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. 2019 May;369(2):182–187. doi: 10.1124/jpet.118.253583

Fig. 1.

Fig. 1.

Acetylcholine receptors regulate platelet activation. (A) Carbachol inhibits platelet activation. Human platelets were isolated and treated with phosphate-buffered saline or carbachol, stimulated with PBS or 10 μM TRAP, and analyzed for surface expression of P-selectin using flow cytometry. (n = 4 ± S.D., *P < 0.05 for TRAP vs. TRAP + carbachol). (B) Acetylcholine inhibits platelet activation. Human platelets were treated with PBS or ACh, stimulated with PBS or 10 μM TRAP, and analyzed as already described. (n = 4 ± S.D., *P < 0.05 for TRAP vs. TRAP + ACh). (C) Carbachol inhibits platelet activation over a range of TRAP doses. Platelets were stimulated with varying concentrations of TRAP and analyzed for surface expression of P-selectin as desccribed (n = 4 ± S.D., *P < 0.05 for the indicated concentration of TRAP vs. TRAP + carbachol). (D) Carbachol inhibits platelet activation by ADP. (E) Carbachol inhibits platelet activation by U46619. (F) Carbachol inhibits platelet activation by convulxin. For (D–G), isolated human platelets were treated with PBS or 10 nM carbachol, then stimulated with various agonists, and analyzed via flow cytometry (n = 4 ± S.D., *P < 0.05 for agonist vs. agonist + carbachol). (G) Carbachol inhibits platelet-dense granule release. Platelets were isolated and treated with 10 nM carbachol, 100 μM pyridostigmine bromide or 100 nM pancuronium bromide, and then stimulated with PBS or TRAP and analyzed for surface expression of P-selectin (n = 4 ± S.D. *P < 0.05 for TRAP vs. TRAP and indicated compound). (H) Carbachol inhibits GPIIbIIIa activation as measured by FITC-fibrinogen binding to platelets. Platelets were isolated and treated with 10 nM carbachol and then stimulated with the indicated concentrations of TRAP and analyzed for surface expression of P-selectin (n = 4 ± S.D., *P < 0.05 for TRAP vs. TRAP + carbachol). (I) Treatment with the nAChRα7-selective agonist PNU-282987 inhibits P-selectin exposure. Platelets were treated with PNU-282987 at the indicated concentrations, then stimulated with TRAP6, and analyzed for surface expression of p-selectin (*P < 0.05 for TRAP6 + vehicle vs. TRAP6 + indicated concentration of PNU). (J) PNU inhibits GPIIbIIIa activation. Platelets were treated with PNU-282987 at the indicated concentrations, then stimulated with TRAP6, and analyzed for activation of GPIIbIIIa as described (*P < 0.05 for TRAP6 + vehicle vs. TRAP6 + indicated concentration of PNU).