Assessment of P2Y12 Inhibition by Clopidogrel in Feline Platelets Using Flow Cytometry Quantification of Vasodilator-Stimulated Phosphoprotein Phosphorylation.

Ronald H L Li,Nghi Nguyen,Karl Jandrey,Tommaso Rosati

doi:10.3389/fvets.2020.00267

Abstract

The primary objective of this study was to evaluate a novel flow cytometry-based assay of quantifying platelet phosphorylation of vasodilator-stimulated phosphoprotein (P-VASP) in cats that received clopidogrel treatment. Eight healthy cats received 18.75 mg PO q24h of clopidogrel for 7 days. Prior to and after clopidogrel treatment, blood was collected for ADP-induced light transmission aggregometry (LTA) and P-VASP measurement by flow cytometry. Flow cytometry measurement of P-VASP levels was used to derive platelet reactivity index (PRI) before and after clopidogrel treatment. Based on P-VASP and LTA findings, platelet response to ADP was significantly attenuated after 7 days of clopidogrel treatment. By eliciting the competing platelet pathways of P2Y12 and cAMP using ADP and PGE1, respectively, ADP had no effect on P-VASP levels following clopidogrel treatment (p = 0.94). Clopidogrel also significantly decreased PRI from 28.84 ± 28.52% to 1.69 ± 12.39% (p = 0.0078). PRI on day 8 correlated moderately with the degree of slope inhibition on LTA (r = −0.4, p = 0.4). Flow cytometry analysis of P-VASP is effective at monitoring the inhibitory effects of clopidogrel on feline platelets.

Highlights

Hypertrophic cardiomyopathy (HCM) is the most common cardiomyopathy in cats affecting ∼15% of the feline population [1]
We aimed to evaluate the pharmacodynamic response to clopidogrel by correlating flow cytometric measurement of platelet phosphorylation of vasodilator-stimulated phosphoprotein (P-vasodilatorstimulated phosphoprotein (VASP)) to ADP-induced light transmission aggregometry (LTA) in cats treated with 7 days of clopidogrel
After 7 days of clopidogrel treatment, P-VASP in prostaglandin E1 (PGE1)-treated platelets was not significantly different from P-VASP in platelets treated with ADP and PGE1-treated platelets (MFI = 3,787 ± 633.5 vs. 3,803 ± 686.6, p = 0.94)

Summary

Introduction

Hypertrophic cardiomyopathy (HCM) is the most common cardiomyopathy in cats affecting ∼15% of the feline population [1]. Clopidogrel, once metabolized to its active metabolite by hepatic cytochrome P450, inhibits ADP-mediated platelet activation by covalently binding onto one of the two G protein-coupled ADP receptors, P2Y12. This irreversibly prevents ADP from activating the G protein, Gi2a, responsible for downstream signaling that amplifies platelet activation and aggregation [6]. A randomized controlled trial in cats with CATE demonstrated the superiority of clopidogrel over aspirin in reducing recurrent thrombotic events [7]. Despite this favorable outcome, recurrence of CATE remains high, underscoring the highly variable pharmacodynamic response to clopidogrel in clinical HCM cats.

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