British Journal of Haematology 2000, 109, 519±522
Heritability of elevated factor VIII antigen levels in factor V
Leiden families with thrombophilia
P. W. Kamphuisen, 1 R. Lensen, 2 J. J. Houwing-Duistermaat, 4 J. C. J. Eikenboom, 1 M. Harvey, 3
R. M. Bertina 1 and F. R. Rosendaal 1 , 2 1 Haemostasis and Thrombosis Research Centre and the Departments of
2
Clinical Epidemiology and 3 Immunohaematology and Blood Bank, Leiden University Medical Centre, and 4 Institute of
Epidemiology and Biostatistics, Erasmus Medical Centre Rotterdam, The Netherlands
Received 1 November 1999; accepted for publication 10 February 2000
Summary. Factor VIII activity (factor VIII:C) and factor VIII
antigen (factor VIII:Ag) levels above150 IU/dl are associated
with a five- to sixfold increased risk of venous thrombosis
compared with levels , 100 IU/dl. These high levels are
present in 25% of patients with a first episode of deep-vein
thrombosis and in 11% of healthy controls. von Willebrand
factor (VWF) and blood group are important determinants
of the factor VIII level in plasma and therefore contribute to
thrombotic risk, while factor VIII appears to be the final
effector. Previously, we found familial clustering of factor
VIII:C levels in women, which remained after adjustment for
VWF and blood group. In the present study, we analysed the
familial influence on factor VIII:Ag levels exceeding
150 IU/dl in 12 large families with thrombophilia in
which high factor VIII:Ag levels contribute to thrombotic
risk. As expected, blood group was a main determinant of
the plasma factor VIII level: 58 relatives (32%) had factor
Elevated factor VIII activity (factor VIII:C) levels are associ-
ated with an increased risk of venous thrombosis (Koster
et al, 1995). Factor VIII:C levels higher than 150 IU/dl
increase the thrombosis risk five- to sixfold compared with
levels below 100 IU/dl (Koster et al, 1995). von Willebrand
factor (VWF) and blood group are well-known determinants
of the factor VIII level in plasma and so contribute to
thrombotic risk, whereas factor VIII itself appears to be the
final effector in promoting thrombosis (Koster et al, 1995).
Elevated factor VIII:C levels are highly correlated with factor
VIII antigen (factor VIII:Ag) levels (Kamphuisen et al, 1997;
O'Donnell et al, 1997). This suggests that the observed
elevation of factor VIII:C levels reflects a true increase in
Correspondence: Professor F. R. Rosendaal, Clinical Epidemiology,
C0-P, Leiden University Medical Centre, PO Box 9600, 2300 RC
Leiden, The Netherlands. E-mail: f.r.rosendaal@lumc.nl
q 2000 Blackwell Science Ltd
VIII levels above 150 IU/dl and 50 (86%) of these had blood
group non-O. After adjustment for blood group and age, we
found an association between factor VIII:Ag levels in sister
pairs (0´35, P ˆ 0´003), brother pairs (0´35, P ˆ 0´003),
brother±sister pairs (0´35, P , 0´001) and in mother±son
pairs (0´45, P ˆ 0´02), but not in father±daughter or
father±son pairs. The familial aggregation test was strongly
positive for factor VIII:Ag levels (P , 0´001) and remained
so after adjustment for the influence of blood group. We
conclude that high factor VIII:Ag levels are a highly
prevalent risk factor for venous thrombosis and contribute
to risk in families with thrombophilia, and that these high
levels are likely to be genetically determined by factors other
than just blood group.
Keywords: factor VIII, thrombophilia, factor V Leiden,
familial clustering.
factor VIII protein and is not the result of activation of the
coagulation system during the blood collection procedure
(Kamphuisen et al, 1997; O'Donnell et al, 1997). Factor
VIII:Ag levels above 150 IU/dl were, like factor VIII:C levels,
also associated with a fivefold increased risk of venous
thrombosis (Kamphuisen et al, 1997). We have recently
shown that elevated factor VIII levels in thrombosis patients
are not the result of acute phase reactions because elevated
factor VIII levels remained associated with a sixfold increased
risk after adjustment for C-reactive protein (CRP), a sensitive
marker for acute phase processes (Kamphuisen et al, 1999).
These observations lend further support to a causal relation-
ship between high factor VIII levels and venous thrombosis.
High factor VIII levels are common; in our study, 25% of
the patients with a first episode of venous thrombosis and
11% of the healthy controls had factor VIII levels above
150 IU/dl (Koster et al, 1995). Considering the sixfold
519
520 P. W. Kamphuisen et al
increased thrombosis risk and the high prevalence in the
population, factor VIII levels exceeding 150 IU/dl contribute
importantly to deep-vein thrombosis.
We previously reported that factor VIII:C levels show a
familial clustering, which remains after correction for VWF
and blood group (Kamphuisen et al, 1998). That study was
performed in female relatives of probands with documented
haemophilia A who came to the Leiden haemophilia centre
for carriership testing. We designed a study that allowed us to
incorporate a large number of families that were seen over a
period of more than 10 years for carrier testing. However, the
design of this study also had several drawbacks. First, factor
VIII:C levels were measured over a 10-year period, which
might have led to extra variation in the plasma factor VIII
level. Furthermore, only women were tested, which provides
less information on familial clustering than when both
women and men are tested. And, finally, in this group of
women, high levels of factor VIII were not as common as
among patients with thrombosis, and the association
between levels and thrombosis was not studied nor was
the heritability of high levels (above 150 IU/dl) itself.
In the present study, we have analysed the familial
aggregation of factor VIII levels in men and women of 12
large families with thrombophilia who had a proband with
both venous thrombosis and factor V Leiden. In these
families, factor VIII:Ag levels above 150 IU/dl contribute to
the thrombosis risk of factor V Leiden carriers (Lensen et al,
1999). We tested whether clustering of factor VIII:Ag levels
higher than 150 IU/dl occurred and the effect of blood
group on these high levels.
SUBJECTS AND METHODS
Family data. The 12 probands originated from a larger
panel of 28 patients who were referred to our centre for
diagnostic work-up for venous thrombophilia, i.e. patients
with a positive family history of venous thrombosis (in
addition to the proband, at least two symptomatic relatives),
and who did not have deficiencies of protein C, protein S or
antithrombin. These 28 patients were screened for the
presence of the factor V Leiden mutation that was detected
in 12 patients. We invited the siblings, parents and children
of these 12 probands as well as uncles and aunts of the
affected parental side and, if these were carriers, their
children (first cousins of the proband). Family members
under 15 years of age were excluded for practical purposes.
Of the 12 probands, 182 family members (93%) participated
in the study while 12 did not, three because they lived
abroad and nine for reasons unknown.
The probands and family members were not selected on
the basis of factor VIII:Ag levels because these levels were
not known at that time.
Laboratory assays. Blood was collected from the ante-
cubital vein in 0´106 m trisodium citrate. ABO phenotypes
were deduced from the reactions of plasma isoagglutinins to
A1, B, A1B or (as negative control) O test blood cell
suspensions (3% v/v) in a standard spin-tube agglutination
technique carried out at room temperature (Walker, 1990).
All the subjects were considered to be immune competent and
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free from malignancy or infection. The chance of an ABO
misphenotyping because of the absence of the appropriate
isoagglutinin, anomalous occurrence of anti-A or -B or
acquisition of a B-antigen was considered to be unlikely.
Factor VIII antigen was measured by a sandwich type
enzyme-linked immunosorbent assay (ELISA) using two
monoclonal antibodies directed against the light chain of
factor VIII (Kamphuisen et al, 1997). Pooled normal plasma,
calibrated against the WHO standard (91/666) for factor
VIII:Ag, was used as a reference.
Statistical analysis. The distribution of factor VIII:Ag levels
was skewed and was logarithmically transformed for all
statistical analyses. The analysis was performed using linear
regression, with age entered as a continuous variable (in
years), and blood group dichotomized into two groups (0 for
blood group O, 1 for non-O).
To investigate genetic effects on factor VIII:Ag levels, the
residuals of the multiple regression models were used.
Residuals are the differences between the observed level Yi
for person i and the predicted value m i obtained from the
multiple regression model. As a first test for familial effects,
correlations between the residuals (obtained from the
multiple regression model) of pairs of relatives were
calculated. Familial aggregation of factor VIII:Ag levels
was studied using a recently developed method (Houwing-
Duistermaat et al, 1995) that tests the null hypothesis of no
correlation within randomly chosen pedigrees. The correla-
tion of the genetic effects is tested by the weighted sum of
correlations between pairs of relatives within a pedigree Q.
The test for familial aggregation is positive when the
calculated Q is larger than the expected Q-value under the
null hypothesis of no aggregation (for more details, see
Houwing-Duistermaat et al, 1995; Kamphuisen et al, 1998).
The familial clustering of factor VIII levels exceeding
150 IU/dl was tested with the individual factor VIII level
dichotomized into two groups (Yi ˆ 0 for factor VIII:Ag
levels , 150 IU/dl, Yi ˆ 1 for factor VIII:Ag levels
$ 150 IU/dl). In this way, the value of Q will be determined
mainly by pairs of relatives who both have factor VIII levels
higher than 150 IU/dl.
RESULTS
We studied 182 relatives of 12 probands with factor V
Leiden and a positive family history of venous thrombosis.
The mean size of the families was 19 members, with a range
from 3 to 29 members. The mean age at the time of the
study was 40 years (range 15±88 years); 92 (50%) were
men and 90 were women. Of these 182 relatives, 91 were
heterozygous for factor V Leiden, one relative was homo-
zygous and 90 were non-carriers.
Mean factor VIII:Ag level was 137 IU/dl with a range
between 54 and 339 IU/dl. Plasma factor VIII:Ag levels
were not clearly different in factor V Leiden carriers
[142 IU/dl; 95% confidence interval (CI) 132±151 U/dl]
and non-carriers (132 IU/dl; 95% CI 121±142 U/dl).
Factor VIII:Ag levels were higher in the 126 subjects
(69%) with blood group non-O than in the 56 subjects
(31%) with blood group O (150 vs. 107 IU/dl) (mean
 Familial Clustering of High Factor VIII Levels 521
Table I. Correlation coefficients for factor VIII:Ag levels between Table III. Familial aggregation of factor VIII:Ag levels above
first-degree relative pairs.* 150 IU/dl.*

Relationship No. of pairs R P-value Factor VIII:Ag

Father±son 28 0´17 0´24 Unadjusted Adjusted*

Mother±son 26 0´45 0´02
Father±daughter 21 0´14 0´45 Q 1´65 1´56
Mother±daughter 33 0´27 0´13 Exp (Q) 0´97 0´97
Sisters 70 0´35 0´003 P-value , 0´001 , 0´001
Brothers 70 0´35 0´004
Brother±sister 180 0´31 , 0´001
*Adjusted for age and blood group.

*Adjusted for age and blood group.

difference 43 IU/dl; 95% CI 29±57 IU/dl). Factor VIII levels
were above 150 IU/dl in 58 individuals (32%). Of these
individuals, 50 subjects had blood group non-O (86%) and
eight (14%) had blood group O.
We tested the correlations of factor VIII:Ag levels between
all first-degree relative pairs. After adjustment for blood
group and age, the factor VIII levels in siblings showed a
strong association (Table I). In sister pairs, the correlation of
factor VIII levels was 0´35 (P ˆ 0´003) and in brother pairs
it was 0´35 (P ˆ 0´004), whereas in brother±sister pairs
this correlation was 0´31 (P , 0´001). In mother±son
pairs, factor VIII:Ag levels were also highly correlated
(r ˆ 0´45, P ˆ 0´02). The other first-degree relationships
showed no clear correlations (Table I).
We assessed the familial clustering of factor VIII:Ag levels
with the familial aggregation test. Table II shows that when
factor VIII:Ag levels are tested as continuous variables the
familial aggregation statistic Q was clearly higher than the
expected Q under the null hypothesis of no correlation, also
after adjustment for age and blood group. Unadjusted, Q
was 308, whereas the expected value of no correlation was
175 (P , 0´001). Adjusted, Q was 301 with an expected Q
of 172 (P , 0´001). Table III shows the familial clustering
of factor VIII levels above 150 IU/dl as a dichotomized
variable. The familial aggregation test was strongly positive
again. In this test, Q was 1´65 with an expected value of no
correlation of 0´97 (P , 0´001). Adjustment for blood
group and age did not essentially change Q (Table III).
DISCUSSION
Our study showed that within thrombophilia families factor
Table II. Familial aggregation of factor VIII:Ag levels.
Factor VIII:Ag
Unadjusted Adjusted*
Q 308 301
Exp (Q) 175 172
P-value , 0´001 , 0´001
*Adjusted for age and blood group.
VIII:Ag levels are highly aggregated. The families we studied
had thrombophilia, in which high factor VIII levels
contribute to the risk of factor V Leiden carriers (Lensen
et al, 1999). Familial clustering of factor VIII:Ag levels
higher than 150 IU/dl was clearly demonstrated by the
familial aggregation test, and this remained after adjust-
ment for the effects of blood group and age. This suggests a
genetic influence on high factor VIII levels other than just
blood group.
Familial aggregation was prominent, indicating that
factor VIII levels were strongly influenced by familial
factors. The support for familial aggregation of factor VIII
levels was much stronger for the families studied here than
that obtained with the female relatives of haemophilia A
patients tested in our previous study (Kamphuisen et al,
1998). This might have been the result of the larger size of
the families which positively influenced the statistic Q of
familial aggregation. The inclusion of men and women
tested in the present study could also affect the outcome
because the familial aggregation test uses all possible
combinations within pedigrees. Further, factor VIII levels
were measured over a much shorter period than in our
previous study (, 4 weeks), which will reduce interassay
variations in the factor VIII level.
These are families with thrombophilia who will probably
have several risk factors for thrombosis. As high factor VIII
levels are associated with venous thrombosis, we may have
selected for high factor VIII levels in choosing these families.
This is reflected by the higher number of factor VIII levels
above 150 IU/dl in the thrombophilia families (32%) than
in the thrombosis patients in the LETS study (25%) (Koster
et al, 1995). The calculated Q of familial aggregation for
high factor VIII levels is mainly the result of concordant
pairs with high factor VIII levels. The probability that
siblings have both elevated factor VIII levels is more likely in
families that have a high prevalence of elevated factor VIII
levels than in families in which low factor VIII levels are
more common. This means that a selection for high factor
VIII levels in families will underestimate the heritability of
high factor VIII levels.
In this study, correlations of factor VIII:Ag levels between
first-degree relatives only partially confirm the theory that
variation of plasma factor VIII levels is under control of
X-linked alleles (Filippi et al, 1984). After adjustment for
the effect of blood group and age, factor VIII levels
correlated in siblings and in mother±son pairs, but not in

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522 P. W. Kamphuisen et al
mother±daughter, father±daughter or father±son pairs.
Especially the finding that factor VIII levels were not
correlated in father±daughter pairs, who share the same
X-chromosome, does not support an X-linked determinant
of factor VIII levels. We have to consider the possibility that
the correlation of factor VIII between fathers and daughters
is lowered by the maternal X-chromosome of the daughter.
A daughter who has a father with high factor VIII and a
mother with low factor VIII may have an intermediate
factor VIII level. This will negatively influence the correla-
tion between father and daughter. Very recently, Mansvelt
et al (1998) investigated the promoter and 3 0 terminus of
the factor VIII gene for variations associated with high
factor VIII:C levels, but found none. It remains possible that
a part of the variation of factor VIII is determined by genetic
factors located on the X-chromosome, outside the factor VIII
gene. We can also not rule out the possibility that
environmental factors and clustering within families also
contribute to the familial aggregation of high factor VIII.
The mean difference in factor VIII:Ag level between blood
group O and non-O was 43 IU/dl. Among subjects with
factor VIII levels above 150 IU/dl, 86% had blood group
non-O, indicating that a substantial part of the elevation in
factor VIII is attributable to blood group. Most of the effect of
blood group on the factor VIII level is mediated through
VWF (Koster et al, 1995; Kamphuisen et al, 1998), but the
exact mechanism of how ABO blood group influences VWF
is unclear. Blood group A, B and H(O) oligosaccharide
structures have been identified on VWF (Sodetz et al, 1979;
Matsui et al, 1992). As modification of carbohydrates has
been shown to influence the half-life of VWF in the circu-
lation in animal models (Stoddart et al, 1996; Sodetz et al,
1977), it is possible that ABO blood group determinants
affect the clearance of VWF in plasma. VWF is an important
determinant of the factor VIII level in plasma, which is
explained by VWF being the carrier protein for factor VIII
(Tuddenham et al, 1982; Brinkhous et al, 1985). Whether
the different types of ABO blood group also influence the
factor VIII survival in plasma remains to be determined.
We conclude that there is strong support that factor VIII
levels exceeding 150 IU/dl aggregate in families, also after
adjustment for blood group and age. This is further evidence
that the high plasma factor VIII levels that previously were
found to be associated with a thrombotic risk in case±
control and family studies are determined by genetic factors
other than just blood group.
ACKNOWLEDGMENTS
We would like to thank H. de Ronde for determination of the
factor VIII antigen levels. This study was supported by a
grant (no. 950-10-629) from The Netherlands Organization
for Scientific Research (NWO) and by a grant (no. 95.026)
from The Netherlands Heart Foundation (NHS).
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