Background
Dual-antiplatelet therapy with aspirin and clopidogrel is the cornerstone of treatment for patients with cardiovascular disease [
1]. Low response to antiplatelet therapy, characterized as high platelet reactivity (HPR), has been recognized as correlating with adverse events after acute coronary syndromes and percutaneous coronary intervention [
2,
3]. The increased prevalence of low platelet inhibition following clopidogrel treatment and twofold to fourfold increased risk of cardiovascular disease were found in diabetes mellitus (DM) patients compared with non-DM patients [
4‐
9]. Furthermore, platelet dysfunction in DM patients contributes to stent thrombosis and adverse events following percutaneous coronary intervention [
10]. The mechanisms leading to the HPR of DM patients are not fully elucidated. Marin F, et al. demonstrated that single nucleotide polymorphisms (SNPs) were able to explain the variability in antiplatelet agents inefficacy [
11]. Whether this may explain the heterogeneity of dual-antiplatelet therapy response in type 2 diabetes mellitus (T2DM) patients remains unexplored.
Several factors including up-regulation of platelet-signaling pathways have been involved in this interindividual response heterogeneity. Insulin inhibits platelet reactivity in the healthy individuals but not T2DM patients [
4,
12]. T2DM patients have a loss of responsiveness to insulin that leads to increased platelet reactivity and reduced response to antiplatelet agents [
11]. The loss of insulin signaling to Ca
2+ regulating mechanisms in DM platelets was accompanied by abnormal signaling initiated by insulin receptor substrate-1 (IRS-1). IRS-1, as a ligand of insulin receptor tyrosine kinase, is central to the insulin signal transduction pathway [
13]. Studies by Ferreira et al. suggested that insulin may cause platelet inhibition by activating IRS-1, which initiates the association with the Giα subunit coupled to the P2Y
12 receptor [
4]. Angiolillo et al. reported that the C allele of rs956115 in IRS-1 was associated with a hyperreactive platelet phenotype in Caucasian T2DM patients [
14]. However, the associations are not reproduced across other ethnic populations. Whether there is other SNP in IRS-1 related to HPR in coronary artery disease (CAD) and T2DM patients remains to be elucidated.
The aim of this study was to investigate the relationship between the IRS-1 polymorphism and hyperreactive platelet phenotype in CAD patients with T2DM.
Discussion
In healthy subjects, platelets are inhibited by insulin leading to reduced Ca
2+ mobilization and aggregate formation [
8]. Studies have demonstrated that platelet hyperactivity in T2DM is likely to be caused by a defect in the mechanisms through which insulin interferes with signaling by the P2Y
12 receptor. After binding with its receptor, insulin activates IRS-1 through tyrosine phosphorylation, which initiates the association with the Giα-subunit. Then, Giα activity is inhibited and impairs suppression of adenylyl cyclase through P2Y
12. Patients with T2DM are at particularly risk for restenosis and repeat the revascularization procedures because of the increased platelet activation and the reduced platelet inhibition by antiplatelet agents [
22‐
25].
This study suggested that IRS-1 polymorphism has an effect on platelet response to dual-antiplatelet therapy in CAD patients with T2DM. The eight SNPs of IRS-1 gene were examined to study the association between the polymorphisms and the hyperreactive platelet phenotype. We found that the G allele of IRS-1 rs13431554 polymorphism was significant associated with high platelet activity. The variant is located in the 3′UTR region of IRS-1, which is more important for mRNA stability, localization, and translational efficiency.
It has been reported that the binding affinity between miRNA and its target mRNA may be changed by SNP located at miRNA target sites [
26]. SNP variant may lead to the degradation of mRNA and the inhibition of mRNA translation into protein [
27]. Based on our bioinformatics analysis using the microRNA.org database [
28], has-miR-23 and has-miR-130 which binding to the sequence surrounding the variant site were identified. These bioinformatics forecasts indicated that rs13431554 may affect the binding affinity between IRS-1 mRNA and miRNAs, which may alter the expression of IRS-1 gene. It is reasonable to suggest that rs13431554 might influence miRNA biogenesis and function, also contribute to platelet activity. Further functional evaluations are needed to confirm this hypothesis.
The heterogeneity of antiplatelet drug efficacy is known to vary according to the genetic background. Cytochrome P450 (CYP) polymorphism correlates with the diminished antiplatelet efficacy of clopidogrel and the high risk for adverse cardiovascular events following stent implantation [
29‐
32]. The impact of CYP2C19 polymorphism on platelet reactivity to clopidogrel seems to be more significant in non-DM patients compared with DM patients [
33]. Precise clinical phenotypes and functional characterizations of gene variants are currently lacking. An early study reported that T2DM patients with the C allele of the rs956115 marker of the IRS-1 gene have a hyperreactive platelet phenotype and increased risk of major adverse cardiac events [
14]. However, this result was not observed in our study. Possible explanation for these divergent results may lie in the ethnic differences in genetic backgrounds. The rs956115 polymorphism is located in the 5′region of the IRS-1 gene which doesn’t affect amino acid coding and protein function. The rs13431554 polymorphism is located in in the 3′region of the IRS-1 gene, and may be more important to the function of IRS-1. We found that the outcomes were special to DM patients because there was no significant association between IRS-1 polymorphism and platelet activity in non-DM cohort. We did not find the association between the haplotype LD blocks and high platelet activity phenotypes in our study.
Glycemic control has an effect on platelet reactivity in diabetic patients. Previous study showed that a reduction in HbA1c level was associated with a reduction in blood thrombogenicity. Grzegorz Gajos et al. demonstrated that fasting glycemia <4.5 mmol/L was associated with enhanced thrombin formation in T2DM patients, especially when strict glycemic control was achieved (HbA1c <6.0 %) [
34]. Patients in our study had got a good glycemic control and limited variability in HbA1C levels, so the level of HbA1C didn’t affect our study results.
More strategies are being studied to overcome increased platelet reactivity in DM patients. Previous study showed that using a loading dose of clopidogrel rather than small daily doses was not sufficient for adequately overcoming increased platelet reactivity in DM patients, highlighting the need for more effective anti-platelet drugs for such patients [
35]. And, novel P2Y12 receptor blockers such as prasugrel and ticagrelor were found that they were superiors in terms of pharmacodynamics and pharmacokinetic effects than clopidogrel [
36‐
38]. Recent randomized studies also demonstrated that ticagrelor was superior to prasugrel for reducing platelet reactivity in subjects with diabetes [
39].
This is the first study to evaluate the association between IRS-1 polymorphism and high on-treatment platelet reactivity in a population of Chinese CAD patients with DM. Identification of susceptible genes that contribute to the declining platelet response may facilitate prediction, prevention, and development of improved antiplatelet treatments.
Study limitations
There is a lack of standardization identification of optimal cutoff values for defining the HPR [
40]. Standardized definitions of defining individual responsiveness to clopidogrel should be used in further experiments. We assessed platelet reactivity in vitro, that can’t fully represent platelet phenotype in circulating condition. The mechanisms of the rs13431554 variant contributing to the poor response to clopidogrel in T2DM patients remain speculative. Further functional evaluations with larger sample sizes are needed to confirm our findings. Different geographical and ethnic backgrounds of the study participants may affect the results of any association study. Therefore, more patients of different geographical areas and ethnicities should be included to confirm our conclusions in the further studies.
Authors’ contributions
All authors have made substantial contributions. ZD performed the statistical analysis and drafted the manuscript. ZD, LD carried out the molecular genetic studies. ZD, LT, CW contributed to data acquisition. HY, ZX, YC conceived of the study, and participated in its design and critically revised the manuscript. All authors read and approved the final manuscript.