Figure 4.
Homocysteine promotes platelet activation through β-arrestin2. (A) GloSensor cAMP inhibition assay results showing that P2Y12-coupled Gi activity in WT and β-arrestin1/2 knockout HEK293T cell lines was significantly amplified when β-arrestin2 was knocked out. The data are from 4 independent experiments (n = 4). The area under the curve from ADP stimulation to the end of the experiment was used to calculate the desensitization of the cAMP response to P2Y12. (B) The enzyme-linked immunosorbent assay results show a significant decrease in the cAMP level in β-arrestin2 knockout platelets after ADP stimulation. The data are from 4 independent experiments (n = 4). (C-E) The turbidimetric aggregometry results showed that (C) ADP- (5 μM), (D) U46619- (350 nM), and (E) thrombin-induced (0.01 U/mL) platelet aggregation largely decreased in washed platelets from Arrb2–/– mice when compared with those from WT mice, whereas the Hcy- (100 μM) and HTL-dependent (30 μM) increases in platelet aggregation significantly diminished in washed platelets from Arrb2–/– mice when compared with those from WT mice. The data are from 5 independent experiments (n = 5). ∗P < .05; ∗∗P < .01; ∗∗∗P < .001. The bars represent means ± SEMs. The data from panels A and C-E were analyzed using a 1-way ANOVA. The data from panel B were analyzed using a 2-tailed Student t test. β-arr1, β-arrestin1; β-arr2, β-arrestin2.

Homocysteine promotes platelet activation through β-arrestin2. (A) GloSensor cAMP inhibition assay results showing that P2Y12-coupled Gi activity in WT and β-arrestin1/2 knockout HEK293T cell lines was significantly amplified when β-arrestin2 was knocked out. The data are from 4 independent experiments (n = 4). The area under the curve from ADP stimulation to the end of the experiment was used to calculate the desensitization of the cAMP response to P2Y12. (B) The enzyme-linked immunosorbent assay results show a significant decrease in the cAMP level in β-arrestin2 knockout platelets after ADP stimulation. The data are from 4 independent experiments (n = 4). (C-E) The turbidimetric aggregometry results showed that (C) ADP- (5 μM), (D) U46619- (350 nM), and (E) thrombin-induced (0.01 U/mL) platelet aggregation largely decreased in washed platelets from Arrb2–/– mice when compared with those from WT mice, whereas the Hcy- (100 μM) and HTL-dependent (30 μM) increases in platelet aggregation significantly diminished in washed platelets from Arrb2–/– mice when compared with those from WT mice. The data are from 5 independent experiments (n = 5). ∗P < .05; ∗∗P < .01; ∗∗∗P < .001. The bars represent means ± SEMs. The data from panels A and C-E were analyzed using a 1-way ANOVA. The data from panel B were analyzed using a 2-tailed Student t test. β-arr1, β-arrestin1; β-arr2, β-arrestin2.

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