A Synthetic Evaluation Of Genetic and Pharmacological Resistance To Clopidogrel and On Treatment Residual Platelet Aggregation In Patients With Atherothrombosis

Clopidogrel is cornerstone treatment for atherothrombotic patients. The incidence of recurrent thrombotic events raises up to 10% of patients on treatment with clopidogrel. Intestinal absorption via the glycoproteine ABCB1, bioactivation by the cytochrome P450 (CYP) system, binding to P2Y12 receptor and inhibition of platelet aggregation are critical steps for the efficiency of clopidogrel treatment. Prospective independent clinical trials showed that the risk of recurrent thrombotic complications is associated with the presence of common polymorphisms of genes encoding ABCB1 (rs1045642C>T) or the CYP2C19 isoenzyme (*2_rs4244285G>A, *3_rs4986893G>A, *4_rs28399504A>G) or the inefficiency of clopidogrel to decrease intracellular VASP or the presence of high residual platelet reactivity (HRPR).

To evaluate the available pharmacogenetic, pharmacological and functional assays for the diagnosis of the resistance to clopidogrel treatment on the same population of patients with atherothrombosis.

A cohort of 94 out-patients with atherothrombosis receiving clopidogrel treatment (75 mg o.d) for more than 3 months were included. Polymorphisms ABCB1, CYP *2, *3, *4, *17 were assessed with rPCR method. VASP was measured with flow cytometry assay and poor response (PR) to the treatment was diagnosed if VASP index was > 50. Light Transmission Platelet Aggregation (LTPA) triggered by ADP (5 ìM and 10 ìM) was assessed on citrated platelet rich plasma. Multielectrode aggregometry (WBMEA) was performed on citrated whole blood using Multiplate instrument and ADP reagent (ADP-test®). The cut-off point for the diagnosis of HRPR on clopidogrel treatment was the lower value of the maximum aggregation or the AUC observed in the upper quintile of ADP triggered LTPA or WBMEA measurements.

The frequency of heterozygous for CYP*2, *3, *4 and ABCB 1 polymorphisms was 28%, 0%, 1%, and 51% respectively. The frequency of the respective homozygocity was 2%, 0%, 0%, and 20%. Patients heterozygous for the CYP*2 G>A polymorphism showed significantly higher VASP index as compared to the wild type but LTPA and WBMEA were not significantly different. VASP index, LTPA and WBMEA were not significantly different in patients heterozygous or homozygous for the ABCB1 C>T polymorphism as compared to the wild type. The VASP index was significantly correlated with the LTPA (r=0.44, p<0.0001) and WBMEA (r=0.376, p=0.001). In addition LTPA was significantly correlated with the WBMEA (r=0.46, p<0.0001). With the VASP assay 42% of patients were diagnosed as PR to clopidogrel. HRPR was found in 12% and 28% of patients tested with LTPA ADP 5 ìM and 10 ìM respectively and in 19% of patients tested with WBMEA. Among patients with HRPR the frequency of PR with the VASP assay was 60% when they were tested with LTPA ADP 10 ìM, 70% when they were tested with LTPA ADP 5 ìM and 76% when they were tested with WBMEA. Less than 50% of patients with HRPR had at least one of the studied polymorphisms. One patient homozygous for CYP*2 and ABCB1 was PR with the VASP assay and had HRPR with the WBMEA.

The present study performed on a cohort of patients with atherothrombosis on treatment with clopidogrel demonstrates that the genotype of CYP and ABCB1 polymorphisms have limited influence on the pharmacological response to the treatment and on the residual platelet reactivity. Among the studied polymorphisms only the CYP*2G>A was related with increase of VASP index and WBMEA. Pharmacological response to clopidogrel is correlated with the level of residual platelet reactivity. However 25-30% of patients with HRPR showed good pharmacological response to clopidogrel. The present study underlines the need to a synthetic use of bioassays for the evaluation of the risk of failure of clopidogrel treatment to prevent the recurrence of atherothrombosis. The need for the construction of a diagnostic algorithm including the genetic, pharmacological and functional assays is revealed by the present study.

No relevant conflicts of interest to declare.