© 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
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.
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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 Arg
Gln
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
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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.
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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).
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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Discussion |
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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 lipoproteins19 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.
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Acknowledgments |
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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.
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References |
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1. Seligsohn U, Kasper CK, Østerud B, Rapaport SI. Activated factor VII: presence in factor IX concentrates and persistence in the circulation after infusion. Blood. 1979;53:828-837.
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