Christian M. Hackeng

Comparison of Platelet Function Tests in Predicting Clinical Outcome in Patients Undergoing Coronary Stent Implantation

Context High on-treatment platelet reactivity is associated with atherothrombotic events following coronary stent implantation. Objective To evaluate the capability of multiple platelet function tests to predict clinical outcome. Design, Setting, and Patients Prospective, observational, single-center cohort study of 1069 consecutive patients taking clopidogrel undergoing elective coronary stent implantation between December 2005 and December 2007. On-treatment platelet reactivity was measured in parallel by light transmittance aggregometry, Verify Now P2Y12 and Platelet works assays, and the IMPACT-R and the platelet function analysis system (PFA-100) (with the Dade PFA collagen/adenosine diphosphate (ADP) cartridge and Innovance PFA P2Y). Cutoff values for high on-treatment platelet reactivity were established by receiver operating characteristic curve (ROC) analysis. Main Outcome Measurement The primary end point was defined as a composite of all-cause death, nonfatal acute myocardial infarction, stent thrombosis, and ischemic stroke. The primary safety end point included TIMI (Thrombolysis In Myocardial Infarction) criteria major and minor bleeding. Results Kaplan-Meier analysis demonstrated that at 1-year follow-up, the primary end point occurred more frequently in patients with high on-treatment platelet reactivity when assessed by light transmittance aggregometry (52 [11.7%; 95% confidence interval {CI}, 8.9%-15.0%] vs 36 [6.0%;95%CI, 4.2%-8.2%] P.001; n=1049),Verify Now (54 [13.3%; 95% CI, 10.2%-17.0%] vs 37 [5.7%; 95% CI, 4.1%-7.8%]P.001; n=1052), Platelet works (33 [12.6%; 95% CI, 8.8%-17.2%] vs 21 [6.1%;95% CI, 3.8%-9.2%] P=.005; n=606), and Innovance PFA P2Y (18 [12.2%; 95%CI; 7.4%-18.6%] vs 28 [6.3%; 95% CI, 4.3%-8.9%] P=.02; n=588). ROC-curve analysis demonstrated that light transmittance aggregometry (area under the curve[AUC], 0.63; 95% CI, 0.58-0.68), Verify Now (AUC, 0.62; 95% CI, 0.57-0.67), and Platelet works (AUC, 0.61; 95% CI, 0.53-0.69) had modest ability to discriminate between patients with and without primary end point at 1-year follow-up. The IMPACT-R(n=905) and the Siemens PFA Collagen/ADP (n=812) were unable to discriminate between patients with and without the primary end point at 1-year follow-up (all AUCs included 0.50 in the CI). None of the tests identified patients at risk for bleeding. Conclusions Of the platelet function tests assessed, light transmittance aggregometry,Verify Now, Platelet works, and Innovance PFA P2Y were significantly associated with the primary end point. However, the predictive accuracy of these 4 tests was only modest. None of the tests provided accurate prognostic information to identify patients at higher risk of bleeding following stent implantation. Trial Registration clinical trials.gov Identifier: NCT00352014 [corrected].

Paraoxonase-1 is a major determinant of clopidogrel efficacy

Clinical efficacy of the antiplatelet drug clopidogrel is hampered by its variable biotransformation into the active metabolite. The variability in the clinical response to clopidogrel treatment has been attributed to genetic factors, but the specific genes and mechanisms underlying clopidogrel bioactivation remain unclear. Using in vitro metabolomic profiling techniques, we identified paraoxonase-1 (PON1) as the crucial enzyme for clopidogrel bioactivation, with its common Q192R polymorphism determining the rate of active metabolite formation. We tested the clinical relevance of the PON1 Q192R genotype in a population of individuals with coronary artery disease who underwent stent implantation and received clopidogrel therapy. PON1 QQ192 homozygous individuals showed a considerably higher risk than RR192 homozygous individuals of stent thrombosis, lower PON1 plasma activity, lower plasma concentrations of active metabolite and lower platelet inhibition. Thus, we identified PON1 as a key factor for the bioactivation and clinical activity of clopidogrel. These findings have therapeutic implications and may be exploited to prospectively assess the clinical efficacy of clopidogrel.

Impaired bioavailability of clopidogrel in patients with a ST-segment elevation myocardial infarction.

a Department of Cardiology, St Antonius Hospital, Nieuwegein, the Netherlands b Department of Pharmacology, University Hospital, University of Cologne, Cologne, Germany c Department of Clinical Chemistry, St Antonius Hospital Nieuwegein, the Netherlands d Department of Cardiology, Deutsches Herzzentrum and 1. Medizinische Klinik rechts der Isar, Technische Universitat Munchen, Munich, Germany e Department of Cardiology, UMC Nijmegen, The Netherlands

CYP2C19*2 and CYP2C9*3 alleles are associated with stent thrombosis: a case–control study

AIMS despite treatment with clopidogrel on top of aspirin, stent thrombosis (ST) still occurs being the most serious complication after percutaneous coronary interventions (PCIs). In this study, we aimed to determine the effect of variations in genes involved in the absorption (ABCB1 C1236T, G2677T/A, C3435T), metabolism (CYP2C19*2 and *3, CYP2C9*2 and *3, CYP3A4*1B and CYP3A5*3), and pharmacodynamics (P2Y1 A1622G) of clopidogrel on the occurrence of ST. METHODS AND RESULTS the selected genetic variants were assessed in 176 subjects who developed ST while on dual antiplatelet therapy with aspirin and clopidogrel and in 420 control subjects who did not develop adverse cardiovascular events, including ST, within 1 year after stenting. The timing of the definite ST was acute in 66, subacute in 87, and late in 23 cases. The presence of the CYP2C19*2 and CYP2C9*3 variant alleles was significantly associated with ST (OR(adj): 1.7, 95% CI: 1.0-2.6, P = 0.018 and OR(adj): 2.4, 95% CI: 1.0-5.5, P = 0.043, respectively). The influence of CYP2C19*2 (OR(adj): 2.5, 95% CI: 1.1-5.5, P = 0.026) and CYP2C9*3 (OR(adj): 3.3, 95% CI: 1.1-9.9, P = 0.031) was most strongly associated with subacute ST. No significant associations of the other genetic variations and the occurrence of ST were found. CONCLUSION carriage of the loss-of-function alleles CYP2C19*2 and CYP2C9*3 increases the risk on ST after PCI.

A head-to-head comparison between the VerifyNow P2Y12 assay and light transmittance aggregometry for monitoring the individual platelet response to clopidogrel in patients undergoing elective percutaneous coronary intervention.

Clopidogrel on top of aspirin lowers the rate of thrombotic events in patients undergoing percutaneous coronary intervention (PCI) with coronary stenting. However, a number of ex vivo studies have demonstrated that the response to aspirin and clopidogrel is not uniform, and poor response has been associatedwith an increased risk for cardiovascular events [1,2]. Light transmittance aggregometry (LTA) is considered to be the gold standard for determining the effects of antiplatelet therapy on platelet function, but the logistical demands make it impossible to use in daily practice. Therefore, the VerifyNow P2Y12 assay was recently introduced as a rapid point-of-care test; however, there is a lack of experimental evidence for the usefulness of this assay in monitoring clopidogrel’s efficacy. In the present study, we prospectively studied the platelet response to clopidogrel (and aspirin) in 211 consecutive patients undergoing elective PCI with stenting. Owing to different protocols in referring hospitals, all patients received different but adequate clopidogrel pretreatment. Regimens were defined as maintenance therapy of 75 mg for > 5 days (n 1⁄4 116) or a loading dose of 300 mg at least 24 h before PCI (n 1⁄4 75) or 600 mg at least 4 h before PCI (n 1⁄4 20). All patients were on ‡ 80 mg of aspirin for at least 7 days. The local institutional review board approved the protocol, and written informed consent was obtained before elective PCI. Classic LTA induced by 20 lmol L adenosinediphosphate (ADP) was quantified in non-adjusted platelet rich plasma (PRP) at maximal (peak) aggregation and after 600 s (late aggregation). The VerifyNow P2Y12 test cartridge system (Accumetrics, San Diego, CA, USA) was used as described previously [3]. Given the fact that the majority of the studies linking low clopidogrel response to atherothrombotic events have used absolute post-treatment aggregation as the measure of clopidogrel response, we preferred using absolute P2Y12 Reaction Units (PRUs) over the reported BASE values of the VerifyNow P2Y12 assay in the present study. A P-value of < 0.05 of the Pearson correlation coefficient was considered statistically significant in comparing the results obtained from both tests. The three regimen groups were not significantly different with respect to both demographic and clinical characteristics (data not shown). Peak aggregation and late aggregation were significantly more strongly inhibited by the 600 mg loading dose of clopidogrel than by the 300 mg loading dose or a chronic daily 75 mg maintenance dose (Fig. 1). Furthermore, the absolute number of PRUs was also significantly lower in patients who had received a 600 mg loading dose than in Correspondence: J. W. van Werkum, Department of Cardiology, St Antonius Hospital, 3435 CM Nieuwegein, The Netherlands. Tel.: +31 306099111; fax: +31 306034420; e-mail: w.van.werkum@ antonius.net

Which platelet function test is suitable to monitor clopidogrel responsiveness? A pharmacokinetic analysis on the active metabolite of clopidogrel

Summary.  Background: Multiple platelet function tests claim to be P2Y12‐pathway specific and capable of capturing the biological activity of clopidogrel. Objectives: The aim of the present study was to determine which platelet function test provides the best reflection of the in vivo plasma levels of the active metabolite of clopidogrel (AMC). Patients/methods: Clopidogrel‐naive patients scheduled for elective percutaneous coronary intervention (PCI) received a 600 mg loading dose of clopidogrel and 100 mg of aspirin. For pharmacokinetic analysis, blood was drawn at 0, 20, 40, 60, 90, 120, 180, 240 and 360 min after clopidogrel loading and peak plasma concentrations (Cmax) of the AMC were quantified with liquid chromatography‐tandem mass spectrometry (LC‐MS/MS). Platelet function testing was performed at baseline and 360 min after the clopidogrel loading. Results: The VASP‐assay, the VerifyNow P2Y12‐assay and 20 μmol L−1 adenosine diphosphate (ADP)‐induced light transmittance aggregometry (LTA) showed strong correlations with Cmax of the AMC (VASP: R2 = 0.56, P < 0.001; VerifyNow platelet reactivity units (PRU): R2 = 0.48, P < 0.001; VerifyNow %inhibition: R2 = 0.59, P < 0.001; 20 μmol L−1 ADP‐induced LTA: R2 = 0.47, P < 0.001). Agreement with Cmax of the AMC was less evident for 5 μmol L−1 ADP‐induced LTA or whole blood aggregometry (WBA), whereas the IMPACT‐R ADP test did not show any correlation with plasmalevels of the AMC. Conclusion: The flow cytometric VASP‐assay, the VerifyNow P2Y12 assay and, although to a lesser extent, 20 μmol L−1 ADP‐induced LTA correlate best with the maximal plasma level of the AMC, suggesting these may be the preferred platelet function tests for monitoring the responsiveness to clopidogrel.

High on‐aspirin platelet reactivity as measured with aggregation‐based, cyclooxygenase‐1 inhibition sensitive platelet function tests is associated with the occurrence of atherothrombotic events

Summary.  Background: High on‐aspirin platelet reactivity (HAPR) is associated with atherothrombotic events following percutaneous coronary intervention (PCI). The aim of the present study was to identify the platelet function test sensitive for platelet cyclooxygenase‐1 inhibition that best predicts atherothrombotic events. Methods and results: Nine hundred and fifty‐one consecutive patients on dual antiplatelet therapy undergoing elective PCI were enrolled. On‐aspirin platelet reactivity was measured in parallel by arachidonic acid (AA)‐induced light transmittance aggregometry (AA‐induced LTA), the VerifyNow® Aspirin Assay (VerifyNow® Aspirin Assay), the arachidonic acid prestimulated IMPACT‐R (IMPACT‐R AA) and the PFA‐100 collagen/epinephrine cartridge (PFA COL/EPI). Cut‐offs for HAPR were established by receiver‐operator characteristic curve analysis. At 1‐year follow‐up, the composite of all‐cause death, non‐fatal acute myocardial infarction, stent thrombosis and ischemic stroke occurred more frequently in patients with HAPR when assessed by LTA [10.1% vs. 6.0%, P = 0.020 (n = 925)] and VerifyNow® [13.3% vs. 5.9%, P = 0.015 (n = 422)]. The VerifyNow® ASA assay (AUC = 0.78) and, to a lesser extent, AA‐induced LTA (AUC = 0.73) added significantly to a model consisting of clinical and procedural risk factors in predicting atherothrombotic events. In contrast, the IMPACT‐R (n = 791) and the PFA Collagen/Epinephrine (n = 719) were unable to discriminate between patients with and without primary endpoint at 1‐year follow‐up. None of the platelet function tests was able to identify patients at risk for bleeding. Conclusions: AA‐induced LTA and the VerifyNow® ASA test were able to identify aspirin‐treated patients undergoing PCI with stenting at risk for atherothrombotic events. The VerifyNow® Aspirin Assay had the highest predictive accuracy. None of the tests was able to identify patients at higher risk of bleeding.

Variability in on-treatment platelet reactivity explained by CYP2C19*2 genotype is modest in clopidogrel pretreated patients undergoing coronary stenting

Background An inadequate response to clopidogrel is mainly attributable to the variable formation of its active metabolite. The CYP2C19*2 loss-of-function polymorphism leads to reduced generation of the active metabolite and is, similarly to high on-treatment platelet reactivity (HPR), associated with recurrent atherothrombotic events following coronary stent implantation. Aim To determine the relative contribution of CYP2C19*2 genotype to HPR. Methods and results CYP2C19*2 genotyping and platelet function testing using 5 and 20 μmol/l ADP-induced light transmittance aggregometry (LTA), the PlateletWorks assay and the VerifyNow P2Y12 assay, were performed in 1069 clopidogrel pretreated patients undergoing elective coronary stenting (POPular study, http://clinicalTrials.gov/ NCT00352014). The relative contributions of CYP2C19*2 genotype and clinical variables to the interindividual variability of on-treatment platelet reactivity and the occurrence of HPR were established using multivariate regression models. CYP2C19*2 carrier status was associated with a more frequent occurrence of HPR. CYP2C19*2 genotype alone could explain 5.0%, 6.2%, 4.4% and 3.7% of the variability in 5 and 20 μmol/l ADP-induced LTA, the PlateletWorks assay and the VerifyNow P2Y12 assay, respectively, which increased to 13.0%, 15.2%, 5.6% and 20.6% when clinical variables were considered as well. Besides the CYP2C19*2 genotype, multiple clinical variables could be identified as independent predictors of HPR, including age, gender, body mass index, diabetes mellitus, clopidogrel loading dose regimen, use of amlodipine and platelet count. Conclusion The CYP2C19*2 loss-of-function polymorphism is associated with a more frequent occurrence of HPR. However, the part of the interindividual variability in on-treatment platelet reactivity explained by CYP2C19*2 genotype is modest.

The influence of clinical characteristics, laboratory and inflammatory markers on 'high on-treatment platelet reactivity' as measured with different platelet function tests.

High on-clopidogrel platelet reactivity (HCPR) and high on-aspirin platelet reactivity (HAPR) are independently associated with an increased risk of atherothrombotic events. However, despite this positive correlation, the definitions of both HCPR and HAPR vary largely throughout studies and between different platelet function assays. The aim of the present study was to explore clinical and laboratory parameters that are associated with HCPR and HAPR as measured with different platelet function tests. 530 clopidogrel and aspirin pre-treated patients undergoing elective PCI (percutaneous coronary intervention) were enrolled. Platelet function measurements were performed with: optical aggregometry, the VerifyNow device and PFA-100 cartridges (including the novel INNOVANCE P2Y assay). HCPR as measured with Adenosin-Di-Phospate-induced (ADP) aggregation based tests was associated with clinical factors such as older age, female gender and Diabetes mellitus (DM). The VerifyNow P2Y12 assay was significantly influenced by haemoglobin and haematocrit levels. HAPR as measured with aggregation based tests was significantly influenced by the presence of malignancy, BMI (Body-Mass Index), older age and increased levels of hsCRP (high sensitivity c-reactive proteine). The PFA-100 COL/EPI (collagen-epinephrine) and COL/ADP (collagen-ADP) cartridges were significantly influenced by monocyte count, hs-CRP, MPV (mean platelet volume), vWF-antigen (von Willebrand factor) and vWF-activity. HCPR as measured with the novel INNOVANCE P2Y cartridge was associated with clinical determinants such as BMI, female gender, impaired LVEF (left ventricular ejection fraction), renal failure and dosing of clopidogrel. Laboratory markers that were associated with HCPR as measured with INNOVANCE P2Y were platelet count, white blood cells (WBC), hsCRP and fibrinogen. Both HCPR and HAPR are highly dependent on the type of platelet function assay. Each platelet function assay, in turn, is significantly influenced by distinct clinical and laboratory variables.

Besides CYP2C19*2, the variant allele CYP2C9*3 is associated with higher on-clopidogrel platelet reactivity in patients on dual antiplatelet therapy undergoing elective coronary stent implantation

Introduction The prodrug clopidogrel plays an important role in the prevention of thrombotic events in patients undergoing coronary stenting. However, a substantial number of atherothrombotic events still occur, which can partially be explained by heightened residual platelet reactivity. Several studies report that the genetic variation in CYP2C19 (*2) is associated with an impaired response to clopidogrel. Objectives To evaluate the effect of genetic variants affecting clopidogrels absorption (ABCB1 C1236T, G2677T/A, C3435T), metabolism (CYP2C9*2, *3, CYP2C19*3, CYP3A4*1B, and CYP3A5*3), and pharmacodynamics (P2Y1 A1622G) on top of the influence of CYP2C19*2 on platelet reactivity in patients undergoing elective coronary stenting on dual antiplatelet therapy. Methods Platelet function was assessed by light transmittance aggregometry and VerifyNow P2Y12 assay in 428 consecutive patients. Patients were either on chronic clopidogrel maintenance therapy (75 mg/day for >5 days before the intervention) or received a 300 mg clopidogrel loading dose (1–5 days before the intervention, followed by 75 mg/day). Linear and logistic regression models were used to assess the associations between genetic variants and platelet reactivity and poor responder status. Results In both the treatment groups, CYP2C19*2-carriage was associated with higher platelet reactivity (P<0.03) and poor responder status; 75 mg group: adjusted odds ratio (ORadj): 3.8, 95% confidence interval (CI): 2.0–7.2, 300 mg group: ORadj: 4.1, 95% CI: 1.6–10.4. In the 300 mg group, CYP2C9*3-carriage was associated with higher platelet reactivity (P<0.05) and poor responder status (ORadj: 11.1, 95% CI: 1.6–78.8, P=0.016). Conclusion Besides CYP2C19*2, the variant allele CYP2C9*3 plays an important role in the response to clopidogrel in patients on dual antiplatelet therapy undergoing coronary stenting.