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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1995;15:655-664.)
© 1995 American Heart Association, Inc.

Articles

Coagulation Factor VII Mass and Activity in Young Men With Myocardial Infarction at a Young Age

Role of Plasma Lipoproteins and Factor VII Genotype

Elisabeth Moor; Angela Silveira; Ferdinand van't Hooft; Anna Maija Suontaka; Per Eriksson; Margareta Blombäck; Anders Hamsten

From the Division of Cardiology (E.M.) and the Atherosclerosis Research Unit, King Gustaf V Research Institute (A.S., F. van't H., P.E., A.H.), Department of Medicine, and the Division of Blood Coagulation Research, Department of Laboratory Medicine (A.M.S., M.B.), Karolinska Hospital, Karolinska Institute, Stockholm, Sweden.

Correspondence to Dr Elisabeth Moor, Department of Cardiology, Karolinska Hospital, S-171 76 Stockholm, Sweden.

	Abstract

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Abstract Factor VII (FVII) coagulant activity has been proven to be associated with the risk of future fatal coronary heart disease (CHD) in middle-aged men. Recent studies have emphasized the role of triglyceride-rich lipoproteins and FVII genotype in determining plasma levels of FVII protein and activity. The present study was undertaken to examine whether FVII activity state and protein concentration in fasting plasma are altered in young men with proven myocardial infarction (MI) and examined the relations of FVII to subfractions of apo B–containing lipoproteins and the ArgGln polymorphism in the FVII gene. Activated FVII (FVIIa) was determined by a clotting assay using soluble, recombinant, truncated tissue factor. A total of 94 men with a first MI before the age of 45 (mean age±SD, 39.6±4.5 years) were included in the study along with 99 population-based, age-matched control subjects. In addition to FVIIa and FVII antigen (FVII:Ag), a panel of FVII activity assays were included for comparison with previous work in this field. The plasma level of FVII:Ag was higher in patients than in control subjects when the entire groups were compared (537±128 versus 479±93 ng/mL, P<.001), the differences being accounted for by patients with hypertriglyceridemic lipoprotein phenotypes. In contrast, FVIIa was similar in patients and control subjects (4.6±1.4 versus 4.3±1.3 ng/mL, NS), which means that the proportion of FVIIa molecules was unaltered or even lower in the patients. As expected, the ArgGln polymorphism significantly influenced both FVII mass and activity levels. In addition, presence of the Gln allele appeared to be associated with a lower proportion of fully active FVII molecules. The polymorphism also affected the relation between the plasma concentration of VLDL and FVII:Ag. The triglyceride content and particle number of all VLDL subfractions, irrespective of particle size, correlated fairly strongly with FVII mass determinations but not at all with FVIIa. HDL cholesterol concentration, on the other hand, presumably reflecting the efficiency of lipoprotein lipase–mediated lipolysis of VLDL, related significantly to the FVIIa level. The ArgGln polymorphism, independent of lipoprotein effects, explained 5% to 10% of the variation in FVII mass and activity. In conclusion, the present findings speak against a role of FVII as a risk factor for CHD, because a significantly increased potential for activation of coagulation (ie, raised basal concentration of FVIIa) was not observed among young postinfarction patients. Prospective epidemiological studies including specific determination of FVIIa are needed to resolve the issue of whether FVII activity is a risk factor for CHD.

Key Words: coagulation factor VII • myocardial infarction • genotype • lipoproteins

	Introduction

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Factor VII (FVII) is the first enzyme involved in the extrinsic pathway of blood coagulation. The major proportion of FVII circulates in plasma in the zymogen form. However, low but significant levels of activated FVII (FVIIa) are also present and appear to serve as a primer for triggering the clotting cascade.^{1 2 3 4 5} Accordingly, measurement of FVIIa is highly relevant in understanding the role of FVII in coronary heart disease (CHD). Unfortunately, previous studies in this area have been performed using assays with different relative sensitivities of FVII mass to FVIIa.⁶ This situation has contributed to the prevailing controversy over whether elevated levels of FVIIa per se or of total FVII plus FVIIa are more closely related to CHD.

The prospective Northwick Park Heart Study has indicated that FVII coagulant activity (FVIIc) is independently associated with the risk of future fatal coronary events in middle-aged men.^{7 8} Several cross-sectional studies have also reported increased FVIIc in groups with manifest CHD or at risk of CHD,^{9 10 11 12} primarily due to a combined increase of FVII protein and activity, with unaltered or lowered specific activity of FVII. The corollary of this observation seems to be that an increase in FVIIc in high-risk individuals or in patients with manifest CHD is due to an increase in zymogen FVII and not to the presence of a larger fraction of FVIIa molecules in plasma. However, there is evidence of FVII activation in unstable angina pectoris,¹³ and it remains controversial whether the important factor in elevated FVIIc is an increase in FVIIa or in total FVII mass.

Hypertriglyceridemia appears to be a procoagulant state. FVIIc correlates directly with plasma triglyceride concentration,^{7 8} and treatment of hypertriglyceridemia with diet, drugs, or both results in a lowering of FVIIc.^{14 15 16} Dietary studies have further emphasized the connection between plasma lipoproteins and FVII. Addition of fat to the diet has been shown to cause a rapid increase in FVIIc.¹⁷ The character of the FVII response to fat intake has suggested that an association with postprandial lipemia exists, and that the activity state rather than the plasma concentration per se of the protein is affected. Accordingly, FVII activation was recently demonstrated during alimentary lipemia.^{18 19} However, a long-term increase in FVIIc, such as occurs in hypertriglyceridemia, appears to be associated with a rise in FVII protein concentration.²⁰

A common polymorphism in the FVII gene has a strong impact on FVIIc.²¹ The base change that causes the polymorphism is a G-to-A substitution in the second position of the codon for amino acid 353, which leads to the substitution of arginine in the protein product of the G allele (designated FVII-Arg) for glutamine in the product of the A allele (FVII-Gln). Heterozygotes for the ArgGln polymorphism account for approximately 20% of various populations and have FVIIc levels 20% to 25% below those of individuals who possess only the Arg allele.^{21 22 23} Interestingly, there is also evidence of genotype-specific differences in the relationship between triglycerides and FVIIc, the expected positive association being confined entirely to individuals with the Arg/Arg genotype.^{22 23}

The present study addressed the issue of whether FVII activity state and protein concentration in fasting plasma are altered in young men with proven myocardial infarction (MI) and examined the relations of FVII to plasma lipoproteins and FVII genotype. FVIIa was determined by a clotting assay with soluble recombinant truncated tissue factor (TF), which by being selectively deficient in promoting FVII activation and retaining FVII cofactor function, allows direct quantification of FVIIa in plasma.^{4 5 24 25} In addition, a panel of tests for measuring FVII activity was included for comparison with previous work in this field. Young postinfarction patients were selected for the study, because they are heterogeneous with respect to angiographic disease severity, a thrombotic component appears to predominate in a substantial proportion of these patients, and the prevalence of hyperlipoproteinemia is high.

	Methods

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Subjects
A total of 94 men with a first MI before the age of 45 were included in the study. They belonged to a consecutive series of 131 male patients who were admitted to the 10 hospitals in the greater Stockholm area with a coronary care unit between April 1989 and April 1991. The patients were subsequently referred to the Karolinska Hospital for metabolic, hemostatic, and cardiological investigations.

Of the initial 131 patients, 3 died in the early postinfarction period. Patients who were being treated with oral anticoagulants or who had heterozygous familial hypercholesterolemia (n=3), severely impaired renal function (n=5), or insulin-dependent diabetes mellitus (n=4) were excluded. Of the remaining 116 patients, 2 were excluded because of extensive ischemic stroke in connection with the MI, 13 declined participation, and 7 were excluded for technical reasons (unavailable to the research team, deficient laboratory capacity, or referral later than 6 months after the infarction).

All patients were examined 4 to 6 months after the acute event, when it was expected that acute-phase reactions due to the MI had declined. No patient in the study was receiving lipid-lowering drugs. They had all received dietary instructions aiming at a diet low in fat, rich in complex carbohydrates, and with a limited alcohol intake in connection with their first visit to the outpatient department 6 weeks after the acute event.

Ninety-nine healthy men with the same age distribution were examined as control subjects. They were selected at random from a register containing all permanent residents in Stockholm County. Of those initially invited, 81% agreed to participate in the research program. All of the men were interviewed to exclude individuals with a history of MI, angina pectoris, or any other severe illness.

Basic characteristics for patients and control subjects are given in Table 1. A history of smoking and hypertension were much more common in the patient than the control group. The patients also had a higher body mass index (BMI). Of note, a majority of patients had hyperlipoproteinemia, with a predominance of hypertriglyceridemic phenotypes, whereas the control subjects were generally normolipidemic. However, postheparin plasma lipoprotein lipase (LPL) and hepatic lipase (HL) activities did not differ significantly between the two groups (P=.07 for LPL).

View this table: [in this window] [in a new window]   Table 1. Basic Characteristics of Patients and Control Subjects

Blood Sampling
Antecubital vein puncture with a 1.4-mm Wasserman needle (diameter, 45 mm; TSK Laboratories) was performed between 8 and 9 AM after a 12-hour fast. After the first 2 mL had been discarded, the blood was allowed to run freely into the tubes, starting with samples for analysis of hemostatic function. Blood was next drawn for analysis of plasma lipoproteins and for DNA preparation.

For the coagulation analyses, venous blood was drawn into 10-mL plastic tubes containing 1 mL of 0.13 mol/L trisodium citrate, pH 7.5. The tubes were immediately centrifuged for 20 minutes (2200g, room temperature), and plasma was dispensed into plastic tubes and frozen at -70°C. Samples for the lipid and lipoprotein analyses were drawn into 10-mL precooled sterile tubes containing 0.12 mL of 0.34 mol/L tripotassium EDTA (Vacutainer, Becton Dickinson) and kept on ice until they were centrifuged. Blood for subsequent FVII genotyping was drawn into the same type of sterile tubes and immediately frozen at -70°C. Procedures for blood sampling and preparation of citrated plasma samples have previously been described in detail.²⁶

FVII Assays
Five different methods were used for measuring plasma FVII. The FVII protein concentration (FVII:Ag) was determined as FVII antigen by using an enzyme immunoassay (Novoclone FVII EIA kit, a kind gift from Novo Nordisk A/S). FVII amidolytic activity (FVII:Am) was measured with a commercially available kit that included the chromogenic peptide substrate S-2765 (Coa-set VII, Chromogenix). In addition to the specific assay for FVIIa, two FVII clotting assays were performed, one using bovine (FVIIa:B) and one using human (FVIIc) thromboplastin. The clotting assay for FVIIa used soluble, recombinant, truncated TF, s-TF (lot CICC III-94, a kind gift from Dr Peter Wildgoose, Novo Nordisk A/S). FVIIa levels were measured in an ACL-300 automated coagulation instrument (Instrumentation Laboratories), operating in research mode, as previously described in detail.²⁷ Coagulation times were converted to FVIIa concentration by comparison with a standard curve constructed from varying concentrations of purified recombinant FVIIa (a kind gift from Dr Ulla Hedner, Novo Nordisk A/S). For technical reasons, FVIIa was measured in only 80 patients and 90 control subjects. In the FVIIa:B method that used bovine thromboplastin, FVII activity was determined essentially according to the method of van Deijk et al²⁸ in an Electra 900c coagulation analyzer (Medical Laboratory Automation Inc). Briefly, 50 µL of FVII-deficient plasma (Helena Laboratories), 50 µL of human bovine brain thromboplastin solution (a kind gift from Dr Ken Denson, Diagnostic Reagents), and 50 µL of diluted plasma sample were incubated for 30 seconds at 37°C. Then, 50 µL of 25 mmol/L CaCl₂ was added and the clotting time measured. Seven dilutions of our own reference plasma (pooled plasma from normal blood donors) were used for the standard curve, and three dilutions of each plasma sample were tested. The same assay was used for FVIIc, except that the incubation time was 120 seconds and human brain thromboplastin was used for activation (prepared according to Owren and Aas²⁹ ). Two dilutions of each plasma sample were tested.

Results were expressed either in units per milliliter (for FVII:Am, FVIIc, and FVIIa:B), one unit being the activity of FVII present in 1 mL of a standard pooled plasma, or in nanograms per milliliter (for FVII:Ag and FVIIa).

The within- and between-run coefficients of variation were, respectively, 1.8% and 2.9% for FVII:Ag, 3.6% and 3.8% for FVII:Am, 3.9% and 9.1% for FVIIa, 4.8% and 6.1% for FVIIa:B, and 4.7% and 6.1% for FVIIc.

TF Pathway Inhibitor Activity Assay
TF pathway inhibitor (TFPI) activity was determined by a three-stage chromogenic assay³⁰ adapted for microplates.

DNA Procedures
Nucleated cells from frozen whole blood were prepared according to Sambrook et al,³¹ and DNA was extracted by a salting-out method.³² Enzymatic amplification was performed by polymerase chain reaction (PCR) with 50 ng to 1 µg genomic DNA and thermostable Taq polymerase (Boehringer Mannheim Scandinavia) according to the manufacturer's instructions. Oligonucleotide primers for PCR were obtained from Pharmacia. The PCR reactions were performed in a GeneAmp PCR System 9600 (Perkin-Elmer). Oligonucleotide primers, cycle times, temperatures, and conditions for Msp I digest and electrophoresis have been described in detail.²¹ The common M1 allele, coding for Arg₃₅₃, gave bands of 205 and 67 bp, and the M2 allele, coding for Gln₃₅₃, gave a band of 272 bp as described previously.²¹

Determination of Major Plasma Lipoproteins and Postheparin Plasma Lipase Activities
The major plasma lipoproteins, ie, VLDL, LDL, and HDL, were determined by a combination of preparative ultracentrifugation and precipitation of apo B–containing lipoproteins, followed by lipid analyses.³³ The cutoff limits for lipoprotein phenotyping were set to the 90th percentiles of the VLDL triglyceride (1.95 mmol/L) and LDL cholesterol (4.70 mmol/L) concentrations in the control population.

Lipase activity determinations were made on fasting plasma samples drawn before and 15 minutes after intravenous injection of heparin (100 U/kg body weight; Heparin, Pharmacia). Postheparin plasma LPL activity was measured after addition of polyclonal antibodies directed against HL.³⁴ Conversely, LPL was inhibited by addition of NaCl to measure postheparin plasma HL activity.

VLDL, IDL, and LDL Fractionation
Plasma levels and chemical compositions of subfractions of apo B–containing lipoproteins were determined in the first 62 consecutive patients. VLDL subfractions (Svedberg flotation rate [Sf] >100, Sf 60 to 100, and Sf 20 to 60) and an IDL fraction (Sf 12 to 20) were prepared by cumulative rate ultracentrifugation in a density gradient.³⁵ LDL subfractions were separated by a modification³⁶ of the density gradient ultracentrifugation procedure described by Chapman et al.³⁷

Lipid and Apolipoprotein Analyses
Free and esterified cholesterol (14106-14108 Merck Diagnostica), triglycerides (877557 Boehringer Mannheim Diagnostica), and phospholipids (9990-54008 Wako Chemicals) were determined in triplicate in plasma and the lipoprotein subfractions with enzymatic methods. Total and soluble protein contents were measured with the Lowry technique using bovine albumin as the protein standard.³⁸ All samples, including the standards, were extracted with chloroform after color development to remove any turbidity. Soluble protein in the VLDL subfractions and IDL was estimated after extraction with isopropanol.³⁹ The content of apo B was calculated as the difference between total and soluble protein.

Statistical Methods
Statistical significance for differences in continuous variables were tested either by Student's unpaired t test or by ANOVA with the Scheffé F test to identify differences between groups when the overall F statistic was significant. Logarithmic transformation was performed on the individual values of skewed variables, and a normal distribution of values was confirmed before statistical computation and significance testing. A ² test was used to compare the observed numbers of each FVII genotype with those expected for a population in Hardy-Weinberg equilibrium. Allele frequencies for the ArgGln polymorphism in patients and control subjects were compared by gene counting and ² analysis. Spearman rank correlation coefficients were calculated between plasma lipoprotein and FVII levels.

Ethical Considerations
The experimental protocol was approved by the ethics committee of the Karolinska Hospital, Stockholm. All subjects gave their informed consent to participate in the study.

	Results

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FVII Levels in Patients and Control Subjects
Plasma levels of FVII measurements in patients and control subjects are shown in Table 2. FVII:Ag, FVII:Am, and FVIIc were higher in patients than control subjects when the entire groups were compared. The case-control differences were accounted for by patients with hypertriglyceridemic lipoprotein phenotypes. Patients with combined hyperlipidemia (lipoprotein phenotype IIB) had increased levels of FVIIc and FVII:Ag compared with both normolipidemic patients and control subjects. They also had higher FVII:Am values than normolipidemic control subjects. Similarly, patients with isolated hypertriglyceridemia (lipoprotein phenotype IV) had higher levels of FVII:Ag than both normolipidemic patients and control subjects, and their FVIIc levels were elevated compared with normolipidemic control subjects. In contrast, levels of activated FVII, measured with either bovine thromboplastin (FVIIa:B) or soluble, recombinant, truncated TF (FVIIa), neither differed significantly between all patients and control subjects nor between patients grouped according to lipoprotein phenotype and normolipidemic control subjects. However, FVIIa concentrations tended to be elevated in patients with IIA and IIB lipoprotein phenotypes. The proportion of FVIIa did not differ between all patients and control subjects, nor between patient groups and normolipidemic control subjects.

View this table: [in this window] [in a new window]   Table 2. Factor VII Levels in Patients and Control Subjects Grouped According to Lipoprotein Phenotype

Influence of the ArgGln Polymorphism on FVII Levels in Patients and Control Subjects
Fig 1 is a photograph of Msp I–digested PCR products from individuals of different genotypes. Both the patient and control populations were found to be in Hardy-Weinberg equilibrium for the ArgGln polymorphism. The frequency of the rare Gln allele was .07 in patients and .10 among control subjects (²=.88, NS). One patient was found to be homozygous for the Gln allele. As expected, his FVII protein and activity levels were strikingly low (FVII:Ag, FVII:Am, FVIIc, FVIIa:B, and FVIIa values were 319 ng/mL; 0.50, 0.52, and 0.28 U/mL; and 2.0 ng/mL, respectively). Control subjects with the Arg/Arg genotype had significantly higher plasma levels of all FVII measurements than did control subjects with the Arg/Gln genotype (Table 3). Similarly, all FVII measurements except FVII:Am differed significantly between patients with the Arg/Arg and those with the Arg/Gln genotype.

View larger version (93K): [in this window] [in a new window]   Figure 1. Photograph of Msp I–digested polymerase chain reaction products from individuals of different genotypes. 11 indicates a homozygote for the common allele, coding for Arg353, with bands of 205 and 67 bp; 22, homozygote for the rare allele, coding for Gln353, with a band of 272 bp; 12, heterozygotes. Lane at left includes molecular weight markers.

View this table: [in this window] [in a new window]   Table 3. Factor VII Levels in Patients and Control Subjects With Arg/Arg and Arg/Gln Genotypes

The activity state of the FVII molecule appeared to differ between Arg/Arg and Arg/Gln genotypes among control subjects, the presence of the Gln allele being associated with a lower proportion of fully active FVII molecules. This was not evident among patients, presumably due to the limited number of patients with Arg/Gln genotype.

Correlations Between Major Plasma Lipoprotein and FVII Levels in Patients and Control Subjects With Arg/Arg and Arg/Gln Genotypes
In patients with the Arg/Arg genotype, all FVII measurements except FVIIa showed fairly strong positive correlations with VLDL cholesterol and triglyceride content as well as with LDL and HDL triglyceride concentrations. In contrast, FVIIa correlated significantly with LDL and HDL cholesterol concentrations as well as with HDL triglyceride level (Table 4). Among control subjects with the Arg/Arg genotype, weaker positive correlations were found for FVII:Ag, FVII:Am, and FVIIc with both cholesterol and triglyceride concentrations in VLDL and LDL, but not with HDL triglycerides, except for FVII:Am. In contrast to patients with the Arg/Arg genotype, FVIIa:B was not associated with plasma lipoprotein levels among control subjects with this genotype, with the exception of LDL triglycerides. The lipoprotein relationships for FVIIa also appeared to be different among the control subjects, with significant correlations with LDL triglycerides and HDL cholesterol. In the subsets of patients and control subjects with the Arg/Gln genotype, significant correlations between plasma lipoproteins and FVII variables were generally not observed. However, a strong positive relation was noted between LDL lipid concentrations and FVII:Am in patients with the Arg/Gln genotype (r=.86, P<.05). The genotype-specific difference for the relation between VLDL triglycerides and FVII:Ag in the patient group is illustrated in Fig 2.

View this table: [in this window] [in a new window]   Table 4. Correlations Between Major Plasma Lipoprotein and Factor VII Levels in Patients and Control Subjects With Arg/Arg and Arg/Gln Genotypes

View larger version (18K): [in this window] [in a new window]   Figure 2. Semilog scatterplot of relations of VLDL triglyceride concentration to factor VII antigen level in patients with Arg/Arg (n=76) and Arg/Gln (n=10) genotypes. Arg indicates arginine; Gln, glutamine.

Relations of VLDL, IDL, and LDL Subfractions to FVII Measurements in Patients With the Arg/Arg Genotype
The relations between subfractions of apo B–containing lipoproteins and FVII levels were examined in detail among patients with the Arg/Arg genotype, but sample size precluded this analysis among Arg/Gln subjects. There were fairly strong positive correlations between triglyceride content and particle number (apo B concentration) of the three VLDL subfractions and FVII:Ag, FVII:Am, and FVIIc levels (Table 5). In contrast, the VLDL subfraction correlations with FVIIa:B were less consistent and weaker, and no relationships at all existed for FVIIa. VLDL particle number was most closely related to FVII:Ag concentration. The strength of these relationships did not differ significantly between VLDL particles of varying size.

View this table: [in this window] [in a new window]   Table 5. Correlations Between VLDL Subfraction and Factor VII Levels in Patients With Arg/Arg Genotype

IDL triglyceride, cholesteryl ester, and apo B concentrations showed moderately strong associations with all FVII measurements except FVIIa (Table 6). Among the various determinations of light and dense LDL, the triglyceride content of the two subfractions appeared to be related to FVII:Am, FVIIc, and FVIIa:B levels, but the correlations were generally fairly weak. The number of light and dense LDL particles in plasma, on the other hand, correlated significantly with neither protein concentration nor activity state of the FVII molecule. No single IDL or LDL subfraction determination correlated significantly with FVIIa level.

View this table: [in this window] [in a new window]   Table 6. Correlations of IDL and LDL Subfractions With Factor VII Levels in Patients With Arg/Arg Genotype

Multivariate Analyses
The relationships between plasma lipoproteins and the ArgGln polymorphism in the FVII gene locus to FVII were determined by multiple stepwise regression analyses in the patient group, with FVII:Ag and FVIIa as dependent variables (Table 7). Age and TFPI activity level were first entered in the regression equation as forced variables. In the first set of models, BMI and major plasma lipoproteins were used as independent variables, along with the ArgGln polymorphism. VLDL triglycerides (increase in multiple R²=.27) and the ArgGln polymorphism (increase in multiple R²=.10) were found to relate significantly to FVII:Ag independent of TFPI, BMI, and other plasma lipoproteins, together accounting for 37% of the variation in FVII:Ag. HDL cholesterol and the ArgGln polymorphism were independently associated with FVIIa, the respective increases in multiple R² being .13 and .05.

View this table: [in this window] [in a new window]   Table 7. Multiple Stepwise Regression Analysis of Determinants of Factor VII Antigen and Activity Levels in Patients

In a second set of models, age, TFPI, BMI, VLDL subfractions, IDL, and HDL were selected as independent variables along with the ArgGln polymorphism. These analyses showed that the combination of age, TFPI activity, VLDL Sf 20 to 60 apo B concentration, and ArgGln polymorphism predicted 61% of the variation in FVII:Ag level. Age, TFPI, and HDL cholesterol accounted for 43% of the variation in FVIIa.

Addition of other lipoprotein variables to these models did not significantly increase the value of the multiple R². Of note, the ArgGln polymorphism related significantly to FVII:Ag and FVIIa, independent of which major plasma lipoproteins were included in the multivariate analysis. Inclusion of subfractions of triglyceride-rich lipoproteins in the analysis diminished the number of patients in the analysis, a consequence of which was that the ArgGln polymorphism was not included in the final regression model.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
Formation of the FVII/TF complex is generally thought to represent the primary stimulus for blood coagulation. Activation of FVII occurs by conversion of the single-chain zymogen to a disulfide-linked double-chain enzyme, a process that in vitro appears to be catalyzed by factors IXa, Xa, and thrombin.^{40 41} FVII can also be activated by FXIIa, which is generated upon activation of the contact system of coagulation⁴² as well as by autoactivation.⁴³ Activation of blood coagulation by formation of an activated FVII/TF complex is counteracted by the inhibitor now named TFPI.^{44 45} TFPI provides feedback inhibition of the complex in the presence of its activation product, factor Xa.

A number of different methods have been used for measurement of FVIIa in previous studies, most of which have employed ratios of two different determinations. Accordingly, the ratio of FVIIa:B, as measured with bovine thromboplastin, to FVIIc, as measured with human thromboplastin, has been used to indicate FVII activation,^{28 46 47} because clotting activity assays using bovine thromboplastin are essentially insensitive to the concentration of the FVII zymogen but show some sensitivity to FVIIa. Alternatively, ratios of clotting activity to amidolytic activity or antigen determination have been used.^{47 48 49} However, all clotting activity assays using bovine or human native thromboplastin suffer from interference by the FVII zymogen because the active component of thromboplastin is TF, which promotes the conversion of FVII to FVIIa. Accordingly, both FVII mass and FVIIa contribute to the results obtained by these clotting activity assays.^{6 46} However, FVIIa levels can now be quantified by using a TF mutant that is selectively deficient in promoting FVII activation but retains the FVII cofactor function.^{4 5 24 25}

The present study addressed the issue of whether FVII activity state and protein concentration in fasting plasma are altered in young men with proven MI and examined the relations of FVII to plasma lipoproteins and FVII genotype. In addition to the specific assay for FVIIa and the enzyme immunoassay for FVII:Ag, three conventional methods for measuring FVII activity were used to allow comparison with previous data. The retrospective design of this study conferred a selection bias. It cannot be excluded that the patients who died during the acute phase of the MI had the highest FVII mass and/or activity levels. In addition, alterations in the hemostatic system may have taken place in the early postinfarction period. These restrictions notwithstanding, FVII mass was found to be higher in young men with recent MI than in population-based control subjects, the case-control difference being primarily accounted for by patients with combined hyperlipidemia or hypertriglyceridemia. In contrast, FVIIa was similar in patients and control subjects, which means that the proportion of FVIIa molecules was unaltered or lower in the patients. The triglyceride content and particle number of all VLDL subfractions, irrespective of particle size, correlated fairly strongly with FVII mass determination but not at all with FVIIa. The HDL cholesterol concentration, on the other hand, presumably reflecting the efficiency of LPL-mediated lipolysis of VLDL, related significantly to FVIIa level. As expected, the ArgGln polymorphism significantly influenced both FVII mass and activity levels. In addition, presence of the Gln allele appeared to be associated with a considerably lower proportion of fully active two-chain FVII molecules. The polymorphism also affected the relation between plasma concentrations of VLDL and FVII antigen. Furthermore, the ArgGln polymorphism, independent of VLDL effects, accounted for 5% to 10% of the variation in FVII mass and activity. Because the frequency of the rare Gln allele ranges between .06 and .11 in healthy populations,^{21 22} the present study, because of sample size, was not designed to detect a difference in allele frequencies between cases and control subjects.

To the best of our knowledge, this is the first study of FVII in CHD that has used a specific assay for FVIIa. However, our findings still agree with several previous cross-sectional studies of subjects with manifest CHD or at high risk of contracting CHD, suggesting an unaltered or lowered specific activity of FVII.^{9 10 11 12} Thus, the raised FVIIc levels previously demonstrated in CHD patients might be partially explained by an elevated FVII protein concentration rather than by an increase in FVIIa.

The concept that FVII activity is influenced by the efficiency of the metabolism of triglyceride-rich lipoproteins^{19 50} accords with the findings in the present study. Based on in vitro experiments and studies of the hypercholesterolemic rabbit, it has been hypothesized that large, triglyceride-rich particles, such as chylomicrons, VLDL, and their remnants that carry the appropriate free fatty acid (FFA) at a sufficient density of negative charge, activate FVII through the intrinsic coagulation pathway and FXIIa.^{51 52 53} The generation and subsequent transfer of FFAs from large, triglyceride-rich lipoproteins through the action of LPL play an important role in this sequence of events. This was elegantly shown in a recent study of familial LPL deficiency, in which high plasma concentrations of triglyceride-rich lipoproteins increased FVIIc only in the presence of LPL.⁵⁴ Postheparin plasma LPL activity, which is assumed to reflect the LPL available at the endothelial surface, tended to be lower in young postinfarction patients, which could provide one explanation why our measurement of FVIIa was unaltered despite raised VLDL concentration and FVII mass. Furthermore, we observed a positive correlation between HDL cholesterol level, which would be expected to reflect lipolytic activity, and FVIIa level.

The notion that possession of the allele for FVII-Gln₃₅₃ confers protection against CHD by reducing FVII activity in plasma was not contradicted by the present study. Furthermore, the existence of genotype-specific differences in the relationships between triglycerides and FVII was shown to be specifically accounted for by VLDL and FVII:Ag, FVII:Am, and FVIIc, the positive associations being confined to individuals with the Arg/Arg genotype. The Arg/Gln heterozygotes in the present study had lower basal specific activity values of FVII. This suggests that the ArgGln polymorphism is located in a region of the FVII molecule that is involved in the interaction with triglyceride-rich lipoproteins. There is the possibility that the ArgGln polymorphism is in linkage disequilibrium with other polymorphisms located in the FVII gene. It is possible that the altered conformation of the FVII-Gln₃₅₃ molecule may affect intracellular processing in hepatocytes, which would lead to reduced secretion of FVII. Alternatively, the ArgGln substitution may alter the stability of the protein in plasma or its rate of removal from the circulation.

In summary, the present findings speak against a role of FVII as a risk factor for CHD, as a significantly increased potential for activation of coagulation (ie, raised basal concentration of FVIIa) was not observed among young postinfarction patients. The associations between FVII activity measurements and CHD observed in previous epidemiological and clinical studies might have been accounted for by the association between FVII protein elevation and hypertriglyceridemia. Prospective epidemiological studies including specific determination of FVIIa are needed to resolve the issue of whether FVII activity is a risk factor for CHD.

	Acknowledgments

 
This study was supported in part by grants from the Swedish Medical Research Council (520 and 8691), the Swedish Heart-Lung Foundation, the Marianne and Marcus Wallenberg Foundation, King Gustaf V 80th Birthday Fund, the Thuring Foundation, the Tore Nilsson Foundation, and Professor Nanna Svartz' fund. Dr Hamsten is a Career Investigator of the Swedish Heart-Lung Foundation.

Received February 15, 1995; accepted February 21, 1995.

	References

Top Abstract Introduction Methods Results Discussion References

 
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