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 



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- 



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 


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 




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, 



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 


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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