaling pathway mediating Akt phosphorylation in response to thrombin receptors.

Figure 1. Akt phosphorylation in response to thrombin or AYPGKF in P2Y₁₂ deficient platelets

(A–E), Washed platelets from P2Y₁₂ deficient mice and littermate wild type controls were incubated with increasing concentrations of thrombin (Throm) (A), ADP (C), or AYPGKF (AY) (D) at 37°C in a platelet aggregometer for 5 min, and solubilized with SDS-PAGE sample buffer. Phosphorylation of Akt was detected by Western blotting with rabbit monoclonal antibodies specifically recognizing the phosphorylated Akt residues Ser⁴⁷³ or Thr³⁰⁸. A rabbit polyclonal antibody against total Akt was used to verify equal loading. Immunoblotting results as in experiments described in panels (A) and (D) were scanned and quantitated. Values were normalized with respect to wild type platelets stimulated with lowest concentration of thrombin (B) or AYPGKF (E) for each immunoblot and are expressed as relative phosphorylation (mean ± SD from 3 separate experiments). (F–G) Washed platelets from P2Y₁₂ deficient mice and littermate wild type controls were incubated with AR-C69931MX (1 μM) for 5 min, and then added with thrombin (F), or AYPGKF (G) at 37°C in a platelet aggregometer for 5 min, and solubilized with SDS-PAGE sample buffer. (H), Washed platelets (1 × 10⁸/ml) from P2Y₁₂ deficient or wild type mice were pre-incubated with thrombin (0.25 U/ml) or AYPGKF (0.5 mM) at 37 °C for 5 min. These platelets were treated for 5 min with forskolin (10 μM). cAMP concentrations were determined by using a cAMP immunoassay kit.

Figure 2. Akt phosphorylation in recombinant CHO cells and the role of integrin outside-in signaling in P2Y₁₂-independent Akt phosphorylation

(A), CHO cells were transfected with human PAR4 cDNA or pCDNA 3.1/Zeocin (−) (Vector). Expression of PAR4 was shown by Western blotting using an anti-human PAR4 polyclonal antibody. (B), Vector- or PAR4-transfected CHO cells were incubated with buffer or AYPGKF (0.5 mM) at 37 °C for 2 or 5 min. Phosphorylation of Akt was detected by Western blot. (C) CHO cells were transfected with human P2Y₁₂ cDNA or pCDNA 3.1/Zeocin (−) (Vector). CHO cells expressing vector or P2Y₁₂ were incubated with ADP (10 μM) for 5 min. The cells were then incubated with forskolin (10 μM) for 5 min. cAMP concentrations were determined by using a cAMP immunoassay kit. (D), Vector- or P2Y₁₂-transfected CHO cells were incubated with buffer or ADP (10 μM) at 37 °C for 5 min. Phosphorylation of Akt was detected by Western blot. (E), Washed platelets from P2Y₁₂ deficient mice or wild type controls were pre-incubated with RGDS peptide (2 mM) for 5 min, and then stimulated with thrombin (0.25 U/ml) or AYPGKF (0.5 mM) at 37°C in a platelet aggregometer for 5 min. (F), Washed platelets from β₃ deficient mice or wild type controls were pre-incubated with 2MeSAMP or AR-C69931MX, and then stimulated with thrombin at 37°C in a platelet aggregometer for 5 min.

Figure 3. The role of Ca²⁺ and PKC in P2Y₁₂-independent Akt phosphorylation induced by thrombin or AYPGKF

(A–B), Washed platelets from P2Y₁₂ deficient mice were pre-incubated with dimethyl-BAPTA (10 μM) or DMSO for 5 min and followed by treated with 0.25 U/ml thrombin (A) or 0.5 mM AYPGKF (B) for 5 min at 37°C in a platelet aggregometer. (C), Washed platelets from wild type or P2Y₁₂ deficient mice were stimulated with increasing concentrations of A23187. (D–E), Washed platelets from P2Y₁₂ deficient mice were pre-incubated with PKC inhibitors Ro-31-8220 (1 μM), Gö6976 (1 μM) for 5 min and followed by treatment with AYPGKF (D) or PMA (100 ng/ml) (E) for 5 min at 37°C in a platelet aggregometer.

Figure 4. TXA2-induced Akt phosphorylation and calcium mobilization

(A–B), Washed platelets from P2Y₁₂ deficient mice and littermate wild type controls were incubated with increasing concentrations of thrombin or U46619 at 37°C in a platelet aggregometer for 5 min (A). Immunoblotting results were scanned and quantitated. Values were normalized with respect to wild type platelets stimulated with lowest concentration of U46619 (B) for each immunoblot and are expressed as relative phosphorylation (mean ± SD from 3 separate experiments). (C), Washed platelets from P2Y₁₂ deficient or wild type mice were labeled with 12.5 μM Fura-2/AM/0.2% Pluronic F-127 and resuspended in Tyrode’s solution. Platelets were then stimulated with increasing concentration of U46619. Changes in the intracellular free calcium level were measured every 2 s and expressed as a ratio of fluorescence (FL) detected at 509 nm emission with an excitation wavelength of 340 nm and 380 nm. Statistical data from three experiments are shown. (D–F), Washed platelets from P2Y₁₂ deficient mice were labeled with 12.5 μM Fura-2/AM/0.2% Pluronic F-127. Platelets were then stimulated with increasing concentration of U46619 (E) or thrombin (F). Statistical data from three experiments are shown in (D), and representative data are shown (E and F).

Figure 5. The role of PI3K and Src family kinase in P2Y₁₂-independent Akt phosphorylation in response to thrombin, AYPGKF, or A23187

(A–C), Washed platelets from P2Y₁₂ deficient mice were pre-incubated with LY294002 (20 μM) or wortmannin (100 nM), stimulated with AYPGKF (0.5 mM) (A), thrombin (0.25 U/ml) (B), or A23187 (0.5 μM) (C). (D–F), Washed platelets from P2Y₁₂ deficient mice were pre-incubated with PP2 (10 μM) or DMSO, stimulated with thrombin (D), AYPGKF (E), or A23187 (F). G and H, Washed platelets from P2Y12 deficient mice were pre-incubated with BAPTA (10 μM) or DMSO, stimulated thrombin (G), or AYPGKF (H). Src phosphorylation was detected by Western blotting with a rabbit monoclonal antibody specifically recognizing the phosphorylated Src residue Tyr⁴¹⁶.