Nauder Faraday

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.

Cardiac Troponin I Predicts Short-Term Mortality in Vascular Surgery Patients

Background—Cardiac troponin I (cTnI) is a highly sensitive and specific marker for myocardial injury that predicts outcomes in patients with acute coronary syndromes. Cardiovascular complications are the leading cause of morbidity and mortality in patients who have undergone vascular surgery. However, postoperative surveillance with cardiac enzymes is not routinely performed in these patients. We evaluated the association between postoperative cTnI levels and 6-month mortality and perioperative myocardial infarction (MI) after vascular surgery. Methods and Results—Two hundred twenty-nine patients having aortic or infrainguinal vascular surgery or lower extremity amputation were included in this study. Blood samples were analyzed for cTnI immediately after surgery and the mornings of postoperative days 1, 2, and 3. An elevated cTnI was defined as serum concentrations >1.5 ng/mL in any of the 4 samples. Twenty-eight patients (12%) had postoperative cTnI >1.5ng/mL, which was associated with a 6-fold increased risk of 6-month mortality (adjusted OR, 5.9; 95% CI, 1.6 to 22.4) and a 27-fold increased risk of MI (OR, 27.1; 95% CI, 5.2 to 142.7). Furthermore, we observed a dose-response relation between cTnI concentration and mortality. Patients with cTnI >3.0 ng/mL had a significantly greater risk of death compared with patients with levels ≤0.35 ng/mL (OR, 4.9; 95% CI, 1.3 to 19.0). Conclusions—Routine postoperative surveillance for cTnI is useful for identifying patients who have undergone vascular surgery who have an increased risk for short-term mortality and perioperative MI. Further research is needed to determine whether intervention in these patients can improve outcome.

Genome-Wide Meta-Analyses Identifies Seven Loci Associated with Platelet Aggregation in Response to Agonists

Platelet function mediates both beneficial and harmful effects on human health, but few genes are known to contribute to variability in this process. We tested association of 2.5 million SNPs with platelet aggregation responses to three agonists (ADP, epinephrine and collagen) in two cohorts of European ancestry (N ≤ 2,753 in the Framingham Heart Study, N ≤ 1,238 in the Genetic Study of Atherosclerosis Risk). We identified associations of seven loci with platelet aggregation near or within GP6 (P = 4.6 × 10−13), PEAR1 (P = 3.4 × 10−12), ADRA2A (P = 3.3 × 10−11), PIK3CG (P = 3.1 × 10−9), JMJD1C (P = 1.6 × 10−8), MRVI1 (P = 2.0 × 10−8) and SHH (P = 4.5 × 10−8). Six of these loci replicated at P < 0.05 in an additional African-American cohort (N ≤ 840 in the Genetic Study of Atherosclerosis Risk). These results provide insights into platelet aggregation pathways and may suggest new antiplatelet therapeutic targets.

Heritability of platelet function in families with premature coronary artery disease

Summary.  Background: Variations in platelet function among individuals may be related to differences in platelet‐related genes. The major goal of our study was to estimate the contribution of inheritance to the variability in platelet function in unaffected individuals from white and African American families with premature coronary artery disease.Methods: Platelet reactivity, in the absence of antiplatelet agents, was assessed by in vitro aggregation and the platelet function analyzer closure time. Heritability was estimated using a variance components model.Results: Both white (n = 687) and African American (n = 321) subjects exhibited moderate to strong heritability (h2) for epinephrine‐ and adenosine diphosphate‐induced aggregation (0.36–0.42 for white and >0.71 for African American subjects), but heritability for collagen‐induced platelet aggregation in platelet‐rich plasma was prominent only in African American subjects. Platelet lag phase after collagen stimulation was heritable in both groups (0.47–0.50). A limited genotype analysis demonstrated that the C825T polymorphism of GNB3 was associated with the platelet aggregation response to 2 μM epinephrine, but the effect differed by race.Conclusions: Considering the few and modest genetic effects reported to affect platelet function, our findings suggest the likely existence of undiscovered important genes that modify platelet reactivity, some of which affect multiple aspects of platelet biology.

In vitro hypothermia enhances platelet GPIIb-IIIa activation and p-selectin expression

Background A clinical bleeding diathesis is associated with hypothermia. Inhibition of platelet reactivity is the purported cause of this coagulopathy despite inconsistent evidence to support this hypothesis. To clarify the effect of temperature on intrinsic platelet function, platelet GPIIb‐IIIa activation and P‐selectin expression were assesed under normothermic and hypothermic conditions in vitro. Methods Blood was obtained by venipuncture from healthy volunteers. Platelet activation was assessed by aggregometry and by cytometric analysis of platelet binding of fibrinogen, PAC‐1, and P‐selectin antibodies. Measurements were made at normothermia (37 [degree sign]C), moderate hypothermia (33 [degree sign]C), and profound hypothermia (22 [degree sign]C) after stimulating samples with adenosine diphosphate (ADP), collagen, or thrombin receptor activating peptide. Results Agonist‐induced platelet aggregation and fibrinogen binding were significantly greater at 22 [degree sign]C and 33 [degree sign]C than at 37 [degree sign]C. Platelet fibrinogen binding values to 20 [micro sign]M ADP were 23,400, 14,300, and 9,700 molecules/platelet at 22 [degree sign]C, 33 [degree sign]C, and 37 [degree sign]C, respectively. The aggregation responses of platelets that were cooled and rewarmed were indistinguishable from those of platelets maintained at 37 [degree sign]C throughout the study. Platelet binding of PAC‐1 and P‐selectin antibodies was greater under hypothermic conditions. Conclusions Aggregation, fibrinogen binding, PAC‐1 binding, and P‐selectin antibody binding studies showed that platelet GPIIb‐IIIa activation and [Greek small letter alpha]‐granule release were enhanced at hypothermic temperatures. Thus hypothermia appears to increase the ability of platelets to respond to activating stimuli. The coagulopathy associated with hypothermia is not likely to be the result of an intrinsic defect in platelet function.

A Novel Variant in the Platelet Endothelial Aggregation Receptor-1 Gene Is Associated With Increased Platelet Aggregability

Objective—Platelet endothelial aggregation receptor-1 (PEAR1) is a recently identified platelet transmembrane protein that becomes activated by platelet contact. We looked for novel genetic variants in PEAR1 and studied their association with agonist-induced native platelet aggregation and with the inhibitory effect of aspirin on platelets. Methods and Results—We genotyped PEAR1 for 10 single nucleotide polymorphisms (SNPs), selected for optimal gene coverage at a density of 4 kb, in 1486 apparently healthy individuals from two generations of families with premature CAD. Subjects had a mean age of 45 years; 62% were white and 38% black. Platelet aggregation to collagen, epinephrine, and ADP was measured in platelet rich plasma, at baseline and after 2 weeks of aspirin (ASA, 81 mg/d), and genotype-phenotype associations were examined separately by ethnicity using multivariable generalized linear models adjusted for covariates. The C allele of SNP rs2768759 [A/C], located in the promoter region of the gene, was common in whites and uncommon in blacks (allele frequency 70.2% versus 17.7%). The C allele was generally associated in both ethnic groups with increased aggregation of native platelets to each agonist. After ASA, the associations were stronger and more consistent and remained significant when post-ASA aggregation was adjusted for baseline aggregation, consistent with a relationship between the C allele and reduced platelet responsiveness to ASA. The PEAR1 SNP explained up to 6.9% of the locus specific genetic variance in blacks and up to 2.5% of the genetic variance in whites after ASA. Conclusion—PEAR1 appears to play an important role in agonist-induced platelet aggregation and in the response to ASA in both whites and blacks.

Sensitivity of routine intensive care unit surveillance for detecting myocardial ischemia.

ObjectiveTo assess the effectiveness of routine intensive care unit surveillance compared with frequent 12-lead electrocardiogram monitoring for detecting electrocardiogram evidence suggestive of prolonged myocardial ischemia in vascular surgery patients. DesignProspective cohort trial. SettingIntensive care unit. ParticipantsWe studied 149 patients undergoing elective infrainguinal or aortic vascular surgery who were admitted to the intensive care unit postoperatively. InterventionsPatients were simultaneously monitored with a 10-electrode/12-lead electrocardiogram obtained every 2 mins (criterion standard) and routine intensive care unit surveillance that included standard monitoring (five-electrode/two-lead electrocardiogram with ST segment trends and routine 12-lead electrocardiogram) and clinical assessment for detecting myocardial ischemia. The results of the criterion standard were not available to the caregivers. Measurements and Main ResultsWe measured the ability of routine intensive care unit surveillance to detect the first 20 mins of electrocardiogram evidence suggestive of myocardial ischemia, defined as ST segment depression or elevation of ≥1 mm in two consecutive leads, during the first postoperative day. Seventeen patients (11%) had electrocardiogram evidence suggestive of prolonged myocardial ischemia, the majority of which occurred in leads V2–V4. The sensitivity of routine intensive care unit surveillance for detecting the first episode of electrocardiogram evidence suggestive of prolonged myocardial ischemia in a patient was 12% (95% confidence interval, 7–17%), and the specificity was 98% (95% confidence interval, 95–100%) with a positive predictive value of 40% (95% confidence interval, 32–48%), a negative predictive value of 90% (95% confidence interval, 85–94%), a positive likelihood ratio of 6, and a negative likelihood ratio of 1. The sensitivity of routine intensive care unit surveillance for detecting all episodes was 3% (95% confidence interval, 2–3%) and the specificity 99% (95% confidence interval, 99–100%) per 20-min monitoring interval, with a positive predictive value of 17% (95% confidence interval, 16–18%), negative predictive value of 95% (95% confidence interval, 95–96%), positive likelihood ratio of 3, and negative likelihood ratio of 1. ConclusionsRoutine intensive care unit surveillance has low sensitivity for detecting electrocardiogram evidence suggestive of prolonged myocardial ischemia compared with frequent 12-lead electrocardiograms. Because detecting electrocardiogram evidence suggestive of prolonged postoperative myocardial ischemia is important, physicians should consider alternative strategies to detect myocardial ischemia.

Hemostatic effects of stress hormone infusion

BackgroundSurgery causes changes in hemostasis, leading to a hypercoagulable state. This postoperative increase in hemostatic function is attenuated In patients receiving regional anesthesia compared with those receiving general anesthesia. Regional anesthesia also decreases the neuroendocrine response to surgery compared with general anesthesia, and this effect Is hypothesized to be responsible for the differences in hemostasis. To test the hypothesis that neuroendocrine hormones cause changes in hemostasis, we infused stress hormones into normal volunteers and measured hemostatic function. MethodsAfter drug screening, 12 normal volunteers were studied. On two admissions, volunteers randomly received either stress hormone (epinephrine, cortisol, or glucagon) or placebo infusion for 24 h. During infusion, patients remained at bed rest and received controlled meals. Blood was obtained from indwelling venous catheters before infusion and 2, 8, and 24 h after the start of infusion. Blood was analyzed for neuroendocrine hormone concentrations, glucose, complete blood count, coagulation proteins, platelet reactivity, and activity of the fibrinolytic system. ResultsIn the stress hormone group, concentrations of epinephrine, norepinephrine, cortisol, glucagon, and insulin were increased during the infusion period compared with those in the placebo group. Glucose concentrations and white blood cell counts were increased in the stress hormone group compared with those in the placebo group. Circulating fibrinogen concentrations increased 30% and ex vivo collagen-induced platelet reactivity increased 123% (aggregation) and 103% (dense granule release) in the stress hormone infusion group, whereas there was no change in the placebo group. Fibrinolytic proteins were similar in both groups, demonstrating a decrease in plasminogen activator inhibitor-1 activity at 8 and 24 h (196% in the hormone group vs. 199% in the placebo group). ConclusionsInfusion of stress hormones to concentrations found during surgery is safely tolerated and causes metabolic changes observed with surgery. Stress hormone infusion Increases ex vivo platelet reactivity and fibrinogen concentrations that resemble changes seen postoperatively but does not recreate the postoperative decrease in fibrinolytic activity. Differences in neuroendocrine response between types of anesthesia may explain some postoperative changes in platelet function and acute phase reactivity, but additional uncharacterized factors are responsible for the differences in fibrinolysis.

Leukocytes can enhance platelet-mediated aggregation and thromboxane release via interaction of P-selectin glycoprotein ligand 1 with P-selectin.

BackgroundPlatelet–leukocyte conjugates have been observed in patients with unstable coronary syndromes and after cardiopulmonary bypass. In vitro, the binding of platelet P-selectin to leukocyte P-selectin glycoprotein ligand-1 (PSGL1) mediates conjugate formation; however, the hemostatic implications of these cell–cell interactions are unknown. The aims of this study were to determine the ability of leukocytes to modulate platelet agonist–induced aggregation and secretion in the blood milieu, and to investigate the role of P-selectin and PSGL-1 in mediating these responses. MethodsBlood was drawn from healthy volunteers for in vitro analysis of platelet agonist–induced aggregation, secretion (adenosine triphosphate, &bgr;-thromboglobulin, and thromboxane), and platelet–leukocyte conjugate formation. Experiments were performed on live cells in whole blood or plasma to simulate physiologic conditions. Whole-blood impedance and optical aggregometry, flow cytometry, and enzyme-linked immunosorbent assays were performed in the presence and absence of blocking antibodies to P-selectin and PSGL1. The platelet-specific agonists, thrombin receptor activating peptide and adenosine diphosphate, were used to elicit platelet activation responses. ResultsInhibition of platelet–leukocyte adherence by P- selectin and PSGL1 antibodies decreased agonist–induced aggregation in whole blood. The presence of leukocytes in platelet-rich plasma increased aggregation, and this increase was attenuated by P-selectin blocking antibodies. Data from flow cytometry confirmed that platelet–leukocyte conjugate formation contributed to aggregation responses. Blocking antibodies reduced platelet agonist–induced thromboxane release but had no impact on adenosine triphosphate and &bgr;-thomboglobulin secretion. ConclusionsLeukocytes can enhance platelet agonist– induced aggregation and thromboxane release in whole blood and platelet-rich plasma under shear conditions in vitro. Interaction of platelet P-selectin with leukocyte PSGL1 contributes substantially to these effects.

Identification of a specific intronic PEAR1 gene variant associated with greater platelet aggregability and protein expression.

Genetic variation is thought to contribute to variability in platelet function; however, the specific variants and mechanisms that contribute to altered platelet function are poorly defined. With the use of a combination of fine mapping and sequencing of the platelet endothelial aggregation receptor 1 (PEAR1) gene we identified a common variant (rs12041331) in intron 1 that accounts for ≤ 15% of total phenotypic variation in platelet function. Association findings were robust in 1241 persons of European ancestry (P = 2.22 × 10⁻⁸) and were replicated down to the variant and nucleotide level in 835 persons of African ancestry (P = 2.31 × 10⁻²⁷) and in an independent sample of 2755 persons of European descent (P = 1.64 × 10⁻⁵). Sequencing confirmed that variation at rs12041331 accounted most strongly (P = 2.07 × 10⁻⁶) for the relation between the PEAR1 gene and platelet function phenotype. A dose-response relation between the number of G alleles at rs12041331 and expression of PEAR1 protein in human platelets was confirmed by Western blotting and ELISA. Similarly, the G allele was associated with greater protein expression in a luciferase reporter assay. These experiments identify the precise genetic variant in PEAR1 associated with altered platelet function and provide a plausible biologic mechanism to explain the association between variation in the PEAR1 gene and platelet function phenotype.