Characterization of Patients With Angioscopically-Detected In-Stent Mural Thrombi

– Genetics of Clopidogrel Responsiveness and Generations of Drug-Eluting Stents –

Abstract

Background: The loss-of-function genotype of cytochrome P450 2C19 (CYP2C19) has been proposed as a risk factor for stent thrombosis in patients with drug-eluting stent implantation. The aim of this study was to clarify the clinical features of patients with angioscopically-detected in-stent mural thrombi (ISMT).

Methods and Results: Enrolled were 100 stented segments in 55 patients with stable angina (20 bare-metal stents; 39 Cypher sirolimus-eluting stents [SES]; 26 Endeavor zotarolimus-eluting stents [ZES]; 13 Xience V everolimus-eluting stents; and 2 Nobori biolimus-eluting stents). Dual antiplatelet therapy (100 mg aspirin+75 mg clopidogrel once daily) had been continued since stenting. A poor metabolizer (PM) of clopidogrel was defined as a homozygote of CYP2C19 loss-of-function alleles. Coronary angioscopy revealed ISMT in 6 patients (5 SES, 1 ZES). Between the ISMT group and control group (n=49), there were no significant differences with regards to the VerifyNow P2Y₁₂ platelet function assay or in-stent endothelial coverage grade. Exact logistic regression analyses with stepwise forward selection at a significance level of 0.10 were performed to reveal predictive variables for ISMT (respectively: odds ratio, 95% confidence interval, P value: CYP2C19 PM genotype (3.28, 0.88–24.80, 0.09), SES implantation (3.37, 0.90–28.09, 0.08), and presence of yellow plaque (3.69, 1.14–25.70, 0.02).

Conclusions: Patients with ISMT were characterized by SES implantation, poor clopidogrel metabolism, and in-stent yellow plaque. (Circ J 2015; 79: 85–90)

Stent thrombosis (ST) is a serious issue, especially with 1st-generation drug-eluting stents (DES) such as the sirolimus-eluting stent (SES).^1,2 To prevent this life-threatening event, long-term dual antiplatelet therapy (DAPT: aspirin+clopidogrel) has been strongly recommended.³ The newer generations of DES appeared to be safer in terms of very late ST.⁴

Editorial p 41

Clopidogrel is a pro-drug that is activated by a liver enzyme, cytochrome P450 2C19 (CYP2C19).⁵ Because the activity of CYP2C19 depends on its loss-of-function polymorphism,⁶ the genotype of CYP2C19 is a proposed risk factor for ST.⁷ Proton-pump inhibitors (PPIs) slow down clopidogrel activation because of their competitive metabolism by CYP2C19.⁸

In this study, we characterized patients with in-stent mural thrombus (ISMT) using coronary angioscopy and we discuss the genetics of clopidogrel responsiveness and the generations of DES.

Methods

Patients

From June 2011 to October 2012, 567 patients underwent coronary angiography and of them, 299 consecutive patients with previous coronary stenting were candidates for this study. DAPT (100 mg aspirin+75 mg clopidogrel once daily) had been continued since the stenting procedure. Coronary angiography was performed at least 6 months after the stenting. Written informed consent was obtained from 63 patients prior to coronary angiography. Blood was sampled for genetic analysis and VerifyNow P2Y₁₂ assay on the morning of the day of coronary angiography. Eight patients were withdrawn because angiography revealed in-stent restenosis, so coronary angioscopy was performed subsequently in 55 patients with stable angina (43 males, 12 females; mean age 67±14 years; Figure 1). Prevalence of conventional coronary risk factors was 38% for diabetes mellitus, 87% for hypertension, 84% for dyslipidemia, and 49% for current smoking. Patients were optimally treated with antidiabetic agents, antihypertensive agents, and/or statins; 24 patients (44%) had been administered PPIs (ie, omeprazole, lansoprazole, or rabeprazole) (Table 1). The study protocol was approved by the Ethical Committee of Higashi-osaka City General Hospital.

Figure 1.

Flow diagram of the enrollment of patients. Of 299 consecutive patients with previous coronary stenting, written informed consent was given by 63 patients prior to follow-up coronary angiography. On the same day (blue shaded box), blood was sampled for genetic analysis and VerifyNow P2Y₁₂ assay in the morning of the day of angiography. Excluded were 8 patients in whom angiography revealed in-stent restenosis and who underwent subsequent coronary revascularization. Coronary angioscopy was performed in 55 patients.

Table 1. Clinical Features of 55 Patients Implanted With 100 Stents

	CYP2C19 genotype			P value
	EM	IM	PM
n	15	29	11
Age (years)	70.3±7.2	66.9±10.5	73.1±5.5	0.13
Sex (M/F)	11/4	24/5	8/3	0.69
Diabetes mellitus [n (%)]	5 (33)	10 (34)	6 (55)	0.46
Hypertension [n (%)]	14 (93)	24 (83)	10 (91)	0.60
Dyslipidemia [n (%)]	12 (80)	25 (86)	9 (82)	0.86
Current smoking [n (%)]	9 (60)	14 (48)	4 (36)	0.49
PPI administration [n (%)]	7 (47)	12 (41)	5 (45)	0.85
VerifyNow P2Y12 (PRU)	236±87	232±81	282±62	0.20

P values were obtained for categorical variables by chi-square test and for continuous variables by one-way analysis of variance.

EM, extensive metabolizer of clopidogrel; IM, intermediate metabolizer; PM, poor metabolizer; PPI, proton-pump inhibitor; PRU, P2Y₁₂ reaction units.

In total, 100 stents were successfully implanted: 20 bare-metal stents (BMS); 39 SES (Cypher^®, Cordis Co, Bridgewater, NJ, USA); 26 zotarolimus-eluting stents (ZES) (Endeavor^®, Medtronic Co, Minneapolis, MN, USA); 13 everolimus-eluting stents (EES) (Xience-V^®, Abbott-vascular Co Abbott Park, IL, USA); and 2 biolimus-eluting stents (BES) (Nobori^®, Terumo, Tokyo, Japan) (Table 2). Plural stents were sometimes implanted in tandem with minimal overlap and in such cases (1) the same type of DES was implanted in the coronary artery or (2) a proximal BMS was combined with a distal DES. The number of stents implanted per patient ranged from 1 to 5 (mean, 1.8 stents).

Table 2. Characteristics of the Stented Segments in 55 Patients on Dual Antiplatelet Therapy

	BMS	SES	ZES	EES	BES
n	20	39	26	13	2
Stent diameter (mm)*	3.68±0.49	3.09±0.36	3.06±0.38	3.27±0.31	3.5, 2.5
Stent length (mm)†	18.6±6.3	24.5±6.5	20.0±6.6	20.4±7.8	14, 14
Years after stenting‡	1.3 (1, 3.3)	3 (1, 6.1)	1 (0.6, 3)	1 (0.5, 1.2)	1, 1
RCA/LMT/LAD/LCX§	12/2/4/2	11/1/22/5	12/1/7/6	5/0/5/3	0/0/1/1
Endothelial CG**	3 (2, 3)	1 (0, 3)	2.5 (1, 3)	1 (1, 3)	1.5 (0, 1)
Yellow plaque	1	10	5	0	0
In-stent mural thrombus	0	5	1	0	0

Data are expressed as mean±SD except for years after stenting and endothelial CG (median, maximum and minimum). For BES (n=2), raw data are shown. Coronary angioscopy revealed the presence of yellow plaque, endothelial CG, and in-stent mural thrombus.

*P values calculated by Tukey’s test. BMS vs. SES, <0.0001; BMS vs. ZES, <0.0001; BMS vs. EES, 0.02; SES vs. ZES, 0.99; SES vs. EES, 0.48; and ZES vs. EES, 0.39. ^†P values calculated by Tukey’s test. BMS vs. SES, 0.01; BMS vs. ZES, 0.88; BMS vs. EES, 0.87; SES vs. ZES, 0.05; SES vs. EES, 0.23; and ZES vs. EES, 1.00. ^‡P values calculated by Steel-Dwass test. BMS vs. SES, 0.019; BMS vs. ZES, 0.005; BMS vs. EES, 0.060; SES vs. ZES. <0.0001; SES vs. EES, <0.0001 and ZES vs. EES, 0.99. ^§Fisher’s exact test revealed no significant difference among the stent groups (P=0.11). **P values calculated by Steel-Dwass test. BMS vs. SES, <0.0001; BMS vs. ZES, 0.017; BMS vs. EES, <0.0001; SES vs. ZES, 0.0007; SES vs. EES, 0.846; and ZES vs. EES, 0.051. Because of the small number, BES was excluded from the comparative analyses of the stent groups.

BES, biolimus-eluting stent; BMS, bare-metal stent; CG, coverage grade; EES, everolimus-eluting stent; LAD, left anterior descending artery; LCX, left circumflex artery; LMT, left main trunk coronary artery; RCA, right coronary artery; SES, sirolimus-eluting stent; ZES, zotarolimus-eluting stent.

Coronary Angioscopy

Coronary angioscopy (Visible^®, Fibertech, Tokyo, Japan) was performed safely with manual pullback under transparent low-molecular-weight dextran solution flush as described previously.^9–11 Coronary angioscopy is the most powerful technology for characterizing the inner surface of the human coronary artery in vivo. It visualizes not only ISMT but also the presence of yellow plaque. Angioscopy also semiquantifies the extent of endothelial coverage on stent struts, which we classified into 4 grades:^12,13 grade 0, no coverage, struts barely exposed in the vessel lumen; grade 1, thin coverage, struts visible transparently; grade 2, thick coverage, struts still bulging into vessel lumen and visible translucently; and grade 3, struts embedded in thick neointima and angioscopically invisible.

Genotyping of CYP2C19

Enzyme activity of CYP2C19 is affected by 2 single-nucleotide polymorphisms (SNPs) at 636 in exon 4 and 681 in exon 5. Wild-type allele *1 is defined as G at 636 and G at 681. The loss-of-function (LOF) allele *2 is defined as G at 636 and A at 681. LOF allele *3 is defined as A at 636 and G at 681. An extensive metabolizer (EM) of clopidogrel is defined as a homozygote of wild-type alleles (*1*1), a poor metabolizer (PM) is defined as a homozygote of LOF alleles (*2*2, *2*3, or *3*3), and an intermediate metabolizer (IM) is defined as a heterozygote of wild-type and LOF alleles (*1*2, *1*3). We requested BML Co (Tokyo, Japan) to analyze the SNPs by the Invader assay.¹⁴

VerifyNow P2Y₁₂ Assay

We used the VerifyNow P2Y₁₂ assay kit (Accumetrics, San Diego, CA, USA) to evaluate residual platelet aggregation activity in blood sampled from the patients receiving DAPT. Briefly, ADP-triggered platelet aggregation was monitored through light transmittance according to the manufacturer’s instruction. P2Y₁₂ reaction units (PRU) >256 has been proposed as the cut-off level to detect carriers of the CYP2C19 LOF alleles in Japanese.¹⁵

Statistical Analysis

Continuous variables are expressed as mean±SD or median (minimum and maximum). Ordinal variables are expressed as median (minimum and maximum). Categorical data are expressed as the number of patients and percentages, and compared by chi-square test. Differences among more than 3 groups were compared by Fisher’s exact test, Tukey’s test, and Steel-Dwass test, or one-way analysis of variance (ANOVA). Univariate and multivariate exact logistic regression analyses were performed to assess predictive variables for ISMT. For the multivariate analysis, variables were selected by forward stepwise manner at a significance level of 0.10. In addition, generalized estimating equations were performed to assess the robustness of the conclusion from multivariate logistic regression analysis (data not shown). P<0.05 was considered statistically significant. Statistical analyses were performed using SAS software for Windows version 9.3 (SAS Institute, Cary, NC, USA).

Results

Characteristics of Patients With Each CYP2C19 Genotype

In total, 55 patients with coronary stents had been administered DAPT; 20% of them were PMs of clopidogrel (Table 1). Residual platelet aggregation activity, evaluated by VerifyNow P2Y₁₂ assay, tended to be higher in the PM group (282±62 PRU) than in the IM group (232±81 PRU, P=0.07) or EM group (236±87 PRU, P=0.15).

Characteristics of Stented Coronary Segments

A total of 100 stents of the previously described types were implanted in the 55 patients (Table 2). Although the number of years after stenting was longest in the SES group (3 years as median), coronary angioscopy found that the endothelial coverage grade for SES was less than that for BMS (P<0.0001) and ZES (P=0.0007). Angioscopy also found yellow plaque and ISMT in the SES and ZES groups, but not in the BMS, EES, or BES groups. The incidence of yellow plaque was highest in the SES group (10/39) compared with the ZES group (5/26, P<0.01), as was the incidence of ISMT (SES group 5/39 vs. ZES group 1/26; P<0.01).

Characteristics of Patients With ISMT

We found ISMT in 6 patients (Table 3), 2 of whom are shown in Figure 2. The 6 patients comprised 5 SES and 1 ZES; 3 PM, 2 IM and 1 EM of clopidogrel; 4 with and 2 without yellow plaque; and 3 with concomitant use of PPI and 3 without. There was no significant difference in the VerifyNow P2Y₁₂ assay between the ISMT group (238±101, n=6) and patients without ISMT (244±78 PRU, n=49). There was no significant difference in endothelial coverage grade between stents with mural thrombi (1, 0 –3; n=6) and those without thrombi (2, 0 –3; median, minimum –maximum, n=94; P=0.24).

Table 3. Clinical Features of the 6 Patients With In-Stent Mural Thrombus

	Case
	1	2	3	4	5	6
Stent type	SES	SES	SES	SES	ZES	SES
PPI administration	No	Yes	Yes	No	Yes	No
VerifyNow P2Y12 (PRU)	131	317	156	156	358	310
CYP2C19 genotype	IM	EM	IM	PM	PM	PM
Years after stenting	3	3	3.5	3	1	3
Endothelial CG	1	3	1	2	3	1
Yellow plaque	Yes	No	Yes	Yes	Yes	No
Hypertension	No	Yes	No	Yes	Yes	Yes

Coronary angioscopy revealed subclinical stent thrombosis in 5 patients implanted with SES and in 1 patient implanted with a ZES. Abbreviations as in Tables 1,2.

Figure 2.

Representative cases (nos. 4 and 6 in Table 3) of in-stent mural thrombi on coronary angiography (A,C) and angioscopy (B,D). Left coronary arteries were viewed from the left anterior oblique 45° and cranial 30° (A) and from the right anterior oblique 30° and caudal 30° (B). White lines, crossing the left anterior descending in (A) and left main trunk in (C), indicate the stenting sites observed on coronary angioscopy (B,D, respectively). Angioscopy found yellow plaque in case 4 (B), glossy stent strut in case 6 (D, double arrowheads), and in-stent mural thrombi attached to the vessel wall (arrowhead in B,D).

To assess the independent predictors of ISMT, we first conducted univariate exact logistic regression analysis for the 100 stented segments (Table 4). The examined parameters included CYP2C19 genotype, residual platelet activity, PPI administration, angioscopic findings, stent type, years after stenting, and conventional coronary risk factors (diabetes mellitus, hypertension, dyslipidemia, and current smoking). For the multivariate analysis, 3 predictive variables were selected at a significance level of 0.10 (odds ratio, 95% confidence interval; P value): CYP2C19 PM genotype (3.28, 0.88–24.80, 0.09), SES implantation (3.37, 0.90–28.09, 0.08), and presence of yellow plaque (3.69, 1.14–25.70, 0.02).

Table 4. Exact Logistic Regression Analysis to Assess Predictors of In-Stent Mural Thrombus

	Univariate			Multivariate
	OR	95% CI	P value	OR	95% CI	P value
VerifyNow P2Y12 >256 PRU	0.94	0.33–2.63	1.00
CYP2C19 PM	2.11	0.74–6.01	0.19	3.28	0.88–24.80	0.09
PPI administration	0.53	0.05–3.87	0.76
BMS	0.68	0.00–1.61	0.50
SES	2.94	0.96–20.59	0.06	3.37	0.90–28.09	0.08
ZES	0.74	0.11–2.31	1.00
EES	0.89	0.00–2.11	0.85
BES	2.57	0.00–7.61	1.00
Yellow plaque	3.61	1.29–12.61	0.01	3.69	1.14–25.70	0.02
Years after stenting (per year)	1.24	0.73–1.98	0.40
Endothelial CG (per unit)	0.62	0.21–1.62	0.39
Diabetes mellitus	0.81	0.24–2.19	0.96
Hypertension	0.41	0.14–1.46	0.18
Dyslipidemia	0.97	0.31–6.92	1.00
Current smoking	1.04	0.37–2.92	1.00

We confirmed the result by generalized estimating equations. CI, confidence interval; OR, odds ratio. Other abbreviations as in Tables 1,2.

Discussion

ST after DES implantation is a serious and sometimes fatal event. In this study, we characterized patients with ISMT detected by coronary angioscopy. Among 55 patients with implantation of various types of coronary stent, we found 6 patients with ISMT (Table 3), who included 3 PMs of clopidogrel, 4 with in-stent yellow plaque, 5 with SES implantation, and 4 with hypertension.

The effect of clopidogrel resistance on clinical outcome in patients with DES implantation has been controversial.^16–18 We used the VerifyNow P2Y₁₂ assay to assess how CYP2C19 PM genotype affected residual platelet aggregation activity in patients with DAPT. Residual platelet function tended to be higher in patients with the PM genotype than in patients with the other genotypes (Table 1). Bhatt et al reported that concomitant use of omeprazole in a Caucasian population did not increase cardiovascular events in patients with coronary artery disease who received DAPT.¹⁹ Prevalence of the CYP2C19 PM genotype, however, is much higher in Asian populations than in Caucasian populations (≈20% vs. 2%).^20,21 Thus, the effect of CYP2C19 genotype on Japanese patients with concomitant use of clopidogrel and PPI remains to be evaluated in a larger population.

Not only platelet aggregation activity but also in-stent luminal characteristics might cause very late ST.²² DES consist of metal struts, a biosuppressive drug, and solvent polymer. The drug is slowly released from the polymer, mainly into the perivascular tissue where it suppresses cell proliferation. Coronary angioscopy visualizes delayed endothelialization on the stent struts of DES^12,13,23 (see SES in Table 2). Uncovered stent struts might be thrombogenic. The solvent polymer remains on the stent struts after drug release and can cause chronic inflammation in the vessel wall, which facilitates in-stent neoatherosclerosis.²⁴ Rupture of the lipid-rich neoatherosclerosis might cause very late ST.^10,25 Coronary angioscopy also visualizes in-stent yellow plaque, especially in the SES-implanted patients of the present study (Table 2), which is comparable with results in previous reports.^26,27 Our logistic regression analyses revealed that the presence of yellow plaque but not the endothelial coverage grade was an independent predictor for ISMT (Table 4).

Certain generations of DES are detrimental for the development of ISMT. Optical coherence tomography (OCT) also can be used to detect intracoronary thrombus and Sawada et al reported that the prevalence of ISMT by OCT was 31% in patients implanted with a 1st-generation DES.²⁸ Recent work in the same laboratory found that OCT underestimated subclinical in-stent thrombi in comparison with coronary angioscopy.²⁹ We angioscopically-detected 5 cases of ISMT in 39 SES (12.8%, Table 2). The discrepancy in prevalence might be attributed to the still immature definition of thrombus by OCT. We reported only 1 case of ISMT in 41 newer generation DES (2.4%, Table 2), which is comparable to a recent report that the newer generations of DES are safer than the 1st generation.³⁰

Study Limitations

First, we observed ISMT using coronary angioscopy, but ST is not exactly the same phenomenon as ISMT. Second, the number of years after stenting differed among the 5 stent groups. Multivariate exact logistic regression analysis indicated that this parameter did not significantly affect ISMT (Table 4). Third, the number of stents implanted per patient varied (mean, 1.8) and to take into consideration the effect of multiple stenting in a single patient on the incidence of ISMT, we performed generalized estimating equations, which gave a similar result to that obtained by logistic regression analysis. Finally, we studied a relatively small number of patients, especially those implanted with BES. Further study of a larger number of patients will be needed for the newer generations of DES.

In conclusion, patients with ISMT were characterized by SES implantation, poor clopidogrel metabolism, and in-stent yellow plaque.

Acknowledgments

We thank Drs Tsuyoshi Nakata, Kenji Kawai, and Takashi Takagi, and Shohei Yoshima for their encouragement to pursue this study. This work was supported by a research grant from Higashi-osaka City General Hospital to Drs Kijima and Ichikawa.

Disclosures

Authors have no conflicts of interest to be disclosed.

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