Aspirin is a cost-effective and widely prescribed antiplatelet agent for primary and secondary prevention of ischemic stroke, worldwide 29. Even though aspirin is beneficial for the secondary prevention of cerebrovascular and cardiovascular disease, new thromboembolic events are common in individuals using aspirin. This might be due to the phenomenon of platelet reactivity that persists after aspirin administration, making them non-responsive to the treatment 30. The molecular mechanism of on-aspirin platelet reactivity is complex, poorly understood, and most likely multifactorial 31. Despite the challenges, research on the platelet response to aspirin therapy in various cardiovascular and cerebrovascular diseases is ongoing 32333435. Our study is one such effort, in which we assessed the influence of genetic factors on on-aspirin platelet reactivity in ischemic stroke patients.
Non-compliance with the treatment regime appears to be one of the more common causes of aspirin non-response [17][18]. A large percentage of people whose platelets showed no aspirin effect (57%) acknowledge not taking their prescription aspirin [19].. Hence, we thought it would be appropriate to measure the serum levels of salicylate to ensure patient compliance.. We found that 63 (21.5%) patients had serum salicylate levels below the therapeutic level. These patients were excluded from our analysis.
In our study, based on the ‘ADP + AA-combined’ criteria we have found that 13.91% of ischemic stroke patients had high on-aspirin platelet reactivity categorizing them as aspirin Non-Responders and an additional 33% were Semi-Responders. We grouped the 'Non-Responders' and 'Semi-Responders', and formed a new group named aspirin 'Inadequate-Responders’ because our major goal was to identify all patients with on-aspirin platelet reactivity.
Out of seven genes selected, the polymorphisms in COX1, COX2, GPIIb/IIIa, P2RY1, PEAR1, and ITGB3 represent the genes of the platelet aggregation pathway and UGT1A6 is an aspirin metabolizing gene. We found that the ‘T’ allele of P2RY1 (rs1371097 C > T) polymorphism of platelet aggregation pathway was significantly associated with on-aspirin platelet reactivity, and increased the risk of inadequate response to aspirin by 3.46 times, after adjusting for the covariates like age, gender, smoking, tobacco, alcohol, hypertension and diabetes. In a previous study of Chinese ischemic stroke patients, the P2RY1 (rs1371097 C > T) was found to be associated with aspirin non-responsiveness with the ‘ADP + AA-combined’ classification 4. In another study the interaction of P2RY1 (rs1371097) with TXA2R and GPIIIa was linked to higher platelet activation and associated with an increased risk of carotid stenosis in patients with ischemic stroke 36. The minor allele of P2RY1 (rs1371097) was linked to TXB2 levels < 1.0 ng/mL assessed as a function of platelet reactivity while on aspirin therapy in patients with coronary artery disease 6. Consistent with the previous findings, in our study the distribution of the ‘T’ allele of P2RY1 (rs1371097) was significantly higher in ischemic stroke patients with on-aspirin platelet reactivity, probably making them inadequately responsive to aspirin therapy.
A previous study has shown that patients with coronary artery disease who were homozygous for the P2RY1 (rs701265) minor allele had a 8.5-fold increased risk of platelet aggregation in response to arachidonic acid 6. Further, the minor allele of P2RY1 (rs701265) polymorphism was associated with increased platelet aggregation in response to ADP in healthy individuals 37. Similarly, like the P2RY1 (rs1371097 C > T), the P2RY1 (rs701265 A > G) polymorphism was also linked to TXB2 levels < 1.0 ng/mL, assessed as a function of platelet reactivity while on aspirin therapy, in patients with coronary artery disease 6. However, in our study we did not find any significant difference among the groups in P2RY1 (rs701265 A > G) variant.
P2RY1 is a purinergic receptor that binds to the nucleotides such as ATP and ADP which are present outside the cell. Previously, haemostatic abnormalities were associated with mutations in this receptor 383940. Mutations in the P2RY1 gene were associated with a 3-fold increase of aspirin non-responsiveness in coronary artery disease 11. Our study shows the involvement of the P2RY1 (rs1371097 C > T) polymorphisms with the risk of inadequate-response in ischemic stroke patients. Hence it can be concluded that variations in the P2RY1 gene might have a significant role in determining the on-aspirin platelet reactivity leading to decreased responsiveness to aspirin therapy in our population.
Surprisingly, candidate gene polymorphisms studied in other genes, including COX1, COX2, PEAR1, GPIIb/IIIa, and ITGB3 of platelet aggregation pathway, were not significantly associated with the phenomenon of on-aspirin platelet reactivity in our population 41 42. Even the polymorphism in aspirin metabolizing gene UGT1A6 failed to show a significant difference between the groups in our cohort. Previously, one study has shown that genotypes in the UGT1A6 gene can modify the protective effects of aspirin 10.
In conclusion, as a step towards pharmacogenetics-based personalised medicine, our study is one of the large studies to explore the influence of genetic predispositions on platelet response to aspirin therapy in ischemic stroke. Most importantly, the phenomenon of on-aspirin platelet reactivity was found to be influenced by the polymorphisms in the P2RY1 gene. Our study adds to the complexity of the phenomenon of aspirin non response, and in the future, larger number of genetic markers for on-aspirin platelet reactivity could be identified employing high-throughput Next Generation Sequencing (NGS) techniques. Further, studying genes in alternative pathways of platelet aggregation might provide further insights about on-aspirin platelet reactivity leading to aspirin non-response.