Figure 1.
2MeSAMP and Cangrelor inhibit platelet aggregation and secretion through P2Y12-dependent mechanism.
(A-B) Washed platelets from wild-type mice (A) or P2Y12 deficient mice (B) were pre-incubated with PGI2 (1 µM) or forskolin (10 µM) at 37°C for 5 min, and added with AYPGKF 250 µM to induce ATP release and aggregation. (C–D) Washed platelets (3×108/ml) from P2Y12 deficient mice and littermate wild-type controls were pre-incubated with Cangrelor (1 µM) (Can) or 2MeSAMP (10 µM) (2Me) at 37°C for 5 min, and added with AYPGKF 60 µM (C) or 250 µM (D) to induce ATP release and aggregation. Data shown are representative of three independent experiments.
Figure 2.
2MeSAMP and Cangrelor failed to stimulate cAMP production.
(A) Washed human platelets were incubated with 2MeSAMP (2Me), Cangrelor (Can), or forskolin (Forsk) at 37°C for 5 min. The reactions were stopped by the addition of an equal volume of 12% (w/v) trichloroacetic acid. cAMP concentrations were determined by using a cAMP immunoassay kit. Statistical differences were examined by Student t test. Data are mean ± SD. *P<0.005 versus platelets in the absence of antagonists and forskolin. (B) Washed platelets from P2Y12 deficient mice or wild-type controls were pre-incubated with 2MeSAMP, Cangrelor, or forskolin for 5 min. The reactions were stopped by the addition of an equal volume of 12% (w/v) trichloroacetic acid. cAMP concentrations were determined by using a cAMP immunoassay kit. Statistical differences were examined by Student t test. Data are mean ± SD. *P<0.001 versus platelets of same genotype in the absence of antagonists and forskolin. (C) Washed platelets from wild-type mice were pre-incubated with 2MeSAMP, Cangrelor, or forskolin for 5 min. cAMP concentrations were determined by HPLC electrospray ionization tandem mass spectrometry as described under Experimental Procedures. Statistical differences were examined by Student t test. Data are mean ± SD. *P<0.001 versus platelets in the absence of antagonists and forskolin.
Figure 3.
2MeSAMP and Cangrelor failed to stimulate VASP phosphorylation in platelets.
(A) Washed human platelets were incubated with 2MeSAMP (2Me), Cangrelor (Can), or forskolin (Forsk) at 37°C for 5 min. The reactions were stopped by adding equal volume of 2×SDS sample buffer. Phosphorylation of VASP was detected by Western blotting with mouse monoclonal antibodies specifically recognizing the phosphorylated VASP residues Ser239. (B–C) Washed platelets from wild-type (B) or P2Y12 deficient mice (C) were pre-incubated with DMSO, forskolin (10 µM), 2MeSAMP (10 µM), or Cangrelor (1 µM) for 5 min. Reactions were stopped by adding equal volume of 2×SDS sample buffer. Phosphorylation of VASP was detected by Western blotting with mouse monoclonal antibodies specifically recognizing the phosphorylated VASP residues Ser157 or Ser239.
Figure 4.
The PKA activators, but not 2MeSAMP and Cangrelor, inhibited Ca2+ mobilization in P2Y12 deficient platelets.
Washed platelets from P2Y12 deficient mice were labeled with 12.5 µM Fura-2/AM/0.2% Pluronic F-127 and resuspended in Tyrode’s solution at 3×108/ml. Platelets were preincubated with Cangrelor (1 µM), 2MeSAMP (10 µM), forskolin (10 µM), or PGI2 (1 µM), and then stimulated with AYPGKF (500 µM). Changes in the intracellular Ca2+ levels 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 (A). Summarized data from three experiments are shown (B). Statistical differences were examined by Student t test. Data are mean ± SD. *P<0.001 versus DMSO.
Figure 5.
The PKA activators, but not 2MeSAMP and Cangrelor, inhibited Akt phosphorylation and Rap1b activation in P2Y12 deficient platelets.
(A-B) Washed platelets from P2Y12 deficient mice were pre-incubated with 2MeSAMP (10 µM) (2Me), Cangrelor (1 µM) (Can), or forskolin (10 µM) (Forsk) at 37°C for 5 min, and then stimulated with thrombin (0.25 U/ml) (A) or AYPGKF (500 µM) (B). Akt phosphorylation was detected by Western blotting with a rabbit monoclonal antibody specifically recognizing the phosphorylated Akt residue Ser473 or Thr308. A mouse monoclonal antibody against β-actin (Sigma-Aldrich) was used to verify equal loading. (C–D) Washed platelets from P2Y12 deficient mice were pre-incubated with 2MeSAMP (10 µM) (2Me), Cangrelor (1 µM) (Can), or forskolin (10 µM) (Forsk) at 37°C for 5 min, and then stimulated with thrombin (0.25 U/ml) (C) or AYPGKF (500 µM) (D). GTP-bound Rap1 was precipitated with GST-RalGDS RBD bound to glutathione-agarose beads and detected by Western blot.
Figure 6.
Cangrelor inhibited thrombus formation in wild-type but not P2Y12 deficient mice.
(A–D) FeCl3-induced carotid artery injury was performed and time to occlusive thrombus formation recorded as described under Experimental Procedures. Wild-type (A and B) or P2Y12 deficient mice (C and D) were injected with saline (A and C) or Cangrelor (B and D) 5 min prior to the carotid artery injury. (E) Mice were calculated by time length of forming occlusive thrombi (<10 min, 10–30 min, >30 min). Statistical differences were examined by Fisher exact test.