Pierre Fontana

Cell-derived microparticles in haemostasis and vascular medicine

Considerable interest for cell-derived microparticles has emerged, pointing out their essential role in haemostatic response and their potential as disease markers, but also their implication in a wide range of physiological and pathological processes. They derive from different cell types including platelets - the main source of microparticles - but also from red blood cells, leukocytes and endothelial cells, and they circulate in blood. Despite difficulties encountered in analyzing them and disparities of results obtained with a wide range of methods, microparticle generation processes are now better understood. However, a generally admitted definition of microparticles is currently lacking. For all these reasons we decided to review the literature regarding microparticles in their widest definition, including ectosomes and exosomes, and to focus mainly on their role in haemostasis and vascular medicine.

Influence of CYP2C19 and CYP3A4 gene polymorphisms on clopidogrel responsiveness in healthy subjects

P . FONTANA,* J . S . HULOT , P . DE MOERLOOS E* and P . GAUSSEM *Division of Angiology and Haemostasis, Department of Internal Medicine, Faculty of Medicine and University Hospitals of Geneva, Geneva, Switzerland; AP-HP, Hôpital Pitié-Salpêtrière, Service de Pharmacologie, Unité de Pharmacogénétique, Université Pierre et Marie Curie-Paris6, UMR S 621 (INSERM), Paris; and AP-HP, Hôpital Européen Georges Pompidou, Service d Hématologie Biologique, INSERM Unité 765, Université Paris-Descartes, Paris, France

Use of the PFA-100 closure time to predict cardiovascular events in aspirin-treated cardiovascular patients: a systematic review and meta-analysis

Summary.  Background: PFA‐100™ is a point‐of‐care assay that evaluates platelet reactivity in high‐shear‐stress conditions by measuring the closure time (CT) of a membrane aperture. When determined with a collagen/epinephrine cartridge (CEPI), the CT is usually prolonged by aspirin. Studies of the predictive value of a short PFA‐100™CTCEPI for ischemic events in aspirin‐treated patients have given variable results. Objectives: To conduct a systematic review and meta‐analysis of studies on the clinical predictive value of a short PFA‐100™CTCEPI in aspirin‐treated cardiovascular patients. Patients and methods: Relevant studies were identified by scanning electronic databases. Studies were selected if they included aspirin‐treated patients with symptomatic atherosclerosis, measured the PFA‐100™CTCEPI, used a CT cut‐off value to define aspirin ‘responders’ and ‘non‐responders’, and reported ischemic events. Results: We selected seven non‐prospective studies (1466 patients) and eight prospective studies (1227 patients). In non‐prospective studies, the PFA‐100™CTCEPI was performed after the ischemic clinical endpoint, and a publication bias was identified. In prospective studies, the global odds ratio (OR) for the recurrence of an ischemic event in ‘aspirin non‐responders’ relative to ‘aspirin responders’ was 2.1 [95% confidence interval (CI) 1.4–3.4, P < 0.001]. Pooled analysis with a random effect model revealed no heterogeneity (Q Cochran P = 0.36 and I2 = 9.4%). Conclusions: A short PFA‐100™CTCEPI is associated with increased recurrence of ischemic events in aspirin‐treated cardiovascular patients. This finding needs to be confirmed in stable ischemic patients, and the PFA‐100™CTCEPI cut‐off needs to be refined in these patients.

Clinical implications of clopidogrel non-response in cardiovascular patients: a systematic review and meta-analysis

Summary.  Background: Previous studies have shown an important risk of cardiovascular events in patients with clopidogrel biological non‐response, and data have shown considerable, unexplored heterogeneity. Objectives: To evaluate the magnitude of cardiovascular risk associated with clopidogrel non‐response and to explore heterogeneity. Methods: This was a systematic review and meta‐analysis of prospective studies of patients treated with clopidogrel for symptomatic atherothrombosis, evaluated by light transmission aggregometry with ADP and monitored prospectively for clinical ischemic events. Results: Fifteen studies were included, totaling 3960 patients, of whom 25% were considered to be clopidogrel non‐responders. The global relative risk (RR) for recurrent ischemic events in clopidogrel non‐responders was 3.5 [95% confidence interval (CI) 2.4–5.2, P < 0.0001]. The results of the different studies were heterogeneous (Cochran P = 0.01 and I2 = 52%). The most recent studies yielded lower RRs [global RR = 2.9 (95% CI 2.3–3.8) after 2007, and global RR = 6.6 (95% CI 3.7–11.9) before 2007, P = 0.01]. Heterogeneity was present in the group of studies in which more than 10% of patients took glycoprotein (GP)IIb–IIIa inhibitors [Cochran P = 0.003 and I2 = 70%; RR = 3.8 (95% CI 2.9–5.1)] and was absent in the other studies [Cochran P = 0.88 and I2 = 0; RR = 2.5 (95% CI 1.7–3.6)]. The RR was significantly higher in studies using higher ADP maximal aggregation cut‐offs (> 65%) for clopidogrel non‐response than in studies using lower cut‐offs [RR = 5.8 (95% CI 3.2–10.3) and RR = 2.9 (95% CI 2.2–3.7), respectively, P = 0.03]. Conclusions: The risk of ischemic events associated with clopidogrel non‐response is now more precisely defined. The risk is heterogeneous across studies, possibly because of an interaction with GPIIb–IIIa inhibitors and the use of different cut‐offs to identify non‐responders.

Biological effects of aspirin and clopidogrel in a randomized cross‐over study in 96 healthy volunteers

Summary.  Background: Some data suggest that biological ‘resistance’ to aspirin or clopidogrel may influence clinical outcome. Objective: The aim of this study was to evaluate the relationship between aspirin and clopidogrel responsiveness in healthy subjects. Methods: Ninety‐six healthy subjects were randomly assigned to receive a 1‐week course of aspirin 100 mg day−1 followed by a 1‐week course of clopidogrel (300 mg on day 1, then 75 mg day−1), or the reverse sequence, separated by a 2‐week wash‐out period. The drug effects were assessed by means of serum TxB2 assay, platelet aggregation tests, and the PFA ‐100® and Ultegra RPFA ‐Verify Now® methods. Results: Only one subject had true aspirin resistance, defined as a serum TxB2 level > 80 pg μL−1 at the end of aspirin administration and confirmed by platelet incubation with aspirin. PFA‐100® values were normal in 29% of the subjects after aspirin intake, despite a drastic reduction in TxB2 production; these subjects were considered to have aspirin pseudo‐resistance. Clopidogrel responsiveness was not related to aspirin pseudo‐resistance. Selected polymorphisms of platelet receptor genes were not associated with either aspirin or clopidogrel responsiveness. Conclusions: In healthy subjects, true aspirin resistance is rare and aspirin pseudo‐resistance is not related to clopidogrel responsiveness.

Antiplatelet Drug Response Status Does Not Predict Recurrent Ischemic Events in Stable Cardiovascular Patients Results of the Antiplatelet Drug Resistances and Ischemic Events Study

Background— The biological response to antiplatelet drugs has repeatedly been shown to predict the recurrence of major adverse cardiovascular events (MACEs). However, most studies involved coronary artery disease patients with recent vessel injury shortly after the initiation of antiplatelet therapy. Data on stable cardiovascular patients are scarce, and the added predictive value of specific assays (the vasodilator phosphoprotein assay for the clopidogrel response and serum thromboxane B2 for the aspirin response) and aggregation-based assays relative to common predictors has rarely been addressed. Methods and Results— Stable cardiovascular outpatients participating in the Antiplatelet Drug Resistances and Ischemic Events (ADRIE) study (n=771) were tested twice, at 2 separate visits, with specific and aggregation-based assays. Follow-up lasted 3 years, and <1% of patients were lost to follow-up. MACEs were adjudicated by an independent committee. Multivariate survival analyses included relevant variables identified in univariate analysis and platelet function test results. The C-index was used to express the prognostic value of various multivariate models. MACEs, the primary end point, occurred in 16% of patients. Hypertension, smoking, older age, and elevated low-density lipoprotein cholesterol were predictive of MACE recurrence, with a C-index of 0.63 (P<0.001). Neither the specific nor the aggregation-based assays added significant predictive value for the primary end point. Conclusions— Biological antiplatelet drug responsiveness, measured with specific or aggregation-based assays, has no incremental predictive value over common cardiovascular risk factors for MACE recurrence in stable cardiovascular outpatients. These results do not support platelet function testing for MACE risk evaluation in stable cardiovascular patients. Clinical Trial Registration— URL: http://www.clinicaltrials.gov. Unique identifier: NCT00501423.

Clinical predictors of dual aspirin and clopidogrel poor responsiveness in stable cardiovascular patients from the ADRIE study

Summary.  Background: Poor response to both aspirin and clopidogrel (dual poor responsiveness [DPR]) is a major risk factor for recurrent ischemic events. Objectives: The aim of this study was to identify factors associated with DPR, defined with specific tests, and derive a predictive clinical score. Methods: We studied 771 consecutive stable cardiovascular patients treated with aspirin (n = 223), clopidogrel (n = 111), or both drugs (n = 437). Aspirin responsiveness was evaluated by serum thromboxane (Tx)B2 assay, and clopidogrel responsiveness by calculating the platelet reactivity index (PRI) on the basis of the phosphorylation status of the vasodilator phosphoprotein. The analysis was focused on patients treated with both drugs, and on independent predictors of DPR. Results: Among patients on dual therapy, there was no relevant correlation between TxB2 levels and PRI values (r = 0.11). Sixty‐seven patients (15.4%) had DPR. Diabetes [odds ratio (OR) 1.89, 95% confidence interval (CI) 1.06–3.39], high body weight (> 86 kg vs. < 77 kg, OR 4.74, 95% CI 2.49–9.73), low aspirin dose (75–81 mg vs. ≥ 160 mg, OR 0.12, 95% CI 0.09–0.93) and high C‐reactive protein (CRP) level (> 1.6 mg L−1 vs. < 0.6 mg L−1, OR 3.66, 95% CI 1.74–8.72) were independently associated with DPR, via increased TxB2 levels, increased PRI, or both. These associations with TxB2 and PRI were reproduced across the whole population. With use of a factor‐weighed score (c‐index = 0.74), the predicted prevalence of DPR was 57% in the highest strata of the score as compared with < 4% for the lowest strata. Conclusions: Diabetes, body weight, the aspirin dose and CRP levels are readily available independent predictors of DPR, and some are potential targets for reducing its prevalence.

Antiplatelet Drug Response Status Does Not Predict Recurrent Ischemic Events in Stable Cardiovascular Patients: Results of the ADRIE Study

Background— The biological response to antiplatelet drugs has repeatedly been shown to predict the recurrence of major adverse cardiovascular events (MACEs). However, most studies involved coronary artery disease patients with recent vessel injury shortly after the initiation of antiplatelet therapy. Data on stable cardiovascular patients are scarce, and the added predictive value of specific assays (the vasodilator phosphoprotein assay for the clopidogrel response and serum thromboxane B2 for the aspirin response) and aggregation-based assays relative to common predictors has rarely been addressed. Methods and Results— Stable cardiovascular outpatients participating in the Antiplatelet Drug Resistances and Ischemic Events (ADRIE) study (n=771) were tested twice, at 2 separate visits, with specific and aggregation-based assays. Follow-up lasted 3 years, and <1% of patients were lost to follow-up. MACEs were adjudicated by an independent committee. Multivariate survival analyses included relevant variables identified in univariate analysis and platelet function test results. The C-index was used to express the prognostic value of various multivariate models. MACEs, the primary end point, occurred in 16% of patients. Hypertension, smoking, older age, and elevated low-density lipoprotein cholesterol were predictive of MACE recurrence, with a C-index of 0.63 (P<0.001). Neither the specific nor the aggregation-based assays added significant predictive value for the primary end point. Conclusions— Biological antiplatelet drug responsiveness, measured with specific or aggregation-based assays, has no incremental predictive value over common cardiovascular risk factors for MACE recurrence in stable cardiovascular outpatients. These results do not support platelet function testing for MACE risk evaluation in stable cardiovascular patients. Clinical Trial Registration— URL: http://www.clinicaltrials.gov. Unique identifier: NCT00501423.

Influence of the paraoxonase-1 Q192R genetic variant on clopidogrel responsiveness and recurrent cardiovascular events: a systematic review and meta-analysis

Summary.  Background:  A poor biological response to clopidogrel is associated with an increased risk of major cardiovascular ischemic events (MACE). Paraoxonase 1 (PON1) enzyme activity is modulated by the PON1‐Q192R variant (rs662) and was recently suggested to be strongly involved in clopidogrel bioactivation, but the influence of the PON1‐Q192R variant on the risk of MACE in clopidogrel‐treated patients is controversial.

Relationship between paraoxonase-1 activity, its Q192R genetic variant and clopidogrel responsiveness in the ADRIE study.

*Division of Angiology and Hemostasis, Division of Endocrinology, Diabetology and Nutrition, Department of Medical Specialties, GenevaUniversity Hospitals and Faculty of Medicine, Geneva, Switzerland; Hematology Laboratory, §Division of Cardiology, Be´ziers Hospital, Be´ziers;–Hematology Laboratory, Montpellier University Hospital, Montpellier, France; **Division of Laboratory Medicine, Department of Genetics andLaboratory Medicine, Geneva University Hospitals, Geneva; and Division of General Internal Medicine, Geneva University Hospital and Facultyof Medicine, Geneva, SwitzerlandTo cite this article: Fontana P, James R, Barazer I, Berdague´ P, Schved JF, Rebsamen M, Vuilleumier N, Reng JL. Relationship betweenparaoxonase-1 activity, its Q192R genetic variant and clopidogrel responsiveness in the ADRIE study. J Thromb Haemost 2011; 9: 1664–6.