© 2000 American Heart Association, Inc.
Thrombosis |
Factor V Antigen Levels and Venous Thrombosis
Risk Profile, Interaction With Factor V Leiden, and Relation With Factor VIII Antigen Levels
From the Hemostasis and Thrombosis Research Center (P.W.K., F.R.R., J.C.J.E., R.M.B.) and the Department of Clinical Epidemiology (F.R.R.), Leiden University Medical Center, and the Gaubius Laboratory (R.B.), TNO Prevention and Health, Leiden, the Netherlands. Dr Bos is now at Pharming Technologies, Leiden, the Netherlands.
Correspondence to Rogier M. Bertina, Hemostasis and Thrombosis Research Center, Leiden University Medical Center, C2-R, PO Box 9600, 2300 RC Leiden, Netherlands. E-mail bertina{at}hematology.azi
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Abstract |
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Abstract—Clotting factor V has a dual function in coagulation: after activation, procoagulant factor V stimulates the formation of thrombin, whereas anticoagulant factor V acts as a cofactor for activated protein C (APC) in the degradation of factor VIII/VIIIa, thereby reducing thrombin formation. In the present study, we evaluated whether plasma factor V levels, either decreased or increased, are associated with venous thrombosis. High procoagulant factor V levels may enhance prothrombinase activity and increase the thrombosis risk. Low anticoagulant factor V levels could reduce APC-cofactor activity in the factor VIII inactivation (APC-resistant phenotype), which might also promote thrombosis. Low factor V levels in combination with factor V Leiden could lead to a more severe APC-resistant phenotype (pseudohomozygous APC resistance). To address these issues, we have measured factor V antigen (factor V:Ag) levels in 474 patients with thrombosis and 474 control subjects that were part of the Leiden Thrombophilia Study (LETS). Factor V:Ag levels increased by 7.6 U/dL for every successive 10 years of age. Mean factor V:Ag levels were 134 (range 41 to 305) U/dL in patients and 132 (range 47 to 302) U/dL in controls. Neither high nor low factor V:Ag levels were associated with venous thrombosis. We found that factor V:Ag and factor VIII antigen levels in plasma were correlated, but factor V did not modify the thrombotic risk of high factor VIII levels. The normalized APC ratio was not influenced by the factor V:Ag level in subjects with or without factor V Leiden. We conclude that neither low nor high factor V:Ag levels are associated with venous thrombosis and that factor V:Ag levels do not mediate the thrombotic risk associated with high factor VIII levels.
Key Words: factor V • venous thrombosis • factor V Leiden • factor VIII
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Introduction |
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Deep-vein thrombosis is a common disorder with an incidence in the general population of
1 in 1000 individuals per
year.1 2 Despite growing insight into inherited as well as
acquired risk factors for thrombosis, the cause of many thrombotic
episodes remains unknown. A single point mutation in the factor V gene
(G1691A, R506Q, factor V Leiden)3 results in a reduced
sensitivity of plasma factor Va to inactivation by activated
protein C (APC)4 and is present in 20% of
white patients with deep-vein thrombosis.5
Coagulation factor V has a central role in procoagulant and
anticoagulant pathways: factor V is activated by factor Xa or
thrombin6 7 8 and markedly accelerates the activation of
prothrombin by factor Xa before factor Va is degraded by
APC.9 10 In addition, factor V (but not factor Va) has
been reported to act as a cofactor of APC in the proteolytic
degradation of both factor VIII and factor
VIIIa.4 11 12 13 Whether the level of factor V in plasma affects the risk of thrombosis is unclear, although, hypothetically, high as well as low factor V levels may increase the thrombotic risk. Elevated factor VIII levels are associated with an increased risk for thrombosis,14 and factor V is, like factor VIII, an important cofactor in one of the steps of the coagulation cascade. Therefore, high plasma levels of factor V might lead to an increased prothrombinase activity and increased risk of thrombosis. In 1966, Gaston15 reported a family with an association between elevated factor V levels and venous thrombosis.
Low factor V levels are associated with a reduced APC cofactor activity in the inactivation of factor VIII/VIIIa.11 12 13 This results in an APC-resistant phenotype and therefore might be associated with an increased risk of thrombosis, especially when the factor V/factor VIII ratio is low. In that case, low factor V levels would increase the risk of thrombosis.
Finally, we have to consider the influence of factor V levels on the thrombotic risk of carriers of factor V Leiden. Reduced expression of factor V by the non–factor V Leiden allele (low factor V levels) might result in a more severe APC-resistant phenotype, as has been reported for patients pseudohomozygous for APC resistance (combined heterozygous factor V deficiency and heterozygous factor V Leiden mutation).16 17 18 19 Similarly, enhanced expression of factor V by the non–factor V Leiden allele (high factor V levels) might result in a less severe APC-resistant phenotype.
To address these issues, we measured the factor V antigen (factor V:Ag) level in 474 patients with thrombosis and 474 healthy control subjects that were part of the Leiden Thrombophilia Study (LETS).20
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Methods |
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Study Population
The patients and control subjects included in the present study came from a population-based case-control study of venous thrombosis (LETS).20 The present study includes 474 unselected consecutive outpatients aged <70 years who were treated with anticoagulants after a first episode of objectively confirmed deep-vein thrombosis and who did not have an underlying malignancy. The median time between the occurrence of the deep-vein thrombosis and blood collection was 18 months (range 6 to 48 months). Each thrombotic patient provided his or her own sex- and age-matched healthy control subject according to predefined criteria.14 The mean age for patients and controls was 47 years (range 16 to 70 years for patients and 16 to 73 years for controls).
Blood Collection and Plasma Assays
Blood was collected in tubes containing 0.106 mmol/L
trisodium citrate. Plasma was prepared by
centrifugation for 10 minutes at 2000g at
room temperature and stored at -70°C in 1.5 mL aliquots. Factor V:Ag
was measured by an in-house–developed sandwich-type ELISA with 2
monoclonal antibodies, both with a high affinity for
(activated) factor V. The monoclonal antibodies, denoted V-6
and V-9, were selected from a panel of antibodies isolated on the light
chain of factor V, as demonstrated by Western blot analysis
with the use of purified factor Va under reducing conditions.
Ninety-six–well plates were coated with monoclonal antibody V-6 (at a
concentration of 3 mg/mL in 0.1 mol/L NaHCO3 and
0.5 mol/L NaCl, pH 9.0) and left overnight at 4°C. Plasma samples
were diluted 1/100 to 1/400 in 0.05 mol/L triethanolamine, 0.1 mol/L
NaCl, 0.01 mol/L EDTA, and 0.1% Tween 20, pH 7.5, and 100 µL per
sample was incubated for 3 hours in the coated wells. Monoclonal
antibody V-9, labeled with horseradish peroxidase (Zymed Laboratories
Inc) and diluted to 2 mg/mL in 0.05 mol/L triethanolamine, 0.1 mol/L
NaCl, 0.01 mol/L EDTA, and 0.1% Tween 20, pH 7.5, was used for the
detection of immobilized factor V (2-hour incubation). In
the final step, 0.42 mmol/L 3,3',5,5'-tetramethylbenzidine, 0.1
mol/L sodium acetate, and 1.1 mol/L
H2O2, pH 5.5, were added,
and the reaction was stopped after 20 minutes with 100 µL
H2SO4. After every step,
plates were washed 4 times with 0.05 mol/L triethanolamine, 0.1 mol/L
NaCl, and 0.1% Tween 20, pH 7.5. Pooled normal plasma (PNP), prepared
from the plasma of 60 healthy volunteers (mean age 40 years) and
diluted 1/50 to 1/3200, was used as a reference and defined to contain
100 U/dL. In each analytical run, we included 2 samples of an in-house
PNP and 2 samples of a commercial high factor V plasma (Mallinckrodt
Baker) as controls. PNP contained, on average, 103±8 U/dL factor V:Ag
(interassay variation 8.2%, intra-assay variation 6.7%). High factor
V plasma contained, on average, 136±12 U/dL factor V:Ag (interassay
variation 8.9%, intra-assay variation 5.2%). Results were the same
for plasma and serum samples (after correction for the dilution with
the anticoagulant) of the same individual and not substantially altered
by repeated freezing and thawing.
Factor VIII antigen levels have been measured by a sandwich-type ELISA with two monoclonal antibodies directed against the light chain of factor VIII.21 CLB Cag 117 was used as a catching antibody, and CLB-A was used as a tagging antibody. Both antibodies were kindly provided by Dr J.A. van Mourik (Department of Blood Coagulation, CLB, Sanquin Blood Supply Foundation, Amsterdam, the Netherlands). PNP, calibrated against the World Health Organization standard (91/666 ratio) for factor VIII antigen (factor VIII:Ag), was used as a reference. The sensitivity of plasma to APC was determined as previously described22 and expressed in a normalized APC sensitivity ratio.22
Statistical Analysis
An unconditional logistic model was used to calculate odds
ratios (ORs) as a measure of relative risk with 95% CIs derived from
the model. This OR estimates the risk of venous thrombosis in the
presence of a risk factor relative to the absence of the particular
risk factor, the reference category. Factor V and factor VIII levels
were entered into the logistic model as categorized variables. The
cutoff points were 100, 125, and 150 U/dL for factor VIII and 110, 130,
and 150 U/dL for factor V. These cutoff points corresponded
approximately to quartiles.
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Results |
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Factor V:Ag Levels
The total mean factor V:Ag level was 132 U/dL, with a range between 41 to 305 U/dL. The factor V:Ag levels were normally distributed. In multiple regression, age and smoking were associated with the plasma factor V:Ag level. For every successive 10 years of age, the factor V:Ag level increased 7.6 (95% CI 6.1 to 9.1) U/dL. Smokers had 8.3 (95% CI 4.0 to 12.6) U/dL higher factor V:Ag levels than did nonsmokers.
Factor V:Ag Levels and Venous Thrombosis
The mean factor V:Ag level was 134 (range 41 to 305) U/dL for the
patients and 132 (range 47 to 302) U/dL for the controls. Table 1
presents the ORs of patients and
controls after stratification of the factor V:Ag levels into 4 groups.
The relative risk of factor V:Ag levels 
150 U/dL was 1.3 (95% CI 0.9
to 1.8) compared with the reference category (factor V:Ag level <110
U/dL). The data of Table 1 also show that reduced factor V
levels are not associated with an increased risk of thrombosis. The
influence on thrombosis of either low or high factor V:Ag levels was
analyzed in more detail by stratification of the patients and
controls into 10 groups; we calculated the relative risk for the
patients with high factor V:Ag compared with the lowest category of
factor V:Ag (<93 U/dL). Even the highest factor V:Ag level (>171
U/dL) was not associated with an increased risk of thrombosis compared
with the reference category (Figure 1).
The same was true for the lowest factor V:Ag category; even factor V
levels <70 U/dL did not increase the risk of thrombosis.
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Interaction Factor V:Ag Levels and Factor V Leiden
Factor V Leiden was determined in 945 of the 948 subjects. In 3
patients, DNA was unavailable. One hundred six subjects carried the
factor V Leiden mutation, of whom 8 were homozygous. The factor V:Ag
level (mean±SD) was 132±34 U/dL for wild-type factor V, 137±33 U/dL
for heterozygous factor V Leiden, and 146±42 U/dL for homozygous
carriers of factor V Leiden. These groups were entered in a regression
model (0 represents wild-type factor V; 1, heterozygous factor
V Leiden; and 2, homozygous factor V Leiden) with factor V:Ag as a
dependent variable; the regression coefficient ß was 5.1 (95% CI
-1 to 11). Further analyses explored the association between
factor V Leiden and venous thrombosis for different factor V:Ag levels.
Because only 8 subjects were homozygous for factor V Leiden,
calculations for factor V Leiden carriers were based on homozygous and
heterozygous carriers as a single group. For carriers of factor V
Leiden who had factor V:Ag levels 150 U/dL, the risk of venous
thrombosis increased nearly 13-fold (95% CI 3.8 to 41) compared with
carriers of wild-type factor V with factor V:Ag levels <110 U/dL
(Table 2). (This risk is not very
different from the risk of factor V Leiden carriers with factor V
antigen levels <150 U/dL, especially when we realize that the
calculations in Table 2 are based on relatively low numbers in
the control group.
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Relation Between Factor V:Ag and Factor VIII:Ag Levels
Factor V and factor VIII are related proteins and probably share
common biosynthetic pathways, as reflected by the identification of
patients with combined factor V and factor VIII
deficiency.23 24 25 It was of interest to analyze to
what extent factor V and VIII levels in plasma are correlated. We found
that factor VIII levels and factor V levels are correlated
(r=0.26, P<0.001). For carriers of the wild-type
factor V, factor VIII:Ag rose by 3.5 U/dL for every 10-U/dL increase in
factor V:Ag, whereas for factor V Leiden carriers, the factor VIII:Ag
level increased by 4.6 U/dL. Adjustment for age and separate
analysis in thrombotic patients and controls yielded the same
results.
Next, we investigated whether the effect of high factor VIII levels on
thrombosis is conditional on the factor V level. Because factor V
Leiden itself is a risk factor for thrombosis, analysis was
restricted to carriers of the normal factor V genotype. For
these calculations, we took the highest quartiles of factor V and
factor VIII and compared them with the lowest quartiles. Table 3 shows that factor VIII levels >150
U/dL in combination with factor V levels <110 U/dL gave an OR of 2.2.
The combination of factor V and VIII levels >150 U/dL increased the
thrombotic risk 6.0 (95% CI 2.6 to 12) times compared with factor V
levels <110 U/dL and factor VIII levels <100 U/dL (Table 3).
This risk is similar to the previously reported 6-fold increase in the
risk of thrombosis for unadjusted factor VIII:Ag levels >150
U/dL,21 indicating that high factor VIII:Ag does not
interact with high factor V:Ag levels in promoting thrombosis.
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Factor V:Ag Levels and APC Ratio
The association of factor V:Ag levels and the normalized APC ratio
in subjects with normal factor V and factor V Leiden is illustrated in
Figure 2. Patients using oral
anticoagulants (n=48) or with a lupus anticoagulant (n=4) were
excluded from this analysis because they have a prolonged
activated partial thromboplastin time in the absence of APC,
which gives unreliable results in the APC resistance test. In factor V
Leiden and factor V wild-type subjects, factor V:Ag levels had no
effect on the APC ratio (r=0.01 for factor V Leiden,
r=-0.04 for wild-type factor V).
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) in relation to
factor V:Ag levels. |
Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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The present study evaluates the relation between factor V levels and venous thrombosis in a large population-based case-control study. Factor V:Ag levels were found to be influenced by age and smoking. The 7.6-U/dL increase in factor V that we found for every successive 10 years of age is in agreement with the findings of Brozovic et al,26 who reported an increase in factor V activity of 0.6% per year (
6 U/dL per decade). Factor V:Ag levels were measured by ELISA with the use of 2 monoclonal antibodies against the light chain of factor V. The mean factor V:Ag level of the control group was 132 U/dL higher than we would have expected. Our PNP contained, on average, 103 U/dL factor V. The intra-assay and interassay variation were relatively low and very acceptable for this type of test.
Neither low nor high factor V:Ag levels in plasma were associated with venous thrombosis. Apparently, unlike factor VIII,14 high levels of plasma procoagulant factor V do not promote venous thrombosis, nor do reduced levels of anticoagulant factor V. It is possible that this is related to a balance between the procoagulant and anticoagulant functions of factor V; a high factor V level may enhance prothrombinase activity, as does factor V Leiden,27 but at the same time, high anticoagulant factor V may result via its APC-cofactor activity in an increased degradation of factor VIII/VIIIa.11 12 13 The present study provides no information on the role of platelet factor V in the pathogenesis of venous thrombosis. Platelet factor V has been implicated as an important regulator of hemostasis in vivo.28 Recently, however, the origin of platelet factor V (biosynthesis in platelets or endocytosis of plasma factor V by platelets) has been questioned.29 30 More information is required to determine how platelet factor V concentrations relate to plasma factor V levels before we can discuss an independent role of platelet factor V in thrombosis.
The conclusion that low factor V levels are not associated with venous thrombosis is not a complete surprise because of the lack of reports on thrombotic events in heterozygous factor V–deficient family members of patients with severe factor V deficiency. Very recently, Redondo et al31 found that high factor V activity levels are associated with arterial thrombosis. No mechanism for this relation was provided.
Carriers of factor V Leiden with factor V:Ag levels >150 U/dL had a 13-fold increased risk compared with those with wild-type factor V with factor V:Ag levels <110 U/dL. Because this result is based on only 3 controls, we do not believe that this indicates a higher risk for this group of factor V Leiden carriers.
Factors V and VIII are related proteins and share common biosynthetic pathways, as reflected by recent studies of Nichols and colleagues23 25 and Neerman-Arbez et al24 in combined factor V and VIII deficiencies. In the present study, we found that factor V:Ag levels correlate with plasma factor VIII:Ag levels, suggesting that common posttranslational modifications23 explain a small part of the large variation in plasma factor V levels and VIII levels. The thrombosis risk of high factor VIII:Ag levels was not affected by factor V levels. The combination of high factor V and high factor VIII levels did not result in a higher thrombotic risk than that of high factor VIII levels by themselves; thus, factor V does not modify the thrombotic risk of elevated factor VIII levels.
The normalized APC ratio was not influenced by the factor V:Ag level in subjects with or without factor V Leiden. Freyburger et al32 found slightly lower mean APC ratios for thrombotic patients with factor V:C levels >100% compared with those with levels <100%. In healthy pregnant women, results are conflicting: Walker et al33 found no relation between the APC ratio and factor V activity levels, whereas Clark et al34 observed a relation (r=-0.33, P=0.03) after excluding subjects with factor VIII:C levels >120 U/dL. In our subjects, after adjustment for the influence of elevated factor VIII:Ag levels, no relation between factor V and the normalized APC sensitivity ratio could be observed. Our data suggest that the synthesis of factor V in subjects with factor V Leiden is normal and that the elevation of the factor V:Ag level does not affect the APC ratio in heterozygotes and therefore cannot be considered as a cause of acquired APC resistance. We also found no relation between low factor V levels and the APC sensitivity ratio, which is explained by the fact that a reduction of the APC ratio occurs only at very low factor V levels (<25%).35
In conclusion, factor V:Ag levels are not associated with a risk of venous thrombosis. Factor V levels and factor VIII levels are correlated in plasma, but factor V does not mediate the thrombotic risk of high factor VIII levels. There seems to be no clear relation between factor V:Ag levels and the normalized APC ratio in subjects with and without factor V Leiden.
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Acknowledgments |
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This study was supported by a grant (No. 950-10-629) from the Netherlands Organization for Scientific Research (NWO). We would like to thank C.J.M. van Leuven for technical support in the manufacturing of the monoclonal antibodies.
Received August 13, 1999; accepted November 3, 1999.
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