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A Genetic Polymorphism in P2RY1 Impacts Response to Clopidogrel in Cats with Hypertrophic Cardiomyopathy

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A Genetic Polymorphism in P2RY1 Impacts Response to Clopidogrel in Cats with Hypertrophic Cardiomyopathy UC Davis UC Davis Previously Published Works Title A genetic polymorphism in P2RY1 impacts response to clopidogrel in cats with hypertrophic cardiomyopathy. Permalink https://escholarship.org/uc/item/1sw4m9cb Journal Scientific reports, 11(1) ISSN 2045-2322 Authors Ueda, Yu Li, Ronald HL Nguyen, Nghi et al. Publication Date 2021-06-15 DOI 10.1038/s41598-021-91372-3 Peer reviewed eScholarship.org Powered by the California Digital Library University of California www.nature.com/scientificreports OPEN A genetic polymorphism in P2RY1 impacts response to clopidogrel in cats with hypertrophic cardiomyopathy Yu Ueda1, Ronald H. L. Li2, Nghi Nguyen2, Eric S. Ontiveros3, Samantha L. Kovacs3, Maureen S. Oldach2, Karen M. Vernau2, Michael H. Court4 & Joshua A. Stern3* Clopidogrel is converted to its active metabolite by cytochrome P450 isoenzymes and irreversibly inhibits platelet activation by antagonizing the adenosine-diphosphate (ADP) receptor. It is frequently used in cats with hypertrophic cardiomyopathy (HCM) to prevent thromboembolic complications. However, signifcant interpatient variability of the response to clopidogrel therapy has been suspected. In this study, we assessed the impact of single nucleotide polymorphisms (SNPs) within ADP receptor (P2RY1, P2RY12) and cytochrome P450 isoenzyme (CYP2C41) genes on platelet inhibition by clopidogrel administration in cats with HCM. Forty-nine cats completed the study, and blood samples were obtained before and after clopidogrel therapy to assess the degree of platelet inhibition based on fow cytometry and whole blood platelet aggregometry. Plasma concentrations of clopidogrel metabolites were measured after the last dose of clopidogrel. Whole blood platelet aggregometry revealed a signifcant reduction of platelet inhibition by clopidogrel in cats with the P2RY1:A236G and the P2RY12:V34I variants. The association with the P2RY1:A236G variant and clopidogrel resistance remained signifcant after adjustment for multiple comparisons. This study demonstrated that a genetic polymorphism in the P2RY1 gene altered response to clopidogrel therapy and suggests that clinicians may consider alternative or additional thromboprophylactic therapy in cats with the P2RY1:A236G variant. Arterial thromboembolism (ATE) is a common complication with high morbidity and mortality in cats with cardiomyopathy. Previous studies showed that 6–17% of cats with hypertrophic cardiomyopathy (HCM) or other cardiac diseases develop ATE with an associated mortality rate of 61–67%1–6. Clopidogrel is a commonly prescribed antiplatelet medication used in people as well as in veterinary patients. Clopidogrel, once metabolized to active metabolite by cytochrome P450 enzymes, irreversibly inhibits one of the platelet adenosine diphos- phate (ADP) receptors, P2Y12. Several large human clinical trials showed that clopidogrel signifcantly reduces the chance of developing thromboembolic diseases 7–9. In cats, one recent randomized controlled trial reported clopidogrel administration in cats with cardiogenic ATE signifcantly reduced the chance of developing recurrent thromboembolic events compared to cats receiving aspirin 10. However, despite broad usage of clopidogrel in both human and veterinary patients, signifcant interpatient variability of pharmacodynamic response has been reported. In humans, clopidogrel resistance, defned by decreased inhibition of ADP-induced platelet aggregation in response to clopidogrel, was reported in 5–30 percent of people11–13. Te aforementioned clinical trial in cats also showed frequent recurrence of ATE despite clopidogrel therapy and underscores the potential for variable response to this drug in clinical patients 10. In addition to these fndings, recent studies also reported interindi- vidual variability of platelet inhibition by clopidogrel assessed by platelet aggregometry and fow cytometry in cats with and without hypertrophic cardiomyopathy (HCM)14,15. Tere are various intrinsic and extrinsic causes of clopidogrel resistance. Genetic polymorphisms in both platelet ADP receptor genes (P2RY1, P2RY12) and the cytochrome P450 enzyme gene have been reported as 1Department of Clinical Sciences, College of Veterinary Medicine, North Carolina State University, Raleigh, NC, USA. 2Department of Veterinary Surgical and Radiological Sciences, School of Veterinary Medicine, University of California – Davis, Davis, CA, USA. 3Department of Medicine and Epidemiology, School of Veterinary Medicine, University of California – Davis, Davis, CA, USA. 4Department of Veterinary Clinical Sciences, College of Veterinary Medicine, Washington State University, Pullman, WA, USA. *email: [email protected] Scientifc Reports | (2021) 11:12522 | https://doi.org/10.1038/s41598-021-91372-3 1 Vol.:(0123456789) www.nature.com/scientificreports/ Genes Location Mutations Polymorphisms Distributions n/49 (%) HWE (Chi2) c/c 16/49 (32.7) P2RY1 C2.110,685,285 p.A236G c/g 25/49 (51.0) 0.11 g/g 8/49 (16.3) g/g 23/49 (46.9) P2RY12 C2.112,204,070 p.V34I g/a 22/49 (44.9) 0.16 a/a 4/49 (8.2) g/g 20/49 (40.9) CYP2C41 D2.54,712,168 p.H231R g/a 23/49 (46.9) 0.54 a/a 4/49 (8.2) Table 1. Frequencies of genetic polymorphisms in three targeted genes (P2RY1, P2RY12, and CYP2C41). HWE; Hardy–Weinberg equilibrium. inciting causes of clopidogrel resistance in people16–24. In cats, multiple non-synonymous single nucleotide polymorphisms (SNPs) in the P2RY1 (NCBI ID: 101090795) and P2RY12 genes (NCBI ID: 101094044) were identifed via whole genome sequencing and Sanger sequencing verifcation, and two non-synonymous SNPs, P2RY1:A236G and P2RY12:V34I, were found with relatively high minor allele frequencies in a population of cats with HCM 25. Te cytochrome P450 2C (CYP2C) genes, were reported 26,27. One recent study also reported that one of the variants in the feline CYP2C41 gene (CYP2C41 (NCBI ID: 101089396) was associated with increased clopidogrel active metabolite (CAM) concentration afer administration of a single dose of clopidogrel in healthy cats28. Tis fnding supports the contention that these SNPs could alter the response to clopidogrel in cats. It is, however, unknown if these variants in the P2RY1, P2RY12, and CYP2C41 genes were associated with signifcant interindividual variability in cats with HCM, especially afer long-term clopidogrel therapy. A better understanding of the impact of genetic polymorphisms on the inhibitory efects of clopidogrel could open doors for precision medicine and optimization of antithrombotic strategies in cats at risk of ATE. In this study, we hypothesized that genetic polymorphisms in the genes encoding the feline platelet ADP receptors, P2RY1:A236G and P2RY12:V34I and the feline cytochrome P450 2C enzyme, CYP2C41:H231R, are common and responsible for clopidogrel resistance in cats with HCM. To test this hypothesis, the frequen- cies of the non-synonymous SNPs in client-owned cats with HCM were determined. Next, pharmacodynamic response to clopidogrel was evaluated by fow cytometry and whole blood platelet aggregometry before and afer a 10–14 day course of clopidogrel therapy. Plasma concentrations of clopidogrel and clopidogrel metabolites including CAM were measured at the completion of the study period to determine if clopidogrel resistance were associated with altered metabolism of clopidogrel by cytochrome P450 enzymes. Te results were then compared in HCM cats with and without the nonsynonymous SNPs identifed in the targeted genes. Results Signalment and patient demographics. A total of 51 client-owned cats with HCM were enrolled and 49 cats completed the study. Of the 49 cats, 38 were Domestic Short Hair, 7 were Domestic Long Hair, 2 were Sphynx, 1 was Maine Coon, and 1 was Siamese. One cat was excluded from the study due to poor animal compli- ance resulting in the failure to administer clopidogrel at home. Another cat was excluded as there was no concen- tration of clopidogrel detected in plasma afer completion of the study, suggesting either poor animal compliance or a failure to follow the drug administration protocol. Te mean and standard deviation (SD) of age was 6.5 (± 4.5) years old with 35 castrated males and 14 spayed females. Te mean body weight (± SD) was 5.6 (± 1.4) kg. No adverse efects of clopidogrel administration were reported by the owners throughout the study period. Missense SNPs in P2RY1, P2RY12, and CYP2C41 genes. In the present study, the frequencies of the missense SNPs in the P2RY1, P2RY12, and CYP2C41 genes were successfully determined in all cats and the results are listed in Table 1. Te obtained genotype distributions did not deviate from Hardy–Weinberg equilib- rium (Table 1). Adenosine diphosphate-induced platelet aggregation is inhibited by clopidogrel ther- apy. Te mean area under the curve (AUC), maximum aggregation (AU), and aggregation velocity (AU/ min) before clopidogrel therapy were 140 ± 40.2 (AU*min), 225.3 ± 63.3 (AU), and 42.9 ± 14.2 (AU/min), respec- tively. Afer clopidogrel therapy, mean AUC, aggregation, and velocity were signifcantly reduced at 35.9 ± 15.2 (AU*min), 73.3 ± 28.9 (AU), 7.4 ± 2.3 (AU/min), respectively (Fig. 1). Te percent inhibition of AUC, maximum aggregation, and aggregation velocity were 73.6 (± 11.3) %, 66.6 (± 12.0) %, and 81.5 (± 7.8) %, respectively. Te Multiplate variables were all signifcantly decreased afer 10–14 days of clopidogrel therapy compared to pre- treatment values (P < 0.0001) (Fig. 1). Clopidogrel inhibits platelet P2Y12 by activating cyclic adenosine monophosphate and subse- quent phosphorylation of vasodilator-stimulated
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