Review article
Factor V: a combination of Dr Jekyll and Mr Hyde
Kenneth G. Mann and Michael Kalafatis
Introduction
The desirable process of hemostasis and the undesirable phenomena of thromboses are unequivocally consequences of the generation of thrombin by the prothrombinase complex. This stoichiometric complex of factor Xa and factor Va assembled on a platelet
membrane converts membrane-bound prothrombin into its active
products by 2 sequential peptide-bond cleavages. The functionality
and durability of the prothrombinase complex is subject to multiple
regulatory processes that may enhance or eliminate its function.
Factor Va, an essential component of prothrombinase, is both
produced and destroyed by proteolytic events. The absence or
dysfunction of factor Va leads to hemorrhagic disease, whereas
excessive longevity of the active species is associated with
thrombosis. Factor V is thus required for a good outcome (Dr
Jekyll) but also is a potential source of disaster (Mr Hyde). In this
review, we summarize the current state of knowledge with respect
to the good and the bad aspects of factor V functionality
and durability.
Background
The factor Va molecule is generated by cleavage of its precursor
factor V. It contributes to the blood clotting reaction by binding
with factor Xa on a membrane surface to form the prothrombinase
complex. This complex is the essential activator of prothrombin to
thrombin during the blood clotting process and effectively increases the activity of factor Xa by 5 orders of magnitude.
Factor V was discovered by Paul Owren in 19431 when, using
relatively primitive technology, he was able to deduce the existence
of a fifth component required for fibrin formation that he named
“factor V,” thus beginning the era of Roman numerology for
coagulation factors. This work, published in Lancet after War
World II,1 also identified the pro-cofactor nature of factor V and the
requirement for its activation by thrombin as well as its participation in the generation of thrombin. Dr Owren’s work defined factor
V as the activity in normal plasma that corrected the prothrombin
time (PT) of the plasma of a patient with factor V deficiency.1,2
Following a long period of controversy, factor V was isolated in
1979.3-6 The isolation established the “biochemistry” period of
blood clotting complexes and launched the paradigm of membranebound complexes contributing to the blood clotting process.
Human plasma factor V circulates at a concentration of 20 nM,
as a large single-chain pro-cofactor with an Mr of 330 000.4-6 In
From the Department of Biochemistry, University of Vermont, College
of Medicine, Burlington; the Department of Chemistry, Cleveland
State University, Cleveland, OH; and the Department of Molecular
Cardiology, The Lerner Research Institute, The Cleveland Clinic Foundation, Cleveland, OH.
Submitted February 14, 2002; accepted July 29, 2002. Prepublished online as
Blood First Edition Paper, August 8, 2002; DOI 10.1182/blood-2002-01-0290.
20
addition, approximately 20% of the total human factor V found in
whole blood is contained in the platelet ␣-granules.7 The significance of platelet factor V is underscored by either patients with a
deficiency in platelet factor V (Factor V Quebec) who exhibit a
bleeding diathesis or by asymptomatic individuals with potent
circulating inhibitory factor V antibodies that do not have access to
platelet factor V.8-13 The platelet fraction of factor V is both
synthesized in the megakaryocyte and absorbed from plasma and
sequestered in the ␣-granules.14 Platelet factor V is partially
fragmented, stored in association with multimerin, and secreted
during platelet activation. It requires further proteolytic activation
to fully participate in prothrombinase function.15-20
The binding protein multimerin is found in platelets and
endothelial cells and appears to be a carrier for platelet factor V. It
contains an RGDS motif as well as an epidermal growth factor
domain.19,20 The multimerin monomers are linked via interchain
disulfide bonds to produce one of the largest proteins found in
platelets with a mass exceeding approximately 1 000 000 Da.
Following platelet activation, both factor V and multimerin are
released and dissociate from one another.19,20
The physiologic significance of factor Va for clot formation is
clearly demonstrated in mice in which complete deficiency of
factor V results in massive hemorrhage and death.21 However,
humans who are deficient in plasma factor V (⬍ 2% factor V
clotting activity) although rare (prevalence of 1 in 1 million) show
variable bleeding tendencies.11,12,22 The physiologic relevance of
factor Va in the pathology of thrombosis is assured because of case
reports of familial thrombophilia associated with factor V mutations and defects in the protein C pathway.23,24 In this review, we
describe the structure, activation, function, and inactivation of
factor V and discuss pathologic states associated with genetic
mutations of the factor V molecule.
Structure
The 80-kb factor V gene is located on chromosome 1 at q21-2525
and contains 24 introns. Following transcription it gives rise to a
6.8-kb mRNA.25-28 Synthesis of factor V has been demonstrated by
bovine aortic endothelial cells,29 by a human hepatocarcinoma cell
line HepG2,30 and by human and guinea pig megakaryocytes.31 The
liver appears to be the principal source of factor V in plasma and
platelets.7
Supported by Merit Award R37 HL34575 from the National Institutes of Health
(K.G.M.) and by Established Investigator Award 0040100N from the American
Heart Association (M.K.).
Reprints: Kenneth G. Mann, Department of Biochemistry, Given Building,
Health Science Complex, University of Vermont, College of Medicine,
Burlington, VT 05405; e-mail: [email protected].
© 2003 by The American Society of Hematology
BLOOD, 1 JANUARY 2003 䡠 VOLUME 101, NUMBER 1
BLOOD, 1 JANUARY 2003 䡠 VOLUME 101, NUMBER 1
The human factor V molecule (Figure 1A) is secreted after
pre-propeptide processing as a 2196 amino acid protein. The
molecule is composed of triplicated A domains, a B domain, and
duplicated C domains.26-28 The A domains are homologous to those
found in plasma ceruloplasmin and factor VIII, each of which are
members of a copper-binding family.32 The C domains are homologous to the slime mold protein discoidin.26 The B domain is poorly
conserved among the various species of factor V that have been
studied. The B domain of human factor V that is ultimately released
in the form of 2 heavily glycosylated fragments of Mr 150 000 and
71 000 contain 44 semiconserved repeats of the form DLSQTT/
NLSP. This highly glycosylated region also contains 2 conserved
repeats of 17 amino acids each.26
The factor V molecule undergoes multiple posttranslational
alterations, including sulfation, phosphorylation, and glycosylation
(Figure 1A; Table 1).33-39 Inhibition of tyrosine sulfation by sodium
chlorate results in a cofactor molecule with one fifth the activity of
the native molecule. Thus, sulfation appears important for factor
V/Va function.34,35 Factor Va is also phosphorylated by a membraneassociated platelet casein kinase II (CKII) enzyme on the heavy
chain at Ser692 and by a platelet-derived protein kinase C isoform
on 2 sites of the light chain (Figure 1A).36-38 Phosphorylation of the
COAGULATION FACTOR V
21
Table 1. Sites of posttranslational modifications of the factor V molecule
Phosphorylation
Ser692, Ser804, Ser1506 for platelet casein kinase II; at
Sulfation
Tyr665, Tyr696, Tyr698, Tyr1494, Tyr1510, Tyr1515,
least 2 sites for protein kinase C on the light chain
Tyr1565
N-glycosylation
Heavy chain
Asn23, Asn27, Asn211, Asn269, Asn354, Asn432, Asn440,
B region
Asn713, Asn724, Asn732, Asn748, Asn754, Asn793,
Asn526, Asn639
Asn910, Asn949, Asn1046, Asn1055, Asn1075,
Asn1078, Asn1175, Asn1193, Asn1229, Asn1238,
Asn1265, Asn1283, Asn1310, Asn1319, Asn1347,
Asn1356, Asn1451, Asn1471, Asn1531
Light chain
Asn1675, Asn1982, Asn2182
Disulfide bridges
Heavy chain
Cys139-Cys165, Cys220-Cys301, Cys472-Cys498,
B region
No disulfides
Light chain
Cys1697-Cys1723, Cys1879-Cys2033, Cys2038-Cys2193
Cys575-Cys656
Free cysteines
Heavy chain
Cys539, Cys585
B region
Cys1085
Light chain
Cys1960, Cys2113
See also Figure 1A for the positions.
heavy chain of factor Va at Ser692 increases the rate of inactivation
of the cofactor by activated protein C.38 The platelet CKII
responsible for factor Va phosphorylation is composed of ␣ and ␤
subunits. The CKII␣ subunit is the product of an intronless gene
located on chromosome 11.40 The factor V molecule has multiple
potential N-linked glycosylation sites in the B region and on the
heavy and light chains that influence secretion (Figure 1A; Table
1).26,39 The N-linked carbohydrate chains on the factor V molecule
include a complex array, ranging from biantenary, bisialyted chains
without fucose addition to tetrantenary, tetrasialyated with multiple
fucose attachments.39
Factor V has 19 cysteine residues (Table 1 and Figure 1A show
locations).41,42 Five of the cysteines are present as free ⫺SH,
whereas the remaining 14 are involved in disulfide bridges forming
several loops: three 26 amino acid residue ␣-loops are present, one
in each A domain; the A1 and A2 domains each contain one ␤-loop
that is composed of 82 amino acids (Table 1); 2 ␥-loops that are
composed of 154 and 155 residues, that represent the C1 and C2
domains (Figure 1A) each with a free ⫺SH. The cysteine residue at
position 539 appears to be very reactive.
Models of the A domains of the molecule based on the
coordinates of ceruloplasmin have been described43-45; however,
these hypothetical models of factor Va domains should be only
provisionally interpreted until the true crystal structure of factor Va
becomes available. The crystal structures of the recombinant C2
domain of the molecule have been reported.46 Bovine factor Va has
been crystallized,47 and a preliminary account of the crystal
structure of bovine factor Va has been reported.48
Figure 1. Human factor V molecule. (A) Diagram of the organization of the human
factor V molecule. The arrows on the top represent activation cleavages by
␣-thrombin, factor Xa, and RVV-V activator. The arrows at the bottom indicate
inactivation cleavages by APC and plasmin. The positions of posttranslational
modifications are also shown. (B) Active factor V (factor Va). Following activation by
␣-thrombin, the active cofactor is a heterodimer composed of a heavy chain divalent
cation associated with light chain. The B region of the cofactor is released as 2
fragments. (C) Inactive factor Va (factor Vai). The upper part shows inactivation of
factor Va by APC, resulting in the dissociation of the A2 domain as 2 fragments, A2N
and A2C. The lower part shows inactivation of factor Va by ␣-thrombin following
cleavage of the heavy chain at Arg643.
Activation
Factor V is the inactive precursor of factor Va that contributes
factor Xa receptor and catalytic effector functions to prothrombinase. The complex formation between factor Xa and factor Va
membrane to form prothrombinase increases the rate at which
prothrombin is converted to ␣-thrombin by 300 000-fold relative to
the rate of the reaction catalyzed by factor Xa acting alone.49 The
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MANN and KALAFATIS
pro-cofactor is activated by a number of proteases, including
thrombin, factor Xa, and (transiently) plasmin. The thrombin
cleavage is the most significant with respect to the biologic
function of the molecule and occurs as an early event during the
blood coagulation process.50-53 Thrombin cleaves sequentially at
Arg709, Arg1018, and Arg1545 to generate the noncovalently
associated light and heavy chains of factor Va.54 The excised B
region dissociates as 2 fragments of Mr 71 000 and 150 000
(residues 710-1018 and 1019-1545, respectively) (Figure 1B). The
human factor Va heterodimer is composed of a heavy chain of Mr
105 000 and a light chain of Mr 74 000,4-6,26 with the noncovalent
association requiring divalent metal ions (Figure 1B).54 The heavy
chain (residues 1-709) is composed of 2 A domains (residues 1-303
and 317-656),26,27 connected by a region rich in basic amino acids
(Figure 1A,B). The COOH-terminal portion of the heavy chain
(residues 657-709) is rich in acidic amino acids. The light chain of
the cofactor (residues 1546-2196) is composed of one A domain
(residues 1546-1877) and 2 C domains (residues 1878-2036 and
2037-2196).26,27 A snake venom enzyme, the RVV-factor V activator, cleaves factor V at Arg1545 and Arg1018, resulting in an active
molecule. The cleavage at Arg1545 is critical for factor Va function.
Single-chain factor V does not bind factor Xa. Thus, the
activation of factor V to factor Va is essential to its biologic
function. During tissue factor–initiated blood coagulation, thrombin generation can be divided into 2 phases; an initiation phase
during which very small amounts of thrombin are produced (⬃ 1%,
5 nM) and clotting is observed; during a subsequent propagation
phase 700- to 900-nM thrombin is produced.50,55-57 The identity of
the initial activator of factor V has been a subject of controversy.
Although factor Xa-phospholipid, thrombin, and plasmin are all
capable of activating factor V to factor Va, the kinetic evaluation of
tissue factor–initiated blood clotting reactions shows that the initial
activator of factor V is ␣-thrombin that is initially produced by
factor Xa and phospholipid during the initiation phase of the blood
coagulation process.50,52,53,55-57 Although factor Xa-phospholipid is
an effective activator of factor V, the concentrations of factor Xa,
which exist during the initiation phase of coagulation, are insufficient to explain the activation of factor V that is observed. It is
noteworthy that factor Va function is usually measured by its ability
to contribute to ␣-thrombin generation. Thus, bioassays of factor
V/Va depend on clotting, which requires the generation of ␣-thrombin and thus the presence of both factor Va and factor Xa. Because
factor Xa will itself activate factor V, the relative concentration of
the pro-cofactor (intact factor V) cannot be reliably analyzed in an
activity-based assay. Plasmin will both activate and inactivate
factor V, and these processes may contribute to the pathology of
thrombosis.58-61
Substrates for thrombin include platelets, fibrinogen, factor XI,
factor V, factor VIII, factor VII, and factor XIII. During the process
of prothrombin activation by prothrombinase, 2 forms of thrombin
are produced, meizothrombin and ␣-thrombin.62-66 Cleavage of
prothrombin at Arg320 produces meizothrombin, in which all
elements of the prothrombin molecule are associated covalently.
Subsequently, cleavage at Arg271 gives rise to ␣-thrombin and
fragment 1●2. Controversy has existed with respect to whether
␣-thrombin or meizothrombin is the activator of factor V. Recent
data obtained by using both natural and recombinant ␣-thrombin
and meizothrombin suggest that the kinetic activation of factor V is
more favorable through the ␣-thrombin mechanism. In either case
the fully active factor Va ultimately depends on cleavage at residue
Arg1545, which liberates the light chain of the cofactor.
Function
Cofactor for factor Xa in prothrombinase
The membrane-binding site of the factor V molecule, which is
crucial to its function, is contributed by elements of the C2 and A3
domains contained within the light chain of the molecule.67-71 The
binding of the light chain to a membrane surface involves both
electrostatic and hydrophobic interactions with anionic/neutral
membranes and penetration of the molecule into the membrane
bilayer.67-72 The hydrophobic binding site of factor Va located on
the middle portion of the A3 domain is suggested to interact with
phospholipid and penetrate into the bilayer.67-69,72 The binding site
of the cofactor located on the C2 domain of factor Va and reported
to interact with anionic phospholipid was found to be exposed on
the surface of the molecule as expected for a hydrophilic amino
acid sequence.70,71,73,74 Two studies using recombinant factor V and
the factor V C2 domain have demonstrated that most of factor V
autoantibody inhibitors are directed to the C1-C2 domain of factor
V and interfere with its binding to phosphatidyl serine–containing
membranes.73,74 Kim et al73 using alanine-scanning mutagenesis
demonstrated that several amino acid substitutions at the COOHterminus of the light chain resulted in molecules with impaired
binding to phosphatidyl serine.73 Because proper interaction of the
cofactor with the membrane surface is required for the expression
of factor Va cofactor activity, it must be concluded that these
residues located at the carboxyl-terminal portion of factor V are
critical for the interaction of the activated molecule with the cell
surface. Overall, the data suggest that initial rapid factor Va–
membrane interaction is most likely first mediated through the
anionic lipid-binding domain on the C2 domain. Following this
diffusion rate-limited interaction,75 a hydrophobic interaction,
potentially involving the A3 domain of the molecule,67-69 tightly
anchors the molecule to the membrane core.72 Binding and
penetration of the molecule into the membranes result in a stable,
high affinity (Kd ⬃ 2.5 nM) factor Va–phospholipid interaction.67
The physiologically relevant membranes for blood coagulation are
provided by platelets and vascular cells.76,77 The membranes of
these cells are lipid-protein complexes composed principally of
phosphatidyl serine, phosphatidyl choline, and phosphatidyl ethanolamine.78-80 The selective qualities associated with cell binding
sites for prothrombinase remain poorly understood.
A fundamental contribution of factor Va to prothrombinase
function is the retention of factor Xa on the membrane surface.
Stopped-flow reaction kinetic data show that both factor Va and
factor Xa interact with phosphatidyl-choline phosphatidyl-serine
(PCPS) vesicles at diffusionally controlled rates (107-108 M⫺1
sec⫺1).75,81-84 However, factor Xa has an affinity (Kd) for membranes of approximately 0.1 ␮M, and this corresponds to a
dissociation rate constant of approximately 3.3 sec⫺1. As a
consequence, the duration of retention of factor Xa alone on a
membrane is short lived even though the membrane association
process is rapid. Biologically relevant factor Xa concentrations
probably never exceed 0.15 ␮M; however, membrane-dependent
complex assembly occurs such that membrane-bound factor Xa
binds tightly to membrane-bound factor Va. The rate of this
2-dimensional process can only be approximated as more than 109
M⫺1sec⫺1 as an equivalent solution rate (modeled as a secondorder process). The interaction of factor Va with the membrane
surface and factor Xa effectively increases the affinity of the
enzyme for the phospholipid bilayer.75,82 The 2 polypeptide chains
BLOOD, 1 JANUARY 2003 䡠 VOLUME 101, NUMBER 1
of the factor Va molecule are also in dynamic self-association with
a dissociation constant in the range of 3.6 nM.54 Thus, at the
physiologic concentrations that occur early during the coagulation
reaction, dissociation (and inactivation) of the factor Va molecule
can also occur. The factor Va–factor Xa protein-protein interaction
is relatively weak in the absence of a membrane surface (Kd ⬃ 0.8
␮M) and probably not relevant at biologic concentrations.85 When
coupled with the interactions of both proteins with the membrane,
both the light and heavy chains of factor Va interact with factor Xa;
each contributing to dissociation constants in the millimolar range,
resulting in a tight association of the 2 proteins (Kd ⬃ 1 nM) when
coupled with membrane binding.81,82,86
The membrane-bound factor Va–factor Xa complex cleaves
membrane-bound prothrombin to produce ␣-thrombin only in the
local environment of membrane-bound complex (principally the
immobilized activated platelet membrane surface). As a consequence of the common membrane binding of both substrate and
enzyme, the effective Km for prothrombin activation is decreased
by 2 orders of magnitude. Owing to alterations in factor Xa,
prothrombin, or both, the kcat for the membrane-localized reaction
is increased by 3 orders of magnitude. As a consequence, the
amount of prothrombin produced by the prothrombinase complex
formed on an appropriate membrane is increased by 5 orders of
magnitude compared with the activation of prothrombin by factor
Xa alone. Thus, the amount of ␣-thrombin formed in 1 minute by
the prothrombinase complex would take 6 months if factor Xa
acted alone. The process of prothrombinase activation of prothrombin is altered from that observed with factor Xa alone bound to a
membrane surface. Prothrombinase cleaves prothrombin initially at
Arg320 to yield meizothrombin, with subsequent cleavage at
Arg271, yielding ␣-thrombin and fragment 1●2.81,87 These 2
products continue to self-associate in a noncovalent manner (Kd ⬃
10⫺8 M).87 In contrast, factor Xa–membrane cleaves prothrombin
first at Arg271 to give rise to prethrombin 2; subsequent cleavage of
this intermediate at Arg320 gives rise to ␣-thrombin, with fragment
1●2 as the other product of the reaction. It is not clear whether the
enhancement in kcat for the enzymatic reaction is a consequence of
alterations in the factor Xa active site, prothrombin as a substrate,
or alterations in both enzyme and substrate when bound to factor
Va. Recent data demonstrate channeling of ␣-thrombin intermediates during activation of prothrombin by prothrombinase.88,89 As a
consequence, the data suggest that all intermediates leading to
␣-thrombin stay attached to the prothrombinase complex until the
end products (␣-thrombin and F 1●2) are released in solution.
Cofactor for the APC/protein S complex
in the inactivation of factor VIII
Shen and Dahlbäck90 reported that the intact pro-cofactor, single
chain factor V, can act as a cofactor, contributing to the acceleration
of inactivation of factor VIIIa by the activated protein C (APC)/
protein S complex. Our laboratory demonstrated that factor V can
accelerate the rate of factor VIII inactivation by APC, only when
protein S was present.91 Under the conditions used, factor V was
completely cleaved by APC early during the inactivation reaction
and before 10% to 20% factor VIII activity was lost.91 APC-treated,
membrane-bound factor V as well as ␣-thrombin–activated factor
Va (inclusive of the B domain) produced an increase in the rate of
inactivation of factor VIII by the APC/protein S complex by 2-fold.
In contrast, purified factor Va (lacking the B domain) even at high
concentrations does not show any cofactor effect on the rate of the
inactivation of factor VIII (⫾ protein S).91 These observations
suggest that the B region fragments of factor V may be responsible
COAGULATION FACTOR V
23
for the cofactor effect of factor V during the inactivation of factor
VIII by the APC/protein S complex.
Inactivation
Activated protein C (APC) down-regulates ␣-thrombin generation
and, as a consequence, the coagulation process by proteolytically
inactivating factor Va and factor VIIIa, the cofactors of prothrombinase and intrinsic factor Xase, respectively.92 Under physiologic
conditions, inactivation of factor VIIIa occurs spontaneously in the
absence of APC following dissociation of its A2 domain; as a
consequence, APC appears not to be essential for factor VIIIa
inactivation.91,93,94 In contrast, proteolytic cleavage of factor Va by
APC is required for its inactivation.95-97
Protein C is activated to APC by ␣-thrombin in the presence of
the vascular cofactor thrombomodulin.98,99 APC binds to the light
chain of factor Va in a competitive manner with factor Xa.100-103
Conversely, factor Xa impairs APC cleavage and inactivation of the
cofactor.102,103 Thus, APC provides inhibition of coagulation by
noncovalent competition with factor Xa and by cleavage and
inactivation of factor Va.95,96,101-103 APC cleaves human factor Va
heavy chain at Arg506, Arg306, and Arg679 with the kinetic order
of these cleavages generally as specified (Figure 1A,1C upper
part).96,101 The cleavages at Arg506 and Arg306 are influenced by a
membrane surface with the cleavage at Arg306 being virtually
dependent on the presence of an anionic phospholipid-containing
membrane.96 Following cleavage at Arg506, the resulting cofactor
(factor VaR506) displays lessened apparent affinity for factor Xa.104
As a consequence, prothrombinase with factor VaR506 has reduced
efficiency in catalyzing the activation of prothrombin to ␣-thrombin, prolonging the prothrombin time. However, factor VaR506 is
still “active,” providing binding and effector functions for factor
Xa, albeit reduced.
During a one-stage plasma-clotting assay, the end point clot is
produced when only approximately 10% of the total factor V
present in plasma has been activated, approximately 7 pM of factor
Xa is available, and less than 5% of the prothrombin activation
process has occurred.53,56 When measured in a clotting assay, most
of the activity of factor Va is perceived to be lost by cleavage at
Arg506, because cleavage at Arg506 reduces the apparent affinity
for factor Xa, and the binding of factor VaR506 to limiting amounts
of factor Xa available becomes a major determinant of the activity
of the factor Va molecule.105 However, when the reaction is
measured at saturating concentrations of factor Xa, the cleavage at
Arg506 of factor Va leads to the loss of only 25% to 40% of the
factor Va cofactor activity. In contrast, cleavage at Arg306 leads to
the ultimate inactivation of factor Va.96,105 Physical studies using
ultracentrifugation and light scattering show that the products of
cleavage of factor Va by APC at Arg306 and Arg506 dissociate to
produce fragments, which correspond to the A1 domain of the
molecule, noncovalently associated with the A3C1C2 (light chain),
and fragments A2N and A2C that correspond to the amino and
carboxyl termini of the A2 domain (Figure 1C, upper part).104 The
inactivation rate of factor Va when explored as a function of APC
concentration becomes independent of enzyme concentration, a
clear signal that the final inactivation step involves not only
cleavage but also dissociation of the A2N and A2C fragments from
the rest of the molecule.101,104 The human pro-cofactor, factor V, is
also inactivated by APC but only when bound to a membranesurface. Human factor V is inactivated by cleavages at Arg306,
24
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MANN and KALAFATIS
Arg506, Arg679, and Lys994. In this instance, cleavage at Arg306
that occurs first appears to be the sole requirement for inactivation.96
In studies of the ability of endothelial cells to complement the
activation of protein C and the inactivation of factor Va, an
APC-independent cleavage was observed that resulted in an
inactive product.105,106 In the presence of endothelial cells, ␣-thrombin appears to cleave factor Va at Arg643, resulting in an inactive
molecule (Figure 1C, lower part). The product of this cleavage,
initially observed in vitro, was also observed in vivo in studies of
blood exuding from microvascular wounds in the skin surface.107
The mechanism of factor Va inactivation because of Arg643mediated cleavage is unclear but appears to be the result of reduced
affinity between the 2 chains of factor Va106 (Figure 1C, lower part).
Plasmin has also been shown to inactivate factor Va with the
cleavages being dependent on the presence of a membrane
surface.59,60 Plasmin inactivation of factor Va involves cleavages at
Lys309, Lys310, Arg313, and Arg348 and most likely results in the
dissociation of the A2 domain of the cofactor from the rest of the
molecule (Figure 1A).60
Table 2. Selected examples of point mutations in the factor V gene, resulting
in no effect, partial factor V (FV) deficiency, or total FV deficiency
Mutation in the DNA
Amino acid change
Consequence
7111G ⬎ A (exon 1)
Gly 3 Asp
FV deficiency
36602C ⬎ T (exon 6)
Ala 3 Val
Partial FV deficiency
38166G ⬎ T (exon 7)
Lys 3 Stop
FV deficiency
40708C ⬎ G (intron 7)
Splice defect
FV deficiency
43580G ⬎ A (exon 10)
Arg 3 Lys
Conserved substitution
43642C ⬎ T (exon 10)
Arg 3 Stop
FV deficiency
49020T ⬎ C (exon 12)
Cys 3 Arg
FV deficiency
50340G ⬎ A (exon 13)
Gly 3 Glu
FV deficiency
50582C ⬎ T (exon 13)
Arg 3 Stop
FV deficiency
50765C ⬎ T (exon 13)
Gln 3 Stop
FV deficiency
50937A ⬎ G (exon 13)
Lys 3 Arg
Conserved substitution
51452C ⬎ T (exon 13)
Arg 3 Stop
FV deficiency
56883G ⬎ A (exon 14)
Glu 3 Lys
FV deficiency
62649A ⬎ G (exon 15)
Tyr 3 Cys
FV deficiency
63672C ⬎ G (exon 16)
Leu 3 Val
Conserved substitution
63846G ⬎ A (exon 16)
Ala 3 Thr
FV deficiency
65414G ⬎ A (exon 17)
Trp 3 Stop
FV deficiency
75001C ⬎ T (exon 23)
Arg 3 Cys
FV deficiency
75002G ⬎ A (exon 23)
Arg 3 His
FV deficiency
77925T ⬎ C (exon 24)
Met 3 Thr
Conserved substitution
Pathology
Hemorrhagic pathology: parahemophilia
Factor V deficiency is extremely uncommon, occurring either
because of a homozygous inheritance or because of a combination
of defective alleles. However, humans with factor V deficiency are
alive,8,11,12 whereas in mice the total lack of factor V is not
consistent with survival.21 In empirical models conducted with
purified reaction components and computer models of the blood
coagulation system, ␣-thrombin cannot be generated in the absence
of factor V when all inhibitors are present in the reaction
system.101,108,109 Conversely, humans with severe truncations in the
factor V molecule that do not allow any synthesis of factor V by
known conventional mechanisms have been observed to have
either manageable or no pathology associated with bleeding. These
observations suggest that there may be compensatory mechanisms
present in human blood, which either bypass or reduce the need for
factor V in generating levels of ␣-thrombin required for survival.
Point mutations in the factor Va gene resulting in abnormal
factor V function are associated with amino acid substitutions
located on the entire coding region of factor V (Table 2).
Deletions of entire portions of the molecule, nonsense mutations, and mutations of the splice sites for the introns have also
been reported. However, multiple mutations resulting in conserved substitutions (polymorphisms) also exist and have no
apparent consequence on factor V function. A compendium
database on factor V mutations, most of which result in
parahemophilia, was constructed by Dr Hans L. Vos (Hemostasis and Thrombosis Research Center, Leiden University Medical
Center, Leiden, The Netherlands; e-mail: [email protected]) and
is available on request. Several representative mutations are
shown in Table 2. Mutations in the factor V gene result either in
low circulating levels of the protein or in the complete
deficiency of factor V if the mutation results in a stop codon in
the middle of the reading frame (Table 2).
Defects in the inactivation of factor V/Va
associated with thrombosis
APC deficiency. Mature protein C is composed of several conserved domains, each of which is characteristic for the vitamin
K-dependent proteins. Human protein C is encoded by an autosomal gene of approximately 10 kb located on chromosome 2q1314.110,111 Absence of the protein or a defective circulating protein in
plasma, which is the result of more that 160 different mutations, has
severe consequences for the individuals bearing the mutations.112
Individuals who are heterozygous for a protein C mutation,
resulting in low levels of circulating protein C because one of the 2
alleles does not produce the normal protein, variably suffer from
venous thrombosis.113 However, homozygous protein C deficiency,
characterized by the complete absence of circulating protein C, is a
very serious condition with life-threatening thrombotic symptoms.
This severe condition usually presents immediately after birth, in
the form of purpura fulminans with the only remedy being the
infusion of protein C.114-116
Congenital APC resistance. Factor VLEIDEN. In 1993, Dahlbäck et al23 observed that plasma from some individuals with
venous thrombosis had a reduced response to APC.23 When APC
is introduced into normal plasma, there is a prolongation of
clotting time. In plasma from some patients, higher concentrations of APC were required to obtain similar prolongations of
clotting time. This condition was called APC resistance. This
observation coupled with data that identified the Arg505/Arg506
APC cleavage site in the bovine and human factor Va molecules95,96,117 led to the identification of factor VLEIDEN.118 This
polymorphism occurs as a consequence of G1691A in the factor
V gene, leading to the substitution of an Arg506Gln of the
human factor V molecule.118 Arg506 is the initial cleavage site
for APC on the heavy chain of human factor Va and facilitates
the subsequent lipid-dependent inactivation cleavage at
Arg306.96,101,105 Because this abnormal molecule does not
possess the APC-cleavage site at Arg506, factor VaLEIDEN is
inactivated by APC at approximately one tenth the rate of
normal factor Va.119 However, inactivation still proceeds through
cleavage at Arg306.97,119-124 Because elimination of the latent
activity of membrane-bound factor V occurs because of an
initial cleavage at Arg306, factor VLEIDEN is inactivated by APC
at the same rate as normal factor V.119 Platelet–factor Va and
factor VaLEIDEN inactivation by APC proceed at similar rates and
BLOOD, 1 JANUARY 2003 䡠 VOLUME 101, NUMBER 1
are delayed when compared with their plasma equivalents.125,126
Thus, on the surface of platelets there is little difference in the
inactivation rates of the 2 molecules, both of which are protected
from inactivation. Two groups have reported patients with
thrombotic disorders that were heterozygous for an amino acid
substitution at the Arg306 cleavage site.127,128
As a consequence of the factor VLEIDEN mutation, the partial
loss of activity of the normal factor Va molecule that occurs
following cleavage at Arg506, resulting in diminished interaction between factor Va and factor Xa, is not observed. The
pathology of factor VLEIDEN is associated in vivo with thrombotic manifestations, which are observed in both homozygous
and heterozygous individuals.129-133 Venous thrombosis is more
common among those individuals homozygous for the Arg506Gln
substitution. Statistical analyses suggest that the relative risk of
a thrombotic episode in individuals heterozygous for factor
VLEIDEN is 7-fold higher that the risk for healthy individuals.134
In contrast, homozygous factor VLEIDEN individuals have an
80-fold higher risk of thrombosis than individuals with the normal
factor V gene.133,134 No correlation has been observed between
arterial thrombosis and the existence of factor VLEIDEN.135-137 The
frequency of factor VLEIDEN in patients with arterial thrombi is
approximately 5%, a value similar to the frequency of the mutation
present in the normal population. An explanation for this phenomenon is potentially found in the composition of the clot. The
composition of a thrombus is dependent on the surface-promoting
coagulation and the hemodynamic factors regulating blood circulation. Although venous thrombi are most commonly formed in areas
of stasis and are composed of fibrin with few platelets, arterial
thrombi are formed in areas of high blood flow and are composed
mainly of platelets with less fibrin. It is possible that plasmaderived factor Va and factor VaLEIDEN are the predominant cofactors
for prothrombinase assembly on the venous side. In contrast, on the
arterial side, the predominant cofactor pool participating in prothrombinase at the site of injury may be platelet factor Va (or
platelet factor VaLEIDEN). Because both platelet factor VaLEIDEN and
normal factor Va cofactors are equally resistant to inactivation by
mutation, APC resistance would not be selectively associated with
arterial thrombosis. Many studies have shown that the thrombotic
risk in homozygous patients carrying the factor VLEIDEN mutation is
influenced by other acquired and congenital risk factors but less
dramatic than the risk of thrombosis for patients homozygous for
protein C and protein S deficiencies.138-142
HR2 haplotype. The factor V allele His1299Arg (also known
as R2 haplotype) is marked by A4070G in the factor V gene.143
This mutation (in the B region) is a marker that cosegregates
with several other polymorphisms encoding several amino acid
changes in the factor V molecule.143-146 It has been reported that
individuals carrying the factor V His1299Arg mutation have
mild APC resistance and an increase in the risk of venous
thrombosis. Thus, this ensemble of polymorphisms represents
per se a potential thrombotic risk factor.146,147 We have described
a thrombotic family with the R2 haplotype and several symptomatic members.147 One of these individuals was found to be
doubly heterozygous for the factor V HR2 haplotype and for the
Tyr1702Cys mutation. The latter mutation was shown to be at
the origin of factor V deficiency, resulting in the absence of
circulating normal factor V.147,148 Thus, because the factor V
allele predicting the Tyr1702Cys substitution and encoding for a
non-R2 haplotype factor V is not expressed at the protein level,
COAGULATION FACTOR V
25
the plasma of this patient contains only molecules encoded by
the R2 haplotype.148 This individual had also mild APC
resistance and was classified as a pseudohomozygous R2
haplotype. We have found in this individual that the factor V
molecule that is characterized by the mutations included in the
R2 haplotype is resistant to APC inactivation because of
impaired cleavage by APC at both Arg506 and Arg306.148 These
findings suggest the possibility that one or more of the amino
acid substitutions, which are predicted by the R2 haplotype,
impair factor Va cleavage by APC at Arg506/Arg306. The
patient studied was also a carrier of the Asp2194Gly substitution
that is tightly associated with the R2 haplotype and is the most
likely candidate for the observed APC resistance of factor Va.
Studies by Kim et al73 using alanine-scanning mutagenesis
suggested that several amino acid residues within the COOHterminal portion of the C2 domain of factor V are crucial for the
interaction of the cofactor with phosphatidyl serine.73 Among
those, the Asp2194Ala substitution had impaired binding to
phosphatidyl serine and defective cofactor activity. However,
the factor V molecule from carriers of the R2 haplotype
possesses normal procoagulant activity. The R2 haplotype
demonstrates resistance to APC because of impaired cleavage at
both Arg506 and/or Arg306.148 Thus, it is possible that the factor
Va mutations surrounding Asp2194 impair its inactivation by APC.
Acquired APC resistance. Activated protein C resistance
may also be observed with elevated levels of homocysteine.149,150 Factor V contains 5 unpaired cysteines, 2 in the
heavy chain, 2 in the light chain, and 1 in the B region41,42 (Table
1). These cysteines can incorporate homocysteine at physiologically relevant concentrations associated with hyperhomocystinemia and, as a consequence, display a form of acquired APC
resistance.151
Influence of factor Va on fibrinolysis. Regulation of factor V
activity also contributes to the function of the fibrinolytic
cascade.152-154 A fibrinolysis inhibitor named thrombin-activatable fibrinolysis inhibitor (TAFI) is activated by the ␣-thrombin–
thrombomodulin complex to generate an enzyme with carboxypeptidaselike activity, which functions by excision of
COOH-terminal Lys and Arg residues from plasmin-cleaved
fibrin.155 These desLys fibrin degradation products are poor
cofactors for the activation of plasminogen by tissue plasminogen activator (tPA). Thus, thrombin activation of TAFI provides
a connection between impaired thrombin generation and unstable fibrin clots. During clotting in plasmas deficient in factor
VIII, factor IX, or factor XI, thrombin generation is reduced
versus the normal case. However, although all the fibrinogen is
quantitatively converted to fibrin in these deficient plasmas, the
clots exhibit a greater tendency toward lysis than do normal
clots, because TAFI activation is also impaired by reduced
prothrombin activation.
It has been demonstrated that clot lysis is delayed in plasma
from individuals homozygous for the factor VLEIDEN mutation.156
These data suggest that resistance of factor VaLEIDEN to inactivation by APC, which results in sustained ␣-thrombin formation,
also results in enhanced activation of TAFI and impaired
removal of the fibrin clot. These data also suggest that the
enhanced activation of prothrombin associated with factor
VLEIDEN may increase the resistance of the fibrin clot to
lysis156,157 in individuals with hemophilia A, producing a milder
hemostatic defect.
26
MANN and KALAFATIS
BLOOD, 1 JANUARY 2003 䡠 VOLUME 101, NUMBER 1
Figure 2. Participation of the factor V molecule in the hemostatic process. Four stages of the participation of the factor V molecule in the hemostatic process are illustrated
in in cartoon form. (A) The factor V molecule is presented with the activation peptide “B domain” illustrated as a loop structure which connects the heavy (A1A2) and light chain
(A3C1C2) domains of the factor Va molecule. The single chain factor V interacts with membranes through its carboxyl terminal region. (B) Factor V is activated to Factor Va by
thrombin (IIa), which excises the B domain, leaving the noncovalently associated light and heavy chains of the factor Va molecule. (C) The membrane-bound factor Va molecule
binds factor Xa through regions of both the light and heavy chains. These interactions together with factor Xa membrane binding provide the tightly associated enzymatic
complex (prothrombinase) which converts prothrombin (II) to thrombin (IIa). (D) Activated protein C (APC) cleaves at 3 sites of the heavy chain of the factor Va molecule,
resulting in the dissociation of the A2 domain as 2 fragments, A2N and A2C. The resulting product (factor Vai), composed of the A1 domain noncovalently associated with the
membrane-bound light chain, binds APC but will no longer function efficiently in generating thrombin.
Potential influence of combined pathology
associated with factor V
TFPI and APC
The dynamic process of factor V inactivation by APC is
synergistic with the tissue factor pathway inhibitor (TFPI)
inhibition during the tissue factor–initiated blood coagulation
process.158-162 This inhibition occurs because the ␣-thrombin
activation of factor V is a sequential phenomenon with cleavage
at Arg709 occurring first to produce the heavy chain. However,
factor Va activity is associated with cleavage at Arg1545 and
formation of the light chain, which occurs more slowly. Once
cleaved at Arg709, the partially activated membrane-bound
factor V molecule can be rapidly cleaved by APC at Arg306 and
Arg506, leading to inactivation. If the inactivation process is
sufficiently vigorous, it can occur prior to the formation of the
light chain of the molecule; hence, factor Va activity is
nullified.162 These regulatory processes display synergy between
the dynamic APC system and the stoichiometric TFPI inactivation system.162 In vitro experimental data suggested that a
combination of factor VLEIDEN and low normal levels of TFPI
could produce unregulated generation of thrombin because of
the absence of the appropriate synergy between 2 dynamic
regulatory processes.162,163 Transgenic mice obtained by crossing mice with reduced levels of TFPI with mice homozygous for
factor VLEIDEN resulted in mice with a lethal phenotype with
fibrin deposition in multiple organs.164 The observation of
disseminated thrombosis in mice homozygous for the factor
VLEIDEN mutation and low levels of TFPI is in keeping with the
hypothesis suggested by in vitro data.163 Thus, reduced TFPI
levels may be a contributor to thrombosis in association with
factor VLEIDEN.
Hemophilia and factor VLEIDEN
The importance of the timely regulation of factor Va cofactor
activity is illustrated by a potentially beneficial effect of factor
VLEIDEN for individuals with severe hemophilia A. The net effect of
factor VIII deficiency is reduced ␣-thrombin formation rate;
conversely, individuals with the factor VLEIDEN mutation have
sustained ␣-thrombin formation. Significant differences in pathology
BLOOD, 1 JANUARY 2003 䡠 VOLUME 101, NUMBER 1
COAGULATION FACTOR V
between unrelated patients carrying the same factor VIII missense
mutations have been reported.165 Although these factor VIII
mutations most commonly result in a phenotype characterized by
severe bleeding in individuals carrying the normal factor V gene, 2
patients with hemophilia heterozygous for the factor VLEIDEN
mutation have been reported who presented with mild/moderate
bleeding episodes. More recently, an individual with severe
hemophilia B (⬍ 1% factor IX activity) but heterozygous for the
factor VLEIDEN mutation presented with only a mild bleeding
diathesis.166 Because factor VaLEIDEN is inactivated by APC more
slowly, a hemophiliac patient with the factor VLEIDEN gene may
have a milder bleeding syndrome because of the increased
durability of prothrombin activation.167 These data suggest that
the variability in the expression of single defects associated with
hemophilia A and B may in part be because of the high
frequency of the factor VLEIDEN mutation in the normal population and that hemophiliac patients should also be regularly
screened for the Arg506Gln mutation.
Parahemophilia and factor VLEIDEN
A pseudohomozygous APC resistance, predisposing to thrombosis,
may occur as a consequence of the combination of 2 defects in
factor V that are associated with a quantitative reduction in factor V
and heterozygous inheritance of the factor VLEIDEN mutation. The
main laboratory findings are the presence of a low APC sensitivity
ratio (APC-SR) and low plasma values of factor V antigen and
activity.168-171 In this case the normal factor V allele is silent, and
only the factor VLEIDEN allele is expressed. As a consequence, the
patient who is genetically heterozygous for factor VLEIDEN is
phenotypically homozygous for the mutation.171
27
Summary
Overall, factor V can be thought of as a Dr Jekyll and Mr Hyde. Dr
Jekyll, the good doctor, provides a useful support for individuals
(hemostasis) and Mr Hyde, the rapacious monster, causes destruction (thrombosis). Four stages of the participation of the factor V
molecule in the hemostatic process are illustrated in Figure 2 in
cartoon form. The factor V molecule is presented with the
activation peptide “B domain” illustrated as a loop structure which
connects the heavy and light chain (A2–A3) domains of the factor
Va molecule. The single chain factor V interacts with membranes
through its carboxyl terminal region. Factor V is activated to Factor
Va by thrombin (IIa) which excises the B domain leaving the
noncovalently-associated light and heavy chains of the factor Va
molecule. The membrane bound factor Va molecule binds factor Xa
through regions of both the light and heavy chains. These
interactions together with factor Xa membrane binding provide the
tightly associated enzymatic complex (prothrombinase) which
converts prothrombin (II) to thrombin (IIa). APC binds competitively with factor Xa to factor Va, primarily through light chain
interactions. APC cleaves at 3 sites of the heavy chain of the factor
Va molecule resulting in the dissociation of the A2 domain as 2
fragments, A2N and A2C. The resulting product (factor Vai)
composed of the A1 domain noncovalently associated with the
membrane-bound light chain binds APC but will no longer function
efficiently in generating thrombin. We need factor V to have a
normal blood clotting physiology; however, the unmanaged evolution of factor V activation can cause severe pathology. However,
sometimes Mr Hyde can abrogate the damage caused by another
monster (hemophilia A or B), providing overall a beneficial support
to the individual.
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