Homocysteine promotes platelet activation and thrombosis through N-homocysteinylation. (A) Western blots showing that AHT (2 mM) and NAC (2 mM) inhibited the increased cellular N-homocysteinylation caused by Hcy (100 μM). The data are from 4 independent experiments (n = 4). (B) Western blot analysis revealed that incubation with HTL (30 μM) largely improved the N-homocysteinylation level in platelets. The data are from 4 independent experiments (n = 4). (C) Turbidimetric aggregometry revealed that the ADP- (5 μM), U46619- (350 nM), and thrombin-induced (0.01 U/mL) aggregation of washed platelets was significantly enhanced after preincubation with Hcy (100 μM), whereas coincubation with AHT (2 mM) and NAC (2 mM) largely diminished the Hcy-induced improvement in platelet aggregation. The data are from 5 independent experiments (n = 5). (D) Turbidimetric aggregometry revealed that ADP- (5 μM), U46619- (350 nM), and thrombin-induced (0.01 U/mL) aggregation of washed platelets was significantly enhanced by preincubation with HTL (30 μM). The data are from 5 or 6 independent experiments (n = 5/6). (E) Enzyme-linked immunosorbent assay results showing that the level of TXB2, which is a TXA2 metabolite, was greatly increased in washed platelets treated with Hcy (100 μM), whereas this increase was inhibited by AHT (2 mM) and NAC (2 mM). The data are from 6 independent experiments (n = 6). (F) Occlusion time for male Apoe–/– mice subjected to FeCl3-induced carotid artery thrombosis and pretreated with phosphate-buffered saline, Hcy (100 mg/kg), or AHT (500 mg/kg) (n = 6 mice per group). (G) Tail bleeding time and total amount of blood loss in male Apoe–/– mice pretreated with phosphate-buffered saline, Hcy (100 mg/kg), or AHT (500 mg/kg) (n = 6 mice per group). (H) Representative OCTA image of FeCl3-induced mesenteric artery thrombosis at different time points for each group. The white arrows indicate the blood vessel where the clot occurred and red arrows indicate the ischemic area by thrombosis. (I) Vessel diameter indices of the different groups (n = 3 mice per group). Vessel diameter refers to the diameter length of the vessel, and the relative vessel diameter index refers to the degree of vascular blockage; the higher the index value, the narrower the blood vessel. (J-L) Turbidimetric aggregometer show that ADP- (J), U46619- (K), and thrombin (L)-induced platelet aggregation rate of washed platelets derived from HHcy patients is significantly higher than platelets from healthy individuals. The data are from 3 independent experiments (n = 3). ∗P < .05; ∗∗P < .01; ∗∗∗P < .001. The bars represent the means ± standard errors of the mean (SEMs). The data from panels A, C, and E-I were analyzed using the 1-way ANOVA. The data from panels B, D, and J-L were analyzed using the a 2-tailed Student t test.

Homocysteine promotes platelet activation and thrombosis through N-homocysteinylation. (A) Western blots showing that AHT (2 mM) and NAC (2 mM) inhibited the increased cellular N-homocysteinylation caused by Hcy (100 μM). The data are from 4 independent experiments (n = 4). (B) Western blot analysis revealed that incubation with HTL (30 μM) largely improved the N-homocysteinylation level in platelets. The data are from 4 independent experiments (n = 4). (C) Turbidimetric aggregometry revealed that the ADP- (5 μM), U46619- (350 nM), and thrombin-induced (0.01 U/mL) aggregation of washed platelets was significantly enhanced after preincubation with Hcy (100 μM), whereas coincubation with AHT (2 mM) and NAC (2 mM) largely diminished the Hcy-induced improvement in platelet aggregation. The data are from 5 independent experiments (n = 5). (D) Turbidimetric aggregometry revealed that ADP- (5 μM), U46619- (350 nM), and thrombin-induced (0.01 U/mL) aggregation of washed platelets was significantly enhanced by preincubation with HTL (30 μM). The data are from 5 or 6 independent experiments (n = 5/6). (E) Enzyme-linked immunosorbent assay results showing that the level of TXB2, which is a TXA2 metabolite, was greatly increased in washed platelets treated with Hcy (100 μM), whereas this increase was inhibited by AHT (2 mM) and NAC (2 mM). The data are from 6 independent experiments (n = 6). (F) Occlusion time for male Apoe–/– mice subjected to FeCl3-induced carotid artery thrombosis and pretreated with phosphate-buffered saline, Hcy (100 mg/kg), or AHT (500 mg/kg) (n = 6 mice per group). (G) Tail bleeding time and total amount of blood loss in male Apoe–/– mice pretreated with phosphate-buffered saline, Hcy (100 mg/kg), or AHT (500 mg/kg) (n = 6 mice per group). (H) Representative OCTA image of FeCl3-induced mesenteric artery thrombosis at different time points for each group. The white arrows indicate the blood vessel where the clot occurred and red arrows indicate the ischemic area by thrombosis. (I) Vessel diameter indices of the different groups (n = 3 mice per group). Vessel diameter refers to the diameter length of the vessel, and the relative vessel diameter index refers to the degree of vascular blockage; the higher the index value, the narrower the blood vessel. (J-L) Turbidimetric aggregometer show that ADP- (J), U46619- (K), and thrombin (L)-induced platelet aggregation rate of washed platelets derived from HHcy patients is significantly higher than platelets from healthy individuals. The data are from 3 independent experiments (n = 3). ∗P < .05; ∗∗P < .01; ∗∗∗P < .001. The bars represent the means ± standard errors of the mean (SEMs). The data from panels A, C, and E-I were analyzed using the 1-way ANOVA. The data from panels B, D, and J-L were analyzed using the a 2-tailed Student t test.

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