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

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.

Keywords: cyclic AMP; hypertrophic cardiomyopathy; light transmission aggregometry; platelet activation and signaling; vasodilator stimulated phosphoprotein.

Figure 1

Representative scatter plot diagrams and histograms of flow cytometric analysis of intraplatelet vasodilator-stimulated phosphoprotein phosphorylation in a cat before and after clopidogrel therapy. (A) Platelets were identified based on their forward (FS) and side scatter (SS) properties. (B) Fluorescent minus one control and isotype control in PGE1-treated platelets were used to determine the P-VASP gate. P-VASP positive platelets were labeled as P-VASP +ve. (C) In PGE₁-treated platelets, 90.1% of platelets expressed the phosphorylated form of vasodilator-stimulated phosphoprotein (P-VASP). (D) A histogram illustrating the number of platelets expressing P-VASP in PGE1-treated platelets in presence (Blue) or absence of ADP (Red) prior to clopidogrel therapy. Note that P2Y₁₂ activation by ADP resulted in loss of P-VASP as the population shifts to the left. (E) Following clopidogrel therapy, regardless of ADP activation, PGE₁ resulted in VASP phosphorylation due to irreversible inhibition of P2Y₁₂.

Figure 2

Scatter dot plots of intraplatelet vasodilator-stimulated phosphoprotein phosphorylation (P-VASP) in eight cats before and after 7 days of clopidogrel therapy. Platelet rich plasma from eight cats treated with PGE1, ADP or a combination of PGE1 and ADP was evaluated for P-VASP expression, as quantified by mean fluorescence intensity (MFI) using flow cytometry. (A) Before clopidogrel therapy, ADP-induced platelet activation in the presence of PGE₁ resulted in significant decrease in P-VASP expression compared to PGE-1 treated platelets in the absence of ADP. (B) Following clopidogrel therapy, ADP in the presence of PGE₁ did not result in significant decrease in P-VASP expression compared to PGE₁-treated platelets. ADP-induced platelet activation resulted in significant decrease in P-VASP expression regardless of clopidogrel treatment. Middle line represents the median and upper and lower lines represent the 25th and 75th quartile, respectively. *p < 0.05.

Figure 3

Before-and-after dot plots of light transmission aggregometry from eight cats before and after 7 days of clopidogrel treatment. ADP or thrombin-induced platelet aggregometry was measured as maximum amplitude (%), slope (no unit) and area under the curve (AUC). Thrombin-induced platelet aggregation served as positive control. ADP-induced aggregation, as measured by percent of maximum amplitude (A), slope (B) and AUC (C), was significantly decreased on day 8 (D8) compared to day 0 (D0). In contrast, thrombin did not result in significant changes in all three measurements on light transmission aggregometry. Red dots represent cats that were resistant to clopidogrel therapy. *p < 0.05.

Figure 4

Platelet reactivity index (PRI) based on flow cytometric quantification of intraplatelet P-VASP. (A) Before-and-after dot plots of PRI in 8 cats following 7 days of clopidogrel treatment. PRI was significantly lower after clopidogrel treatment. (B) Prior to clopidogrel treatment (Day 0), PRI appeared to be higher among non-responders compared to responders. (C) However, on day 8, PRI was similar among the two groups. *p < 0.05.