Contribution of the P2Y12 receptor-mediated pathway to platelet hyperreactivity in hypercholesterolemia

Abstract

Background: In hypercholesterolemia, platelets demonstrate increased reactivity and promote the development of cardiovascular disease.

Objective: This study was carried out to investigate the contribution of the ADP receptor P2Y12-mediated pathway to platelet hyperreactivity due to hypercholesterolemia.

Methods: Low-density lipoprotein receptor-deficient mice and C57Bl/6 wild-type mice were fed on normal chow and high-fat (Western or Paigen) diets for 8 weeks to generate differently elevated cholesterol levels. P2Y12 receptor-induced functional responses via G(i) signaling were studied ex vivo when washed murine platelets were activated by 2MeSADP and PAR4 agonist AYPGKF in the presence and absence of indomethacin. Platelet aggregation and secretion, α(IIb)β(3) receptor activation and the phosphorylation of extracellular signal-regulated protein kinase (ERK) and Akt were analyzed.

Results: Plasma cholesterol levels ranged from 69 ± 10 to 1011 ± 185 mg dL(-1) depending on diet in mice with different genotypes. Agonist-dependent aggregation, dense and α-granule secretion and JON/A binding were gradually and significantly (P < 0.05) augmented at low agonist concentration in correlation with the increasing plasma cholesterol levels, even if elevated thromboxane generation was blocked. These functional responses were induced via increased levels of G(i) -mediated ERK and Akt phosphorylation in hypercholesterolemic mice vs. normocholesterolemic animals. In addition, blocking of the P2Y12 receptor by AR-C69931MX (Cangrelor) resulted in strongly reduced platelet aggregation in mice with elevated cholesterol levels compared with normocholesterolemic controls.

Conclusions: These data revealed that the P2Y12 receptor pathway was substantially involved in platelet hyperreactivity associated with mild and severe hypercholesterolemia.

Fig. 1

2MeSADP and AYPGKF-induced platelet aggregation in WT and LDLR^−/− mice on chow (c), Western (w) and Paigen (p) diets in response to a low and a sub-maximal concentration of agonists. Isolated murine platelets were activated by 30 nM and 100 nM 2MeSADP (A & B) and 100 and 250 μM AYPGKF (C & D) for 3.5 min at 37°C in stirring condition without indomethacin pre-treatment. The tracings are representative of results from 3 independent experiments. The maximal extent (E & G) and the slope (F & H) of aggregation tracings were quantified and depicted in graph. ‘Filled’ line represents data from aggregation by sub-maximal agonist concentration, while ‘slash’ line means at low concentration of agonist (2MeSADP: E & F; AYPGKF: G & H). *P < 0.05 versus normocholesterolemic mice by using Student’s t test. Mean ± SEM.

Fig. 2

Comparison of the levels of dense and α-granule secretion as well as α_IIbβ₃ receptor activation downstream of P2Y12receptor pathway in mice on chow (c), Western (w) and Paigen (p) diets. (A) Simultaneous dense granule secretion was measured during aggregation and expressed as ATP secretion (nmol/10⁸ platelets). (B) Isolated platelets from LDLR^−/− and WT mice were stimulated with 2MeSADP (30 nM) and PAR4 agonist AYPGKF (100 μM) for 15 min at 37°C in non-stirring condition in the presence of FITC-labeled anti-mouse-P-selectin (CD62) antibody to detect α-granule release. Reactions were terminated by fixing platelets with (1%) PFA and then analyzed by flow cytometry. (C) We also tested the JON/A binding to measure the level of activatedα_IIbβ₃ receptors in the same samples. *P < 0.05 versus normocholesterolemic mice by using Student’s t test. The bars represent the results of 3 independent experiments. Mean ± SEM.

Fig. 3

The level of TxB₂ generation in WT and LDLR−/− mice on chow (c), Western (w) and Paigen (p) diets in response to a low and a sub-maximal concentration of P2Y12 receptor agonists. Isolated murine platelets were stimulated by 2MeSADP and AYPGKF in an aggregometer. The stimulation was performed for 3.5 min and the reaction was stopped by quickly freezing the sample in a dry ice-methanol bath. Samples were processed in ELISA kit to measure the level of TXB₂. *P < 0.05 versus normocholesterolemic mice by using Student’s t test. The data represent mean ± SEM of 3 independent experiments.

Fig. 4

2MeSADP and AYPGKF-induced aggregation of platelets after preincubation with indomethacin in WT and LDLR^−/− mice on chow (c), Western (w) and Paigen (p) diets. Isolated murine platelets were pre-treated with 10 μM indomethacin for 10 min and activated by 30 nM 2MeSADP (A) and 100 μM AYPGKF (B) for 3.5 min at 37°C in stirring condition. The maximal extent (C & E) and the slope (D & F) of aggregation tracings were quantified and depicted in graph. ‘Slash’ line depicts values in the presence of TXA₂ production, and ‘filled’ line represents data when TXA₂ generation was blocked (2MeSADP: C & D; AYPGKF: E & F). *P < 0.05 versus normocholesterolemic mice by using Student’s t test. (G) Washed murine platelets were also stimulated in response to 100 μM AYPGKF in the presence of 10 μM indomethacin for 10 min and 100 nM AR-C69931MX added prior to platelet aggregation. The tracings are representative of results from 3 independent experiments. Mean ± SEM.

Fig. 5

ERK and Akt phosphorylation in response to 2MeSADP and AYPGKF in normo-and hypercholesterolemic mice. WT and LDLR^−/− mice with differently high cholesterol levels were pre-treated with 10 μM indomethacin for 10 min and then stimulated with 30 nM and 100 nM 2MeSADP (A & B) as well as 100 μM and 250 μM AYPGKF (C & D) at 37°C in stirring condition for 1 min for activating ERK, and for 3 min in order to induce Akt phosphorylation. The reaction was stopped by the addition of 3x SDS sample buffer. The phosphorylation level of proteins was analyzed by Western blot analysis with anti-phospho(Thr202/Tyr204)-ERK and anti-phospho(Ser473)-Akt antibodies. The total amount of proteins was measured with anti-ERK and anti-Akt antibodies as lane loading. Phosphorylation data were quantified by densitometry and analyzed in fold increase over control (mean ± SEM). Results are representative of 3 independent experiments.