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Keywords:

  • biological resistance to clopidogrel;
  • coronary angioplasty;
  • major adverse cardiovascular events;
  • platelet reactivity;
  • vasodilator-stimulated phosphoprotein phosphorylation

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Disclosure of Conflict of Interests
  9. References

Summary. Background: Despite dual antiplatelet therapy, the rate of major adverse cardiovascular events (MACE) after percutaneous coronary angioplasty remains high. Studies have shown interindividual variations in response to clopidogrel. Furthermore, there is an apparent link between clinical outcomes and clopidogrel resistance. Objectives: To investigate the value of platelet reactivity index (PRI), assessed by vasodilator-stimulated phosphoprotein (VASP) phosphorylation analysis, for predicting MACE after percutaneous coronary intervention (PCI) with stent implantation.Methods: A prospective monocentric study was performed on 144 patients undergoing PCI. PR was evaluated by VASP phosphorylation analysis 24 h after they received a 300-mg loading dose of clopidogrel. MACE were recorded during a 6-month follow-up. Patients were divided into quintiles according to PRI, as assessed by VASP analysis. The receiver operating characteristic (ROC) curve served to determine the optimal cut-off value of VASP analysis to detect MACE.Results: Of the 144 patients, 34% had stable angina pectoris, 40% silent ischemia, and 26% low-risk non-ST-segment elevation acute coronary syndrome. During the follow-up, 21 MACE were observed. Patients in quintile 1 of VASP analysis had a significantly lower risk of MACE as compared with those among the four higher quintiles (0 vs. 21, P < 0.01). ROC curve analysis of VASP showed an optimal cut-off value of 50% PR to exclude MACE. The negative predictive value of the test was 100%.Conclusions: VASP phosphorylation analysis can evaluate the individual response to clopidogrel loading dose prior to PCI and predict postprocedural MACE.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Disclosure of Conflict of Interests
  9. References

During the last decade, angioplasty has become the most used form of coronary revascularization. Systematic stent delivery has diminished the occurrence of procedure-related complications, including occlusive dissections and in-stent restenosis [1]. In addition, dual antiplatelet therapy with clopidogrel and aspirin has decreased the number of major adverse cardiovascular events (MACE) after percutaneous coronary intervention (PCI) [2–4].

Despite these improvements, the incidence of MACE after PCI remains high [2–4]. These clinical findings have raised concern about antiplatelet therapy. Aspirin resistance is acknowledged [5]. More recently, the concept of biological resistance to clopidogrel has also emerged. Clopidogrel is a platelet ADP receptor antagonist that selectively and irreversibly inhibits the activation of the ADP-dependent P2Y12 receptor. This action indirectly inhibits the activation of the glycoprotein (GP)IIb/IIIa receptor, which participates in the final common pathway of platelet aggregation [6]. Moreover, interindividual variability in platelet response to clopidogrel is known to be large. Bad responders represent between 10% and 40% of patients receiving therapy, depending on the tests and thresholds used [7–9].

More recently, studies have reported a link between biological resistance to clopidogrel and therapeutic failure, defined by stent thrombosis or MACE, in coronary artery disease (CAD) patients undergoing PCI with stent implantation [10–13]. These studies used light-transmission aggregometry, which is the gold standard for the assessment of platelet function. However, there is no consensus about the concentration of ADP agonist and the definition of clopidogrel resistance [11–13]. Moreover, this test is not specific for the P2Y12 ADP receptor and is technically challenging.

In contrast, a flow cytometric assay based on the measurement of the level of the vasodilator-stimulated phosphoprotein (VASP) phosphorylation/dephosphorylation ratio was recently validated [14,15]. VASP is an intracellular actin-regulatory protein, which is a substrate of both cAMP-dependent and cGMP-dependent protein kinases. Analysis of the VASP phosphorylation ratio can be used for measuring various signal transduction processes, including dephosphorylation following P2Y12 ADP receptor activation and its reversal by P2Y12 antagonists. The degree of VASP phosphorylation correlates very closely with the inhibition of platelet aggregation. This test therefore specifically assesses the inhibition of platelet reactivity index (PRI) linked to P2Y12 ADP receptor inhibition [14,15].

The aim of the study was to determine the value of PR, assessed by VASP analysis, for predicting MACEs after PCI with stent implantation in CAD patients.

Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Disclosure of Conflict of Interests
  9. References

Study design and patients

A monocentric, prospective study was undertaken in the cardiology department of the university hospital Nord of Marseille. The principle of the study was in accordance with the declaration of Helsinki, and the protocol was approved by the local ethics committee. Informed consent was obtained from all participants.

Consecutive patients admitted for PCI between November 2004 and November 2005 in our institution were eligible. Inclusion criteria were the need for PCI with stent implantation for: (i) silent ischemia or stable angina pectoris confirmed by stress test or thallium-201 myocardial scintigraphy; (ii) clinical symptoms compatible with low-risk non-ST-segment elevation acute coronary syndrome (NSTE-ACS), defined according to the ACC/AHA guidelines: myocardial ischemia within 12 h before admission and at least one of the following: a new finding of ST-segment depression >0.05 mV, T-wave inversion >0.3 mV in at least two leads, or coronary disease documented by a history of catheterization, revascularization, or myocardial infarction [16].

The exclusion criteria were a history of bleeding diathesis, persistent ST-segment elevation ACS, high-risk non-ST-segment elevation ACS according to ACC/AHA guidelines [16], elevated levels of cardiac markers, New York Heart Association functional class III or IV, contraindications to antiplatelet therapy, platelet count <100 g L−1, creatinine clearance <25 mL min−1, and GPIIb/IIIa inhibitor use.

Blood samples

Blood samples for PRI testing were drawn in the catheterization unit through an atraumatic venipuncture of the antecubital vein before PCI and at least 24 h after administration of a clopidogrel loading dose and aspirin. The first few milliliters of blood were discarded to avoid platelet activation induced by needle puncture, and blood was immediately collected in vacutainer tubes containing 3.8% trisodium citrate, filled to capacity. The tubes were then inverted three to five times for gentle mixing, and taken immediately to the hemostasis unit.

Angioplasty procedure

PCI was carried out according to international guidelines, using a standard technique, through the femoral route [16]. The interventional cardiologist was not aware of VASP analysis results when performing coronary angioplasty. Systematic stent implantation was conducted in all patients. Either eluting stents or bare metal stents were used, according to the French Cardiology Society guidelines. An i.v. bolus of UFH (100 IU kg−1) was administered at the beginning of the procedure. The sheath was removed immediately after the end of the procedure. Routine care was given before and after the procedure to all patients, including pretreatment with a loading dose of clopidogrel (300-mg initial oral bolus) 24 h before the procedure, followed by 75 mg per day for at least 6 months. In addition, all patients received aspirin 160 mg daily, starting at least 12 h before stenting.

VASP phosphorylation analysis

VASP phosphorylation analysis was performed within 24 h by an experienced investigator. VASP phosphorylation was determined with Platelet VASP kits according to the manufacturer’s instructions (Diagnostica Stago, Asnières, France). In brief, blood samples collected on sodium citrate were incubated in vitro with ADP and/or prostaglandin E1 (PGE1) before fixation, according to the manufacturer’s instructions. Indirect immunolabeling on each sample was performed with a first incubation with 16C2 monoclonal antibody, followed by staining with a goat antimouse fluorescein isothiocyanate polyclonal reagent (BioCytex, Marseille, France). Flow cytometric analysis was performed on a Coulter EpicsXL cytometer. The platelet population was identified by its forward and side scatter distribution, and 3000 platelet events were gated and analyzed for mean fluorescence intensity (MFI), using epicsxl software. An MFI corresponding to each experimental condition (ADP, ADP + PGE1) was determined to establish a ratio that was directly correlated with VASP phosphorylation state. A PR index (PRI VASP) was calculated from the MFI of samples incubated with PGE1 or PGE1 and ADP according to the formula: PRI VASP = [(MFI(PGE1) – MFI(PGE1) + ADP)/MFI(PGE1)] × 100. The PRI is expressed as a percentage. The intra-assay coefficient of variation (CV) was <5%, and the interassay CV was <8%. The normal value of the PRI in healthy subjects is higher than 70% [15]. PR measurement using VASP analysis was carried out 24 h after the clopidogrel loading dose was administered and before angioplasty.

Clinical endpoint

Clinical follow-up was performed 6 months after PCI. All MACE were collected, including cardiovascular death, ischemic stroke, recurrent ACS, and repeated revascularization either by coronary angioplasty or bypass surgery. ACS was defined according to the ACC/AHA guidelines [16]. We recorded any occurrence of major bleeding, defined as intracranial bleeding or clinically overt bleeding associated with a decrease in hemoglobin of 5 g dL−1, according to the Thrombolysis in Myocardial Infarction criteria [17]. The treating physician and the investigators who evaluated the clinical endpoints were not aware of the results of platelet testing.

Statistical analysis

Statistical analysis was performed using spss 10.1 (SPSS Inc., Chicago, IL, USA). Continuous variables are expressed as mean ± SD. Categorical variables are expressed as frequencies and percentages. Comparison between categorical variables was performed using the χ2-test or Fischer’s exact test as appropriate. anova was used to compare means of quantitative variables.

Multiple logistic regression was used to identify factors potentially associated with MACE. To compare individuals with lower VASP levels (in the first quintile) to those in the four higher VASP quintiles, VASP was taken into account as one of the independent factors.

The receiver operating characteristic (ROC) curve served to determine the optimal cut-off value of VASP analysis to detect MACE.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Disclosure of Conflict of Interests
  9. References

Patients’ demographic, biological and angiographic characteristics

One hundred and 44 patients were included in our study. Demographic, biological and angiographic characteristics are summarized in Table 1. The mean age was 68 ± 10 years, and 79% of the patients were men. As expected, a high incidence of multiple cardiovascular risk factors was observed. In particular, 40% of patients had diabetes mellitus.

Table 1.   Patients’ demographic, biological and angiographic characteristics
  1. CV, cardiovascular; CAD, coronary artery disease; VASP, vasodilator-stimulated phosphoprotein; PCI, percutaneous coronary intervention; ACS, acute coronary syndrome; MACE, major adverse cardiovascular events.

Age (years) (mean ± SD)68 ± 10
Male sex (n, %)114 (79)
CV risk factors (n, %)
 Smoking94 (50)
 Dyslipidemia76 (53)
 Hypertension72 (50)
 Diabetes mellitus58 (40)
 Family history of CAD45 (31)
 Personal history of CAD56 (39)
Treatment (n, %)
 β-Blockers67 (47)
 Calcium channel blockers16 (11)
 Statins86 (60)
Platelet reactivity % (mean ± SD)
 VASP analysis66 ± 20
PCI indication (n, %)
 ACS38 (26)
 Positive stress test49 (34)
 Silent ischemia55 (40)
MACE (n, %)21 (14)

On admission, 60% of patients were receiving statin therapy, and 19% were receiving chronic clopidogrel therapy (75 mg daily). Indications for PCI were well balanced between the three inclusion criteria: 34% of patients were admitted for symptoms of angina pectoris confirmed by positive stress test, 40% for silent ischemia on thallium scintigraphy, and 26% for low-risk NSTE-ACS. A mean of 1.7 ± 0.8 stents was implanted per patient, with 100% procedural success. Thirty per cent of the patients had at least one drug-eluting stent implantation during the procedure.

Platelet reactivity index

We analyzed post-treatment platelet function using the flow cytometric assessment of VASP phosphorylation at a mean time of 25 ± 3 h after patients received a 300-mg loading dose of clopidogrel.

The mean value of PRI was 66% ± 20%. According to our preliminary data, 48% of the patients had a PR lower than the normal range (70%) after the loading dose of clopidogrel. Large interindividual variability was observed in the biological response to clopidogrel (from 9% to 98%) (Fig. 1). Patients receiving chronic clopidogrel therapy had a PRI similar to those who were not receiving it (66.5% ± 19% vs. 66% ± 20%, P = 0.9).

image

Figure 1.  Platelet reactivity 24 h after a 300-mg loading dose of clopidogrel in the population.

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According to their PRI, patients were stratified into quintiles (Table 2). The mean PRI results were, respectively, 32% ± 12%, 58% ± 5%, 72% ± 3%, 80% ± 3%, and 90% ± 3% from the first to the fifth quintile. The range of PRI values obtained using VASP phosphorylation analysis for the first quintile was 9–47%. There was no significant difference between the first quintile and the four higher quintiles in terms of demographic, clinical, biological or angiographic data. In particular, no relationship was observed between VASP analysis and smoking (P = 1), diabetes (P = 1), or body mass index (P = 0.7). Indications for PCI were not different between the quintiles. Moreover, the numbers of stents per patient and of drug-eluting stents per patient did not differ between the quintiles (P = 0.4 and P = 0.2, respectively).

Table 2.   Baseline characteristics and endpoints according to quintiles of vasodilator-stimulated phosphoprotein (VASP) analysis
  Quintile 1 (n = 28) Quintile 2 (n = 29) Quintile 3 (n = 30) Quintile 4 (n = 28) Quintile 5 (n = 29)Quintile 1 vs. quintiles 2–5, P-value
  1. CAD, coronary artery disease; BMI, body mass index; CRP, C-reactive protein; PCI, percutaneous coronary intervention; ACS, acute coronary syndrome; LVEF, left ventricular ejection fraction.

Age (years) (mean ± SD)69 ± 1168 ± 1066 ± 1069 ± 1069 ± 120.6
Sex (F/M)6 (21)8 (28)5 (17)6 (21)3 (10)0.8
Risk factors (n, %)
 Smoking19 (68)18 (62)21 (70)18 64)19 (66)1
 Dyslipidemia13 (46)18 (62)16 (53)16 (57)13 (45)0.5
 Hypertension13 (46)20 (69)18 (60)10 (36)12 (41)0.7
 Diabetes11 (39)13 (45)15 (50)10 (36)10 (35)1
 Damily history of CAD11 (39)9 (31)11 (37)8 (29)12 41)0.7
 Personal history of CAD12 (43)10 (34)13 (43)9 (32)12 (41)0.8
 BMI (kg m−2)25 ± 325 ± 225 ± 324 ± 325 ± 40.7
Treatment (n, %)
 β-Blockers15 (54)12 (41)15 (50)15 (54)10 (35)0.5
 Calcium channel blockers5 (18)5 (17)2 (7)2 (7)4 (14)0.3
 Clopidogrel4 (14)6 (21)9 (30)3 (11)4 (14)0.8
 Statins17 (61)15 (52)13 (43)17 (61)15 (52)0.4
Biology (mean ± SD)
 Platelet count (g L−1)220 ± 79204 ± 97195 ± 88194 ± 62230 ± 600.4
 Fibrinogen (g L−1)4 ± 14.5 ± 1.14.3 ± 1.23.7 ± 14.1 ± 1.30.7
 Creatinine (μmol L−1)85 ± 2476 ± 2178 ± 2393 ± 3091 ± 340.8
 CRP (mg L−1)16 ± 1414 ± 1613 ± 1611 ± 99 ± 60.2
PCI indication (n, %)
 ACS6 (21)7 (24)9 (30)10 (36)7 (24)0.7
 Positive stress test11 (39)10 (35)9 (30)7 (25)12 (41)0.7
 Silent ischemia11 (39)12 (41)12 (40)11 (39)10 (35)0.7
Angiography
 LVEF (mean ± SD)52 ± 1356 ± 1455 ± 1556 ± 1256 ± 130.3
 Number of diseased vessel (mean ± SD)1.5 ± 0.61.4 ± 0.71.6 ± 0.71.5 ± 0.61.5 ± 0.60.7
 Number of stents per patient (mean ± SD)1.8 ± 0.91.8 ± 0.71.7 ± 0.71.7 ± 0.91.8 ± 0.90.4
 Number of drug-eluting stents (mean ± SD)0.5 ± 0.50.4 ± 0.60.4 ± 0.50.5 ± 0.70.5 ± 0.60.2

Clinical endpoints during follow-up

All patients completed the 6-month follow-up. Twenty-one patients (14%) had a major cardiovascular event during this period. Four patients died during the follow-up (Table 3). Among these, three deaths were of cardiac origin. One patient died of cancer. Eighteen patients had an ischemic event during the follow-up, consisting of ACS as defined by the AHA guidelines [16]. None of the patients had major bleeding.

Table 3.   Major adverse cardiovascular events (MACE) during the follow-up
 Quintile 1 (n = 29)Quintiles 2–4 (n = 115)
  1. ACS, acute coronary syndrome; PCI, percutaneous coronary intervention.

  2. *P = 0.01 (Fischer’s exact test).

Death of cardiovascular origin (n, %)03 (2.6)
ACS (n, %)018 (15.6)
Urgent revascularization (n, %)
 PCI016 (13.9)
 Bypass surgery02 (1.7)
MACE (n, %)021 (14.5)*

Relationship between major cardiovascular events and PR

Quintile 1 of VASP analysis was associated with a lower risk of MACE as compared to the four higher quintiles (0 vs. 21, P < 0.01, Fisher’s exact test) (Table 3). Patients in the first quintile had no MACE during the 6-month follow-up. In the four higher quintiles, there was no gradual increase in MACE.

The ROC curve of PRI measured using VASP analysis for prediction of MACE is shown in Fig. 2. The optimal threshold of this test was 50% of PRI. With this cut-off value, the assay had 100% sensitivity and 25% specificity. The area under the curve was 0.55. With this threshold, only 20% of the population had an optimal inhibition of PRI 24 h after the oral loading dose of clopidogrel.

image

Figure 2.  Receiver operating characteristic curve for prediction of major adverse cardiovascular events according to platelet reactivity using vasodilator-stimulated phosphoprotein analysis.

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There was no relationship between PRI and time of the ischemic recurrence or type of MACE. The negative predictive value of VASP analysis for prediction of MACE within 6 months after PCI was 100%.

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Disclosure of Conflict of Interests
  9. References

Our study demonstrates that, in CAD patients undergoing PCI, biological resistance to clopidogrel, evaluated by analysis of VASP phosphorylation state, is associated with an increased rate of MACE. Moreover, this test has a very high negative predictive value for MACE, and thus could be of clinical interest for the optimization of therapy for complex coronary angioplasty.

Recent reports have demonstrated a link between biological resistance to clopidogrel and MACE [10–13]. However, the definition of biological resistance to clopidogrel is not established. Moreover, there is debate about the methods used to quantify PRI and the extent of platelet inhibition after clopidogrel treatment. Light-transmission aggregometry is the ‘gold standard’, and as such is the most commonly used. However, there is no consensus on the ADP agonist concentrations and the definition of PRI. Moreover, ADP-induced aggregometry is technically demanding and not standardized.

In our study, we analyzed the predictive value of a standardized whole blood assay that measures VASP phosphorylation. This assay is a standardized flow cytometric analysis that is reproducible and not affected by P2Y1 receptor inhibition or by aspirin treatment. VASP analysis is highly specific for P2Y12 ADP receptor inhibition, and therefore for the effect of clopidogrel [7]. This test was shown in this study to have high negative predictive value for MACE.

Recent studies have demonstrated that high post-treatment platelet activity is a better estimate of thrombotic risk than pretreatment PRI or the difference between pretreatment and post-treatment values [18,19]. Moreover, Gurbel et al. [9] showed that PRI after a 300-mg loading dose of clopidogrel occurs within 24 h. Accordingly, VASP analysis was performed 24 h after the loading dose was administered in our study.

We observed a high frequency of bad responders to clopidogrel. Only 48% of our patients had PRI lower than the normal range (<70%). Finally, 20% of our patients had PRI lower than 50%. There are several possible explanations for the low number of patients that could be considered as ‘good responders’. First, we defined biological resistance according to the optimal value of the assay for detecting clinical events. In contrast, previous studies used empirically defined thresholds. Second, the high number of patients with diabetes mellitus could also be implicated. In fact, patients with diabetes mellitus are more often bad responders to clopidogrel [20]. Finally, numerous studies have demonstrated the ability of a 600–900-mg loading dose to decrease the frequency of biological resistance to clopidogrel [21–23]. The ALBION trial revealed that clopidogrel doses higher than 300 mg can provide faster onset of action and greater levels of inhibition of PRI in patients presenting with NSTE-ACS [24]. Therefore, our loading dose based on recent guidelines [16] may be insufficient. It is uncertain, however, whether these higher doses of clopidogrel have a clinical impact.

In this prospective study, the ROC curve showed that the optimal value for predicting MACE was 50% inhibition of PRI. This result is consistent with a previous report showing an association between stent thrombosis and PRI inhibition lower than 50% using VASP analysis [10].

In cases of clopidogrel resistance, authors have recommended ticlopidine use [25] or increased loading doses of clopidogrel [21–23], but large prospective trials are needed to verify these recommendations.

Our study is the first prospective study to demonstrate a link between clopidogrel resistance, assessed by VASP phosphorylation analysis, and MACE following PCI. This test could be a very useful clinical tool in the setting of high-risk PCI, to optimize therapy and prevent complications, given its very high negative predictive value for MACE.

VASP analysis has an optimal negative predictive value (100%) but a low specificity. This finding supports the hypothesis that clopidogrel resistance is a triggering factor rather than a causal factor for MACE after PCI. This low specificity is consistent with the multifactorial pathophysiology of atherothrombosis and PCI complications such as stent thrombosis.

A multicenter study evaluating whether a VASP-adjusted loading dose of clopidogrel would decrease the frequency of MACE after interventions is now necessary.

Conclusions

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Disclosure of Conflict of Interests
  9. References

Our results demonstrate a link between biological resistance to clopidogrel after a 300-mg loading dose, evaluated by VASP phosphorylation analysis, and MACE in patients undergoing PCI for various clinical settings of CAD. VASP analysis has a 100% negative predictive value, and therefore allows the exclusion of ischemic events after PCI. This assay could therefore be used to evaluate the individual response to a clopidogrel loading dose in patients prior to PCI in order to prevent postprocedural MACE.

Disclosure of Conflict of Interests

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Disclosure of Conflict of Interests
  9. References

The authors state that they have no conflict of interest.

References

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  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Disclosure of Conflict of Interests
  9. References
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