Effect of platelet antigen polymorphism on platelet inhibition by aspirin, clopidogrel, or their combination.

OBJECTIVES: We studied the modifier effect of platelet antigen polymorphism (PlA2) on platelet inhibition by acetylsalicylic acid (ASA, i.e., aspirin), clopidogrel, or their combination in patients with coronary heart disease. BACKGROUND: Clopidogrel, when administered with ASA, was shown to significantly improve the outcome of patients with acute coronary syndromes compared with patients receiving only ASA. We have shown previously that the effect of ASA on platelets is modified by the glycoprotein IIIa single nucleotide polymorphism PlA2. Hence, an important pharmacogenetic question remains whether the antiplatelet effect of clopidogrel is uniform for all patients or, like acetylsalicylic acid, more selective. METHODS: Thirty PlA1/A1 and 30 PlA1/A2 patients were assigned randomly to ASA 325 mg/day, clopidogrel 75 mg/day, or both. After 10 days, platelet function was studied. RESULTS: Clopidogrel provided stronger platelet inhibition than ASA with adenosine diphosphate as the agonist, and combination therapy resulted in greater inhibition than either inhibitor used alone (p < 0.0001). The use of ASA resulted in greater inhibition compared with clopidogrel with epinephrine (p < 0.0001) and collagen as agonists (p < 0.0001). With collagen as the agonist, platelets from PlA1/A2 donors were markedly and significantly less inhibited by ASA (p = 0.005). In contrast, with clopidogrel, no significant difference could be detected between inhibition of Pl(A1/A1) and Pl(A1/A2) platelets. CONCLUSIONS: The combination of ASA and clopidogrel appears superior to either agent alone in inhibiting platelet function. Pl(A2) functions as an important modifier for platelet responsiveness to ASA but not to clopidogrel. These findings could have significant impact on the future design of pharmacogenetic antithrombotic strategies for patients with coronary heart disease.

A polymorphism of the endothelial nitric oxide synthase promoter is associated with an increase in autonomic imbalance in patients with congestive heart failure.

BACKGROUND: In addition to its well-recognized role in the regulation of vascular tone, nitric oxide modulates sympathetic and parasympathetic nervous system activities. Abnormalities of both autonomic control and nitric oxide synthase activity are known to occur in patients with congestive heart failure. Recently, a polymorphism of the promoter of the endothelial nitric oxide synthase (eNOS) gene has been associated with a reduction of eNOS activity. This study tested the hypothesis that patients with congestive heart failure who are homozygous for this polymorphism will have a more advanced imbalance of autonomic activity. METHODS: Patients who have congestive heart failure were tested for the presence of an eNOS promoter polymorphism (thymidine to cytosine transition [T(-786)C]). Spectral analysis of heart rate variability was performed to quantify sympathetic and parasympathetic autonomic activity, which were compared between subjects homozygous for the polymorphism and all other subjects. RESULTS: Patients homozygous for the polymorphism of the eNOS promoter had a greater autonomic imbalance as reflected by significant differences in high- and low-frequency heart rate variability. These differences in autonomic function were noted in the absence of intergroup differences in patterns of respiratory variability, demographic features, and despite a higher mean ejection fraction in patients homozygous for the polymorphism. CONCLUSIONS: Patients with congestive heart failure who are homozygous for this polymorphism of the eNOS promoter were found to have a more advanced autonomic imbalance. This polymorphism may serve as a marker for patients at increased risk for sudden death and more rapid progression of disease.

Molecular classification of parathyroid neoplasia by gene expression profiling.

The current classification of sporadic parathyroid neoplasia, specifically the distinction of adenoma from multiple gland neoplasia (double adenoma and nonfamilial primary hyperplasia) is problematic and results in a relatively high rate of clinical error. Oligonucleotide microarrays (Affymetrix U133A) were used to evaluate parathyroid samples from 61 patients; 35 adenomas, 10 nonfamilial multiple gland neoplasia, 3 familial primary hyperplasia, 8 renal-induced hyperplasia, and 5 from patients without parathyroid disease (normals). A multiclass comparison using supervised clustering identified distinct gene signatures for each class of parathyroid samples. We developed a predictor model that correctly identified 34 of 35 cases of adenoma, 9 of 10 cases of nonfamilial multiple gland neoplasia, and identified a minimum set of 11 genes for the distinction of adenoma versus multiple gland neoplasia. All methods of unsupervised clustering showed two related but different types of parathyroid adenomas that we have arbitrarily designated as type 1 and type 2 adenomas. Multiple gland parathyroid neoplasia, which represents either synchronous or asynchronous autonomous growth in two, three, or all four parathyroid glands, is a distinct molecular entity and does not represent the molecular pathogenesis of adenoma occurring in multiple glands.

Dietary sodium regulates angiotensin AT1a and AT1b mRNA expression in mouse brain.

Previous results showed that angiotensin (Ang) AT1a and AT1b receptor mRNA are expressed in mouse hypothalamus (HYP), brainstem (BS) and anterior pituitary (PIT). To extend these findings, we developed a real-time polymerase chain reaction (PCR) method to differentiate and quantify Ang AT1a and AT1b mRNA in mouse brain. An experiment was conducted in male C57Bl/6J mice to determine the effects of low and high dietary salt (0.04 or 8% NaCl for 2 weeks) on mRNA expression. Physiological measurements showed that high salt increased water intake (15.1 +/- 0.6 ml/day), whereas low salt decreased water intake (3.2 +/- 0.1 ml/day). There were no significant changes in body weight, hematocrit or plasma osmolality. Real-time PCR was effective in distinguishing AT1a and AT1b receptor mRNA. The PCR efficiencies for AT1a, AT1b and 18S ribosome were tested to be identical, making it possible to quantify mRNA levels. There were differences in angiotensin receptor expression, related to diet and brain region. In hypothalamus, both the high salt and low salt diet decreased AT1a expression (to 63 +/- 4% and 62 +/- 1%), although there were no changes in AT1b. In brainstem, there was a marked increase in AT1a (to 365 +/- 60%) and AT1b (to 372 +/- 23%) after high salt, although there was only a marked decrease for AT1b (to 23 +/- 5%) after low salt. In anterior pituitary, both high salt and low salt diet increased AT1a expression (to 152 +/- 8% and 123 +/- 9%), although there were no changes in AT1b. Results document that both AT1 receptor subtypes are present in mouse hypothalamus, brainstem and anterior pituitary, and that there is differential regulation of expression in response to changes in dietary salt.

Effect of male sex and obesity on platelet arachidonic acid in spontaneous hypertensive heart failure rats.

Sexual dimorphism is observed in the progression to congestive heart failure and, ultimately, in longevity in spontaneously hypertensive heart failure (SHHF) rats. As platelet activation may impact development of cardiovascular diseases, we studied the effects of obesity and sex on platelet polyunsaturated fatty acid (PUFA) profile and its relationship to platelet aggregation in 6-month-old SHHF rats. After a 24-hr fast, blood was obtained for measurement of platelet phospholipid omega-6 (n-6) and omega-3 (n-3) PUFA. Collagen-induced platelet aggregation was measured by whole-blood impedance aggregometry. Obese male (OM) SHHF had significantly more platelet arachidonic acid (AA) and total n-6 PUFA than lean males (LMs), lean females (LFs), or obese females (OFs). Platelet aggregation was enhanced in males compared to females, with OMs by 45% compared to OFs and with LMs by 28% compared to LFs. Though no difference was found between OFs and LFs, platelet aggregation was increased in OMs by 20% compared to LMs. Though not significantly different, lag time to initiate platelet aggregation tended to be shortest in OMs and then, in increasing duration, LMs, LFs, and OFs, suggesting that platelets from male rats were quicker to aggregate than those from females. Platelet aggregation was correlated with platelet AA and total n-6 PUFA content. There was no relationship between n-3 PUFA and platelet aggregation. In SHHF rats, elevated AA and n-6 PUFA levels in platelets are associated with enhanced platelet aggregation. This relationship is potentiated by obesity and male sex.

DNA microarray technology in nutraceutical and food safety.

The quality and quantity of diet is a key determinant of health and disease. Molecular diagnostics may play a key role in food safety related to genetically modified foods, food-borne pathogens and novel nutraceuticals. Functional outcomes in biology are determined, for the most part, by net balance between sets of genes related to the specific outcome in question. The DNA microarray technology offers a new dimension of strength in molecular diagnostics by permitting the simultaneous analysis of large sets of genes. Automation of assay and novel bioinformatics tools make DNA microarrays a robust technology for diagnostics. Since its development a few years ago, this technology has been used for the applications of toxicogenomics, pharmacogenomics, cell biology, and clinical investigations addressing the prevention and intervention of diseases. Optimization of this technology to specifically address food safety is a vast resource that remains to be mined. Efforts to develop diagnostic custom arrays and simplified bioinformatics tools for field use are warranted.

Potential thrombophilic mutations/polymorphisms in patients with no flow-limiting stenosis after myocardial infarction.

BACKGROUND: Although inherited thrombophilias are more common in patients with venous thromboembolism, their influence on the development of myocardial infarction (MI) requires clarification. METHODS AND RESULTS: To determine whether there are increased frequencies of mutations/polymorphisms in 14 genes potentially causing thrombophilia in patients with no flow-limiting stenoses after MI compared with patients with > or =1 flow-limiting stenosis of >50%, we studied 395 patients (60 with no flow-limiting stenosis) who underwent angiography at approximately 1 month. The mutations/polymorphisms studied included Factor V Leiden, prothrombin variant G20210A, beta-fibrinogen 448 (G/A), endothelial protein C receptor (23-base pair insertion), methyl tetrahydrofolate reductase 677 (C/T), platelet glycoprotein IIIa PlA1/A2, plasminogen activator inhibitor-1 4G/5G, angiotensin II type 1 receptor (A/C), hemochromatosis gene 282 (G/A), nitric oxide synthase (NOS) (3 forms: eNOS, eNOS3, eNOS4), p22 phox of NADPH oxidase C242T, and angiotensin-converting enzyme insertion/deletion polymorphism. The frequencies of Factor V Leiden and the beta-fibrinogen 448 A allele were higher in patients with no flow-limiting stenosis than in patients with > or =1 stenosis (11.7% vs 3.6%, odds ratio [OR] 3.6, 95% CI 1.3-9.4, P =.015; and 42% vs 27%, OR 2.0, 95% CI 1.1-3.5, P =.018, respectively), and there was a trend toward an increased frequency of prothrombin variant G20210A (6.7% vs 2.1%, OR 3.4, 95% CI 0.95-11.8, P =.069). However, in patients with no flow-limiting stenosis after MI the frequencies of the other gene mutations/polymorphisms were not increased. Also, there were no significant interactions between any of these 14 mutation/polymorphisms, major cardiovascular risk factors, and the absence of any flow-limiting stenosis, except for Factor V Leiden and hypertension (OR 6.34, 95% CI 2.67-100, P =.004). CONCLUSIONS: Patients with no flow-limiting stenosis after MI had increased frequencies of 2 inherited thrombophilias (Factor V Leiden and beta-fibrinogen 448 A allele), and there was a trend toward an increased frequency of prothrombin variant G20210A compared with patients with > or =1 stenosis. These data suggest that polymorphisms/mutations in some gene products influencing coagulation may influence the pathogenesis of MI.