Factor VII, Cholesterol, and Triglycerides
The CARDIA Study
Cross-sectional studies have shown that factor VII coagulant activity (VIIc) is positively associated with plasma total cholesterol (TC), LDL cholesterol, and triglycerides (TG) as well as body mass index (BMI) and diastolic blood pressure. To determine whether changes in VIIc parallel changes in coronary risk factors over a period of 2 years, we examined data from 1514 participants in the Coronary Artery Risk Development in Young Adults Study (CARDIA), an ongoing investigation of lifestyles and evolution of cardiovascular risk factors. Subjects were 23 to 35 years old at the year 5 examination. Cross-sectional analyses at these examinations showed that VIIc was positively correlated (P<.001) with TC and TG in all race/sex groups except for TC in black women at the year 5 examination. Changes in VIIc over the 2-year period were correlated positively with changes in TC in all except black men and TG in all groups; the association of VIIc change with change in TC and TG was reduced only slightly with adjustment for age and BMI at year 5 and 2-year change in BMI. To determine whether the higher levels of VIIc in subjects with higher lipid values were due to activation of the factor or to an increase in the concentration of the factor VII clotting protein, we measured factor VII antigen (VIIag) in a randomly selected subsample of 223 subjects at the year 7 examination. In all sex/race groups, VIIag correlated with VIIc (r=.69 to 0.81). After adjustment for sex and race, the partial correlation coefficient between TG and VIIc was .28 (P=.0001); between TG and VIIag, .35 (P=.0001); between TC and VIIc, .39 (P=.0001); and between TC and VIIag, 0.43 (P=.0001). No associations were observed between lipid levels and the ratio of VIIc to VIIag. We conclude that the raised VIIc with higher lipid levels occurs in blacks as well as whites, in men and women, persists over time, and represents a true increase in the plasma concentration of this clotting factor.
- Received January 18, 1996.
- Revision received July 23, 1996.
Factor VII is a vitamin K–dependent zymogen that plays a key role in thrombin generation and thrombus formation. Raised levels of factor VII have been recorded in patients with myocardial infarction1 and the relatives of such patients,2 suggesting a possible role for factor VII in the pathogenesis of atherothrombotic disease. An association between factor VII levels and serum lipids has been a consistent finding in a large number of studies,3 4 including the year 5 examination of the CARDIA Study, an ongoing investigation of lifestyles and evolution of cardiovascular risk factors.5 6 To confirm and extend this observation, we restudied CARDIA participants at the year 7 examination. Our goals were to assess the consistency of the cross-sectional correlations of factor VII with cholesterol and triglycerides and to determine whether the changes in factor VII recorded over the 2-year period correlated with changes in cholesterol and triglycerides during this interval.
The determinants of factor VII concentrations in plasma are still incompletely understood. It has been suggested that chylomicrons and VLDL cholesterol might activate factor VII, thereby accounting for the apparently higher levels of the factor postprandially and in patients with hypertriglyceridemia.7 However, factor VII is activated in complexes formed with phospholipid rather than triglycerides.8 9 More recently, direct measurements of VIIa have shown no correlation with fasting levels of triglycerides.10 11 Thus, the increased VIIc observed in subjects with elevated lipids may represent an increased synthesis of factor VII rather than a rise in VIIa. To examine this possibility, we included measurements of VIIag as well as VIIc and studied the relationships between VIIc, VIIag, cholesterol, and triglycerides after controlling for the effects of other variables such as age and BMI.
The subjects of this study were participants in CARDIA, a multicenter, longitudinal study of lifestyles and the evolution of risk factors for cardiovascular diseases in young adults. Two of the four CARDIA Centers, Chicago and Minneapolis, participated in this ancillary study. A total of 1818 persons were examined at year 5, 1791 at year 7, and 1514 (441 white men, 282 black men, 449 white women, and 342 black women) on both occasions. These subjects were 23 to 35 years old at the time of the year 5 examination. Participants were excluded from the analyses if age, cholesterol, BMI, or VIIc data were missing or if their triglyceride values were >500 mg/dL. In addition, triglyceride measurements were not performed if participants had not fasted for at least 8 hours.
Blood was drawn between 8 am and 12 noon from participants who had fasted overnight. A 21-gauge butterfly needle was inserted into a large antecubital vein, the tourniquet was removed, and samples were collected for lipid analyses and chemistries. Then 5 mL of blood was placed into each of two vacuum tubes containing 3.8% sodium citrate, mixed by repeated inversion, and centrifuged at 3000g for 20 minutes at 4°C. This centrifugation was performed within 10 minutes of collection, and within 1 hour the plasma was placed in a −70°C freezer.
To avoid cold-activation of factor VII,12 the plasma was never held at 4°C except during the brief centrifugation. After storage for a maximum of 4 months, samples were thawed at 37°C, kept at room temperature until tested, and analyzed. Samples collected and frozen in Minneapolis were packed in dry ice and sent to Chicago by overnight express.
VIIc was assayed by a one-stage system with reagents from Pacific Hemostasis and George King Biomedical, Inc. The standard curve was prepared by use of universal reference plasma from Curtin Matheson Scientific, and the results were calculated as a percentage of the standard with the data management system of the MLA-Electra 800. VIIag was examined in every 10th participant at year 7 (total, 223) with an ELISA (Asserachrom, American Bioproducts). The technical error of the VIIag assay, based on duplicate analysis of 50 samples, was 1.9%.
The technical error of the VIIc assay was determined by internal and external testing. For internal quality control, blood was obtained from five volunteers, each sample was split into two parts, each aliquot was divided into four parts, and all sample splits were stored at −20°C. Each week for 4 weeks, samples were retrieved and assayed by technicians blinded to the identity of the samples. The technical error as a percentage of the mean was 5.0±2.2%.
In addition, external quality control was assessed in split samples derived from 8% of the study participants (123 pairs). The duplicate samples were given bogus identification numbers, stored for varying periods of time, and assayed as much as 4 months apart. The technical error for VIIc was 13% to 14% of the mean. This higher technical error reflects the greater complexity of this testing procedure and is probably more reflective of actual laboratory accuracy in assays of study samples.
At both examinations, height (to the nearest 0.5 cm) and weight (to the nearest 0.5 lb) were measured with the subject in light clothing and without shoes. BMI was computed as weight in kilograms divided by the square of height in meters. Plasma total cholesterol, HDL-C, LDL-C, and triglycerides were determined by the University of Washington Northwest Lipid Research Clinic Laboratory. Total cholesterol and total triglyceride levels were determined by enzymatic procedures13 on the ABA 200 bichromatic instrument. HDL-C was measured by enzymatic methods after dextran sulfate–magnesium precipitation.14 LDL-C was estimated from the Friedewald equation15 : LDL-C=total cholesterol−(HDL-C)−(triglycerides/5).
Average levels of VIIc, cholesterol, and triglycerides were determined for each sex/race group. Pearson correlations and partial correlations (adjusting for age and BMI) of VIIc with total cholesterol and triglycerides were calculated for each sex/race group. Similarly, the correlation of 2-year change in VIIc with changes in total cholesterol and triglycerides, adjusted for age and BMI at years 5 and 7 and 2-year change in BMI, were computed for each group. To examine the relationship of VIIag to total cholesterol and triglycerides, the four sex/race groups were combined because of the smaller number of participants with VIIag determinations; sex and race were controlled for in the calculation of partial correlations.
Table 1⇓ shows the mean values for VIIc, total cholesterol, and triglycerides in the four race/sex groups and at the two examinations (years 5 and 7), displaying the values for all participants at each examination and for those participating in both examinations. At each examination period, differences in values for those participating in one examination or both examinations were slight, indicating that the population at each year of examination was representative of the whole group. However, possibly in part because of laboratory drift, the mean values for VIIc at the year 7 examination period were about 9% lower in all race/sex groups than those in the earlier period.
The cross-sectional relationship between triglycerides and VIIc is shown in the Figure⇓ and Table 2⇓. In every race/sex group, for each higher quartile of triglyceride concentration there was a corresponding higher VIIc level. Table 3⇓ displays the correlation coefficients of VIIc with triglycerides cross-sectionally at each examination as well as longitudinally over the 2-year period. In the cross-sectional analysis, statistically significant positive correlations were observed in all race/sex groups, which were only slightly reduced by adjustment for age and BMI at the years 5 and 7 examinations. The correlations for changes in triglycerides with changes in VIIc were statistically significant for white men, white women, and black men and of borderline significance for black women. The association of VIIc change with changes in triglycerides was moderately reduced by adjustment for age and BMI at the year 5 examination and 2-year change in BMI.
Table 4⇓ shows the correlation coefficients of VIIc with total cholesterol cross-sectionally at each examination and the changes in cholesterol with changes in VIIc over the 2-year period. Statistically significant positive correlations were observed among all race/sex groups in the cross-sectional analysis, which were only slightly reduced by adjustment for age and BMI. Because levels of cholesterol and triglyceride are highly correlated, the possibility that the relationship between cholesterol and VIIc was due to the triglyceride-VIIc relationship was examined. Adjusting the data for triglyceride concentration had only a minor effect on the strength of the associations. The correlations of changes in cholesterol with changes in VIIc were statistically significant in whites but not blacks and persisted despite adjustment for age, BMI, and triglycerides at year 5 and 2-year change in BMI and triglycerides. The relationships with changes in HDL-C and LDL-C were inconsistent (not shown).
In a subgroup of 223 subjects from the year 7 examination, VIIag (mean±SD) was 87±25%. VIIag correlated closely with VIIc in all race/sex groups (r=.69 to .81). Table 5⇓ shows the partial correlation coefficients of VIIc, VIIag, and the ratio of VIIc to VIIag with total cholesterol and triglycerides, adjusted for race and sex. Statistically significant correlations were observed with VIIc and VIIag but not with the ratio of VIIc to VIIag. Adjustment for BMI and age only slightly weakened the correlations, and they remained statistically significant (for VIIc with triglycerides, P=.006; for VIIag with triglycerides, P=.0001; for either VIIc or VIIag with cholesterol, P=.0001).
This study confirms the positive association of factor VII with triglycerides and total cholesterol. Even though VIIc values were lower at the second examination and a large proportion of the 2-year changes in VIIc might reflect intraindividual variability, the results are consistent with and extend previously reported cross-sectional findings.3 4 5 For example, the Atherosclerosis Risk in Communities Study4 showed positive associations of factor VIIc with plasma triglycerides, LDL-C, HDL-C, and BMI in men and women (P<.01). In our study, the positive correlations of VIIc with triglycerides and cholesterol were observed in each race/sex group and were only slightly reduced by adjustment for age and BMI. In addition, statistically significant correlations of 2-year changes in VIIc with changes in triglycerides were observed in all race/sex groups and with changes in cholesterol in whites. The association of VIIc with total cholesterol persisted despite adjustment for triglycerides in every group (except black women at year 5); the reasons for this apparent independent relationship are unclear and will require further study.
To examine the relationship between factor VII and triglycerides, VIIag was measured in a subset of subjects who were representative of the group as a whole. Both VIIc and VIIag were statistically significantly correlated with total cholesterol and triglycerides. Were factor VII activated by lipids, its coagulant activity would increase and the ratio of VIIc to VIIag would be expected to correlate with the levels of cholesterol and triglycerides, but no such correlations were observed. Rather, the data suggest that the higher levels of VIIc with elevated lipids are accompanied by an increase in the factor VII protein, a concept supported by the strong correlation between VIIc and VIIag. That the higher levels of VIIc were not due to an increase in VIIa could be confirmed by direct measurement of this material.16
Plasma concentrations of factor VII are governed by a variety of influences. At the genetic level, ≈20% of persons studied have a mutant factor VII (Arg353-Gln353), which is associated with less than the normal amount of circulating factor VII.17 Studies examining associations between triglycerides and the various factor VII genotypes have given discrepant results. Saha and colleagues18 reported that the correlation with triglycerides was stronger in carriers of the Gln353 allele than in those homozygous for the more common Arg353 form, whereas Humphries et al19 found such a correlation only in those with the Arg353 form. Perhaps the different populations studied, Dravidian Indians and Englishmen, may have some bearing on these results. Further studies are needed to clarify these differences.
Factor VII also appears to be strongly influenced by dietary constituents. Within hours after an oral fat load, VIIc but not VIIag increases; the factor VII may be activated by factor XIIa induced by free fatty acids resulting from the lipolysis of triglyceride-rich lipoproteins.20 In contrast, when a high-fat diet is habitually eaten, both VIIc and VIIag increase.21 22 The specific fats consumed also influence factor VII levels. Significantly higher levels of factor VII (both VIIc and VIIag) were reported after volunteers ingested a diet enriched in myristic and lauric acids compared with one enriched with stearic acid.23
The observation that not only factor VII but also the other vitamin K–dependent clotting factors are increased by a high-fat diet suggests that regular ingestion of such a diet increases factor VII concentrations rather than just activating factor VII.21 Furthermore, it has been shown that in patients at high risk for ischemic heart disease, triglyceride levels correlate with VIIag levels, and all four vitamin K–dependent clotting factors (factors II, VII, IX, and X) are significantly higher than in subjects at low risk for heart disease.24 Whether dietary fats or serum triglycerides stimulate increased hepatic synthesis of vitamin K–dependent clotting factors or prolong their survival in the circulation is unknown.
In summary, we conclude that raised VIIc with higher blood lipids occurs in blacks as well as whites, in men and in women, and persists over time. This enhanced VIIc is probably due to a true increase in the plasma concentration of this clotting factor.
Selected Abbreviations and Acronyms
|BMI||=||body mass index|
|CARDIA||=||Coronary Artery Risk Development in Young Adults|
|VIIa||=||activated form of factor VII|
|VIIag||=||factor VII antigen|
|VIIc||=||factor VII coagulant activity|
This study was supported by grant HL-43758 and contracts NO1-HC-48049 and NO1-HC-95095 from the National Heart, Lung, and Blood Institute, National Institutes of Health.
Balleisen L, Assmann G, Bailey J, Epping P-H, Schulte H, van de Loo J. Epidemiological study on factor VII, factor VIII and fibrinogen in an industrial population, II: baseline data on the relation to blood pressure, blood glucose, uric acid, and lipid fractions. Thromb Haemost. 1985;54:721-723.
Friedman GD, Cutter G, Donahue RP, Hughes GH, Hulley SB, Jacobs DR, Liu K, Savage PJ. CARDIA: study design, recruitment, and some characteristics of the examined subjects. J Clin Epidemiol. 1988;44:1105-1116.
Green D, Ruth KJ, Folsom AR, Liu K. Hemostatic factors in the Coronary Artery Risk Development in Young Adults (CARDIA) study. Arterioscler Thromb. 1994;14:686-693.
Skartlien AH, Lyberg-Beckmann S, Holme I, Hjermann I, Prydz H. Effect of alteration in triglyceride levels on factor VII-phospholipid complexes in plasma. Arteriosclerosis. 1989;9:798-801.
Mann KG. Factor VII assays, plasma triglyceride levels, and cardiovascular disease risk. Arteriosclerosis. 1989;9:783.
Kario K, Miyata T, Sakata T, Matsuo T, Kato H. Fluorogenic assay of activated factor VII. Arterioscler Thromb. 1994;14:265-274.
Moor E, Silveira A, van't Hooft F, Suontaka AM, Eriksson P, Blomback M, Hamsten A. Coagulation factor VII mass and activity in young men with myocardial infarction at a young age. Arterioscler Thromb Vasc Biol. 1995;15:655-664.
Warnick GR, Benderson J, Albers JJ. Dextran sulphate-Mg2+ precipitation procedure for quantitation of high-density-lipoprotein lipids. Clin Chem. 1982;28:1379-1388.
Friedewald WT, Levy RI, Fredrickson DS. Estimation of the concentration of low-density-lipoprotein cholesterol in plasma without use of the preparative ultracentrifuge. Clin Chem. 1972;18:499-501.
Morrissey JH, Macik BG, Neuenschwander PF, Comp PC. Quantitation of activated factor VII levels in plasma using a tissue factor mutant selectively deficient in promoting factor VII activation. Blood. 1993;81:734-744.
Green F, Kelleher C, Wilkes H, Temple A, Meade T, Humphries S. A common genetic polymorphism associated with lower coagulation factor VII levels in healthy individuals. Arterioscler Thromb. 1991;11:540-546.
Saha N, Liu Y, Heng CK, Hong S, Low PS, Tay JSH. Association of factor VII genotype with plasma factor VII activity and antigen levels in healthy Indian adults and interaction with triglycerides. Arterioscler Thromb. 1994;14:1923-1927.
Humphries SE, Lane A, Green FR, Cooper J, Miller GJ. Factor VII coagulant activity and antigen levels in healthy men are determined by interaction between factor VII genotype and plasma triglyceride concentration. Arterioscler Thromb. 1994;14:193-198.
Silveira A, Karpe F, Blomback M, Steiner G, Walldius G, Hamsten A. Activation of coagulation factor VII during alimentary lipemia. Arterioscler Thromb. 1994;14:60-69.