Factor V and Thrombotic Disease Description of a Janus-Faced ...

Factor V and Thrombotic Disease : Description of a Janus-Faced Protein
Gerry A.F. Nicolaes and Björn Dahlbäck
Arterioscler Thromb Vasc Biol. 2002;22:530-538; originally published online February 7, 2002;
doi: 10.1161/01.ATV.0000012665.51263.B7
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Factor V and Thrombotic Disease
Description of a Janus-Faced Protein
Gerry A.F. Nicolaes, Björn Dahlbäck
Abstract—The generation of thrombin by the prothrombinase complex constitutes an essential step in hemostasis, with
thrombin being crucial for the amplification of blood coagulation, fibrin formation, and platelet activation. In the
prothrombinase complex, the activated form of coagulation factor V (FVa) is an essential cofactor to the enzyme-activated
factor X (FXa), FXa being virtually ineffective in the absence of its cofactor. Besides its procoagulant potential,
intact factor V (FV) has an anticoagulant cofactor capacity functioning in synergy with protein S and activated protein
C (APC) in APC-catalyzed inactivation of the activated form of factor VIII. The expression of anticoagulant cofactor
function of FV is dependent on APC-mediated proteolysis of intact FV. Thus, FV has the potential to function in
procoagulant and anticoagulant pathways, with its functional properties being modulated by proteolysis exerted by
procoagulant and anticoagulant enzymes. The procoagulant enzymes factor Xa and thrombin are both able to activate
circulating FV to FVa. The activity of FVa is, in turn, regulated by APC together with its cofactor protein S. In fact,
the regulation of thrombin formation proceeds primarily through the upregulation and downregulation of FVa cofactor
activity, and failure to control FVa activity may result in either bleeding or thrombotic complications. A prime example
is APC resistance, which is the most common genetic risk factor for thrombosis. It is caused by a single point mutation
in the FV gene (factor V Leiden) that not only renders FVa less susceptible to the proteolytic inactivation by APC but also
impairs the anticoagulant properties of FV. This review gives a description of the dualistic character of FV and describes
the gene-gene and gene-environment interactions that are important for the involvement of FV in the etiology of venous
thromboembolism. (Arterioscler Thromb Vasc Biol. 2002;22:530-538.)
Key Words: factor V � activated protein C resistance � factor V Leiden � thrombosis � protein C
Historically, clinical studies focusing on coagulation factor
V (FV) almost exclusively described bleeding tendencies
as the result of a deficiency of this procoagulant
protein. In fact, the discovery of FV by the Norwegian Paul
Owren1 in 1947 was based on the identification of a patient
having a severe bleeding tendency due to the deficiency of a
previously unknown coagulation factor (parahemophilia, Owren’s
disease). FV deficiency is inherited as an autosomalrecessive
disorder with an estimated frequency of 1 in 1
million. 2–4 Heterozygous cases are usually asymptomatic,
whereas homozygous individuals show variable bleeding
symptoms. A great increase in research interest in FV has
been seen in recent years, but this has not been in the context
of bleeding problems but rather in association with thrombosis
studies. The reason for the explosive increase of clinical
and biochemical interest in FV originates from the discovery
of activated protein C (APC) resistance, a laboratory phenotype
originally identified in a single patient with venous
thrombosis. 5 APC resistance, which is characterized by a
reduced anticoagulant response to APC, was subsequently
found to be the most common risk factor for thrombosis. The
strict correlation of the APC resistance to a single point
Brief Reviews
mutation in the gene for FV (factor V Leiden [FV Leiden] orFV
R506Q), occurring in �3% to 15% of the general Caucasian
population, further established the importance of this FVrelated
abnormality (see reviews 6–10 ). At a time when large
population-based studies in the field of coagulation were set
up and an integrated genetic approach became available to
many research laboratories in the field of thrombosis and
hemostasis, the discoveries of APC resistance and FV Leiden
gave the research of thrombophilia, in general, and coagulation
FV, in particular, a new impulse.
See cover
Coagulation FV is an enzyme cofactor performing central and
pivotal functions in maintaining a normal hemostatic balance. In
the present review, we attempt to shed some light on the role that
it plays in relation to the etiology of thrombotic disease.
Biosynthesis and Structure of FV
FV is a large single-chain glycoprotein of �330 kDa. Its
plasma concentration is �20 nmol/L (�0.007 g/L). 11 Besides
circulating in free form in plasma, FV is also present in the
�-granules of platelets; this form accounts for �25% of the
Received October 2, 2001; revision accepted January 28, 2002.
From the Department of Laboratory Medicine, Division of Clinical Chemistry, Lund University, The Wallenberg Laboratory, University Hospital
Malmö, Malmö, Sweden. Dr Nicolaes is now at the Department of Biochemistry, Cardiovascular Research Institute Maastricht, Maastricht University,
Maastricht, the Netherlands.
Correspondence to Björn Dahlbäck, Department of Clinical Chemistry, Lund University, MAS, The Wallenberg Laboratory, S-205 02 Malmö, Sweden.
E-mail bjorn.dahlback@klkemi.mas.lu.se
© 2002 American Heart Association, Inc.
Arterioscler Thromb Vasc Biol. is available at http://www.atvbaha.org DOI: 10.1161/01.ATV.0000012665.51263.B7
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total FV content in human blood. 11,12 During coagulation,
platelet FV is secreted as a result of platelet activation.
Although several cellular types have been reported to synthesize
FV, it is generally accepted that the principal site of
biosynthesis is the liver, where human FV is synthesized as a
single-chain molecule, undergoing extensive posttranslational
modifications before being secreted into the blood. 13,14 It is
still unclear whether the presence of FV in platelets is the
result of the uptake of exogenous FV from the circulation via
endocytotic processes by megakaryocytes or whether these
cells themselves can account for the FV production. 15–17 The
FV gene (gene locus on chromosome 1q23) spans more than
80 kb and contains 25 exons. The isolated cDNA has a length
of 6672 bp and encodes a preprotein of 2224 amino acids,
including the 28-amino-acid residue long signal peptide. 18,19
FV has a mosaic-like structure, with a domain organization
(A1-A2-B-A3-C1-C2, Figure 1) that is similar to that of
factor VIII (FVIII), 20,21 another essential coagulation cofactor
protein. The A domains of FV and FVIII together with those
of ceruloplasmin 22 have evolved from a common ancestral
protein. Overall, the two coagulation factors (FV and FVIII)
share �40% sequence identity in their A and C domains. 19,23
The 3D structure of ceruloplasmin has been elucidated, and
the homology between the A domains of FV and those of
ceruloplasmin has allowed the creation of molecular models
for the A-domain part of FV. 24,25 In these models, the three A
domains are arranged in a triangular fashion (Figure 1).
Molecular models were also created for the C domains of
FV, 26 and more recently, the 3D structure of the C2 domain
of FV was determined with x-ray crystallography. 27 A preliminary
model for the whole FVa molecule (FVa is the
activated form of FV) has been generated on the basis of the
information of the individual domains. 24,28
Nicolaes and Dahlbäck Factor V and Thrombosis 531
Figure 1. Schematic models of FV illustrating
the mosaic-like structure of the molecule
and the spatial arrangement of the FV
domains. The triple A domains (A1, A2, and
A3) are indicated in blue, and the C-terminal
(C1 and C2) domains are indicated in red.
The large connecting B domain is yellow. a,
On activation of FV, 3 peptide bonds (upper
black arrows) at positions Arg709, Arg1018,
and Arg1545 are cleaved, thereby releasing
the B domain. FV/FVa is subject to APCmediated
proteolysis at Arg306, Arg506,
Arg679, and Arg994, which is indicated by
the lower red arrows. b, In 3D, the 3 A
domains are believed to be arranged in a
triangular fashion, with the 2 smaller C
domains extending from the A3 domain,
thus creating a windmill-like model. c, The
heterodimeric FVa is formed by A1-A2
(heavy chain), which is noncovalently associated
with A3-C1-C2 (light chain). The C2
domain is indispensable for the interaction
of the FVa molecule with phospholipid
membranes (in green). An additional
membrane-binding region might be present
in the A3 domain.
Regulation of Procoagulant FXa-Cofactor
Activity of FV
Circulating single-chain FV is an inactive procofactor, expressing
�1% of the procoagulant enzyme-activated factor X
(FXa)-cofactor activity that it can maximally obtain. 29 An
increase in FXa-cofactor activity is associated with limited
proteolysis of several peptide bonds in FV mediated by
procoagulant enzymes such as thrombin and FXa. 30–32 As a
result, the large connecting B domain (Figure 1) dissociates
from FVa, which is formed by the noncovalently associated
heavy (A1-A2) and light (A3-C1-C2) chains. The prothrombinase
complex comprises FXa and FVa, which in the
presence of calcium ions assemble on negatively charged
phospholipid membranes. FVa is considered an essential FXa
cofactor, inasmuch as its presence in the prothrombinase
complex enhances the rate of prothrombin activation by
several orders of magnitude. 29,33 Homozygous deficiency of
FV in humans is associated with variable severity of bleeding
problems, suggesting that FV deficiency in humans is not a
lethal disease. 3 This stands in contrast to the fatal bleeding
disorder that affects FV knockout mice. 34 Approximately
50% of the affected embryos die during embryonic day 9 to
10, and the remaining full-term mice die from bleeding within
2 hours after birth. The explanation for the discrepancy in
phenotypic severity between humans and mice is unknown.
Downregulation of the procoagulant activity of FVa is
accomplished by APC-mediated proteolysis of FVa at positions
Arg306, Arg506, and Arg679. 35 The cleavages at these
positions are under strict kinetic control, with the cleavage
site at Arg506 being preferred at low concentrations of APC
and FVa. However, the Arg506 cleavage yields only partial
inactivation of FVa, and cleavage at Arg306 is necessary for
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532 Arterioscler Thromb Vasc Biol. April 2002
the complete inactivation of FVa activity. 36 The third cleavage
at Arg679 is likely of lesser importance to FVa inactivation.
Inactivation of FVa is greatly enhanced by protein S,
which is an APC-cofactor protein with high affinity for
negatively charged phospholipid membranes. However,
the cleavages in FVa demonstrate different dependence on
the APC-cofactor activity of protein S. Thus, protein S
does not affect the cleavage rate at the Arg506 site,
whereas the addition of protein S in systems containing
purified coagulation proteins increases the rate of Arg306
cleavage 20-fold. 37 This indicates the importance of protein
S in the regulation of FVa cofactor activity. In
addition, a substantial amount of evidence has been provided
showing the importance of protein S for in vivo
regulation of the anticoagulant protein C system, the
system responsible for the proteolytic regulation of FV and
FVIII. 38,39 Moreover, protein S and protein C deficiencies
are well-recognized risk factors for venous thrombosis,
demonstrating the importance of careful regulation of FV
and/or FVIII activities in vivo. 40 Membrane-bound bovine
FVa has also been shown in in vitro experiments to be
inactivated by plasmin. 41 Whether this is a physiologically
important mechanism in vivo under normal and pathological
conditions remains to be elucidated.
APC Resistance
In 1993, our laboratory found that plasmas from a group of
patients with family histories of thromboembolic disease
showed reduced anticoagulant response to the addition of
APC. Because of the partial or complete resistance toward
APC, we named the phenotype APC resistance and also
demonstrated its inherited nature. 5,42 APC resistance has been
recognized as the most important cause of venous thrombosis,
which is present in 20% to 60% of patients with venous
thromboembolism. In the early studies, it appeared as if
APC-resistant individuals were missing an essential APC
cofactor. This cofactor was subsequently suggested to be
intact FV. Supporting evidence for the cofactor hypothesis
was the observed correction of the phenotype by added FV.
On the basis of these studies, FV was proposed to act as a
cofactor in the APC-catalyzed prolongation of coagulation
times in plasma. 43 The anticoagulant function of FV was
further characterized later in 1994, when it was demonstrated
that intact FV holds the potential to function in synergy with
protein S as an APC cofactor in the inactivation of activated
FVIII (FVIIIa). 44 Thus, FV, protein S, and APC regulate the
activity of the tenase complex, ie, the membrane-associated
complex (comprising activated factor IX and FVIIIa) that
efficiently activates factor X.
The genetic background for the APC resistance phenotype
was also demonstrated in 1994. A single nuclear polymorphism
in the FV gene was found to be associated with
APC resistance. 45–48 At position 1691, a G3A missense
mutation resulted in the replacement of Arg506 by Gln
(FV Leiden). This mutation has an unprecedented high occurrence,
with frequencies in the general population of 2% to
15% and up to 60% in selected patients with venous thromboembolism.
49 This prevalence was 10 times higher than the
sum of frequencies of all hereditary causes of thrombophilia
known at that time. To date, the Arg5063Gln mutation is the
most common genetic risk factor for thrombosis. 10 Notably,
variation in allelic frequencies for FV Leiden is extensive, with
the mutation being present exclusively in populations of
Caucasian descent. Because all FV Leiden alleles have the same
haplotype, it can be concluded that the mutation occurred
only once and that a founder effect has been involved. The
estimated age of the mutation is �30 000 years; 50 ie, it
occurred after the out-of-Africa migration that took place
�100 000 years ago.
Because the FV Arg5063Gln mutation affects one of the
prime target sites for the APC-catalyzed inactivation of FVa (see
above), it appears obvious that impaired downregulation of FXa
cofactor activity of FVa contributes to the increased risk of
thrombosis. However, this is not the sole molecular mechanism
involved, inasmuch as the mutant FV isolated from patients with
APC resistance is much less active as APC cofactor in the
FVIIIa inactivation. 51–53 This was further unequivocally demonstrated
by using recombinant mutant FV. 54 Hampered FVa
inactivation alone did not satisfyingly explain the increased
thrombin generation, 55 because in vitro experiments had shown
that under certain conditions (eg, high FVa in the presence of
protein S and FXa), the APC-catalyzed inactivation of normal
FVa and activated FV Leiden appeared similar. 37 The identification
of the APC-cofactor function of FV reinforced the association
between carriership of the FV Leiden mutation and thrombosis and
contributed pathogenic explanations for the hypercoagulable
state associated with APC resistance. Thus, FV presents itself as
a true Janus-faced protein: In its activated form, it has essential
functions in the procoagulant pathways, without which severe
bleeding tendencies can occur. On the other hand, the nonactivated
precursor protein factor, as it circulates in plasma, possesses
anticoagulant properties functioning as an APC cofactor
in the regulation of FVIIIa activity. Failure to fully express this
anticoagulant function may lead to thrombosis.
Anticoagulant FV
As mentioned above, an integrated account of all the functions
of FV must include its anticoagulant properties as well.
Still, the molecular mechanism by which FV can exert its
APC-cofactor function in the downregulation of FVIIIa
activity is largely unknown. However, some experimental
results that give an insight into the functional requirements of
the anticoagulant FV function are available. Thus, full procoagulant
activation of FV (ie, cleavage at Arg709 and
Arg1545 associated with the release of the B domain) results
in lost anticoagulant APC-cofactor activity of FV, 44,52 suggesting
the involvement of the B domain in this function
(Figure 1). Moreover, studies using recombinant FV variants
have shown the C-terminal part of the B domain (last 70
amino acids) to be essential for the anticoagulant APCcofactor
activity of FV. 56 The APC-mediated cleavage of
intact FV at Arg506 is also involved in generating anticoagulant
FV, as demonstrated with recombinant variants of FV. 54
This explains the reduced cofactor function of FV Leiden,
because it cannot be cleaved at Arg506. These data indicate
that a delicate balance exists between the procoagulant and
anticoagulant properties of FV, with activation leading to a
procoagulant protein that is devoid of anticoagulant proper-
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ties (Figure 2). In contrast, APC-catalyzed proteolysis of
intact FV generates an anticoagulant protein that has virtually
no procoagulant properties. On the basis of discussed observations,
a dual pathway determining the fate of FV in blood
coagulation is emerging. In this pathway, the local concentrations
and availability of procoagulant and anticoagulant
enzymes, such as thrombin, FXa, and APC, determine the
fate of each FV molecule. Thus, FV acts as a local sensor of
procoagulant and anticoagulant forces, inasmuch as it is able
to sustain ongoing reactions through its susceptibility to
limited proteolysis and its ability to function as either a
procoagulant or an anticoagulant cofactor.
In 1993, it was reported that human blood and platelets
contain 2 forms of FV (FV 1 and FV 2) having slightly different
molecular weights and affinities for phospholipid membranes. 57
The membrane-binding properties of the 2 forms affect their
procoagulant and anticoagulant cofactor activities. 52,58 In model
systems mimicking physiological conditions, FV 1 appears to be
the more thrombogenic, yielding up to 7-fold higher thrombin
generation than FV 2. 58 Recently, we demonstrated the molecular
difference of the FV 1FV 2 forms to reside in partial glycosylation
of Asn2181. FV 1 carries a carbohydrate at this residue, in
contrast to FV 2, which does not. 59,60
Venous Thromboembolic Disease
Thrombosis is defined as the pathological presence of a blood
clot (thrombus) in a blood vessel or the heart. Venous thrombosis
and arterial thrombosis are considered distinct disease states
having different pathogenic mechanisms and underlying risk
Nicolaes and Dahlbäck Factor V and Thrombosis 533
Figure 2. Dual pathways of FV are represented in
a simplified and schematic form. The inactive
(blue) precursor molecule FV is subject to limited
proteolysis by procoagulant and/or anticoagulant
enzymes, depending on the local concentrations of
effector proteases. Cleavage of intact FV at Arg506
directs the molecule in an anticoagulant direction.
FVac indicates anticoagulant FV (in green). This
molecule can proceed in 2 directions. It is either
routed through a connecting reaction, representing
cleavage at Arg709, Arg1018, and Arg1545, into a
semiprocoagulant pathway (FVa int, red/blue), yielding
a molecule that still possesses limited cofactor
activity in prothrombin activation, or it is further
cleaved by APC at Arg306, which eliminates the
procoagulant potential (FVi). The cleavage at
Arg1545 is underlined, signifying the importance of
the cleavage at this peptide: on cleavage of
Arg1545, all anticoagulant cofactor activity is lost.
FV is activated directly to a procoagulant cofactor
FVa (orange) via cleavages at Arg709, Arg1018,
and Arg1545, with the underlined Arg1545 again
representing the importance of the cleavage at
Arg1545. Cleavage of this bond in FVa is essential
for the expression of full cofactor activity in prothrombin
activation. Control of FVa procoagulant
activity is achieved via proteolysis by APC; again,
cleavage at Arg506 results in a partially active molecule,
the activity of which is abolished by subsequent
cleavage at Arg306. FVa activity can also be
directly downregulated through initial cleavage at
Arg306 in FVa. Potential cleavage of the inactive
FVi at Arg679 and/or Arg994 yields FVi�, with no
further consequence for the cofactor activity of the
molecule.
factors. 61–63 The estimated annual incidence of venous thromboembolism
in individuals aged �40 years is 1 per 10 000, and
in those aged �75 years, it is 1 per 100. 63–65 Thrombosis is a
complex and episodic disease, with recurrences being common.
In recent years, it has become clear that venous thrombosis is
multigenic 66 and that the pathogenesis often includes several
hereditary factors, which synergistically tip the natural hemostatic
balance between procoagulant and anticoagulant forces. In
addition, acquired and environmental factors modulate the risk
of thrombosis and are often involved in the pathogenesis of the
disease. Thrombosis is believed to develop when a certain
threshold is passed as a result of risk factor interactions, with the
combined total risk exceeding the sum of their separate risk
contributions. Above this threshold, natural anticoagulant systems
are insufficient to balance the procoagulant forces, resulting
in the development of thrombotic disease. 62 Most inherited
risk factors for venous thrombosis are found in the protein C
system, such as APC resistance (FV Leiden), and deficiencies of
protein C and protein S. 9,67 Other less common genetic risk
factors are antithrombin deficiency and the prothrombin 20210A
(PT 20210A) mutation. Acquired risk factors range from prolonged
immobilization, surgery, malignant disease, trauma, use
of oral contraceptives, and pregnancy/puerperium to the presence
of antiphospholipid antibodies. Personal histories of thrombosis
and advanced age have also been acknowledged as risk
factors for venous thrombosis. 68
Role of FV Leiden in Venous Thrombosis
Thromboembolic episodes associated with FV Leiden are almost
exclusively venous in nature. Although some case reports link
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534 Arterioscler Thromb Vasc Biol. April 2002
TABLE 1. Occurrence of FV Leiden and Other Risk Factors for
Venous Thrombosis and the Relative Risks (RR)
They Contribute
Population Thrombosis
Risk Factor
Prevalence, % Patients, % RR
FVLeiden 2–15 20 6.6*
Protein S deficiency 0.03–0.13 1–2 0.7†
Protein C deficiency 0.2–0.4 3 6.5
Antithrombin deficiency 0.02 1 5.0
Prothrombin 20210A 2 6 2.8
High FVIII levels 11 25 4.8
*Risk for heterozygous FVLeiden carriers, risk for homozygous carriers is
approximately 80-fold.
†This low number is derived from case-control studies, which fail to identify
protein S deficiency as a risk factor. However, family studies demonstrate that
protein S deficiency carries similar risk for thrombosis as FVLeiden and protein C
deficiency (reviewed by Zöller et al69 ).
Data adapted from Martinelli, 10 Rees et al, 49 Rosendaal, 62 Rosendaal et al, 70
Koster et al, 71 and Dykes et al. 72
FV Leiden to arterial thrombosis, they are rare, and these
incidents can likely be due to other unrecognized pathogenic
factors. A large number of studies using different approaches,
such as case-control studies, population-based studies, family
studies, and prospective studies, have estimated the risk
increase for venous thrombosis due to FV Leiden to be 5- to
7-fold for heterozygous carriers and 80-fold for homozygous
carriers of the mutation (Table 1). The severity and localization
of thrombosis in carriers of FV Leiden are very diverse.
Common are thromboses in the deep veins of the leg, 70
whereas portal vein thrombosis, superficial vein thrombosis,
and cerebral vein thrombosis are less prevalent. However,
there seems to be no association between FV Leiden and primary
pulmonary embolism 73 or retinal vein thrombosis. 74 Moreover,
most studies do not find a higher risk of recurrent thrombosis in
carriers of heterozygous FV Leiden than in other patients with
thrombosis, whereas homozygous individuals have an increased
risk of recurrence. 75–78 The FV Leiden mutation has been suggested
to be a “gain of function” mutation, 62 with the overall FV levels
and functions being unchanged, which distinguishes it from the
“loss of function” mutations that are seen in protein C or protein
S deficiency. Yet, taking the lost APC-cofactor activity of the
FV Leiden molecule into account, one could still argue that this
mutation induces a deficiency regarding the anticoagulant properties
of FV.
The high prevalence of FV Leiden in the general white
population (Table 1) related to the relatively lower annual
incidence of venous thromboembolism suggests that the
mutation yields a modestly increased risk of thrombosis, per
se. 10 However, because of the high frequency of FV Leiden in the
population, combinations with other hereditary or acquired
risk factors are relatively common. Because risk factors
appear to synergistically increase the risk of thrombosis,
many patients with thrombosis are indeed affected by �1 risk
factor. For instance, the prevalence of the PT 20210A
mutation is �2% in the general population (Table 1), 79
suggesting that double mutations are present in up to 0.1% to
0.3% of white individuals. The multigenetic nature of throm-
TABLE 2. Gene-Environment Interaction: RR of Venous
Thrombosis in Users of Oral Contraceptives Depends on the
Presence or Absence of the FV Leiden Mutation
Population
Non-FVLeiden carriers
Thrombosis Risk RR
Not using OC 0.8 1.0
Using OC
FVLeiden carriers
3.0 3.7
Not using OC 5.7 6.9
Using OC 28.5 34.7
Given are the annual risks of thrombosis per 10 000 people and RR.
Data adapted from Vandenbroucke et al. 85
bosis involving FV Leiden as a risk factor was demonstrated by
the high prevalence of FV Leiden among thrombophilic families
with antithrombin, protein C, or protein S deficiency or the
prothrombin 20210A mutation (25%, 19%, 38%, and 10%,
respectively 80– 84 ). Because these prevalences are much
higher than those in the general population, it can be
concluded that FV Leiden is involved in the development of
thrombosis in these families. In families affected by multiple
genetic risk factors, individuals having �2 genetic defects
suffer from thrombotic events more frequently and earlier in
life than do those with single defects.
One of the most common acquired risk factors associated with
FV Leiden is probably the use of oral contraceptives. It is estimated
that �40% of fertile women in Sweden and the Netherlands use
oral contraceptives. This suggests that many fertile women carry
at least 2 risk factors of thrombosis. Synergistic effects have
been shown for FV Leiden and the use of oral contraceptives, and
the combined relative risk for the development of thrombosis
was much higher than could be foreseen on the basis of the
individual risks (Table 2; compare risk ratios). In this case, the
risk factors clearly interact, although the exact molecular mechanism
of this interaction still needs to be clarified. Of particular
interest in this respect is the demonstration in in vitro experiments
that the use of oral contraceptives or of hormone replacement
therapy, per se, induces an acquired resistance to APC and
also that it enhances APC resistance due to FV Leiden. 55,86–88 It has
been debated whether investigation for APC resistance and/or
FV Leiden should be performed before prescribing oral contraceptives
or hormone replacement therapy, but to date, there is no
consensus in favor of general screening in these situations.
Other FV Haplotypes Contributing to APC
Resistance and Venous Thrombosis
Several allelic variants of the FV gene have been described.
Two particularly interesting variants, FV Arg306Thr (FV
Cambridge 89 ) and FV Arg3063Gly (FV Hong Kong 90 ),
were identified in patients with thrombosis. Both these
mutations result in the loss of the APC cleavage site at
Arg306, which is an important cleavage site in FVa for
complete loss of FVa activity (see above). FV Cambridge was
identified in an individual with unexplained APC resistance
and seems to be an extremely rare mutation. In contrast, FV
Hong Kong is common in certain Chinese populations but
does not appear to be a risk factor for thrombosis. Moreover,
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FV Hong Kong is reportedly not associated with APC
resistance. Intuitively, one would suspect the loss of the
Arg306 cleavage site to be a risk factor of thrombosis and
possibly also to yield APC resistance. In addition, the
replacement of the Arg306 with a Gly and a Thr is expected
to have essentially the same effect on FVa degradation.
Recently, FV Cambridge and FV Hong Kong have been
recreated in vitro with recombinant DNA techniques. In all
functional tests, there were no appreciable differences between
the 2 FV variants, with both phenotypes presenting APC
responses intermediately between normal FV and FV Leiden. 91
Thus, even though an important APC cleavage site is lost in FV
Cambridge and in FV Hong Kong, it is still not known whether
these FV variants are risk factors for venous thrombosis.
Recently, several investigators have associated the socalled
R2 allele (or HR2 polymorphism) with a slightly
increased risk of venous thrombosis, even though no consensus
has been reached regarding whether the R2 allele is really
a risk factor for thrombosis. 92–96 The R2 allele is characterized
by several linked mutations (missense and silent) in the
gene for FV. Exons 13, 16, and 25 encoding the B, A3, and
C2 domains, respectively, carry these mutations. 93 The R2
allele is associated with slightly decreased levels of circulating
FV, which, if combined with FV Leiden, may enhance the
APC-resistance phenotype and increase the risk of thrombosis.
94 Moreover, the plasma of carriers of the R2 allele seems
to contain increased amounts of FV 1, 97,98 the form of FV that
may be more thrombogenic than FV 2. 58
Other conditions linked to the FV gene influencing APC
resistance include combinations of FV Leiden and quantitative
FV deficiency. Heterozygous deficiency of FV combined
with FV Leiden results in a pseudohomozygous state of APC
resistance. 99–104 Because only the FV Leiden allele is expressed,
all circulating FV is mutated, resulting in a phenotype that is
similar to that of homozygous FV Leiden individuals. Taking
into account the dose-response relationship between the in
vitro APC response and the risk of venous thrombosis, 105 it is
believed that pseudohomozygous and homozygous individuals
have a similar risk of thrombosis. Because
pseudohomozygous APC resistance is rare, it will be difficult
to find enough individuals to achieve statistical power in
studies of thrombosis risk in these affected individuals.
However, almost all cases of pseudohomozygous APC resistance
reported to date have clinical manifestations 99,101,104 as
severe as those of homozygous individuals.
Studies have been performed to analyze whether high
levels of circulating FV increase the risk of thrombosis. 106
However, in contrast to reports regarding homologous FVIII,
FV plasma levels did not show any statistically significant
association with thrombosis. In addition, FV levels did not
modify the thrombosis risk associated with high FVIII levels.
In 3 patients, spontaneously developing autoantibodies
against FV were associated with the occurrence of thrombosis.
107 In 1 of these patients, lupus anticoagulant activity
could be detected. A second patient was found to have an
elevated anticardiolipin antibody titer. The molecular mechanisms
that yield the increased risk of thrombosis in the rare
patients with thrombosis are not known. Nonetheless, it is
possible that the autoantibodies in these patients block the
Nicolaes and Dahlbäck Factor V and Thrombosis 535
anticoagulant activity of the FV molecule. This is a rare
phenomenon, inasmuch as most individuals presenting with
anti-FV antibodies show clinical manifestations ranging from
no symptoms to life-threatening hemorrhages. 107
Conclusions and Perspective
Coagulation FV is an essential cofactor protein with important
functions in procoagulant and anticoagulant pathways.
Regulation of FV cofactor activity is of prime importance for
homeostasis of blood coagulation. Besides its activation to a
procoagulant cofactor by activated coagulation enzymes such
as thrombin and FXa, downregulation of FVa activity under
physiological conditions is the result of APC-mediated proteolysis.
In contrast, proteolysis by APC of circulating intact
FV recruits FV to the anticoagulant pathway because APCcleaved
intact FV functions as a synergistic cofactor with
protein S in the APC-mediated regulation of FVIIIa. The
most common hereditary cause of venous thrombosis is a
single point mutation in FV, which results in a phenotype
known as APC resistance. The FV Arg506Gln mutation
(FV Leiden) affects one of the target sites for APC, which results
in impaired efficiency in the APC-mediated degradation of
FVa. In addition, the same mutation impairs the anticoagulant
APC-cofactor activity of FV in the APC-catalyzed inactivation
of FVIIIa. As a result of the mutation, the regulation of
thrombin formation via the protein C pathway is impaired.
Although the mutation confers only a mild to moderate risk of
thrombosis, it is often seen as a contributing factor in patients
with thrombosis, particularly when they are affected by other
genetic or acquired risk factors. Given the key importance of
FV in procoagulant and anticoagulant pathways, it can be
envisioned that any condition affecting FVa inactivation, on
the one hand, or the expression of anticoagulant FV cofactor
activity in the inactivation of FVIIIa, on the other, will
increase the risk of venous thromboembolism. Such mechanisms
have been suggested for the recently described association
between the HR2 polymorphism and thrombosis. Because
of the large size of the FV gene, it is not unlikely that
within the coming years, more FV variants with altered
functions and/or expression levels will be found.
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