William R. Herzog

Association of Cytochrome P450 2C19 Genotype With the Antiplatelet Effect and Clinical Efficacy of Clopidogrel Therapy

CONTEXT Clopidogrel therapy improves cardiovascular outcomes in patients with acute coronary syndromes and following percutaneous coronary intervention by inhibiting adenosine diphosphate (ADP)-dependent platelet activation. However, nonresponsiveness is widely recognized and is related to recurrent ischemic events. OBJECTIVE To identify gene variants that influence clopidogrel response. DESIGN, SETTING, AND PARTICIPANTS In the Pharmacogenomics of Antiplatelet Intervention (PAPI) Study (2006-2008), we administered clopidogrel for 7 days to 429 healthy Amish persons and measured response by ex vivo platelet aggregometry. A genome-wide association study was performed followed by genotyping the loss-of-function cytochrome P450 (CYP) 2C19*2 variant (rs4244285). Findings in the PAPI Study were extended by examining the relation of CYP2C19*2 genotype to platelet function and cardiovascular outcomes in an independent sample of 227 patients undergoing percutaneous coronary intervention. MAIN OUTCOME MEASURE ADP-stimulated platelet aggregation in response to clopidogrel treatment and cardiovascular events. RESULTS Platelet response to clopidogrel was highly heritable (h(2) = 0.73; P < .001). Thirteen single-nucleotide polymorphisms on chromosome 10q24 within the CYP2C18-CYP2C19-CYP2C9-CYP2C8 cluster were associated with diminished clopidogrel response, with a high degree of statistical significance (P = 1.5 x 10(-13) for rs12777823, additive model). The rs12777823 polymorphism was in strong linkage disequilibrium with the CYP2C19*2 variant, and was associated with diminished clopidogrel response, accounting for 12% of the variation in platelet aggregation to ADP (P = 4.3 x 10(-11)). The relation between CYP2C19*2 genotype and platelet aggregation was replicated in clopidogrel-treated patients undergoing coronary intervention (P = .02). Furthermore, patients with the CYP2C19*2 variant were more likely (20.9% vs 10.0%) to have a cardiovascular ischemic event or death during 1 year of follow-up (hazard ratio, 2.42; 95% confidence interval, 1.18-4.99; P = .02). CONCLUSION CYP2C19*2 genotype was associated with diminished platelet response to clopidogrel treatment and poorer cardiovascular outcomes.

The genetic response to short-term interventions affecting cardiovascular function: Rationale and design of the Heredity and Phenotype Intervention (HAPI) Heart Study

BACKGROUND The etiology of cardiovascular disease (CVD) is multifactorial. Efforts to identify genes influencing CVD risk have met with limited success to date, likely because of the small effect sizes of common CVD risk alleles and the presence of gene by gene and gene by environment interactions. METHODS The HAPI Heart Study was initiated in 2002 to measure the cardiovascular response to 4 short-term interventions affecting cardiovascular risk factors and to identify the genetic and environmental determinants of these responses. The measurements included blood pressure responses to the cold pressor stress test and to a high salt diet, triglyceride excursion in response to a high-fat challenge, and response in platelet aggregation to aspirin therapy. RESULTS The interventions were carried out in 868 relatively healthy Amish adults from large families. The heritabilities of selected response traits for each intervention ranged from 8% to 38%, suggesting that some of the variation associated with response to each intervention can be attributed to the additive effects of genes. CONCLUSIONS Identifying these response genes may identify new mechanisms influencing CVD and may lead to individualized preventive strategies and improved early detection of high-risk individuals.

Timing of Magnesium Therapy Affects Experimental Infarct Size

BACKGROUND Controversy exists regarding the use of magnesium in the treatment of acute myocardial infarction (AMI) because of apparent conflicting results from clinical trials. One hypothesis to explain the various clinical observations proposes that the timing of magnesium administration significantly influences its therapeutic effect; ie, supraphysiological levels of Mg2+ must be present at the time of reperfusion for magnesium to produce clinical benefit. METHODS AND RESULTS These experiments evaluated the effect of varying the timing of magnesium administration during AMI. Female Yorkshire swine (34 to 42 kg) underwent thoracotomy and 50 minutes of left anterior descending coronary artery (LAD) occlusion, followed by 3 hours of reperfusion. In the first group, MgSO4 (250 mg of magnesium diluted in 60 cm3 saline) was infused into the LAD over 12 minutes, beginning immediately with the onset of reperfusion (n = 6, Mg-early group). In the second group, MgSO4 was given after 1 hour of reperfusion (n = 6, Mg-late group). Six pigs received saline instead of magnesium and served as the control group. Lethal arrhythmias were significantly reduced in the Mg-early group. Infarct size was determined by vital staining. Infarct size was 0.16 +/- 0.05 g/kg body wt (Mg-early), 0.35 +/- 0.08 g/kg (Mg-late), and 0.42 +/- 0.04 g/kg for the control group. Compared with the control group, significant (P = .029) reduction in infarct size occurred in the Mg-early group but not in the Mg-late group. CONCLUSIONS We conclude that intracoronary MgSO4 delivered during reperfusion can significantly diminish infarct size in swine, but the timing of administration is critical.

Genetic Variation in PEAR1 Is Associated With Platelet Aggregation and Cardiovascular Outcomes

Background—Aspirin or dual antiplatelet therapy with aspirin and clopidogrel is a standard therapy for patients who are at increased risk for cardiovascular events. However, the genetic determinants of variable response to aspirin (alone and in combination with clopidogrel) are not known. Methods and Results—We measured ex vivo platelet aggregation before and after dual antiplatelet therapy in individuals (n=565) from the Pharmacogenomics of Anti-Platelet Intervention (PAPI) Study and conducted a genome-wide association study of drug response. Significant findings were extended by examining genotype and cardiovascular outcomes in 2 independent aspirin-treated cohorts: 227 percutaneous coronary intervention patients and 1000 patients of the International Verapamil SR/Trandolapril Study (INVEST) Genetic Substudy (INVEST-GENES). Results from the genome-wide association study revealed a strong association between single-nucleotide polymorphisms on chromosome 1q23 and post–dual antiplatelet therapyplatelet aggregation. Further genotyping revealed rs12041331 in the platelet endothelial aggregation receptor-1 (PEAR1) gene to be most strongly associated with dual antiplatelet therapy response (P=7.66×10−9). In white and black patients undergoing percutaneous coronary intervention, A-allele carriers of rs12041331 were more likely to experience a cardiovascular event or death compared with GG homozygotes (hazard ratio, 2.62; 95% confidence interval, 0.96–7.10; P=0.059; and hazard ratio, 3.97; 95% confidence interval, 1.10–14.31; P=0.035, respectively). In aspirin-treated INVEST-GENES patients, rs12041331 A-allele carriers had significantly increased risk of myocardial infarction compared with GG homozygotes (odds ratio, 2.03; 95% confidence interval, 1.01–4.09; P=0.048). Conclusion—Common genetic variation in PEAR1 may be a determinant of platelet response and cardiovascular events in patients on aspirin alone or in combination with clopidogrel. Clinical Trial Registration—URL: http://www.clinicaltrials.gov. Unique identifiers: NCT00799396 and NCT00370045

Association of single nucleotide polymorphisms on chromosome 9p21.3 with platelet reactivity: A potential mechanism for increased vascular disease

Background—Genome-wide association studies have identified a locus on chromosome 9p21.3 to be strongly associated with myocardial infarction/coronary artery disease and ischemic stroke. To gain insights into the mechanisms underlying these associations, we hypothesized that single nucleotide polymorphisms (SNPs) in this region would be associated with platelet reactivity across multiple populations. Methods and Results—Subjects in the initial population included 1402 asymptomatic Amish adults in whom we measured platelet reactivity (n=788) and coronary artery calcification (CAC) (n=939). Platelet reactivity on agonist stimulation was measured by impedance aggregometry, and CAC was measured by electron beam CT. Twenty-nine SNPs at the 9p21.3 locus were genotyped using the Affymetrix 500K array. Twelve correlated SNPs in the locus were significantly associated with platelet reactivity (all P⩽0.001). The SNP most strongly associated with platelet reactivity, rs10965219 (P=0.0002), also was associated with CAC (P=0.002) along with 9 other SNPs (all P<0.004). Association of rs10965219 with platelet reactivity persisted after adjustment for CAC, a measure of underlying atherosclerotic burden known to affect platelet reactivity. We then tested rs10965219 for association with platelet function in 2364 subjects from the Framingham Heart Study and 1169 subjects from the Genetic Study of Aspirin Responsiveness. The rs10965219 G allele (frequency ≈51% across all 3 populations) was significantly associated with higher platelet reactivity in the Framingham Heart Study (P=0.001) and trended toward higher reactivity in the Genetic Study of Aspirin Responsiveness (P=0.087); the combined P value for metaanalysis was 0.0002. Conclusions—These results suggest that risk alleles at 9p21.3 locus may have pleiotropic effects on myocardial infarction/coronary artery disease and stroke risk, possibly through their influence on platelet reactivity.

Bivalirudin and Clopidogrel With and Without Eptifibatide for Elective Stenting: Effects on Platelet Function, Thrombelastographic Indexes, and Their Relation to Periprocedural Infarction: Results of the CLEAR PLATELETS-2 (Clopidogrel With Eptifibatide to Arrest the Reactivity of Platelets) Study

OBJECTIVES The primary objective of this study was to compare the effect of therapy with bivalirudin alone versus bivalirudin plus eptifibatide on platelet reactivity measured by turbidometric aggregometry and thrombin-induced platelet-fibrin clot strength (TIP-FCS) measured by thrombelastography in percutaneous coronary intervention (PCI) patients. The secondary aim was to study the relation of platelet aggregation and TIP-FCS to the occurrence of periprocedural infarction. BACKGROUND Bivalirudin is commonly administered alone to clopidogrel naïve (CN) patients and to patients on maintenance clopidogrel therapy (MT) undergoing elective stenting. The effect of adding eptifibatide to bivalirudin on platelet reactivity (PR) and TIP-FCS, and their relation to periprocedural infarction in these patients are unknown. METHODS Patients (n = 200) stratified to clopidogrel treatment status were randomly treated with bivalirudin (n = 102) or bivalirudin plus eptifibatide (n = 98). One hundred twenty-eight CN patients were loaded with 600 mg clopidogrel immediately after stenting, and 72 MT patients were not loaded. The PR, TIP-FCS, and myonecrosis markers were serially determined. RESULTS In CN and MT patients, bivalirudin plus eptifibatide was associated with markedly lower PR at all times (5- and 20-microM adenosine diphosphate-induced, and 15- and 25-microM thrombin receptor activator peptide-induced aggregation; p < 0.001 for all) and reduced mean TIP-FCS (p < 0.05). Patients who had a periprocedural infarction had higher mean 18-h PR (p < 0.0001) and TIP-FCS (p = 0.002). CONCLUSIONS For elective stenting, the addition of eptifibatide to bivalirudin lowered PR to multiple agonists and the tensile strength of the TIP-FCS, 2 measurements strongly associated with periprocedural myonecrosis. Future studies of PR and TIP-FCS for elective stenting may facilitate personalized antiplatelet therapy and enhance the selection of patients for glycoprotein IIb/IIIa blockade. (Peri-Procedural Myocardial Infarction, Platelet Reactivity, Thrombin Generation, and Clot Strength: Differential Effects of Eptifibatide + Bivalirudin Versus Bivalirudin [CLEAR PLATELETS-2]; NCT00370045.

Short-term low dose intracoronary diltiazem administered at the onset of reperfusion reduces myocardial infarct size

BACKGROUND Currently, controversy exists regarding the use of calcium-channel blockers in the treatment of acute myocardial infarction (AMI), due to apparent conflicting results from clinical trials and animal models. One hypothesis to explain such a discrepancy proposes that the timing and duration of drug administration might influence its cardioprotective effect. Pretreatment with calcium-channel blockers or their administration during coronary artery occlusion is associated with the diminished infarct size in animal models. While verapamil failed to reduce infarct size when the drug was given at the onset of reperfusion, similar effects of low dose diltiazem are not known. METHODS AND RESULTS This experiment evaluated the effect of intracoronary short term low dose diltiazem administration given immediately with postischemic myocardial reperfusion. Yorkshire swine underwent thoracotomy and 50 min of left anterior descending (LAD) occlusion, followed by 3 h of reperfusion. In the first group, diltiazem (2.5 mg diluted in 60 cc saline) was infused into the LAD over 12 min, beginning with the onset of reperfusion (n=8). In the second group, animals received saline instead of diltiazem and served as controls (n=6). Infarct size was 0.13+/-0.06 g/kg of body weight for diltiazem group, and 0.42+/-0.04 g/kg for controls (P=0.01). CONCLUSIONS Short-term low dose diltiazem delivered exclusively during early reperfusion can significantly diminish infarct size in swine. Local intracoronary diltiazem may be valuable adjunct in patients subject to myocardial ischemia/reperfusion during coronary artery bypass grafting, primary angioplasty for AMI, or thrombolysis for AMI if given immediately after restoration of coronary blood flow.

Dietary coenzyme Q10 supplementation alters platelet size and inhibits human vitronectin (CD51/CD61) receptor expression.

Improved cardiovascular morbidity and mortality have been observed in several clinical studies of dietary supplementation with coenzyme Q10 (CoQ10, ubiquinone). Several mechanisms have been proposed to explain the effects of CoQ10, but a comprehensive explanation of its cardioprotective properties is still lacking. One attractive theory links ubiquinone with the inhibition of platelets. The effect of CoQ10 intake on platelet size and surface antigens was examined in human volunteers. Study participants received 100 mg of CoQ10 twice daily in addition to their usual diet for 20 days. Receptor expression was measured by flow cytometry with monoclonal murine anti-human antibodies CD9 (p24), CD42B (Ib), CD41b (IIb), CD61 (IIIa), CD41a (IIb/IIIa), CD49b (VLA-2), CD62p (P selectin), CD31 (PECAM-1), and CD51/CD61 (vitronectin). An increase of total serum CoQ10 level (from 0.6 +/- 0.1 to 1.8 +/- 0.3 micrograms/ml; p < 0.001) was found at protocol termination. Fluorescence intensity was higher for the large platelets when compared with the whole platelet population. Significant inhibition of vitronectin-receptor expression was observed consistently throughout ubiquinone treatment. Reduction of platelet size was observed at the end of CoQ10 supplementation. Inhibition of the platelet vitronectin receptor and a reduction of the platelet size are direct evidence of a link between dietary CoQ10 intake and platelets. These findings may not be fully explained by the known antioxidant and bioenergetic properties of CoQ10. Diminished vitronectin-receptor expression and reduced platelet size resulting from CoQ10 therapy may contribute to the observed clinical benefits in patients with cardiovascular diseases.

Effects of magnesium supplementation in a porcine model of myocardial ischemia and reperfusion

We tested the hypothesis that acute, intravenous (i.v.) magnesium (Mg2+) supplementation would protect against myocardial stunning in an in situ swine model of regional ischemia and reperfusion and that a concomitant inhibitory effect on platelet aggregation would be elicited. An open-chest model was used, with transient occlusion of the left anterior descending coronary artery (LAD) for 8 min. Regional contractile function was assessed by measuring wall thickening fraction with epicardial Doppler crystals. One control group (n = 6) and two treatment groups were studied: group I (n = 6) received 750 mg MgSO4 before occlusion; group II (n = 6) received 1 g MgSO4 after the occlusion. Both protocols produced significant hypermagnesemia. In group I, platelet aggregation was measured before and after Mg2+ treatment using platelet-rich plasma (PRP) and various agonists (ADP 5 and 10 mM and collagen 1 mg/ml). As compared with controls, both treatment groups experienced significantly less postischemic dysfunction, with systolic function returning more quickly to baseline. Furthermore, platelet aggregation was significantly decreased immediately after Mg2+ infusion. Inhibition of platelet aggregation induced by Mg2+ treatment occurs concomitantly with significant amelioration of postischemic myocardial dysfunction.

Magnesium deficiency prolongs myocardial stunning in an open-chest swine model

The effect of magnesium deficiency on postischemic myocardial dysfunction (myocardial stunning) in an open-chest swine model was studied. Twelve swine were assigned either to low magnesium diet or control diet. Myocardial stunning was assessed by measuring regional wall thickening by epicardial Doppler before and after brief occlusion (8 min) of the left anterior descending coronary artery. Serum magnesium levels decreased significantly in the experimental group only. Glutathione levels were 42.6% lower in the magnesium deficient swine than in controls. Stunning time was significantly prolonged from 32.8 +/- 3.1 min in the control group to 43.8 +/- 4.6 min in the hypomagnesemic swine. In conclusion, magnesium deficiency is associated with prolonged recovery from myocardial stunning.