The impact of P2Y12 promoter DNA methylation on the recurrence of ischemic events in Chinese patients with ischemic cerebrovascular disease

Abstract

The primary mechanism of clopidogrel resistance is still unclear. We aimed to investigate whether the methylation status of the P2Y12 promoter has effects on platelet function and clinical ischemic events. Patients with ischemic cerebrovascular disease were enrolled into our study. Venous blood samples were drawn for thrombelastograpy (TEG) and active metabolite assay. Patients were divided into a case- or control-group based on the occurrence of ischemic events during a one year follow-up. Two TEG parameters between the case and control groups were statistically significant [ADP inhibition rate (ADP%): P = 0.018; ADP-induced platelet-fibrin clot strength (MA_ADP): P = 0.030]. The concentrations of clopidogrel active metabolite had no significant difference (P = 0.281). Sixteen CpG dinucleotides on P2Y12 promoter were tested. Three CpG sites (CpG11 and CpG12 + 13) showed lower methylation status, which correlated with a strong association with increased risk of clinical events. Changes of MA_ADP and ADP% were also associated with methylation levels of CpG 11 and CpG 12 + 13. Hypomethylation of the P2Y12 promoter is associated with a higher platelet reactivity and increased risk of ischemic events in our patients. Methylation analysis of peripheral blood samples might be a novel molecular marker to help early identification of patients at high risk for clinical ischemic events.

Figure 1. Heatmap generated from TEG parameters, MA, ADP%, MA_ADP and AA% values reflecting the parameter’s difference between cases and controls.

Parameter values (ranged from 0–100) are displayed by a gradient color. The high data values with bright red tones and low data values with light green tones. Mixed tones depict values in-between. MA: maximum amplitude; MA_ADP: ADP-induced platelet-fibrin clot strength; ADP%: ADP inhibition rate; AA%: AA inhibition rate.

Figure 2. Heatmap generated from CpG methylation levels.

Methylation levels are indicated by a gradient map representing the hypo- to hypermethylated status (from green to red respectively) of different CpG sites. CpG 11 and CpG 12 + 13 were significantly hypermethylated in cases as compared to controls. Several methylation levels of CpG sites cannot be tested in our study and are presented using a black color.

Figure 3. The correlation of clopidogrel active metabolite concentration and platelet activity (ADP% and MA_ADP).

The relationship was investigated by linear regression model and the lines are regression lines. R: Correlation coefficient.

Figure 4. The relationship between methylation level (CpG11 and CpG12 + 13) and platelet function index (ADP% and MA_ADP).

The impact of methylation status on platelet function was assessed by linear regression model. The lines in the plots represent the regression lines. R: Correlation coefficient.