Skip to main content
NCBI home page
British Journal of Pharmacology logoLink to British Journal of Pharmacology
. 2019 Apr 21;176(11):1717–1727. doi: 10.1111/bph.14646

Enhanced responsiveness of platelets to vicagrel in IL‐10‐deficient mice through STAT3‐dependent up‐regulation of the hydrolase arylacetamide deacetylase in the intestine

Yu‐Meng Jia 1,2, Huan Zhou 1,2, Ting Tai 1, Tong‐Tong Gu 1, Jin‐Zi Ji 1, Qiong‐Yu Mi 1, Bei‐Bei Huang 1, Yi‐Fei Li 1, Ting Zhu 1,2, Hong‐Guang Xie 1,2,3,✉
PMCID: PMC6514289  PMID: 30825385

Abstract

Background and Purpose

Vicagrel is a novel promising antiplatelet drug designed for overcoming clopidogrel resistance. There is limited evidence indicating that exogenous IL‐10 suppresses CYP3A4 activity in healthy subjects and that IL‐10 knockout (KO) mice exhibit increased clopidogrel bioactivation compared with wild‐type (WT) mice. In this study, we sought to determine whether IL‐10 could play an important role in the metabolism of and platelet response to vicagrel in mice.

Experimental Approach

IL‐10 KO and WT mice were administered vicagrel, then their plasma H4 (active metabolite of vicagrel) concentrations were determined by LC–MS/MS, and inhibition of ADP‐induced whole‐blood platelet aggregation by vicagrel was assessed with an aggregometer. The mRNA and protein levels of several relevant genes between IL‐10 KO and WT mice were measured by qRT‐PCR and Western blots, respectively. Intestinal Aadac protein levels were measured in IL‐10 WT mice injected i.p. with vehicle control, Stattic, or BAY 11‐7082.

Key Results

Compared with WT mice, IL‐10 KO mice exhibited significantly increased plasma levels of H4 and enhanced platelet responses to vicagrel, as well as significantly higher mRNA and protein levels of arylacetamide deacetylase (Aadac) in the intestine. In WT mice, STAT3, not NF‐κB, mediated Aadac expression in the intestine.

Conclusions and Implications

IL‐10 suppresses metabolic activation of vicagrel through down‐regulation of Aadac in mouse intestine in a STAT3‐dependent manner and, consequently, attenuates platelet responses to vicagrel, suggesting that the antiplatelet effect of vicagrel may be modulated by changes in plasma IL‐10 levels in relevant clinical settings.

What is already known

  • Elevated serum IL‐10 levels are associated with attenuated responsiveness to clopidogrel in patients undergoing percutaneous coronary intervention.

  • Exogenous IL‐10 decreases CYP3A4 but increases CYP2C9 catalytic activity in humans.

What this study adds

  • IL‐10 suppresses metabolic activation of and platelet response to vicagrel in mice.

  • IL‐10 down‐regulates the expression of hydrolase Aadac in mouse intestine in a STAT3‐dependent manner.

What is the clinical significance

  • Antiplatelet effects of vicagrel may be varied by changes in plasma IL‐10 levels in humans.

  • Drug–drug interactions may exist between vicagrel and the substrates of hydrolase AADAC.

Abbreviations

AADAC

arylacetamide deacetylase

AUC0 − ∞

the area under the plasma drug concentration‐time curve extrapolated to infinity

AUC0 − t

the area under the plasma drug concentration‐time curve at last time point measured

CES2

carboxylesterase 2

Cmax

maximum plasma drug concentration

CYP

cytochrome P450

H4

clopidogrel (or vicagrel) active metabolite

IS

internal standard

KO

knockout

m/z

ratio of mass to electronic charge

MPB

2‐bromo‐3′‐methoxyacetophenone

WT

wild‐type

1. INTRODUCTION

A large number of relevant studies have demonstrated that inflammatory cytokines are involved in the whole process of atherosclerosis, due to their excessive generation and uninterrupted release in response to inflammatory stimuli (Ramji & Davies, 2015), and that inflammation plays a critical role in the pathogenesis and progression of atherosclerosis (Hansson, 2005, 2009; Libby, 2002; Ross, 1986, 1999). Currently, coronary artery disease is thought to be a chronic inflammatory disease (Gregersen, Holm, Dahl, Halvorsen, & Aukrust, 2016; Pedicino et al., 2018; Raggi et al., 2018; Ruparelia, Chai, Fisher, & Choudhury, 2017; Vaccarezza, Balla, & Rizzo, 2018). Of the all inflammatory cytokines, IL‐10 is a cytokine that receives less attention from the scientists than IL‐6, IL‐1β, TNF‐α, and high‐sensitivity C‐reactive protein. Only a limited number of clinical research studies have indicated that elevated serum IL‐10 levels would predict a worse prognosis in patients with acute coronary syndrome or acute myocardial infarction (Heeschen et al., 2003; Shibata et al., 1997; Yip et al., 2007), which is not consistent with its biological function as a well‐defined anti‐inflammatory cytokine. Clearly, the role of IL‐10 in the pathogenesis of coronary artery disease is more complicated than we thought.

However, there is also evidence indicating that IL‐10 modulates the expression level of some (if not all) drug‐metabolizing enzymes, such as cytochrome P450 (also known as CYP or P450), in the intestine and liver. For example, administration of exogenous IL‐10 decreased CYP3A activity (as measured by midazolam clearance) and increased CYP2C9 activity (as measured by tolbutamide oral clearance and urinary metabolic ratio) and the unbound fraction of tolbutamide in 12 healthy subjects (Gorski et al., 2000). Clinical research studies suggested that elevated plasma IL‐10 levels are associated with an increased risk of attenuated clopidogrel responsiveness in patients undergoing percutaneous coronary intervention (Osmancik, Paulu, Tousek, Kocka, & Widimsky, 2012). Our recent work showed that the formation of clopidogrel active metabolite (CAM) was markedly increased in IL‐10 knockout (KO) mice compared with wild‐type (WT) mice (Q. Yin et al., 2016).

Vicagrel, an inactive prodrug itself and an acetate analogue of clopidogrel, is a promising novel antiplatelet drug that was developed in China to overcome clopidogrel resistance (Jia et al., 2018; X. Li et al., 2018; Shan et al., 2012; Xie, Jia, Tai, & Ji, 2017). As illustrated in Figure 1, the metabolic activation of vicagrel is predominantly catalysed by intestinal arylacetamide deacetylase (AADAC) and carboxylesterase (CES) 2 (Jiang, Chen, & Zhong, 2017), rather than hepatic P450 enzymes (Qiu et al., 2014), different from that of clopidogrel (Xie et al., 2017). However, little is known about whether the presence or absence of IL‐10 could affect the metabolic activation of and platelet responses to vicagrel despite some clinical findings that elevated plasma IL‐10 levels are associated with impaired platelet responses to clopidogrel and also with worse prognosis in patients with coronary heart disease (Heeschen et al., 2003; Osmancik et al., 2012; Shibata et al., 1997; Yip et al., 2007). This study was aimed at delineating the mechanisms involved in this effect using IL‐10 KO mice, which can be used to explain the clinical issues associated with IL‐10 expression levels.

Figure 1.

Figure 1

Open in a new tab

The metabolic pathway of vicagrel and clopidogrel in humans. AADAC: arylacetamide deacetylase; CES2: carboxylesterase 2; CYP: cytochrome P450; H4: active metabolite common for clopidogrel and vicagrel

2. METHODS

2.1. Study animals

All the animal care and experimental procedures were approved by the Experimental Animal Welfare and Ethics Committee, Nanjing Medical University, and conducted in accordance with the Guide for the Care and Use of Laboratory Animals, released by the U.S. National Institutes of Health. All studies involving animals are reported in compliance with the ARRIVE guidelines (Kilkenny, Browne, Cuthill, Emerson, & Altman, 2010). The IL‐10 −/− (KO) mice were supplied from Jackson Laboratory (Bar Harbor, ME, USA) and were maintained through homozygous mating under the specific pathogen‐free state as described elsewhere (Wan et al., 2014; Q. Yin et al., 2016). The C57BL/6 control (WT) mice (matched, with age and gender similar or identical to KO mice) were obtained from the Model Animal Research Centre, Nanjing University, China. All mice, housed in specific pathogen‐free cages at the local animal facility, were maintained in an air‐conditioned room (kept at 22–24°C) with alternating a light to dark cycle every 12 hr (a light cycle from 7 a.m. to 7 p.m.). Male mice (weighed 18 ± 2 g; aged 6–8 weeks) had free access to tap water and standard laboratory chow at all times but were fasted 12 hr before starting the pharmacokinetic or platelet aggregation studies.

Randomization was used to assign mice to each of the experimental groups for all in vivo studies. Data collection and analysis of all in vivo and in vitro were performed in a blind manner.

2.2. Pharmacokinetics study of vicagrel in mice

Male IL‐10 KO and WT mice were each randomly divided into three groups (n = 8 in each) and were administered, by oral gavage, vicagrel at a single dose of 1.25, 2.5, or 5 mg·kg−1, respectively. The dosage of vicagrel was based on our pilot study. A series of blood samples (30 μl each) were withdrawn from the orbital venous plexus at 5, 15, 30, 45, 75, 120, 240, and 480 min after dosing and were collected into heparin‐containing EP tubes pretreated with 2 μl of 500 mM MPB in acetonitrile to keep vicagrel active metabolite H4 stable through immediate derivatization with MPB as described elsewhere (Tai et al., 2016; Q. Yin et al., 2016). Plasma was separated after centrifugation at 1,960× g for 10 min and stored at −80°C until analysis.

2.3. Sample preparation

The samples were prepared as described previously (Jia et al., 2018; Tai et al., 2016; Q. Yin et al., 2016) with slight modifications. In brief, an aliquot of 10 μl plasma was spiked with 100‐μl acetonitrile (containing 10‐ng·ml−1 piroxicam as IS) and then vortex‐mixed for 3 min for protein precipitation. After centrifugation at 1,960× g for 10 min, the supernatant was collected into a clean tube and then centrifuged at 24,100× g for 20 min, and finally, 5 μl of purified aliquot was injected into the LC–MS/MS system for further quantitative analysis.

2.4. LC–MS/MS assay

Chromatographic conditions were as described previously (Jia et al., 2018; Tai et al., 2016; Q. Yin et al., 2016) with some modifications. In brief, separation of each analyte was performed on a Poroshell 120 SB‐C18 column (100 × 2.1 mm, I.D., 2.7 μm) at 40°C. The mobile phase consisted of solvent A (0.1% formic acid in water) and solvent B (0.1% formic acid in acetonitrile). The optimal gradient elution programme was set as follows: phase B was increased from 20% to 35% during the period of 0 to 2 min and from 35% to 60% from 2 to 7 min, and then declined to 20% at 7.01 min, and kept unchanged until the end of elution at 9 min. The total running time was 9 min to separate vicagrel active metabolite H4 and the internal standard (IS). Typical MS conditions were used for the determination of H4 and IS in mouse plasma during electrospray ionization by multiple reaction monitoring in positive ion mode. The multiple reaction monitoring settings for each compound were as shown in Table 1.

Table 1.

Mass spectrometric settings for quantification of H4

Compound Parent ion (m/z) Daughter ion (m/z) Declustering potential (V) Collision energy (V) Collision cell exit potential (V)
H4 504.1 155.0 100 63 10
IS 322.0 95.0 76 47 10
Open in a new tab

Note: H4, vicagrel active metabolite; IS: internal standard; m/z: ratio of mass to electronic charge.

2.5. Inhibition of ADP‐induced platelet aggregation by vicagrel in mice

The doses of vicagrel used for measuring its antiplatelet effect were determined based on the data from our pharmacokinetic study. Male IL‐10 KO and WT mice were each randomly divided into four groups (n = 8 each) and were treated with vicagrel at a single dose of 0 (0.5% sodium carboxyl methyl cellulose as vehicle control), 1.25, 2.5, or 5 mg·kg−1, respectively, by oral gavage. About 10 min before initiating the study, mice were anaesthetized with ketamine (125 mg·kg−1, i.p.), and then whole‐blood platelet aggregation induced by ADP (10 μM as a final concentration in a testing chamber) was measured by the whole‐blood aggregometer (Chrono‐log Model 590‐2D, Chrono‐Log Corp., Havertown, PA, USA) as described previously (Jia et al., 2018; Tai et al., 2016; Q. Yin et al., 2016). The extent of inhibition of ADP‐induced platelet aggregation was calculated as [1 − (value of a treated mouse/value of a control mouse)] × 100%.

2.6. Measurement of mRNA expression of the genes involved in vicagrel bioactivation

As a prodrug, vicagrel exerts its potent antiplatelet effect via a two‐step metabolic pathway to form its active metabolite H4 as shown in Figure 1. Several drug‐metabolizing enzymes are known to be involved in these processes, including AADAC (Jiang et al., 2017) and CES2 (Qiu et al., 2014; Xie et al., 2017) for vicagrel hydrolysis in the intestine, and CYP3A4, CYP2B6, CYP2C19, and CYP2C9 for its second‐step oxidation in the liver (Xie et al., 2011, 2017). To determine whether IL‐10 could significantly modulate the expression levels of these genes, we measured the mRNA expression levels of the mouse‐specific orthologue genes of human genes AADAC (Aadac), CES2 (Ces2c), CYP2B6 (Cyp2b10), CYP2C9 (Cyp2c29 and Cyp2c37), CYP2C19 (Cyp2c50 and Cyp2c54), and CYP3A4 (Cyp3a11) in the intestine and liver of IL‐10 KO or WT mice using qRT‐PCR. The extraction of total RNA and subsequent synthesis of cDNA were as described previously (Tai et al., 2016) according to the protocols from the manufacturers. The primer sequences for the genes tested and an internal reference gene Gapdh are available upon request. PCR amplification and melting analysis were conducted using ABI 7500 real‐time PCR apparatus (Applied Biosystems, Carlsbad, CA, USA) as described previously (Tai et al., 2016). The level of mRNA expression was represented by a Ct value (defined as the number of PCR cycles that have crossed an arbitrarily chosen signal threshold in the log phase of the amplification curve), and relative expression of a target gene was expressed as fold change, which was calculated using the following equation: fold change = 2−∆∆Ct, where ∆Ct = Ct(target) − Ct(Gapdh).

2.7. Western blotting assay of intestinal Aadac and hepatic Cyp3a11 involved in vicagrel bioactivation

To further confirm whether changes in mRNA and protein expression levels of the intestinal hydrolase Aadac and hepatic Cyp3a11 in mice were due to the presence or absence of IL‐10, a Western blotting assay was performed. First, intestinal and hepatic tissue samples were taken from KO and WT mice (n = 5 each), after they had been killed (by cervical dislocation), and were homogenized and extracted in RIPA (KeyGEN BioTECH, Nanjing, Jiangsu, China) supplemented with 1% protease inhibitor cocktail (Thermo, Rockford, IL, USA). Then the protein concentrations were quantified by the BCA protein content kit (KeyGEN BioTECH, Nanjing, Jiangsu, China) according to the manufacturer's instruction. Lysates (protein of 50 μg each) were separated by 10% SDS‐PAGE and then transferred to PVDF membranes (Roche Diagnostics, Indianapolis, IN, USA). After being blocked in 5% nonfat dry milk–TBS–0.1% Tween 20 for 1 hr at ambient temperature, membranes were incubated with primary antibodies against mouse Aadac (1:500; Cat#: sc‐390592; Santa Cruz Biotechnology, Dallas, TX, USA), rabbit β‐actin (1102) polyclonal antibody (1:10,000; Cat#: AP0060; Bioworld Technology, Nanjing, Jiangsu, China), mouse Cyp3a11 (1:1,000; Cat#: MAB10041; Merck‐Millipore, USA), or mouse Gapdh monoclonal antibody (1:5,000; Cat#: 60004‐1‐1g; Proteintech, Group Inc., Chicago, IL, USA) at 4°C overnight. Thereafter, the immunoblots were incubated with secondary antibodies—peroxidase‐conjugated affinipure goat anti‐rabbit IgG (1:100,000; Cat#: 119181; Jackson ImmunoResearch Laboratories, West Grove, PA, USA) or goat anti‐mouse IgG, peroxidase conjugated, H + L (1:80,000; Cat#: BL001A; Biosharp Life Sciences, Hefei, Anhui, China)—for 2 hr at room temperature. Finally, the immunoreactivity was detected with an enhanced chemiluminescence system reagent (Millipore Corporation, Billerica, MA, USA). The density of each immunoblot band was scanned by the software ImageJ (RRID:SCR_003070) from the National Institutes of Health (Bethesda, MD, USA), and the relative expression level of the target protein was normalized to β‐actin or to Gapdh in the corresponding samples, respectively, which was used as a loading control to minimize variances. Data were collected and analysed from five independent samples.

2.8. Effects of STAT3 and NF‐κB on IL‐10‐mediated modulation of intestinal Aadac expression in mice

To explore the potential signalling pathways involved in the IL‐10‐mediated modulation of intestinal Aadac expression, Stattic (an STAT3‐specific inhibitor) and BAY 11‐7082 (an NF‐κB inhibitor) were used to further explore the potential mechanisms of action involved, respectively. Male C57BL/6 mice were randomly divided into three groups (n = 6 in each) and injected i.p. with vehicle control, BAY 11‐7082 (20 mg·kg−1), or Stattic (3.75 mg·kg−1), respectively, every 2 days for up to 7 days as described previously (Bitto et al., 2014; C. Li et al., 2015). Mice were killed after 7 days, and samples of their intestine were harvested for Western blotting analysis. Protein extractions and measurement were performed as mentioned above. Data were collected and analysed from six independent samples. The relative expression level of the target protein was normalized to β‐actin in the corresponding samples.

2.9. Data and statistical analysis

The data and statistical analysis comply with the recommendations on experimental design and analysis in pharmacology (Curtis et al., 2015, 2018). The data are presented as mean ± SD for individual groups. Pharmacokinetic parameters for each individual mouse were calculated using the DAS (drug and statistics) software (version 3.0, the Chinese Society for Mathematical Pharmacology) using a non‐compartmental analysis model. Statistically significant differences between IL‐10 KO and WT mice were analysed using Student's unpaired t test for group comparisons of a single variable or one‐way ANOVA for comparisons of multiple groups. A two‐tailed P value of less than 0.05 was considered statistically significant. All statistical analysis was performed using SPSS software version 22.0 (RRID:SCR_002865; SPSS Inc., Chicago, IL, USA).

2.10. Materials

Vicagrel (purity of 99.0%) and its active metabolite H4 derivative (purity 95.1%) were generously supplied by Jiangsu Vcare Pharmatech Co. Ltd., Nanjing, China. Piroxicam (internal standard [IS]), 2‐bromo‐3′‐methoxyacetophenone (MPB), and ADP were all purchased from Sigma‐Aldrich (St. Louis, MO, USA). Stattic (an STAT3‐specific inhibitor) and BAY 11‐7082 (an NF‐κB‐specific inhibitor) were purchased from MedChem Express (Monmouth Junction, NJ, USA). HPLC‐grade acetonitrile was purchased from Merck (Darmstadt, Germany). Formic acid and other chemicals and solvents were of analytical grade or above. Sodium carboxyl methyl cellulose was purchased from Sinopharm Chemical Reagent Co., Ltd., Shanghai, China. Ultra‐pure water was purified by a Milli‐Q system (Millipore, Milford, MA, USA).

2.11. Nomenclature of targets and ligands

Key protein targets and ligands in this article are hyperlinked to corresponding entries in http://www.guidetopharmacology.org, the common portal for data from the IUPHAR/BPS Guide to PHARAMACOLOGY (Harding et al., 2018), and are permanently archived in the Concise Guide to PHARMACOLOGY 2017/18 (Alexander, Christopoulos et al., 2017;Alexander, Fabbro et al., 2017; Alexander, Kelly et al., 2017).

3. RESULTS

3.1. Increased formation of vicagrel active metabolite H4 in IL‐10 KO mice

After oral administration of vicagrel at three different doses in IL‐10 KO or WT mice, the typical chromatographic profiles and mean plasma H4 concentration‐time curves were illustrated in Figures S1 and 2, respectively, and the main pharmacokinetic parameters were summarized in Table 2. As shown in Table 2, KO mice exhibited 38%, 39%, and 61% increase in maximal plasma H4 concentration (Cmax) than WT mice (all P < 0.05) when treated with vicagrel at a dose of 1.25, 2.5, and 5 mg·kg−1, respectively. Similarly, KO mice displayed 19%, 17%, and 52% (P < 0.05) increase in AUC0 − t of H4 than WT mice at these doses, respectively. Furthermore, KO mice just required a twofold smaller dose (2.5 vs. 5 mg·kg−1) of vicagrel than did WT mice to achieve similar average systemic exposure to H4 (Cmax: 268.6 vs. 285.0; AUC0 − t: 211.0 vs. 234.0). These data suggest that the formation of H4 is markedly elevated in IL‐10 KO mice compared with WT mice.

Figure 2.

Figure 2

Open in a new tab

The mean plasma concentration‐time profiles of H4 in IL‐10 wild‐type (WT) and knockout (KO) mice (n = 8 each) after oral administration of 1.25‐, 2.5‐, and 5‐mg·kg−1 vicagrel as shown in panels (a), (b), and (c), respectively. Data are presented as mean ± SD

Table 2.

Pharmacokinetic parameters of vicagrel active metabolite (H4) in IL‐10 knockout (KO) and wild‐type (WT) mice after oral administration of 1.25‐, 2.5‐, or 5‐mg·kg−1 vicagrel (n = 8 each)

Parameter 1.25 mg 2.5 mg 5 mg
WT KO WT KO WT KO
Cmax (ng·ml−1) 77.4 ± 19.5 107.1 ± 16.7* 193.3 ± 20.5 268.6 ± 70.9* 285.0 ± 76.9 459.7 ± 121.3*
AUC0 − t (ng·hr·ml−1) 78.6 ± 9.7 93.5 ± 23.4 180.2 ± 42.7 211.0 ± 54.8 234.0 ± 48.1 354.8 ± 88.4*
AUC0 − ∞ (ng·hr·ml−1) 82.7 ± 10.4 94.0 ± 23.2 219.1 ± 80.2 211.7 ± 55.1 234.9 ± 48.3 356.1 ± 87.8*
Tmax (min) 16.9 ± 5.3 10.0 ± 5.3 13.6 ± 3.8 15.0 15.0 16.7 ± 5.0
t 1/2 (min) 94.2 ± 37.2 72.2 ± 24.5 71.1 ± 23.6 66.7 ± 25.5 65.3 ± 20.4 70.5 ± 19.3
MRT0 − t (min) 87.3 ± 13.3 62.9 ± 8.6* 90.2 ± 31.1 53.8 ± 3.8* 72.1 ± 11.9 52.1 ± 5.7*
MRT0 − ∞ (min) 121.1 ± 39.1 66.1 ± 8.8* 118.7 ± 79.0 55.7 ± 2.5 74.2 ± 10.7 54.5 ± 5.6*
Open in a new tab

Note: Data are presented as mean ± SD (n = 8 each). Student's unpaired t test. MRT: mean residence time; Tmax: time to reach the peak plasma drug concentration.

*

P < 0.05, versus WT mice.

3.2. Enhanced inhibition of ADP‐induced platelet aggregation by vicagrel in IL‐10 KO mice

As a prodrug, vicagrel exhibits its antiplatelet effects through the blockade of the P2Y12 receptor, exerted by its active metabolite H4. Therefore, increased systemic exposure to H4 could be translated into enhanced antiplatelet response in IL‐10 KO mice compared with WT mice. To test this hypothesis, ADP‐induced whole‐blood platelet aggregation was measured after dosing of 0 (vehicle control), 1.25, 2.5, or 5 mg·kg−1 of vicagrel, respectively, in IL‐10 KO versus WT mice. The typical tracing of ADP‐induced platelet aggregation was depicted in Figure S2, and the inhibition of ADP‐induced platelet aggregation by vicagrel was plotted in Figure 3. As presented in Figure 3a, vicagrel exerted its antiplatelet effects in a dose‐dependent manner in IL‐10 KO or WT mice. At a dose of 1.25 mg·kg−1, vicagrel exerted significantly potent antiplatelet effect in KO mice, not in WT mice, when compared with vehicle control. As shown in Figure 3b, inhibition of ADP‐induced platelet aggregation by vicagrel was more potent in IL‐10 KO mice than in WT mice treated with each of the same doses tested (P < 0.05). These data suggest enhanced antiplatelet effect in KO mice compared with WT mice when treated with vicagrel.

Figure 3.

Figure 3

Open in a new tab

Whole‐blood platelet aggregation induced by ADP (10 μM as a final concentration in a testing chamber) in IL‐10 wild‐type (WT) or knockout (KO) mice treated with vehicle control (sodium carboxyl methyl cellulose, or Ctrl), 1.25‐, 2.5‐, and 5‐mg·kg−1 vicagrel for 1 hr, respectively. (a) Dose‐dependent ADP‐induced platelet aggregation in IL‐10 KO or WT mice treated with vicagrel. The ADP‐induced platelet aggregation was measured as % of platelet aggregation. *P < 0.05 versus Ctrl; # P < 0.05 versus 1.25‐mg·kg−1 vicagrel. n = 8 each. One‐way ANOVA and subsequently, Student's unpaired t test. (b) Inhibition of ADP‐induced whole‐blood platelet aggregation by vicagrel. Inhibition of platelet aggregation by vicagrel was calculated as [1 − (value of a treated mouse/value of a control mouse)] × 100%. * P < 0.05, versus WT mice. n = 8 each, Student's unpaired t test

3.3. IL‐10 modulated the mRNA expression of drug‐metabolizing enzymes in mice

As an analogue of clopidogrel, the major drug‐metabolizing enzymes responsible for vicagrel bioactivation are identified as AADAC and CES2 for its first‐step hydrolysis to generate clopidogrel thiolactone (or called 2‐oxo‐clopidogrel) in the intestine as shown in Figure 1, different from those for clopidogrel bioactivation (Jiang et al., 2017; Qiu et al., 2014; Xie et al., 2017). To determine the potential effects of IL‐10 on the expression of the genes related to vicagrel bioactivation, mRNA expression of eight mouse‐specific, human ortholog genes in the intestine and liver of IL‐10 KO or WT mice was detected. As shown in Figure 4, there was a significant increase in mRNA expression level of intestinal Aadac and hepatic Ces2c but a significant decrease in mRNA expression level of hepatic Cyp3a11 in KO mice compared with WT mice. Other genes like Cyp2b10 exhibited an increased trend in KO mice but did not reach statistical significance due to large interindividual variation.

Figure 4.

Figure 4

Open in a new tab

Differences in mRNA expression levels of genes related to the bioactivation of vicagrel in the liver (a) and intestine (b) of IL‐10 knockout (KO) and wild‐type (WT) mice (n = 8 each group), respectively. *P < 0.05, versus WT mice, Student's unpaired t test

3.4. Up‐regulation of intestinal Aadac protein expression in IL‐10 KO mice

Consistent with the observations of its mRNA expression as shown in Figure 4, the protein expression level of the intestinal Aadac was significantly higher in IL‐10 KO mice than in WT mice, whereas expression of hepatic Cyp3a11 was significantly less in IL‐10 KO mice than in WT mice as shown in Figure 5. In contrast, the band of intestinal Ces2 protein seemed to be very faint, with no marked differences between KO mice and WT mice (data not shown). Similarly, the band of Ces2 protein was not seen in the liver of the two mouse groups (data not shown). These data strongly suggested that significantly greater protein expression level of Aadac in the intestine is the major contributor to increased formation of vicagrel active metabolite H4 in IL‐10 KO mice when compared with WT mice.

Figure 5.

Figure 5

Open in a new tab

The protein expression of intestinal Aadac and hepatic Cyp3a11 of IL‐10 knockout (KO) mice and wild‐type (WT) mice. (a) Representative Western blots and (b) the ratio of intestinal Aadac protein to β‐actin or of hepatic Cyp3a11 protein to Gapdh as measured by the density of the immunoblot bands. Data are presented as mean ± SD (n = 5 each). *P < 0.05, versus WT mice, Student's unpaired t test

3.5. Down‐regulation of intestinal Aadac protein expression in Stattic‐treated mice

As shown in Figure 6, the protein levels of hydrolase Aadac in the intestine were significantly decreased in mice treated with Stattic, not with BAY 11‐7082, when compared with mice treated with vehicle control. These data suggest that STAT3, rather than NF‐κB, mediates Aadac expression in mouse intestine.

Figure 6.

Figure 6

Open in a new tab

The protein expression of intestinal Aadac in IL‐10 wild‐type mice treated i.p. with vehicle control (Ctrl), BAY 11‐7082, or Stattic. (a) Representative Western blots and (b) the ratio of intestinal Aadac protein to β‐actin, as measured by the density of the immunoblot bands. Data are presented as mean ± SD (n = 6 each). *P < 0.05 versus control mice, Student's unpaired t test

4. DISCUSSION

In this study, we compared differences in metabolic activation of and platelet response to vicagrel in mice with or without IL‐10 and linked these variations to the changes in Aadac mRNA and protein levels in the intestine, and established a substantial relationship between plasma H4 concentrations, inhibition of platelet aggregation by vicagrel, Aadac protein levels, and modulation of Aadac expression by STAT3, respectively. To our knowledge, this is the first study revealing that the antiplatelet effect of vicagrel is significantly enhanced in IL‐10 KO mice, due to increased levels of Aadac in a STAT3‐dependent manner. This knowledge would be useful for patient care when seeking the correct personalized dosage of vicagrel, that is if it is ever marketed.

Vicagrel was designed as a novel clopidogrel analogue to overcome clopidogrel resistance (X. Li et al., 2018; Shan et al., 2012; Xie et al., 2017). As illustrated in Figure 1, vicagrel undergoes complete hydrolysis in the intestine by AADAC and CES2 to generate its immediate metabolite 2‐oxo‐clopidogrel (precursor of the active metabolite H4) before it exerts its antiplatelet effect (Jiang et al., 2017; Qiu et al., 2014; Xie et al., 2017). In humans, the relative contribution of AADAC to generating 2‐oxo‐clopidogrel is roughly 53% (Jiang et al., 2017). In this study, Aadac mRNA and protein levels in mouse intestine were significantly higher in IL‐10 KO mice than in WT mice, which is translated to a marked increase in Aadac hydrolase activity in KO mice compared with WT mice. Although the mRNA expression of intestinal Ces2 did not differ much between the two groups, Ces2 may contribute to some extent to vicagrel hydrolysis. In addition, the expression of other known P450s (that are responsible for the formation of H4) was either significantly lower in IL‐10 KO mice than in WT mice, such as Cyp3a11 (a predominant player), or not significantly different from one another, such as Cyp2b10 and Cyp2c.

As a prodrug, clopidogrel exerts its antiplatelet effect through covalent binding of its active metabolite H4 to cysteine residues of ADP purinergic receptor P2Y12 on the platelet surface in an irreversible manner (Ding, Kim, Dorsam, Jin, & Kunapuli, 2003; Savi et al., 2006). In theory, the extent of platelet inhibition by clopidogrel may be proportional to the Cmax of plasma H4 (Chunsangchan, Chariyavilaskul, Ketchart, Prompila, & Wittayalertpanya, 2017; Karazniewicz‐Lada et al., 2014) and thus is persistent for the life span of the affected platelets (at least 5–8 days after ceasing medicine and disappearing in proportion to the platelet turnover; Dorsam & Kunapuli, 2004; Savi et al., 1992; Xie et al., 2017). In this study, the Cmax values of plasma H4 were significantly higher in KO mice than in WT mice treated with vicagrel at each of the doses, which is translated to enhanced antiplatelet effect of vicagrel in IL‐10 KO mice compared with WT mice. As anticipated, significantly more potent antiplatelet effect was seen in KO mice than in WT mice. Moreover, the Cmax of plasma H4 was associated with its antiplatelet effect in a dose‐dependent manner for each of the mouse groups. These data suggest enhanced platelet response to vicagrel in mice due to the absence of IL‐10.

An earlier double‐blind crossover pilot study indicated that exogenous IL‐10 significantly suppresses CYP3A4 activity but increases CYP2C9 activity in healthy subjects (Gorski et al., 2000). Because CYP3A4 is one of the dominant P450s responsible for the generation of H4 (Kazui et al., 2010; Xie et al., 2011), increased H4 formation would be anticipated in IL‐10 KO mice compared with WT mice. As expected, a significant increase in the exposure to H4 was observed in IL‐10 KO mice compared with WT mice (Q. Yin et al., 2016). However, inconsistent with observations derived from exogenous IL‐10 treatment (Gorski et al., 2000), significantly lower levels of Cyp3a11 mRNA were seen in hepatocytes of IL‐10 KO mice than in those of WT mice, but there were no marked differences in mRNA levels for Cyp2c29 as shown in this study. Limited evidence indicated that as an acetate analogue of clopidogrel, vicagrel is different from clopidogrel in their metabolic activation (Kazui et al., 2010; Xie et al., 2017). For example, Aadac and Ces2 hydrolases in the intestine are recently identified as the most predominant enzymes responsible for the complete hydrolysis of vicagrel to generate 2‐oxo‐clopidogrel (Jiang et al., 2017; Qiu et al., 2014), whereas hepatic Cyp3a and Cyp2c are the major P450 enzymes involved in the formation of active metabolite H4 when taking clopidogrel (Kazui et al., 2010; Xie et al., 2011, 2017). Therefore, increased mRNA and protein expression of intestinal Aadac can be used to better explain why platelet inhibition of vicagrel is more potent in IL‐10 KO mice than in WT mice.

The clinical significance of IL‐10 is more complicated and important than originally thought. For example, activated platelets exhibit enhanced IL‐10 production and secretion (Gudbrandsdottir, Hasselbalch, & Nielsen, 2013; Hottz et al., 2014; Linke et al., 2017), and elevated serum IL‐10 levels would be associated with a worse prognosis in patients with acute myocardial infarction or acute coronary syndrome (Heeschen et al., 2003; Shibata et al., 1997; Yip et al., 2007). These links demonstrated that serum IL‐10 levels seem to be proportional to the extent of platelet activation, which reflects the severity of thrombotic diseases as the result of platelet activation. On the other hand, the extent of platelet activation is correlated inversely with the extent of inhibition of platelet aggregation by the antiplatelet drug for patient care. Therefore, elevated serum IL‐10 levels would predict high residual (on‐treatment) platelet reactivity in patients treated with clopidogrel (Osmancik et al., 2012), suggesting an increased risk of attenuated clopidogrel responsiveness in patients who exhibit higher serum IL‐10 levels. Consistent with these clinical observations, we observed that a 23% (significantly) lower mean systemic exposure of active metabolite H4 (i.e., AUC0 − ∞) existed in WT mice than in IL‐10 KO mice in an earlier work (Q. Yin et al., 2016). Consistently, we observed that WT mice exhibited a 34% (significantly) lower mean systemic exposure of H4 than IL‐10 KO mice when treated with vicagrel at a dose of 5 mg·kg−1. These results suggest that elevated serum IL‐10 levels would predict not only a worse prognosis of patients with coronary heart disease but also attenuated platelet responses to vicagrel.

Plasma IL‐10 levels are affected by alcohol (Chien et al., 2017; Yan & Yin, 2007), cigarette smoking (Vogel et al., 2016), camel milk consumption (Arab et al., 2017), exposure to AhR ligand (TCDD) or organic extract of diesel exhaust particles (X. J. Yin, Ma, Antonini, Castranova, & Ma, 2004), and some medicines (see below). For example, significantly increased expression of IL‐10 is seen upon the use of cAMP‐enhancing drugs (Sahay et al., 2018), berberine (Chandirasegaran, Elanchezhiyan, Ghosh, & Sethupathy, 2017; Mohammadi, Seyedhoseini, Asadi, & Yazdani, 2017), or statins (Cicek Ari et al., 2016) or when exposed to docosahexaenoic acid or eicosapentaenoic acid (McDougle et al., 2017). In addition, the −1082G/A polymorphism in the gene IL‐10 (carriers of −1082A/A for low IL‐10 production) may affect between‐subject variability of tacrolimus pharmacokinetics in liver transplant patients (D. Li et al., 2007). It is deduced that carriers of IL‐10 −1082A would exhibit more potent platelet inhibition than non‐carriers when taking vicagrel due to lower IL‐10 production. This means that metabolic activation of and platelet response to vicagrel would vary with the IL‐10 genetic polymorphisms. All these situations could result in changes in plasma IL‐10 and consequently changes in platelet responses to vicagrel.

IL‐10 exerts its biological actions by activating the STAT3‐mediated signalling pathway after binding to IL‐10 receptors on the cell surface (Antoniv & Ivashkiv, 2011; Murray, 2007; Williams, Bradley, Smith, & Foxwell, 2004). In other words, STAT3 mediates the direct activation of IL‐10 target genes (Murray, 2007). However, significantly greater levels of phosphorylated STAT3 (p‐STAT3) were observed in IL‐10 KO mice than in WT mice (Dong et al., 2014; Wang et al., 2016). In this study, compared with WT mice, significantly up‐regulated Aadac in IL‐10 KO mice should be the result of up‐regulated p‐STAT3, as WT mice exhibited significantly decreased Aadac protein levels in the intestine after administration of Stattic (an inhibitor of STAT3 phosphorylation). In contrast, after i.p. injection of BAY 11‐7082 (an inhibitor of NF‐κB inhibitory protein IκB‐α), Aadac levels in the intestine did not change much, indicating that the canonical NF‐κB pathway is not involved in the modulation of Aadac expression. This can be explained by the fact that, in unstimulated cells (like the WT mice used in this study), NF‐κB exists in the cytoplasm as an inactive form bound to an inhibitor protein called IκB, which can be phosphorylated by IκB kinase (Profita et al., 2008), and therefore, use of BAY 11‐7082 could not result in sufficient p65 nuclear translocation and activation of p65 NF‐κB‐dependent transcription. Not limited to STAT3 and NF‐κB measured in this study, other signalling pathways may be involved, such as PI3K‐Akt‐GSK3 (Antoniv & Ivashkiv, 2011).

In conclusion, our results are the first to demonstrate that the absence of IL‐10 is associated with an increased formation of vicagrel active metabolite H4 and enhanced inhibition of ADP‐induced platelet aggregation by vicagrel in mice. One of the major mechanisms underlying these changes is the STAT3‐mediated up‐regulation of the intestinal hydrolase Aadac that catalyses vicagrel bioactivation. Therefore, considerable variations in the metabolic activation of and platelet response to vicagrel would be anticipated when the levels of IL‐10 expression are changed significantly under certain circumstances.

CONFLICT OF INTEREST

The authors declare no conflicts of interest.

AUTHOR CONTRIBUTIONS

Y.‐M.J., H.Z., T.T., T.‐T.G., J.‐Z.J., Q.‐Y.M., B.‐B.H., Y.‐F.L., and T.Z. performed all the experiments. Y.‐M.J. and H.‐G.X. analysed and interpreted the data. Y.‐M.J. prepared all the tables and figures. H.‐G.X. conceived and designed the study and wrote, revised, and finalized the paper. All authors critically reviewed and approved the final version of the manuscript.

DECLARATION OF TRANSPARENCY AND SCIENTIFIC RIGOUR

This Declaration acknowledges that this paper adheres to the principles for transparent reporting and scientific rigour of preclinical research as stated in the BJP guidelines for Design & Analysis, Immunoblotting and Immunochemistry, and Animal Experimentation, and as recommended by funding agencies, publishers, and other organizations engaged with supporting research.

Supporting information

Figure S1.

Typical chromatograms of (A) blank plasma, (B) plasma spiked with standards at lower limit of quantification, plasma samples obtained 15 min after oral administration of vicagrel in (C) IL‐10 +/+ (WT) and (D) IL‐10 −/− (KO) mice. In each panel, H4 and IS go with left and right traces, respectively.

Click here for additional data file. (742.6KB, pdf)

Figure S2.

The representative tracing of ADP‐induced platelet aggregation in IL‐10 WT or KO mice after oral administration of vehicle control (CMC‐Na), 1.25, 2.5, and 5 mg·kg−1 of vicagrel, respectively. Ctrl, control. ADP, used as a final concentration of 10 μM in a testing chamber.

Click here for additional data file. (1.2MB, pdf)

ACKNOWLEDGEMENTS

This work was supported in part by the National Natural Science Foundation of China (Grants 81473286 to H.‐G.X. and 81503114 to Q.‐Y.M.), a launching research Grant 31010300010339 to H.‐G.X., funded by Nanjing First Hospital, and by Nanjing Medical Science and Technique Development Foundation (Grant QRX17069 to Q.‐Y.M.), China. In addition, Dr. Xie is a recipient of the Distinguished Medical Experts of the Province of Jiangsu, China. Jiangsu Vcare Pharmatech Co. Ltd., Nanjing, China, generously provided highly purified vicagrel and vicagrel active metabolite H4 as chemical standards.

Jia Y‐M, Zhou H, Tai T, et al. Enhanced responsiveness of platelets to vicagrel in IL‐10‐deficient mice through STAT3‐dependent up‐regulation of the hydrolase arylacetamide deacetylase in the intestine. Br J Pharmacol. 2019;176:1717–1727. 10.1111/bph.14646

REFERENCES

  1. Alexander, S. P. H. , Christopoulos, A. , Davenport, A. P. , Kelly, E. , Marrion, N. V. , Peters, J. A. , … CGTP Collaborators . (2017). The Concise Guide to PHARMACOLOGY 2017/18: G protein‐coupled receptors. British Journal of Pharmacology, 174(S1), S17–S129. 10.1111/bph.13878 [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Alexander, S. P. , Fabbro, D. , Kelly, E. , Marrion, N. V. , Peters, J. A. , Faccenda, E. , … CGTP Collaborators . (2017). The Concise Guide to PHARMACOLOGY 2017/18: Enzymes. British Journal of Pharmacology, 174(Suppl 1), S272–S359. 10.1111/bph.13877 [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Alexander, S. P. H. , Kelly, E. , Marrion, N. V. , Peters, J. A. , Faccenda, E. , Harding, S. D. , … CGTP Collaborators . (2017). The Concise Guide to PHARMACOLOGY 2017/18: Overview. British Journal of Pharmacology, 174(S1), S1–S16. 10.1111/bph.13882 [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Antoniv, T. T. , & Ivashkiv, L. B. (2011). Interleukin‐10‐induced gene expression and suppressive function are selectively modulated by the PI3K‐Akt‐GSK3 pathway. Immunology, 132, 567–577. 10.1111/j.1365-2567.2010.03402.x [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Arab, H. H. , Salama, S. A. , Abdelghany, T. M. , Omar, H. A. , Arafa, E. A. , Alrobaian, M. M. , & Maghrabi, I. A. (2017). Camel milk attenuates rheumatoid arthritis via inhibition of mitogen activated protein kinase pathway. Cellular Physiology and Biochemistry, 43, 540–552. 10.1159/000480527 [DOI] [PubMed] [Google Scholar]
  6. Bitto, A. , Altavilla, D. , Pizzino, G. , Irrera, N. , Pallio, G. , Colonna, M. R. , & Squadrito, F. (2014). Inhibition of inflammasome activation improves the impaired pattern of healing in genetically diabetic mice. British Journal of Pharmacology, 171, 2300–2307. 10.1111/bph.12557 [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Chandirasegaran, G. , Elanchezhiyan, C. , Ghosh, K. , & Sethupathy, S. (2017). Berberine chloride ameliorates oxidative stress, inflammation and apoptosis in the pancreas of streptozotocin induced diabetic rats. Biomedicine & Pharmacotherapy, 95, 175–185. 10.1016/j.biopha.2017.08.040 [DOI] [PubMed] [Google Scholar]
  8. Chien, Y. W. , Peng, H. C. , Chen, Y. L. , Pai, M. H. , Wang, H. Y. , Chuang, H. L. , & Yang, S. C. (2017). Different dietary proportions of fish oil regulate inflammatory factors but do not change intestinal tight junction ZO‐1 expression in ethanol‐fed rats. Mediators of Inflammation, 2017, 5801768. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Chunsangchan, M. , Chariyavilaskul, P. , Ketchart, W. , Prompila, N. , & Wittayalertpanya, S. (2017). Bioequivalence and pharmacokinetic/pharmacodynamic correlation of clopidogrel in healthy Thai subjects. International Journal of Clinical Pharmacology and Therapeutics, 55, 915–922. 10.5414/CP203054 [DOI] [PubMed] [Google Scholar]
  10. Cicek Ari, V. , Ilarslan, Y. D. , Erman, B. , Sarkarati, B. , Tezcan, I. , Karabulut, E. , … Berker, E. (2016). Statins and IL‐1β, IL‐10, and MPO levels in gingival crevicular fluid: Preliminary results. Inflammation, 39, 1547–1557. 10.1007/s10753-016-0390-7 [DOI] [PubMed] [Google Scholar]
  11. Curtis, M. J. , Alexander, S. , Cirino, G. , Docherty, J. R. , George, C. H. , Giembycz, M. A. , … Ahluwalia, A. (2018). Experimental design and analysis and their reporting II: Updated and simplified guidance for authors and peer reviewers. British Journal of Pharmacology, 175, 987–993. 10.1111/bph.14153 [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Curtis, M. J. , Bond, R. A. , Spina, D. , Ahluwalia, A. , Alexander, S. P. A. , Giembycz, M. A. , … McGrath, J. C. (2015). Experimental design and analysis and their reporting: New guidance for publication in BJP. British Journal of Pharmacology, 172, 3461–3471. 10.1111/bph.12856 [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Ding, Z. , Kim, S. , Dorsam, R. T. , Jin, J. , & Kunapuli, S. P. (2003). Inactivation of the human P2Y12 receptor by thiol reagents requires interaction with both extracellular cysteine residues, Cys17 and Cys270. Blood, 101, 3908–3914. 10.1182/blood-2002-10-3027 [DOI] [PubMed] [Google Scholar]
  14. Dong, J. , Wang, H. , Wu, G. , Zhao, J. , Zhang, L. , Zuo, L. , … Li, J. (2014). Oral treatment with SEW2871, a sphingosine‐1‐phosphate type 1 receptor agonist, ameliorates experimental colitis in interleukin‐10 gene deficient mice. Clinical and Experimental Immunology, 177, 94–101. 10.1111/cei.12304 [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Dorsam, R. T. , & Kunapuli, S. P. (2004). Central role of the P2Y12 receptor in platelet activation. The Journal of Clinical Investigation, 113, 340–345. 10.1172/JCI20986 [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Gorski, J. C. , Hall, S. D. , Becker, P. , Affrime, M. B. , Cutler, D. L. , & Haehner‐Daniels, B. (2000). In vivo effects of interleukin‐10 on human cytochrome P450 activity. Clinical Pharmacology and Therapeutics, 67, 32–43. 10.1067/mcp.2000.103860 [DOI] [PubMed] [Google Scholar]
  17. Gregersen, I. , Holm, S. , Dahl, T. B. , Halvorsen, B. , & Aukrust, P. (2016). A focus on inflammation as a major risk factor for atherosclerotic cardiovascular diseases. Expert Review of Cardiovascular Therapy, 14, 391–403. 10.1586/14779072.2016.1128828 [DOI] [PubMed] [Google Scholar]
  18. Gudbrandsdottir, S. , Hasselbalch, H. C. , & Nielsen, C. H. (2013). Activated platelets enhance IL‐10 secretion and reduce TNF‐α secretion by monocytes. Journal of Immunology, 191, 4059–4067. 10.4049/jimmunol.1201103 [DOI] [PubMed] [Google Scholar]
  19. Hansson, G. K. (2005). Inflammation, atheroscherosis, and coronary artery disease. The New England Journal of Medicine, 352, 1685–1695. 10.1056/NEJMra043430 [DOI] [PubMed] [Google Scholar]
  20. Hansson, G. K. (2009). Inflammatory mechanisms in atherosclerosis. Journal of Thrombosis and Haemostasis, 7(Suppl 1), 328–331. 10.1111/j.1538-7836.2009.03416.x [DOI] [PubMed] [Google Scholar]
  21. Harding, S. D. , Sharman, J. L. , Faccenda, C. , Southan, C. , Pawson, A. J. , Ireland, S. , … NC‐IUPHAR . (2018). The IUPHAR/BPS Guide to PHARMACOLOGY in 2018: Updates and expansion to encompass the new guide to IMMUNOPHARMACOLOGY. Nucleic Acids Research, 46(D1), D1091–D1106. 10.1093/nar/gkx1121 [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Heeschen, C. , Dimmeler, S. , Hamm, C. W. , Fichtlscherer, S. , Boersma, E. , Simoons, M. L. , … CAPTURE Study Investigators . (2003). Serum level of the antiinflammatory cytokine interleukin‐10 is an important prognostic determinant in patients with acute coronary syndromes. Circulation, 107, 2109–2114. 10.1161/01.CIR.0000065232.57371.25 [DOI] [PubMed] [Google Scholar]
  23. Hottz, E. D. , Medeiros‐de‐Moraes, I. M. , Vieira‐de‐Abreu, A. , de Assis, E. F. , Vals‐de‐Souza, R. , Castro‐Faria‐Neto, H. C. , … Bozza, P. T. (2014). Platelet activation and apoptosis modulate monocyte inflammatory responses in dengue. Journal of Immunology, 193, 1864–1872. 10.4049/jimmunol.1400091 [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Jia, Y. M. , Gu, T. T. , Ji, J. Z. , Tai, T. , Zhang, M. R. , Huang, B. B. , … Xie, H. G. (2018). Aspirin attenuates the bioactivation of and platelet response to vicagrel in mice. Journal of Cardiovascular Pharmacology, 72, 252–258. 10.1097/FJC.0000000000000622 [DOI] [PubMed] [Google Scholar]
  25. Jiang, J. , Chen, X. , & Zhong, D. (2017). Arylacetamide deacetylase is involved in vicagrel bioactivation in humans. Frontiers in Pharmacology, 8, 846 10.3389/fphar.2017.00846 [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Karazniewicz‐Lada, M. , Danielak, D. , Burchardt, P. , Kruszyna, L. , Komosa, A. , Lesiak, M. , & Główka, F. (2014). Clinical pharmacokinetics of clopidogrel and its metabolites in patients with cardiovascular diseases. Clinical Pharmacokinetics, 53, 155–164. 10.1007/s40262-013-0105-2 [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Kazui, M. , Nishiya, Y. , Ishizuka, T. , Hagihara, K. , Farid, N. A. , Okazaki, O. , … Kurihara, A. (2010). Identification of the human cytochrome P450 enzymes involved in the two oxidative steps in the bioactivation of clopidogrel to its pharmacologically active metabolite. Drug Metabolism and Disposition, 38, 92–99. 10.1124/dmd.109.029132 [DOI] [PubMed] [Google Scholar]
  28. Kilkenny, C. , Browne, W. , Cuthill, I. C. , Emerson, M. , & Altman, D. G. (2010). Animal research: Reporting in vivo experiments: The ARRIVE guidelines. British Journal of Pharmacology, 160, 1577–1579. 10.1111/j.1476-5381.2010.00872.x [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Li, C. , Iness, A. , Yoon, J. , Grider, J. R. , Murthy, K. S. , Kellum, J. M. , & Kuemmerle, J. F. (2015). Noncanonical STAT3 activation regulates excess TGF‐β1 and collagen I expression in muscle of stricturing Crohn's disease. Journal of Immunology, 194, 3422–3431. 10.4049/jimmunol.1401779 [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Li, D. , Zhu, J. Y. , Gao, J. , Wang, X. , Lou, Y. Q. , & Zhang, G. L. (2007). Polymorphisms of tumor necrosis factor‐α, interleukin‐10, cytochrome P450 3A5 and ABCB1 in Chinese liver transplant patients treated with immunosuppressant tacrolimus. Clinica Chimica Acta, 383, 133–139. 10.1016/j.cca.2007.05.008 [DOI] [PubMed] [Google Scholar]
  31. Li, X. , Liu, C. , Zhu, X. , Wei, H. , Zhang, H. , Chen, H. , … Ding, Y. (2018). Evaluation of tolerability, pharmacokinetics and pharmacodynamics of vicagrel, a novel P2Y12 antagonist, in healthy Chinese volunteers. Frontiers in Pharmacology, 9, 643 10.3389/fphar.2018.00643 [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Libby, P. (2002). Inflammation in atherosclerosis. Nature, 420, 868–874. 10.1038/nature01323 [DOI] [PubMed] [Google Scholar]
  33. Linke, B. , Schreiber, Y. , Picard‐Willems, B. , Slattery, P. , Nüsing, R. M. , Harder, S. , … Scholich, K. (2017). Activated platelets induce an anti‐inflammatory response of monocytes/macrophages through cross‐regulation of PGE2 and cytokines. Mediators of Inflammation, 2017, 1463216 10.1155/2017/1463216 [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. McDougle, D. R. , Watson, J. E. , Abdeen, A. A. , Adili, R. , Caputo, M. P. , Krapf, J. E. , … Das, A. (2017). Anti‐inflammatory ω‐3 endocannabinoid epoxides. Proceedings of the National Academy of Sciences of the United States of America, 114, E6034–E6043. 10.1073/pnas.1610325114 [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Mohammadi, S. , Seyedhoseini, F. S. , Asadi, J. , & Yazdani, Y. (2017). Effects of berberine on the secretion of cytokines and expression of genes involved in cell cycle regulation in THP‐1 monocytic cell line. Iranian Journal of Basic Medical Sciences, 20, 530–537. 10.22038/IJBMS.2017.8677 [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Murray, P. J. (2007). The JAK‐STAT signaling pathway: Input and output integration. Journal of Immunology, 178, 2623–2629. 10.4049/jimmunol.178.5.2623 [DOI] [PubMed] [Google Scholar]
  37. Osmancik, P. , Paulu, P. , Tousek, P. , Kocka, V. , & Widimsky, P. (2012). High leukocyte count and interleukin‐10 predict high on‐treatment platelet reactivity in patients treated with clopidogrel. Journal of Thrombosis and Thrombolysis, 33, 349–354. 10.1007/s11239-011-0659-5 [DOI] [PubMed] [Google Scholar]
  38. Pedicino, D. , Giglio, A. F. , Ruggio, A. , Massaro, G. , D'Aiello, A. , Trotta, F. , … Liuzzo, G. (2018). Inflammasome, T lymphocytes and innate‐adaptive immunity crosstalk: Role in cardiovascular disease and therapeutic perspectives. Thrombosis and Haemostasis, 118, 1352–1369. 10.1055/s-0038-1666860 [DOI] [PubMed] [Google Scholar]
  39. Profita, M. , Bonanno, A. , Siena, L. , Ferraro, M. , Montalbano, A. M. , Pompeo, F. , … Gjomarkaj, M. (2008). Acetylcholine mediates the release of IL‐8 in human bronchial epithelial cells by a NF‐κB/ERK‐dependent mechanism. European Journal of Pharmacology, 582, 145–153. 10.1016/j.ejphar.2007.12.029 [DOI] [PubMed] [Google Scholar]
  40. Qiu, Z. , Li, N. , Song, L. , Lu, Y. , Jing, J. , Parekha, H. S. , … Chen, X. (2014). Contributions of intestine and plasma to the presystemic bioconversion of vicagrel, an acetate of clopidogrel. Pharmaceutical Research, 31, 238–251. 10.1007/s11095-013-1158-5 [DOI] [PubMed] [Google Scholar]
  41. Raggi, P. , Genest, J. , Giles, J. T. , Rayner, K. J. , Dwivedi, G. , Beanlands, R. S. , & Gupta, M. (2018). Role of inflammation in the pathogenesis of atherosclerosis and therapeutic interventions. Atherosclerosis, 276, 98–108. 10.1016/j.atherosclerosis.2018.07.014 [DOI] [PubMed] [Google Scholar]
  42. Ramji, D. P. , & Davies, T. S. (2015). Cytokines in atherosclerosis: Key players in all stages of disease and promising therapeutic targets. Cytokine & Growth Factor Reviews, 26, 673–685. 10.1016/j.cytogfr.2015.04.003 [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Ross, R. (1986). The pathogenesis of atherosclerosis—An update. The New England Journal of Medicine, 314, 488–500. 10.1056/NEJM198602203140806 [DOI] [PubMed] [Google Scholar]
  44. Ross, R. (1999). Atherosclerosis—An inflammatory disease. The New England Journal of Medicine, 340, 115–126. 10.1056/NEJM199901143400207 [DOI] [PubMed] [Google Scholar]
  45. Ruparelia, N. , Chai, J. T. , Fisher, E. A. , & Choudhury, R. P. (2017). Inflammatory processes in cardiovascular disease: A route to targeted therapies. Nature Reviews. Cardiology, 14, 133–144. 10.1038/nrcardio.2016.185 [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Sahay, B. , Bashant, K. , Nelson, N. L. J. , Patsey, R. L. , Gadila, S. K. , Boohaker, R. , et al. (2018). Induction of interleukin 10 by borrelia burgdorferi is regulated by the action of CD14‐dependent p38 mitogen‐activated protein kinase and cAMP‐mediated chromatin remodeling. Infection and Immunity, 86, e00781–17 10.1128/IAI.00781-17 [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Savi, P. , Herbert, J. M. , Pflieger, A. M. , Dol, F. , Delebassee, D. , Combalbert, J. , … Maffrand, J. P. (1992). Importance of hepatic metabolism in the antiaggregating activity of the thienopyridine clopidogrel. Biochemical Pharmacology, 44, 527–532. 10.1016/0006-2952(92)90445-O [DOI] [PubMed] [Google Scholar]
  48. Savi, P. , Zachayus, J. L. , Delesque‐Touchard, N. , Labouret, C. , Herve, C. , Uzabiaga, M. F. , … Herbert, J. M. (2006). The active metabolite of clopidogrel disrupts P2Y12 receptor oligomers and partitions them out of lipid rafts. Proceedings of the National Academy of Sciences of the United States of America, 103, 11069–11074. 10.1073/pnas.0510446103 [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Shan, J. , Zhang, B. , Zhu, Y. , Jiao, B. , Zheng, W. , Qi, X. , … Sun, H. (2012). Overcoming clopidogrel resistance: Discovery of vicagrel as a highly potent and orally bioavailable antiplatelet agent. Journal of Medicinal Chemistry, 55, 3342–3352. 10.1021/jm300038c [DOI] [PubMed] [Google Scholar]
  50. Shibata, M. , Endo, S. , Inada, K. , Kuriki, S. , Harada, M. , Takino, T. , … Hiramori, K. (1997). Elevated plasma levels of interleukin‐1 receptor antagonist and interleukin‐10 in patients with acute myocardial infarction. Journal of Interferon & Cytokine Research, 17, 145–150. 10.1089/jir.1997.17.145 [DOI] [PubMed] [Google Scholar]
  51. Tai, T. , Mi, Q. Y. , Ji, J. Z. , Yin, Q. , Pan, Y. Q. , Zhang, M. R. , … Xie, H. G. (2016). Enhanced platelet response to clopidogrel in Abcc3‐deficient mice due to its increased bioactivation. Journal of Cardiovascular Pharmacology, 68, 433–440. 10.1097/FJC.0000000000000428 [DOI] [PubMed] [Google Scholar]
  52. Vaccarezza, M. , Balla, C. , & Rizzo, P. (2018). Atherosclerosis as an inflammatory disease: Doubts? No more. International Journal of Cardiology Heart & Vasculature, 19, 1–2. 10.1016/j.ijcha.2018.03.003 [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Vogel, C. F. , Chang, W. L. , Kado, S. , McCulloh, K. , Vogel, H. , Wu, D. , … Gershwin, M. E. (2016). Transgenic overexpression of aryl hydrocarbon receptor repressor (AhRR) and AhR‐mediated induction of CYP1A1, cytokines, and acute toxicity. Environmental Health Perspectives, 124, 1071–1083. 10.1289/ehp.1510194 [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Wan, X. , Huang, W. J. , Chen, W. , Xie, H. G. , Wei, P. , Chen, X. , & Cao, C. C. (2014). IL‐10 deficiency increases renal ischemia‐reperfusion injury. Nephron. Experimental Nephrology, 128, 37–45. 10.1159/000366130 [DOI] [PubMed] [Google Scholar]
  55. Wang, H. , Shi, P. , Huang, C. , & Liu, Q. (2016). Maresin 1 ameliorates iron-deficient anemia in IL‐10(‐/‐) mice with spontaneous colitis by the inhibition of hepcidin expression through the IL‐6/STAT3 pathway. The American Journal of Translational Research, 8, 2758–2766. [PMC free article] [PubMed] [Google Scholar]
  56. Williams, L. , Bradley, L. , Smith, A. , & Foxwell, B. (2004). Signal transducer and activator of transcription 3 is the dominant mediator of the anti‐inflammatory effects of IL‐10 in human macrophages. Journal of Immunology, 172, 567–576. 10.4049/jimmunol.172.1.567 [DOI] [PubMed] [Google Scholar]
  57. Xie, H. G. , Jia, Y. M. , Tai, T. , & Ji, J. Z. (2017). Overcoming clopidogrel resistance: Three promising novel antiplatelet drugs developed in China. Journal of Cardiovascular Pharmacology, 70, 356–361. 10.1097/FJC.0000000000000529 [DOI] [PubMed] [Google Scholar]
  58. Xie, H. G. , Zou, J. J. , Hu, Z. Y. , Zhang, J. J. , Ye, F. , & Chen, S. L. (2011). Individual variability in the disposition of and response to clopidogrel: Pharmacogenomics and beyond. Pharmacology & Therapeutics, 129, 267–289. 10.1016/j.pharmthera.2010.10.001 [DOI] [PubMed] [Google Scholar]
  59. Yan, S. L. , & Yin, M. C. (2007). Protective and alleviative effects from 4 cysteine‐containing compounds on ethanol‐induced acute liver injury through suppression of oxidation and inflammation. Journal of Food Science, 72, S511–S515. 10.1111/j.1750-3841.2007.00449.x [DOI] [PubMed] [Google Scholar]
  60. Yin, Q. , Tai, T. , Ji, J. Z. , Mi, Q. Y. , Zhang, M. R. , Huang, W. J. , … Xie, H. G. (2016). Interleukin‐10 does not modulate clopidogrel platelet response in mice. Journal of Thrombosis and Haemostasis, 14, 596–605. 10.1111/jth.13238 [DOI] [PubMed] [Google Scholar]
  61. Yin, X. J. , Ma, J. Y. , Antonini, J. M. , Castranova, V. , & Ma, J. K. (2004). Roles of reactive oxygen species and heme oxygenase‐1 in modulation of alveolar macrophage‐mediated pulmonary immune responses to Listeria monocytogenes by diesel exhaust particles. Toxicological Sciences, 82, 143–153. 10.1093/toxsci/kfh255 [DOI] [PubMed] [Google Scholar]
  62. Yip, H. K. , Youssef, A. A. , Chang, L. T. , Yang, C. H. , Sheu, J. J. , Chua, S. , … Hang, C. L. (2007). Association of interleukin‐10 level with increased 30‐day mortality in patients with ST‐segment elevation acute myocardial infarction undergoing primary coronary intervention. Circulation Journal, 71, 1086–1091. 10.1253/circj.71.1086 [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Figure S1.

Typical chromatograms of (A) blank plasma, (B) plasma spiked with standards at lower limit of quantification, plasma samples obtained 15 min after oral administration of vicagrel in (C) IL‐10 +/+ (WT) and (D) IL‐10 −/− (KO) mice. In each panel, H4 and IS go with left and right traces, respectively.

Click here for additional data file. (742.6KB, pdf)

Figure S2.

The representative tracing of ADP‐induced platelet aggregation in IL‐10 WT or KO mice after oral administration of vehicle control (CMC‐Na), 1.25, 2.5, and 5 mg·kg−1 of vicagrel, respectively. Ctrl, control. ADP, used as a final concentration of 10 μM in a testing chamber.

Click here for additional data file. (1.2MB, pdf)

Articles from British Journal of Pharmacology are provided here courtesy of The British Pharmacological Society

RESOURCES