Plasminogen activator inhibitor and the risk of cardiovascular disease: The Framingham Heart Study.

INTRODUCTION: Although plasminogen activator inhibitor (PAI-1) plays a key regulatory role in fibrinolysis, it has not been clearly shown to independently predict cardiovascular disease (CVD) among individuals without prior CVD. We investigated, in the Framingham Heart Study offspring cohort, whether PAI-1 predicted CVD risk among individuals without prior CVD. METHODS: Plasma PAI-1 antigen and tissue plasminogen activator (TPA) antigen were measured in 3203 subjects without prior CVD between 1991 and 1995; average follow-up of 10 years. PAI-1 was remeasured 4 years after baseline, to determine the effect of serial change on risk. RESULTS: PAI-1 levels (mean ± SD) were 29.1 ng/ml (19.2) versus 22.1 (16.5) for those and without incident CVD; p<0.001, and TPA levels were 12.0 ng/ml (5.7) versus 9.0 (4.7); p<0.001. PAI-1 and TPA antigen levels had a strong unadjusted linear relation with incident CVD (p<0.001). After adjustment for conventional risk factors, the hazard ratios (HRs) for higher quartiles of PAI-1, compared with the lowest, were 1.9, 1.9, 2.6 (linear trend p=0.006), and 1.6, 1.6, 2.9 (p<0.001) for TPA antigen. The adjusted HRs for increasing quartiles of serial change in PAI-1 at 4 years, compared with the lowest, were 0.9, 0.8, 1.3 (p=0.050). C statistic assessment showed that adding PAI-1 or TPA to conventional risk factors resulted in small increases in discrimination and modest reclassification of risk, which was statistically significant for TPA (net reclassification 6.8%, p=0.037) but not PAI-1 (4.8%, p=0.113). CONCLUSION: PAI-1 and TPA antigen levels are predictive of CVD events after accounting for established risk factors. A serial increase in PAI-1 is associated with a further increase in risk. These findings support the importance of fibrinolytic potential in CVD.

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 Pl(A2) polymorphism and the fibrinogen Hind III beta-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.

Association of blood pressure with fibrinolytic potential in the Framingham offspring population.

BACKGROUND: Hypertension is an established risk factor for acute coronary events. Because fibrinolytic and hemostatic factors are also associated with cardiovascular disease, we examined the relations of systolic and diastolic blood pressures (SBP and DBP) to levels of plasminogen activator inhibitor antigen, tissue plasminogen activator antigen, fibrinogen, factor VII, von Willebrand factor, fibrinogen, and plasma viscosity in subjects of the Framingham Offspring Study. METHODS AND RESULTS: We studied 1193 men and 1459 women after the exclusion of subjects with known cardiovascular disease and those receiving anticoagulant or antihypertensive therapy. Linear regression models were used to evaluate SBP and DBP as predictors of fibrinolytic and hemostatic factor levels in separate sex models, with adjustment for age, body mass index, smoking, diabetes, total cholesterol, HDL, triglycerides, alcohol intake, and estrogen use (in women). In both sexes, levels of plasminogen activator inhibitor and tissue plasminogen activator antigen were positively related to SBP and DBP (P<0.001). Plasma viscosity was positively related to SBP (P=0.008) and DBP (P=0.001) in women only. There was no association between SBP or DBP and fibrinogen, factor VII, or von Willebrand factor in either sex. CONCLUSIONS: These data suggest that impaired fibrinolysis may play an important role in the pathogenesis of cardiovascular disease in hypertensive patients.

Platelet glycoprotein IIIa Pl(a) polymorphism, fibrinogen, and platelet aggregability: The Framingham Heart Study.

BACKGROUND: Recent data suggest that the Pl(A2) allele of the platelet glycoprotein IIIa receptor may be a genetic risk factor for cardiovascular disease. We previously reported that the Pl(A2) allele was associated with increased platelet aggregability, as indicated by lower epinephrine threshold concentrations. Paradoxically, however, it has been reported that Pl(A2)-positive platelets have reduced fibrinogen binding. Because fibrinogen mediates platelet aggregability, we hypothesized that plasma fibrinogen levels may interact with Pl(A) genotype in modulating platelet aggregability. Methods and Results-- Glycoprotein IIIa Pl(A) 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 Pl(A1/A1) genotype (P=0.0005 and P=0.03 for epinephrine- and ADP-induced aggregation, respectively) but not for the Pl(A2)-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 Pl(A) genotype and platelet aggregability. Further study of this gene-environment interaction may provide insight into cardiovascular disease risk.

Alcohol consumption and hemostatic factors: analysis of the Framingham Offspring cohort.

BACKGROUND: Moderate alcohol consumers have lower rates of cardiovascular disease than abstainers. One proposed mechanism is a beneficial effect on hemostatic parameters, but previous studies have provided conflicting results. METHODS AND RESULTS: We measured levels of fibrinogen, plasma viscosity, von Willebrand factor, factor VII, plasminogen activator inhibitor antigen-1, and tissue plasminogen activator antigen in a cross-sectional analysis of 3223 adults free of cardiovascular disease enrolled in the Framingham Offspring Study. We assessed their alcohol consumption with a standardized questionnaire. Light-to-moderate alcohol consumption was associated with lower levels of fibrinogen, plasma viscosity, von Willebrand factor, and factor VII. This association was most pronounced for consumers of 3 to 7 drinks weekly for viscosity and 7 to 21 drinks weekly for the other hemostatic measures. Alcohol intake of 7 to 21 drinks weekly or more was associated with impaired fibrinolytic potential, reflected by higher levels of plasminogen activator inhibitor antigen-1 and tissue plasminogen activator antigen. Wine drinkers had lower plasminogen activator inhibitor antigen-1 levels than other drinkers, particularly at 3 to 21 drinks weekly, but beverage type did not otherwise consistently affect the results. CONCLUSIONS: Light-to-moderate alcohol consumption is associated with lower levels of coagulatory factors, but higher intake is associated with impaired fibrinolytic potential. These findings are consistent with the hypothesis that a balance between hemostatic and fibrinolytic activity may contribute to the complex relation of alcohol use with coronary heart disease.

Identification of Mycobacterium tuberculosis and Mycobacterium avium-M. intracellulare directly from primary BACTEC cultures by using acridinium-ester-labeled DNA probes.

Identification of members of the Mycobacterium tuberculosis complex and the M. avium-M. intracellulare complex (MAC) directly from primary BACTEC cultures was evaluated by using acridinium-ester-labeled DNA probes (AccuProbe; GenProbe, Inc., San Diego, Calif.). In preliminary experiments, blood present in samples was found to interfere with the assay because of nonspecific chemiluminescence, which was measured in relative light units (RLUs). There was a direct relationship between the age of the culture and the number of nonspecific RLUs. A protocol using 1% sodium dodecyl sulfate-5 mM EDTA to treat BACTEC broth cultures which, with specimens containing blood, gave on the average a ninefold reduction in nonspecific chemiluminescence was developed. By using this treatment protocol, 120 specimens were tested directly from BACTEC broth cultures with an AccuProbe for the M. tuberculosis complex and/or the MAC. In order to establish the background of the specimen, the patient sample was assayed without probe. The criteria for the inclusion of BACTEC cultures in the evaluation were a growth index of greater than or equal to 100 and a positive smear for acid-fast bacilli directly from the BACTEC broth. For the 120 cultures tested, if a hybridization result of greater than or equal to 30,000 RLUs was considered positive, the sensitivities for detecting the M. tuberculosis complex and the MAC were 47 and 90%, respectively, with a specificity of 100% for both. However, if a ratio of the RLUs obtained with the MAC or the M. tuberculosis complex probe to those obtained with the specimen background of >/= 20 was considered positive, this gave 77% sensitivity and 100% specificity for BACTEC cultures containing M. tuberculosis complex isolates and 96% sensitivity and 100% specificity for those growing MAC isolates.

Replication of double-stranded RNA of the virus-like particles in Saccharomyces cerevisiae.

The mode of replication of the L double-stranded RNA (dsRNA) present in virus-like particles in Saccharomyces cerevisiae was examined by density transfer experiments. After transfer to light medium, significant amounts of fully heavy dsRNA persisted over a number of cell doublings. In addition, very little material of hybrid density was ever formed, and the accumulation of fully light material began as early as 0.5 doubling after transfer to light medium. Our results are compatible with a conservative mode of replication or with a semiconservative mode of replication carried out by a small portion of the total dsRNA population. In additional experiments the synthesis of dsRNA relative to the cell cycle was studied. This was done by determining the ratio of short-term to long-term radioactive label in size-separated cell fractions of a prelabeled exponential culture. The ratio of short-term to long-term label remained constant for all fractions, implying that dsRNA is synthesized throughout the cell cycle, increasing through the cell cycle at an exponential rate.

Biochemical and physiological studies of the yeast virus-like particle.

A study was made of the virus-like particle (VLP) of Saccharomyces cerevisiae S7. This strain contains elevated amounts of P1 double-stranded ribonucleic acid (dsRNA) but no P2 dsRNA. The amount of dsRNA contained in cells grown on a fermentable carbon source (glucose) was compared with that in cells grown on a nonfermentable carbon source (ethanol). It was found that ethanol-grown cells contain higher levels of dsRNA than glucose-grown cells. In the former, the amount of dsRNA increased during the logarithmic phase of growth, whereas in the latter it increased during the transition from the logarithmic to the stationary phase. A method was devised to isolate VLPs from these cells by using CsCl gradients, and the yield was assessed by monitoring the recovery of dsRNA. Three proteins were found to be tightly associated with these particles. They have molecular weights of 75,000, 53,000, and 37,000. Together they account for almost all of the coding capacity of the P1 dsRNA that the VLP contains.

Hypobetalipoproteinemia is associated with low levels of hemostatic risk factors in the Framingham offspring population.

BACKGROUND: Given the importance of thrombosis in causation of acute coronary syndromes, it is possible that the beneficial effect of low lipid levels on the risk of coronary events is achieved by lowering thrombotic potential of the blood. Hypobetalipoproteinemia is characterized by plasma concentrations of apolipoprotein B and LDL cholesterol that are one third of those observed in the general population. The aim of this study was to utilize subjects with hypobetalipoproteinemia to examine the relation between thrombotic potential and low levels of LDL cholesterol. METHODS AND RESULTS: Hemostatic risk factors were measured in 1878 individuals (1003 women and 875 men) participating in cycle 5 of the Framingham Offspring Study. The subjects were divided into five groups on the basis of LDL cholesterol level. Subjects with hypobetalipoproteinemia (LDL cholesterol < 70 mg/dL) had the lowest levels of fibrinogen, plasminogen activator inhibitor-1 antigen, and tissue plasminogen activator antigen. As LDL cholesterol increased, there was a significant increase in the levels of the hemostatic risk factors, with the exception of von Willebrand factor antigen. Adjustment with multivariate regression analyses for the covariates age, sex, body mass index, diabetes mellitus, smoking, alcohol intake, triglyceride level, and use of antihypertensive medication did not materially alter the results. CONCLUSIONS: Decreasing levels of LDL cholesterol are associated with decreasing levels of hemostatic risk factors. Subjects with hypobetalipoproteinemia have the lowest levels of hemostatic risk factors and may be protected against thrombotic complications of atherosclerotic cardiovascular disease because of reduced thrombotic potential. One mechanism by which lipid-lowering therapy may decrease clinical cardiac events is through a reduction in thrombotic tendency.

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.

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 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.