Towards PET imaging of the dynamic phenotypes of microglia.

There is increasing evidence showing the heterogeneity of microglia activation in neuroinflammatory and neurodegenerative diseases. It has been hypothesized that pro-inflammatory microglia are detrimental and contribute to disease progression, while anti-inflammatory microglia play a role in damage repair and remission. The development of therapeutics targeting the deleterious glial activity and modulating it into a regenerative phenotype relies heavily upon a clearer understanding of the microglia dynamics during disease progression and the ability to monitor therapeutic outcome in vivo. To that end, molecular imaging techniques are required to assess microglia dynamics and study their role in disease progression as well as to evaluate the outcome of therapeutic interventions. Positron emission tomography (PET) is such a molecular imaging technique, and provides unique capabilities for non-invasive quantification of neuroinflammation and has the potential to discriminate between microglia phenotypes and define their role in the disease process. However, several obstacles limit the possibility for selective in vivo imaging of microglia phenotypes mainly related to the poor characterization of specific targets that distinguish the two ends of the microglia activation spectrum and lack of suitable tracers. PET tracers targeting translocator protein 18 kDa (TSPO) have been extensively explored, but despite the success in evaluating neuroinflammation they failed to discriminate between microglia activation statuses. In this review, we highlight the current knowledge on the microglia phenotypes in the major neuroinflammatory and neurodegenerative diseases. We also discuss the current and emerging PET imaging targets, the tracers and their potential in discriminating between the pro- and anti-inflammatory microglia activation states.

Regulation of microglial TMEM119 and P2RY12 immunoreactivity in multiple sclerosis white and grey matter lesions is dependent on their inflammatory environment.

Multiple Sclerosis (MS) is the most common cause of acquired neurological disability in young adults, pathologically characterized by leukocyte infiltration of the central nervous system, demyelination of the white and grey matter, and subsequent axonal loss. Microglia are proposed to play a role in MS lesion formation, however previous literature has not been able to distinguish infiltrated macrophages from microglia. Therefore, in this study we utilize the microglia-specific, homeostatic markers TMEM119 and P2RY12 to characterize their immunoreactivity in MS grey matter lesions in comparison to white matter lesions. Furthermore, we assessed the immunological status of the white and grey matter lesions, as well as the responsivity of human white and grey matter derived microglia to inflammatory mediators. We are the first to show that white and grey matter lesions in post-mortem human material differ in their immunoreactivity for the homeostatic microglia-specific markers TMEM119 and P2RY12. In particular, whereas immunoreactivity for TMEM119 and P2RY12 is decreased in the center of WMLs, immunoreactivity for both markers is not altered in GMLs. Based on data from post-mortem human microglia cultures, treated with IL-4 or IFNγ+LPS and on  counts of CD3+ or CD20+ lymphocytes in lesions, we show that downregulation of TMEM119 and P2RY12  immunoreactivity in MS lesions corresponds with the presence of lymphocytes and lymphocyte-derived cytokines within the parenchyma but not in  the meninges. Furthermore, the presence of TMEM119+ and partly P2RY12+ microglia in pre-active lesions as well as in  the rim of active white and grey matter lesions, in addition to TMEM119+ and P2RY12+ rod-like microglia in subpial grey matter lesions suggest that blocking the entrance of lymphocytes into the CNS of MS patients may not interfere with all possible effects of TMEM119+ and P2RY12+ microglia in both white and grey matter MS lesions.

Precision of VerifyNow P2Y12 Assessment of Clopidogrel Response in Patients Undergoing Cerebral Aneurysm Flow Diversion.

BACKGROUND: Dual antiplatelet therapy (DAT), most commonly with aspirin and Clopidogrel, is the standard of care for intracranial stenting, including flow diversion. Clopidogrel response varies by individual. OBJECTIVE: To investigate the real-world precision of VerifyNow P2Y12 assessment (Accumetrics, San Diego, California) of Clopidogrel response. METHODS: Using a prospectively-collected, IRB-approved cerebral aneurysm database 643 patients were identified who were treated with the Pipeline embolization device from 2011 to 2017. Patients with multiple P2Y12 assays drawn within a 24-h window were identified. A single patient could contribute multiple, independent sets. Levels drawn before a 5-d course of DAT and patients who received alternative antiplatelet agents were excluded. Therapeutic range was defined as platelet reaction units (PRU) 60-200. RESULTS: A total of 1586 P2Y12 measurements were recorded; 293 (46%) patients had more than one assay. One hundred forty (22%) patients had multiple P2Y12 measurements within 24 h. These patients accounted for 230 independent 24-h sets. The average P2Y12 fluctuation across all sets was 35 points; the 25th, 50th, and 75th percentiles were 12, 26, and 48 points, respectively. Of the 230 24-h sets of P2Y12 assays, 76% remained within their original therapeutic category: 100 (43%) all therapeutic, 54 (23%) all hypo-responsive, and 21 (9%) all hyper-responsive. Twenty-four percent of patients fluctuated between therapeutic categories when multiple P2Y12 assessments were drawn within a 24-h period: 29 (13%) between hypo-response and therapeutic, 23 (10%) between hyper-response and therapeutic, and 3 (1%) between hypo-response and hyper-response. CONCLUSION: Our experience suggests P2Y12 is an often-imprecise measure, and this should be considered when utilizing P2Y12 levels for clinical decisions.

Identification of new molecular targets for PET imaging of the microglial anti-inflammatory activation state.

Microglia are potential targets for therapeutic intervention in neurological and neurodegenerative diseases affecting the central nervous system. In order to assess the efficacy of therapies aimed to reduce the tissue damaging activities of microglia and/or to promote the protective potential of these cells, suitable pre-clinical and clinical tools for the in vivo analysis of microglia activities and dynamics are required. The aim of this work was to identify new translational markers of the anti-inflammatory / protective state of microglia for the development of novel PET tracers. Methods: New translational markers of the anti-inflammatory/protective activation state of microglia were selected by bioinformatic approaches and were in vitro and ex vivo validated by qPCR and immunohistochemistry in rodent and human samples. Once a viable marker was identified, a novel PET tracer was developed. This tracer was subsequently confirmed by autoradiography experiments in murine and human brain tissues. Results: Here we provide evidence that P2RY12 expression increases in murine and human microglia following exposure to anti-inflammatory stimuli, and that its expression is modulated in the reparative phase of experimental and clinical stroke. We then synthesized a novel carbon-11 labeled tracer targeting P2RY12, showing increased binding in brain sections of mice treated with IL4, and low binding to brain sections of a murine stroke model and of a stroke patient. Conclusion: This study provides new translational targets for PET tracers for the anti-inflammatory/protective activation state of microglia and shows the potential of a rationale-based approach. It therefore paves the way for the development of novel non-invasive methodologies aimed to monitor the success of therapeutic approaches in various neurological diseases.

Impact of platelet reactivity on 5-year clinical outcomes following percutaneous coronary intervention: a landmark analysis.

We investigated the impact of suboptimal platelet reactivity on clinical outcomes after percutaneous coronary intervention (PCI). We enrolled 500 patients with stable coronary artery disease undergoing elective PCI. Platelet reactivity was measured before PCI using the VerifyNow P2Y12 assay. Primary endpoint was the incidence of ischemic or bleeding events at 1 month and 5 years. Patients with high platelet reactivity (HPR) showed significantly higher rates of ischemic events both during the 1st month after PCI (HR 2.06, 95% CI 1.02-4.06), and beyond 1 month compared with patients without HPR (HR 1.73, 95% CI 1.02-2.95). Conversely, compared with patients without low platelet reactivity (LPR), patients with LPR presented significantly higher rates of bleeding only during the 1st month (HR 3.67, 95% CI 1.68-8.02). In conclusion, pre-procedural HPR is associated with ischemic events even beyond the 1st month after PCI. The association of LPR with bleeding events seems to be confined to the periprocedural period.

Clopidogrel IBS Patients Have Higher Incidence of Gastrointestinal Symptoms Influenced by Age and Gender.

BACKGROUND: Clopidogrel is an irreversible antagonist of P2Y12 receptors (P2Y12Rs) used as an antiplatelet drug to reduce risk of thrombosis. P2Y12Rs are expressed in gastrointestinal (GI) tract where they might regulate GI function. AIM: To evaluate if blockade of P2Y12Rs by clopidogrel is associated with higher incidence of GI symptoms in patients with irritable bowel syndrome (IBS). METHODS: A retrospective analysis of our institutional database was conducted for a 13-year period. IBS patients were identified, and their demographics, GI symptoms and clopidogrel therapy were collected. Logistic regression models were used to characterize symptoms in clopidogrel versus no-clopidogrel IBS-groups, adjusting for Age and Sex differences. An additional study characterized the P2Y12R distribution in human gut. RESULTS: The search identified 7217 IBS patients (6761 no-clopidogrel/456 clopidogrel). There were a higher proportion of patients with GI symptoms on clopidogrel (68%) compared to controls (60%, p = 0.0011) that were Females (70 vs. 60%, p = 0.0003) not Males (61 vs. 60%; p = 0.8312). In Females, clopidogrel was associated with higher incidence of GI symptoms (Age adjusted; p < 0.0001) for pain, constipation, gastroparesis (p ≤ 0.0001) and psychogenic pain (p = 0.0006). Age or Sex (adjusted models) influenced one or more GI symptoms (i.e., pain, p < 0.0001; constipation, p < 0.0001/p = 0.008; diarrhea, flatulence, p = 0.01). P2Y12R immunoreactivity was abundant in human ENS; glial-to-neuron ratio of P2Y12Rs expressed in Females â‰« Males. CONCLUSIONS: Irreversible blockade of P2Y12R by clopidogrel is associated with higher incidence of GI symptoms in Female IBS patients, although Age or Sex alone contributes to symptomatology. Prospective studies can determine clinical implications of P2Y12Rs in IBS.

The effect of correcting VerifyNow P2Y12 assay results for hematocrit in patients undergoing percutaneous coronary interventions.

Essentials Platelet reactivity is correlated with thrombotic risk after percutaneous coronary intervention (PCI). Hematocrit (HCT) is associated with platelet reactivity as measured with the VerifyNow P2Y12 assay. We tested a formula proposed to correct VerifyNow measurements for HCT in 978 PCI patients. Correcting platelet reactivity for HCT did not improve the prediction of thrombotic events after PCI. SUMMARY: Background High on-treatment platelet reactivity is predictive for the occurrence of atherothrombotic events following percutaneous coronary interventions (PCIs). A low hematocrit (HCT) value is associated with higher platelet reactivity values, expressed in P2Y12 reaction units (PRU), as measured with the VerifyNow P2Y12 assay. However, it is suggested that this is only an in vitro phenomenon. Objective To determine whether adjusting PRU for HCT improves the predictive value for thrombotic events following PCI. Material and methods The VerifyNow P2Y12 assay was performed in clopidogrel-treated patients undergoing non-urgent PCI included in a prospective cohort study. PRU values were corrected for HCT with a formula proposed in recent literature. Receiver operating characteristic (ROC) curves were made to determine the optimal cut-off values to predict the occurrence of the primary endpoint, a composite of all-cause death and non-fatal myocardial infarction, stent thrombosis and ischemic stroke, during 1 year of follow-up. The chi-squared test was performed to determine whether correcting PRU for HCT improved the prediction of the primary endpoint. Results A total of 978 patients were analyzed. A negative correlation between PRU and HCT was observed (R2 = 0.104). The optimal cut-off value for the corrected PRU was 215. ROC analyses showed that prediction of the primary endpoint did not differ for the corrected PRU (area under the curve, 0.61; sensitivity, 0.57; specificity, 0.64) and the uncorrected PRU (area under the curve, 0.61; sensitivity, 0.69; specificity, 0.53). Conclusion Correcting PRU for HCT does not improve the prediction of thrombotic events following PCI.

P2Y12 Receptor Localizes in the Renal Collecting Duct and Its Blockade Augments Arginine Vasopressin Action and Alleviates Nephrogenic Diabetes Insipidus.

P2Y12 receptor (P2Y12-R) signaling is mediated through Gi, ultimately reducing cellular cAMP levels. Because cAMP is a central modulator of arginine vasopressin (AVP)-induced water transport in the renal collecting duct (CD), we hypothesized that if expressed in the CD, P2Y12-R may play a role in renal handling of water in health and in nephrogenic diabetes insipidus. We found P2Y12-R mRNA expression in rat kidney, and immunolocalized its protein and aquaporin-2 (AQP2) in CD principal cells. Administration of clopidogrel bisulfate, an irreversible inhibitor of P2Y12-R, significantly increased urine concentration and AQP2 protein in the kidneys of Sprague-Dawley rats. Notably, clopidogrel did not alter urine concentration in Brattleboro rats that lack AVP. Clopidogrel administration also significantly ameliorated lithium-induced polyuria, improved urine concentrating ability and AQP2 protein abundance, and reversed the lithium-induced increase in free-water excretion, without decreasing blood or kidney tissue lithium levels. Clopidogrel administration also augmented the lithium-induced increase in urinary AVP excretion and suppressed the lithium-induced increase in urinary nitrates/nitrites (nitric oxide production) and 8-isoprostane (oxidative stress). Furthermore, selective blockade of P2Y12-R by the reversible antagonist PSB-0739 in primary cultures of rat inner medullary CD cells potentiated the expression of AQP2 and AQP3 mRNA, and cAMP production induced by dDAVP (desmopressin). In conclusion, pharmacologic blockade of renal P2Y12-R increases urinary concentrating ability by augmenting the effect of AVP on the kidney and ameliorates lithium-induced NDI by potentiating the action of AVP on the CD. This strategy may offer a novel and effective therapy for lithium-induced NDI.

Characterization of patients with angioscopically-detected in-stent mural thrombi ­ genetics of clopidogrel responsiveness and generations of drug-eluting stents.

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 P2Y12platelet 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.

Development of a multiplex and cost-effective genotype test toward more personalized medicine for the antiplatelet drug clopidogrel.

There has been a wide range of inter-individual variations in platelet responses to clopidogrel. The variations in response to clopidogrel can be driven by genetic polymorphisms involved in the pathway of absorption, distribution, metabolism, excretion, and the target receptor P2Y12. A set of genetic variants known for causing variations in clopidogrel responses was selected, which included CYP2C19*2, *3, *17, CYP2B6*4, *6, *9, CYP3A4*18, CYP3A5*3, MDR1 2677G>T/A, 3435C>T, and P2Y12 H2 (742T>C). The simultaneous detection of these 10 variants was developed by using a multiplex PCR and single-base extension (MSSE) methodology. The newly developed genotyping test was confirmed by direct DNA sequencing in the representative positive control samples and validated in an extended set of 100 healthy Korean subjects. Genotyping results from the developed MSSE exhibited a perfect concordance with the direct DNA sequencing data and all of variants tested in 100 healthy Korean subjects were in agreement with Hardy-Weinberg equilibrium (p>0.05). The present molecular diagnostic studies provide an accurate, convenient, and fast genotyping method for the detection of multiple variants. This would be helpful for researchers, as well as clinicians, to use genetic information toward more personalized medicine of clopidogrel and other antiplatelet drugs in the future.

Hirudin anticoagulation allows more rapid determination of P2Y12 inhibition by the VerifyNow P2Y12 assay.

VerifyNow (VN) P2Y12 is a point-of-care assay used to assess response to P2Y12 inhibitors. Sodium citrate (citrate) is the standard anticoagulant used for this assay but requires a pre-incubation period. Hirudin is an alternative anticoagulant for platelet function studies that maintains physiological divalent cation levels. We investigated whether hirudin anticoagulation might allow more rapid testing of P2Y12 inhibition at the time of percutaneous coronary intervention (PCI). Blood was collected from the arterial sheath of aspirin-treated patients undergoing elective, urgent or emergency coronary angiography±PCI and aliquots were anticoagulated with either citrate or hirudin. For each anticoagulant, VN P2Y12 was performed both immediately and after 20 minutes. A total of 98 patients were included in this study following pre-treatment with clopidogrel (n=88), prasugrel (n=6) or no P2Y12 inhibitor (n=4). PRU with hirudin immediately (PRU_H_Imm) and PRU with citrate 20 minutes post sampling (PRU_C_20) were very strongly correlated (R=0.95) though PRU_H_Imm tended to be lower than PRU_C_20 so that optimal correlation was estimated by the equation PRU_H_Imm=0.95xPRU_C_20 (p<0.001). Bland-Altman plots showed good agreement between PRU_H_Imm and (0.95xPRU_C_20). Platelet reactivity was more stable over the studied time course with hirudin as compared to citrate. We therefore conclude that VN P2Y12 with hirudin anticoagulation can be performed more rapidly and results are strongly correlated with delayed citrate measurements. Further studies are warranted to assess the utility of this method for improving clinical outcomes in patients undergoing PCI.

A comparison of INNOVANCE® PFA P2Y and VerifyNow P2Y12 assay for the assessment of clopidogrel resistance in patients undergoing percutaneous coronary intervention.

INTRODUCTION: VerifyNow P2Y12 is commonly used to measure responsiveness to clopidogrel. We sought to compare the results obtained from novel INNOVANCE® PFA P2Y and VerifyNow P2Y12 assay to assess the clopidogrel resistance in patients undergoing percutaneous coronary intervention. METHODS: A total of 255 patients undergoing percutaneous coronary intervention, preliminarily treated with 100 mg/day of aspirin followed by coadministration of clopidogrel (loading dose, 600 mg; maintenance dose, 75 mg/day), were enrolled in this study. Platelet aggregation was measured by INNOVANCE® PFA P2Y and VerifyNow P2Y12. RESULTS: INNOVANCE® PFA P2Y and VerifyNow P2Y12 assay showed moderate correlations with INNOVANCE® PFA P2Y vs. VerifyNow%inhibition: r = 0.412, P < 0.0001; INNOVANCE® PFA P2Yvs.VerifyNow P2Y12 reaction units (PRU): r = -0.402, P < 0.0001. The agreement between INNOVANCE® PFA P2Y and VerifyNow%inhibition was 85% and that of INNOVANCE® PFA P2Y and VerifyNow PRU was 79%. The k statistics between INNOVANCE® PFA P2Y and VerifyNow%inhibition and PRU were 0.52 and 0.44, respectively. CONCLUSIONS: The sensitivity of INNOVANCE® PFA P2Y in detecting clopidogrel resistance is comparable to that of VerifyNow P2Y12 assay. As the PFA-100® system is already widely used, the new test cartilage may be a useful tool for the assessment of clopidogrel effects. Additional clinical correlation studies are required to validate the effectiveness of INNOVANCE® PFA P2Y in predicting long-term clinical outcomes.

Roles of purinergic receptor P2Y, G protein-coupled 12 in the development of atherosclerosis in apolipoprotein E-deficient mice.

OBJECTIVE: The aim of the study was to evaluate the role of purinergic receptor P2Y, G protein-coupled 12 (P2Y12), an ADP receptor, in the development of atherosclerotic lesions. METHODS AND RESULTS: Apolipoprotein E-null mice were crossed with P2y12(-/-) mice to generate double knockout mice. The double knockout mice and the control apolipoprotein E-null mice were fed a high-fat diet for 20 weeks. Assessment of the atherosclerotic lesions in the control and double knockout mice demonstrated that P2Y12 deficiency caused a diminished lesion area, an increased fibrous content at the plaque site, and decreased monocyte/macrophage infiltration of the lesions. Polymerase chain reaction studies revealed that white blood cells do not express significant levels of P2Y12. Bone marrow transplantation experiments confirmed that P2Y12 expressed on platelets is a key factor responsible for atherosclerosis, but do not exclude a role of smooth muscle cell P2Y12. Supernatant fluid from activated P2y12(+/+) but not P2y12(-/-) platelets was capable of causing monocyte migration. In vitro studies showed that platelet P2Y12 deficiency suppressed platelet factor 4 secretion and P-selectin expression. Further work demonstrated that platelet P2Y12, through inhibition of the cAMP/protein kinase A pathway, critically regulates the release of platelet factor 4, and thereby affects monocyte recruitment and infiltration. CONCLUSIONS: These results demonstrate that P2Y12 modulates atherogenesis, at least in part by augmenting inflammatory cell recruitment via regulation of platelet α-granule release.

Latest evidence in personalized antiplatelet therapy in patients with acute coronary syndromes undergoing percutaneous coronary intervention.

In patients with acute coronary syndromes undergoing percutaneous coronary intervention, the combination of aspirin and clopidogrel, a P2Y12 adenosine diphosphate (ADP) receptor antagonist, is the gold standard of antiplatelet therapy. Two more potent P2Y12 ADP receptor antagonists are now available. Pharmacodynamic studies have revealed a large interindividual variability in the biological response to clopidogrel that is primarily related to variable active metabolite generation, depending on clinical factors, drug-drug interactions, and genetic polymorphisms. Several assays to measure platelet function are available and have revealed a high prevalence of high on-treatment platelet reactivity (HTPR). Patients exhibiting HTPR after a clopidogrel loading dose have a higher risk of thrombotic recurrence after percutaneous coronary intervention. A recent consensus has defined HTPR for the main platelet assays available (using receiver operating characteristic curve analysis) to define the optimal cutoff value for each assay in order to predict thrombotic recurrences. In this article, we present several lines of evidence that suggest a therapeutic window of platelet reactivity inhibition with P2Y12 ADP receptor antagonists. Such a paradigm shift is supported by the results of the Platelet Inhibition and Patient Outcomes (PLATO) trial and the Trial to Assess Improvement in Therapeutic Outcomes by Optimizing Platelet Inhibition with Prasugrel-Thrombolysis in Myocardial Infarction (TRITON-TIMI) 38, which showed the superiority of ticagrelor and prasugrel on thrombotic events compared with clopidogrel; however, these 2 medications had an increased bleeding rate. With the results of these trials, in addition to the evidence of a therapeutic window with P2Y12 ADP receptor antagonists, we summarize the potential of platelet reactivity monitoring and pharmacogenomics to tailor therapy.

Usefulness of platelet response to clopidogrel by point-of-care testing to predict bleeding outcomes in patients undergoing percutaneous coronary intervention (from the Antiplatelet Therapy for Reduction of Myocardial Damage During Angioplasty-Bleeding Study).

Platelet reactivity predicts ischemic outcomes in patients who undergo percutaneous coronary intervention (PCI), but the correlation of heightened platelet response with bleeding has not been characterized. The aim of this study was to evaluate whether low platelet reactivity by point-of-care measurement after clopidogrel administration correlates with bleeding complications of PCI. A total of 310 patients receiving clopidogrel before PCI were prospectively enrolled. Platelet reactivity was measured with the VerifyNow P2Y12 assay. The primary end point was the 30-day incidence of major bleeding or entry-site complications according to quartile distribution of P2Y12 reaction units (PRU). The primary end point occurred more frequently in patients with preprocedural PRU levels in the lowest quartile compared to those in the highest quartile (10.1% vs 1.3%, p = 0.043), due mainly to entry-site hemorrhages. Absolute PRU levels were lower in patients with major bleeding (171 ± 49 vs 227 ± 68 in patients without, p = 0.002). On multivariate analysis, pre-PCI PRU levels in the first quartile were associated with a 4.5-fold increased risk for major bleeding (odds ratio 4.5, 95% confidence interval 1.9 to 25.9, p = 0.01). By receiver-operating characteristic curve analysis, the optimal cutoff for the primary end point was a pre-PCI PRU value ≤ 189 (area under the curve 0.76, 95% confidence interval 0.66 to 0.87, p = 0.001). In conclusion, this study suggests that an enhanced response to clopidogrel may be associated with higher risk for early major bleeding or entry-site complications in patients who undergo PCI. Point-of-care monitoring of platelet reactivity after clopidogrel administration may help identify patients in whom individualized strategies are indicated to limit bleeding complications after coronary intervention.