DaLi Feng

Increased Platelet Aggregability Associated With Platelet GPIIIa PlA2 Polymorphism : The Framingham Offspring Study

The platelet glycoprotein IIb/IIIa (GP IIb/IIIa) plays a pivotal role in platelet aggregation. Recent data suggest that the Pl A2 polymorphism of GPIIIa may be associated with an increased risk for cardiovascular disease. However, it is unknown if there is any association between this polymorphism and platelet reactivity. We determined GPIIIa genotype and platelet reactivity phenotype data in 1422 subjects from the Framingham Offspring Study. Genotyping was performed using PCR based restriction fragment length polymorphism analysis. Platelet aggregability was evaluated by the Born method. The threshold concentrations of epinephrine and adenosine diphosphate (ADP) were determined. Allele frequencies of Pl A1 and Pl A2 were 0.84 and 0.16, respectively. The presence of one or two Pl A2 alleles was associated with increased platelet aggregability as indicated by incremen- tally lower threshold concentrations for epinephrine and ADP. For epinephrine, the mean concentrations were 0.9 µmol/L (0.9–1.0) for homozygous Pl A1 , 0.7 µmol/L (0.7–0.9) for the heterozygous Pl A1 / Pl A2 and 0.6 µmol/L (0.4–1.0) for homozygous Pl A2 individuals, p = 0.009. The increase in aggregability induced by epinephrine remained highly significant (p = 0.007) after adjustment for covariates. For ADP-induced aggregation, the respective mean concentrations were 3.1 µmol/L (3.0–3.2), 3.0 µmol/L (2.9–3.2), and 2.8 µmol/L (2.4–3.3), p = 0.19 after adjustment for covariates. Our findings indicate that molecular variants of the gene encoding GPIIIa play a role in platelet reactivity in vitro. Our observations are compatible with and provide an explanation for the reported association of the Pl A2 allotype with increased risk for cardiovascular disease.

Increased Platelet Aggregability Associated With Platelet GPIIIa PlA2 Polymorphism

The platelet glycoprotein IIb/IIIa (GP IIb/IIIa) plays a pivotal role in platelet aggregation. Recent data suggest that the PlA2 polymorphism of GPIIIa may be associated with an increased risk for cardiovascular disease. However, it is unknown if there is any association between this polymorphism and platelet reactivity. We determined GP IIIa genotype and platelet reactivity phenotype data in 1422 subjects from the Framingham Offspring Study. Genotyping was performed using PCR-based restriction fragment length polymorphism analysis. Platelet aggregability was evaluated by the Born method. The threshold concentrations of epinephrine and ADP were determined. Allele frequencies of PlA1 and PlA2 were 0.84 and 0.16, respectively. The presence of 1 or 2 PlA2 alleles was associated with increased platelet aggregability as indicated by incrementally lower threshold concentrations for epinephrine and ADP. For epinephrine, the mean concentrations were 0.9 micromol/L (0.9 to 1.0) for homozygous PlA1, 0.7 mmol/L (0.7 to 0.9) for the heterozygous PlA1/PlA2, and 0.6 micromol/L (0.4 to 1.0) for homozygous PlA2 individuals, P=0.009. The increase in aggregability induced by epinephrine remained highly significant (P=0.007) after adjustment for covariates. For ADP-induced aggregation, the respective mean concentrations were 3.1 micromol/L (3.0 to 3.2), 3.0 micromol/L (2.9 to 3.2), and 2.8 micromol/L (2.4 to 3.3); P=0.19 after adjustment for covariates. Our findings indicate that molecular variants of the gene encoding GP IIIa play a role in platelet reactivity in vitro. Our observations are compatible with and provide an explanation for the reported association of the PlA2 allotype with increased risk for cardiovascular disease.

Genetic and Environmental Contributions to Platelet Aggregation The Framingham Heart Study

Background—Platelet aggregation plays an important role in arterial thrombosis in coronary heart disease, stroke, and peripheral arterial disease. However, the contribution of genetic versus environmental influences on interindividual variation in platelet aggregability is poorly characterized. Methods and Results—We studied the heritability of platelet aggregation responses in 2413 participants in the Framingham Heart Study. The threshold concentrations of epinephrine and ADP required to produce biphasic platelet aggregation and collagen lag time were determined. Mixed-model linear regression was used to calculate correlation coefficients within sibships and within spouse pairs. Variance and covariance component methods were used to estimate the proportion of platelet aggregation attributable to measured covariates versus additive genetic effects. After accounting for environmental covariates, the adjusted sibling correlations for epinephrine, ADP, and collagen lag time were 0.24, 0.22, and 0.31, respectively (P =0.0001 for each). In contrast, adjusted correlations for spouse-pairs were −0.01, 0.05, and −0.02, respectively (all P >0.30). The estimated heritabilities were 0.48, 0.44, and 0.62, respectively. Measured covariates accounted for only 4% to 7% of the overall variance in platelet aggregation, and heritable factors accounted for 20% to 30%. The platelet glycoprotein IIIa PlA2 polymorphism and the fibrinogen Hind III &bgr;-148 polymorphism contributed <1% to the overall variance. Conclusions—In our large, population-based sample, heritable factors play a major role in determining platelet aggregation, and measured covariates play a lesser role. Future studies are warranted to identify the key genetic variants that regulate platelet function and to lay the groundwork for rational pharmacogenetic approaches.

Hemostatic state and atrial fibrillation (The Framingham Offspring Study)

Atrial fibrillation (AF) is strongly associated with thromboembolic complications, although the mechanism for the increased risk has not been fully explained. To determine whether AF might be associated with a hypercoagulable state, we studied hemostatic factors in subjects with or without AF in the Framingham Heart Study. In 3,577 subjects, we measured fibrinogen, von Willebrand factor antigen, tissue plasminogen activator (tPA) antigen, and plasminogen activator inhibitor-1 antigen. Forty-seven subjects had AF at the index clinic examination and 15 had AF on a prior examination, but not on the current examination. Before matching, the 47 subjects with prevalent AF had higher levels of fibrinogen, von Willebrand factor, and tPA antigen than those without AF, all p < or =0.03. Compared with 167 referent subjects matched for age, sex, and other risk factors, those with AF had higher tPA antigen levels than those without AF, 1 1.8 +/- 4.0 ng/ml versus 10.5 +/- 3.9 ng/ml (p = 0.04). However, when further stratified according to their cardiovascular disease status, the differences in hemostatic factors were no longer significant. We conclude that the prothrombotic profile associated with AF was explained by the risk factors of the subjects and the presence of cardiovascular disease. Nonetheless, the hemostatic changes may contribute toward the propensity for thromboembolic complications in AF. Further prospective studies are needed to evaluate whether measurement of these and other hemostatic factors will identify patients with AF who are at increased risk for thromboembolic complications, and who may therefore benefit from more intensive therapy.

Effect of Short-Term Aspirin Use on C-Reactive Protein

Markers of inflammation, such as C-reactive protein (CRP) and fibrinogen, have been shown to be predictive of cardiovascular disease. In the Physicians Health Study, the magnitude of reduction in the risk of myocardial infarction with aspirin therapy was related to baseline CRP levels, raising the possibility that the protective effect of aspirin may be due to antiinflammatory properties in addition to its antiplatelet effect. We therefore investigated whether aspirin therapy lowers CRP levels. Because heavy physical exertion is a well-known trigger of myocardial infarction, we also investigated the effect of aspirin on CRP levels before and after strenuous exercise. Thirty-two healthy men, aged 29 ± 6 years, were enrolled in a randomized, double-blind, parallel study. Blood samples were obtained immediately before and after maximal treadmill exercise at baseline and following 7 days of aspirin therapy (81 or 325 mg). The levels of CRP, as measured by ELISA, increased by 13% following exercise (P < 0.0001). However, aspirin did not significantly alter CRP levels, either at rest (0.81 ± 0.13 mg/L before aspirin vs. 0.78 ± 0.13 mg/L on aspirin) or following exercise (0.92 ± 0.13 mg/L before aspirin vs. 0.86 ± 0.13 mg/L on aspirin), P = 0.73. When the resting and postexercise data were combined, the levels were 0.87 ± 0.13 mg/L before aspirin and 0.82 ± 0.13 mg/L on aspirin (a nonsignificant 6% reduction, P = 0.20). In conclusion, in healthy male subjects CRP levels were not significantly reduced by short-term aspirin therapy. Our data, taking together with other reports, suggest that aspirin may not affect the levels of inflammatory markers. However, further studies are needed with a longer duration of therapy, among subjects with coronary heart disease, and using additional markers of inflammation besides CRP to determine the long-term effects of aspirin use.

Platelet Glycoprotein IIIa PlA Polymorphism, Fibrinogen, and Platelet Aggregability The Framingham Heart Study

Background—Recent data suggest that the PlA2 allele of the platelet glycoprotein IIIa receptor may be a genetic risk factor for cardiovascular disease. We previously reported that the PlA2 allele was associated with increased platelet aggregability, as indicated by lower epinephrine threshold concentrations. Paradoxically, however, it has been reported that PlA2-positive platelets have reduced fibrinogen binding. Because fibrinogen mediates platelet aggregability, we hypothesized that plasma fibrinogen levels may interact with PlA genotype in modulating platelet aggregability. Methods and Results—Glycoprotein IIIa PlA genotype, fibrinogen level, and platelet aggregability were ascertained in 1340 subjects enrolled into the Framingham Offspring Study. Platelet aggregability was evaluated by the Born method. Higher fibrinogen levels were associated with increased epinephrine-induced aggregation (P =0.002) and a trend for ADP-induced aggregation (P =0.07). The fibrinogen effect was genotype specific, however, in that the increase in platelet aggregability with higher fibrinogen was present for the PlA1/A1 genotype (P =0.0005 and P =0.03 for epinephrine- and ADP-induced aggregation, respectively) but not for the PlA2-positive genotype (P >0.90). Conclusion—Higher fibrinogen levels were associated with increased platelet aggregability. However, the association between fibrinogen and platelet aggregability was genotype specific. This interaction may be responsible for the conflicting findings regarding PlA genotype and platelet aggregability. Further study of this gene-environment interaction may provide insight into cardiovascular disease risk.

Factor VII Gene Polymorphism, Factor VII Levels, and Prevalent Cardiovascular Disease: The Framingham Heart Study

Elevated factor VII levels have been associated with increased cardiovascular risk in some studies. The arginine/glutamine (Arg/Gln) polymorphism of the factor VII gene has been previously shown to modify factor VII levels. However, the presence of a gene/environment interaction on factor VII levels or a link with cardiovascular disease (CVD) remains uncertain. We studied subjects from the Framingham Heart Study to determine (1) the extent to which this genetic polymorphism affects factor VII levels; (2) whether interactions exist between this polymorphism and environmental factors on factor VII levels; and (3) the association between the polymorphism and CVD. Genotype data and factor VII antigen levels were available in 1816 subjects. Factor VII levels differed significantly among genotypes in an additive fashion: Gln homozygous, 82.7+/-2.5%; heterozygous, 92.2+/-0.7%; and Arg homozygous, 100. 5+/-0.4% (P<0.0001). The polymorphism was the strongest, single predictor of factor VII levels, explaining 7.7% of the total variance of factor VII levels, whereas other traditional risk factors combined explained an additional 11.5% of the variance. There was an interaction (P=0.02) between the genotype and total cholesterol on factor VII levels, such that the correlation coefficient and slope (factor VII level/total cholesterol) were greatest in Gln/Gln subjects. Among 3204 subjects characterized for genotype and CVD, there was no significant relationship between the genotype and CVD (P=0.12). In the Framingham Heart Study, the Arg/Gln polymorphism was significantly associated with factor VII antigen levels. The strength of the association suggests that genetic variation plays an important role in determining factor VII levels. However, despite being associated with factor VII levels, the Arg/Gln polymorphism was not associated with prevalent CVD.

A genome-wide search for genes affecting circulating fibrinogen levels in the Framingham Heart Study

INTRODUCTION Circulating levels of fibrinogen are associated with atherosclerosis and predict future coronary heart disease and stroke. Levels of fibrinogen are correlated among family members, suggesting a heritable component. Variants of the beta-fibrinogen gene subunit on 4q28 are associated with fibrinogen levels but explain only a small proportion of the total genetic variability. It remains unknown what role, if any, is played by other genetic variants in the inter-individual variability in levels of fibrinogen in the general population. MATERIALS AND METHODS We conducted a 10-cM spaced genome-wide scan using 402 original cohort subjects and 1193 offspring subjects from 330 extended families of the Framingham Heart Study. Heritability and linkage analyses were carried out using variance component methods. Regression analyses were performed to adjust for traditional risk factors and HindIII beta-148 genotypes. RESULTS AND DISCUSSIONS The total heritability was estimated as 0.24. The highest and second highest LOD scores of linkage were found on chromosomes 2 (LOD=1.5 at 243 cM) and 10 (LOD=2.4 at 87 cM) using only offspring subjects in the analysis, and on chromosomes 2 (LOD=2.1 at 242 cM) and 10(LOD=1.4 at 86 cM), 17 (LOD=1.4 at 96 cM) and 20 (LOD=1.4 at 80 cM) using both original cohort and offspring. These results suggest that there may be influential genetic regions on these chromosomes. While no linkage with genome-wide significance was detected, further research to confirm our findings is warranted.

Effect of Aspirin Dosage and Enteric Coating on Platelet Reactivity

Although aspirin is effective in the prevention and treatment of cardiovascular diseases, the optimal dose remains uncertain. The purpose of this study was to compare the platelet inhibitory and prostacyclin-sparing effects of 2 doses (81 and 325 mg) and forms (enteric-coated and regular) of aspirin. Since platelet reactivity has been reported to increase after strenuous exercise, a known trigger of myocardial infarction, subjects were studied following maximal treadmill exercise as well as at rest. Forty male healthy subjects were evaluated using a randomized, double-blind, parallel study design. Blood samples were obtained before and after maximal treadmill exercise at baseline and after 7 days on aspirin therapy. Both enteric and regular aspirin in 81- and 325-mg dosages markedly inhibited adenosine diphosphate and epinephrine-induced aggregation at rest and after exercise. Aspirin also inhibited the platelet response to collagen as assessed by a longer lag time to aggregation. The prolongation of lag time was greater for 325 mg than for 81 mg (100 +/- 7 vs 91 +/- 7; p = 0.04, after exercise). There were no significant dose-related differences in plasma 6-keto-prostaglandin F1alpha level; however, enteric-coated aspirin inhibited the exercise-induced increase in 6-keto-prostaglandin F1alpha to a lesser extent than regular aspirin. Although both doses (81 and 325 mg) and types (regular and enteric-coated) of aspirin inhibited adenosine diphosphate and epinephrine-induced aggregation equally, the 325-mg dose inhibited collagen-induced aggregation to a greater extent than 81 mg. The greater platelet inhibition observed with 325 mg may be clinically relevant in acute coronary syndromes characterized by plaque rupture with extensive collagen exposure and platelet activation.

The effect of Vitamin C supplementation on coagulability and lipid levels in healthy male subjects

Although dietary intake and plasma levels of vitamin C have been inversely associated with cardiovascular disease, the mechanism through which it may exert its effect has not been fully explained. Since thrombosis plays an important role in the onset of cardiovascular disease, we investigated the effect of vitamin C on measures of hemostasis that have been associated with cardiovascular risk. The effect of vitamin C on lipid levels was also evaluated. In a randomized, placebo-controlled, crossover study, we determined the effect of 2 g daily of vitamin C supplementation on platelet adhesion and aggregation, levels of tissue plasminogen activator antigen, plasminogen activator inhibitor, fibrinogen, plasma viscosity, von Willebrand factor, and lipid levels in 18 healthy male volunteers with low normal vitamin C levels. No striking effects of vitamin C on the hemostatic measures were observed, although tissue plasminogen activator antigen levels were inversely related to Vitamin C levels. Von Willebrand factor levels were slightly higher with vitamin C, although within the normal range. Total cholesterol levels were 10% lower when subjects were receiving vitamin C compared to placebo (167+/-7 mg/dL vs. 184+/-7 mg/dL), P=0. 007), although the total cholesterol/HDL ratio was not significantly different. Higher levels of tissue plasminogen activator antigen, which in the present study were associated with lower vitamin C levels, have been shown in prospective studies to convey an increased risk of cardiovascular events. Further studies of the effect of vitamin C on hemostatic measures are required in higher risk populations or those with known cardiovascular disease.