Homocysteine promotes GPCR-coupled Gi and Gq signaling in platelets through N-homocysteinylation. (A) Western blots showing that ADP- (5 μM), U46619- (350 nM), and thrombin-induced (0.01 U/mL) AKT and PKD2 phosphorylation in washed platelets was significantly augmented by preincubation with HTL (30 μM). The data are from 3 independent experiments (n = 3). (B) Western blots showing that ADP-induced (5 μM) AKT and PKD2 phosphorylation in washed platelets was significantly augmented by preincubation with Hcy (100 μM), whereas the Hcy-induced increase in phosphorylation was largely suppressed by co-incubation with AHT (2 mM) and NAC (2 mM). The data are from 3 independent experiments (n = 3). (C-D) The enzyme-linked immunosorbent assay results show a significant decrease in the cAMP level in platelets incubated with Hcy (100 μM) or HTL (30 μM) upon ADP and prostaglandin I2 (PGI2) stimulation (C) or thrombin and PGI2 stimulation (D), whereas the effect of Hcy was reversed by AHT (2 mM) and NAC (2 mM). The data are from 3 independent experiments (n = 3). (E-F) The Ca²⁺ concentration was measured by assaying changes in the fluorescence intensity, and quantification of transient Ca²⁺ levels was represented by the relative area under the curve. The results show a significant increase in Ca²⁺ concentration in platelets incubated with Hcy (100 μM) or HTL (30 μM) upon ADP (E) and thrombin (F) stimulation, whereas the effect of Hcy was reversed by AHT (2 mM). The data are from 3 independent experiments (n = 3). ∗P < .05; ∗∗P < .01; ∗∗∗P < .001. The bars represent means ± SEMs. Data from panel A were analyzed using the 2-tailed Student t test. The data from panels B-F were analyzed using the1-way ANOVA.