Selective Inhibition of P2Y₁ and P2Y₁₂ Receptor Signal Pathways in Platelet Aggregation in Transgenic Cell Lines and Rats by Potassium 2‐(1‐Hydroxypentyl)‐Benzoate, Puerarin and Salvianolic Acid B

Figures

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  FIGURE 1. Effects of dl‐PHPB, puerarin and salvianolic acid B on 2MeSADP‐induced rat platelet aggregation in vivo and in vitro. For in vivo assessment, rats were treated with dl‐PHPB (A), puerarin (C) and salvianolic acid B (E) or clopidogrel (10 mg/kg) by oral gavage. Rat blood was collected 1 h after orally gavage. The solvents were deionized water for dl‐PHPB and salvianolic acid B, and 0.5% CMC‐Na for puerarin. For in vitro assessment, rat blood was collected and rat platelets were pre‐incubated with dl‐PHPB (B), puerarin (D) and salvianolic acid B (F) for 5 min. The solvents were ddH2O for dl‐PHPB and salvianolic acid B, and DMSO for puerarin (The final concentration of DMSO was 0.1%). Platelet aggregation was induced by 0.1 μM 2‐MeSADP. The aggregation was assessed using a four‐channel aggregometer. n = 4 for (A–E) and n = 5 for (F), *p < 0.05, **p < 0.01 versus control or solvent.

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  FIGURE 2. Identification of protein expression of P2Y1 and P2Y12 transiently transfected in HEK293 cells. The expression of Flag in HEK293 cells was elevated after pcDNA‐P2Y1‐Flag (A) and pcDNA‐P2Y12‐Flag (B) were transfected. The expression of P2Y1 (C) and P2Y12 (D) in HEK293 cells were elevated after pcDNA‐P2Y1‐Flag and pcDNA‐P2Y12‐Flag were transfected. Typical immunofluorescence images after P2Y1 (E) and P2Y12 (F) transfection. Quantified results were normalized to β‐Actin expression. Values were expressed as percentages compared to the control group (set to 100%). n = 3, *p < 0.05, ** p < 0.01 versus control.

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  FIGURE 3. Identification of IP3, cAMP and [Ca2+]i measurements in HEK293 cells transfected with P2Y1 and P2Y12 pcDNA3.1, pcDNA3.1‐P2Y1, pcDNA3.1‐P2Y12 was transfected into HEK293 cells to measure the content of IP3, cAMP and [Ca2+]i in vitro. 0.3 μM P2Y1 antagonist MRS2179 or 0.5 μM P2Y12 antagonist ticagrelor before stimulated with 2MeSADP were used as positive drugs to verify the reliability of the measuements of IP3 (A), cAMP (B) and [Ca2+]i (C). IP3 and cAMP were determined by immunoassay. [Ca2+]i was determined by a flow cytometry. n = 5, *p < 0.05, **p < 0.01 versus control group, # p < 0.05, ## p < 0.01 versus 2MeSADP.

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  FIGURE 4. Effects of dl‐PHPB, puerarin and salvianolic acid B on 2‐MeSADP‐induced IP3 increase in HEK 293 cell transfected with P2Y1. The IP3 content was enhanced by 0.1 μM 2‐MeSADP and could be reversed by 0.5 μM MRS2179. dl‐PHPB (A), puerarin (B) and salvianolic acid B (C) were preincubated before stimulated with 2‐MeSADP. IP3 content was determined by immunoassay. n = 5, *p < 0.05, **p < 0.01 versus control group, # p < 0.05, ## p < 0.01 versus 2MeSADP.

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  FIGURE 5. Effects of dl‐PHPB, puerarin and salvianolic acid B on 2MeSADP‐induced cAMP decrease in HEK 293 cell transfected with P2Y12. The cAMP content was reduced by 0.1 μM 2MeSADP and could be reversed by 0.5 μM ticagrelor. dl‐PHPB (A), puerarin (B) and salvianolic acid B (C) were preincubated before stimulated with 2‐MeSADP. cAMP content was determined by immunoassay. n = 5, *p < 0.05, **p < 0.01 versus control group, # p < 0.05 versus 2MeSADP.

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  FIGURE 6. Effects of dl‐PHPB, puerarin and salvianolic acid B on 2MeSADP‐induced intracellular [Ca2+]i increase in HEK 293 cells transfected with P2Y1. The intracellular [Ca2+]i was increased by 0.1 μM 2MeSADP and could be reversed by 0.5 μM MRS2179. Dl‐PHPB (A), puerarin (C) and salvianolic acid B (E) were preincubated before stimulated with 2‐MeSADP. The intracellular [Ca2+]i was determined by a flow cytometry. Typical images of dl‐PHPB (B), puerarin (D) and salvianolic acid B (F) from flow cytometry were shown. n = 5, **p < 0.01 versus control group, ## p < 0.01 versus 2MeSADP.

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  FIGURE 7. The molecular docking of dl‐PHPB, puerarin and salvianolic acid B with P2Y1 and P2Y12 receptor. The computer molecular docking was shown that the bond energies of dl‐PHPB and puerarin with P2Y1 receptor were − 7.250 and − 6.468 kcal/mol, while the bond energy salvianolic acid B with P2Y12 receptor was −4.039 kcal/mol. (A, B) Two‐dimensional interaction map and three‐dimensional docking pose of dl‐PHPB with P2Y1 receptor. (C, D) Two‐dimensional interaction map and three‐dimensional docking pose of puerarin with P2Y1 receptor. (E, F) Two‐dimensional interaction map and three‐dimensional docking pose of salvianolic acid B with P2Y12 receptor.

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  FIGURE 8. Schematic diagram of the mechanisms of dl‐PHPB, puerarin and salvianolic acid B against platelet aggregation induced by 2‐MeSADP. Dl‐PHPB and puerarin inhibit the Gq‐PLC‐IP3‐Ca2+ signal transduction pathway, reduce intracellular Ca2+ and decrease platelet aggregation. However, salvianolic acid B as well as ticagrelor inhibit the Gi‐AC‐cAMP signal transduction pathway and enhance cAMP and decreases platelet aggregation. This figure was modulated on the basis of our former published study [11].