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From www.bloodjournal.org by guest on October 21, 2014. For personal use only.
2003 102: 4014-4020
doi:10.1182/blood-2003-04-1199 originally published online
July 24, 2003
Coinheritance of Factor V (FV) Leiden enhances thrombin formation and
is associated with a mild bleeding phenotype in patients homozygous
for the FVII 9726+5G>A (FVII Lazio) mutation
Elisabetta Castoldi, José W. P. Govers-Riemslag, Mirko Pinotti, Debora Bindini, Guido Tans, Mauro
Berrettini, Maria Gabriella Mazzucconi, Francesco Bernardi and Jan Rosing
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HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
Coinheritance of Factor V (FV) Leiden enhances thrombin formation and
is associated with a mild bleeding phenotype in patients homozygous
for the FVII 9726⫹5G⬎A (FVII Lazio) mutation
Elisabetta Castoldi, Jose´ W. P. Govers-Riemslag, Mirko Pinotti, Debora Bindini, Guido Tans, Mauro Berrettini,
Maria Gabriella Mazzucconi, Francesco Bernardi, and Jan Rosing
We investigated the role of thrombophilic
mutations as possible modifiers of the
clinical phenotype in severe factor VII
(FVII) deficiency. Among 7 patients homozygous for a cross-reacting material–
negative (CRMⴚ) FVII defect (9726ⴙ5G>A,
FVII Lazio), the only asymptomatic individual carried FV Leiden. Differential
modulation of FVII levels by intragenic
polymorphisms was excluded by a FVII to
factor X (FX) gene haplotype analysis.
The coagulation efficiency in the FV Leiden carrier and a noncarrier was evalu-
ated by measuring FXa, FVa, and thrombin generation after extrinsic activation of
plasma in the absence and presence of
activated protein C (APC). In both patients coagulation factor activation was
much slower and resulted in significantly
lower amounts of FXa and thrombin than
in a normal control. However, more FXa
and thrombin were formed in the plasma
of the patient carrying FV Leiden than in
the noncarrier, especially in the presence
of APC. These results were confirmed in
FV-FVII doubly deficient plasma reconsti-
tuted with purified normal FV or FV Leiden. The difference in thrombin generation between plasmas reconstituted with
normal FV or FV Leiden gradually decreased at increasing FVII concentration.
We conclude that coinheritance of FV
Leiden increases thrombin formation and
can improve the clinical phenotype in
patients with severe FVII deficiency.
(Blood. 2003;102:4014-4020)
© 2003 by The American Society of Hematology
Introduction
Coagulation factor VII (FVII) is a vitamin K–dependent glycoprotein that plays a key role in the initiation of coagulation.1 Following
vascular damage, membrane-bound tissue factor (TF) forms a
Ca2⫹-dependent complex either with FVII (which is then converted
to FVIIa by a single proteolytic cleavage) or directly with
circulating FVIIa, present in blood at a very low concentration.
TF-bound FVIIa activates factor X (FX) to FXa, which, together
with its cofactor factor Va (FVa), converts prothrombin to thrombin. In addition, the TF/FVIIa complex can also activate factor IX
to FIXa, which, after forming a complex with factor VIIIa (FVIIIa),
contributes to FXa generation and thereby to thrombin formation
via the intrinsic pathway. In plasma, the activity of the TF/FVIIa
complex is down-regulated by the tissue factor pathway inhibitor
(TFPI), which acts via the formation of a quaternary complex with
FXa, FVIIa, and TF.2 Thrombin generation is ultimately shut down
by activated protein C (APC), which proteolytically inactivates
FVa and FVIIIa, the essential cofactors of the prothrombin- and
intrinsic FX–activating complexes.3
Severe FVII deficiency4,5 affects about 1 in 500 000 individuals
in the general population and is inherited as an autosomal recessive
trait with variable penetrance. Severely affected patients may
develop life-threatening hemorrhages and require substitutive
treatment with plasma concentrates or recombinant FVIIa.6 Several
intragenic mutations that impair gene expression or protein
secretion (CRM⫺ deficiency) or alter protein function (CRM⫹
deficiency) have been described (FVII Mutation Database,
http://europium.csc.mrc.ac.uk). Moreover, a few intragenic polymorphisms 7-10 that modulate plasma levels of FVII 8,10-12
are known.
The FVII gene is located on chromosome 13 (13q34-qter), next
to the FX gene,13,14 and comprises 9 exons and 8 introns.15 A
polymorphic minisatellite, consisting of a variable number of
37-nucleotide tandem repeats, spans the 3⬘ end of exon 7 and the 5⬘
portion of intron 7,7 and the first repeat contains the donor splice
site for the excision of intron 7. A point mutation (9726⫹5G⬎A,
FVII Lazio) affecting nucleotide ⫹5 of intron 7, which is part of the
splicing consensus sequence, has been identified as a common
cause of CRM⫺ FVII deficiency in central Italy.16 FVII Lazio
homozygous individuals have undetectable FVII levels and are
usually severe bleeders.16 In vitro expression experiments have
shown that the FVII Lazio mutation activates a cryptic splice site in
the second minisatellite repeat, leading to a 37-nucleotide insertion
in the mature transcript, which in turn causes a frameshift and
premature termination of translation. Only 0.2% to 1% of all FVII
Lazio mRNA is spliced correctly, resulting in a very small amount
of normal FVII.17
From the Department of Biochemistry, Cardiovascular Research Institute
Maastricht (CARIM), Maastricht University, Maastricht, The Netherlands; the
Department of Biochemistry and Molecular Biology, University of Ferrara,
Ferrara, Italy; the Haemophilia Centre, University of Perugia, Perugia, Italy;
and the Department of Cell Biotechnology and Haematology, “La Sapienza”
University, Rome, Italy.
E.C. was supported by a Long-Term Fellowship of the European Molecular
Biology Organization (EMBO).
Submitted April 16, 2003; accepted July 13, 2003. Prepublished online as
Blood First Edition Paper, July 24, 2003; DOI 10.1182/blood-2003-04-1199.
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734.
Supported by Telethon (grant GGP02182) and Ministero dell’ Universita` e della
Ricerca Scientifica e Tecnologica (MURST) (Young Investigator Project 2000).
4014
Reprints: Elisabetta Castoldi, Department of Biochemistry, Cardiovascular
Research Institute Maastricht (CARIM), Maastricht University, PO Box 616,
6200 MD Maastricht, the Netherlands; e-mail: [email protected].
© 2003 by The American Society of Hematology
BLOOD, 1 DECEMBER 2003 䡠 VOLUME 102, NUMBER 12
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BLOOD, 1 DECEMBER 2003 䡠 VOLUME 102, NUMBER 12
PROTECTION BY FV LEIDEN IN SEVERE FVII DEFICIENCY
The poor correlation between FVII levels and bleeding tendency4,5,18 in carriers of FVII defects points at the existence of
additional genetic and/or environmental factors that modulate the
clinical phenotype. In particular, coinheritance of common thrombophilic mutations might result in a milder clinical phenotype in
patients with severe FVII deficiency, as it has been shown for
hemophilic disorders.19-23 The FVII Lazio mutation, which is
relatively frequent in central Italy and predicts a severe bleeding
diathesis in the homozygous condition, provides an excellent
model to investigate the effects of thrombophilic mutations on
severe FVII deficiency. Here we report on the identification of an
asymptomatic FV Leiden carrier among 7 patients homozygous for
the FVII Lazio mutation. The amounts of FXa, FVa, and thrombin
generated after extrinsic activation of plasma in the absence and
presence of APC were compared between 2 FVII Lazio homozygotes, the FV Leiden carrier, and a noncarrier. The effect of the FV
Leiden mutation on thrombin generation was further investigated
in a plasma model of FVII deficiency.
Patients, materials, and methods
Patients
There were 7 patients with severe FVII deficiency due to homozygosity for
the FVII Lazio mutation recruited from 2 Italian hemophilia centers. Their
demographic and clinical characteristics are summarized in Table 1.
Genetic analysis (ie, FVII-FX gene haplotype analysis and the screening for
thrombophilic mutations) was performed in all 7 patients. Functional assays
(ie, measurement of FXa, FVa, and thrombin generation in plasma in which
coagulation was triggered via the extrinsic pathway) were performed only
in the patients for whom plasma was available, namely patients no. 3 and
no. 6. Informed consent for participation in the study was obtained from
all patients.
Blood collection
Venous blood was drawn by venipuncture in 129 mM sodium citrate (9:1
vol/vol). Platelet-poor plasma for functional assays was obtained after 2
centrifugation steps. Whole blood was first centrifuged at 3000g for 25
minutes at room temperature, then the supernatant was transferred to a fresh
tube and centrifuged again at 20 000g for 30 minutes at 4°C. Plasma was
aliquoted, snap-frozen, and stored at ⫺80°C.
Coagulation assays
FVII activity (FVII:C) was measured via a one-stage clotting assay in
FVII-deficient plasma, using human thromboplastin (Thromborel S;
DADE-Behring, Marburg, Germany) as a trigger. FVII antigen (FVII:
Ag) was measured with a commercially available enzyme-linked
immunosorbent assay (ELISA) kit (Asserachrom VII:Ag; Diagnostica
Stago, Asnie`res, France).
4015
DNA analysis
Genomic DNA was extracted from peripheral blood leukocytes according
to a standard procedure. The FVII Lazio mutation was detected by
polymerase chain reaction (PCR)–mediated amplification of FVII exon 7
(including splicing junctions) followed by RsaI restriction analysis, as
described.16 Genotyping for the FVII and FX gene polymorphisms included
in the multipoint haplotype analysis (ie, ⫺402G⬎A, ⫺401G⬎T, 5⬘F7,
IVS7 VNTR, and R353Q in the FVII gene, and 5⬘F10 in the FX gene) was
performed as previously described.10,11,24
Carriership of the thrombophilic mutations FV R506Q (FV Leiden), FV
H1299R (FV R2), and prothrombin (PT) 20210G⬎A was ascertained by
DNA amplification followed by restriction analysis, as reported.25-27
Measurement of thrombin, FXa, and FVa generation in
extrinsically triggered plasma
Platelet-poor plasma was defibrinated with 1 U/mL Ancrod (WHO International Laboratory for Biological Standards, Hertfordshire, United Kingdom) and the clot was removed with a plastic spatula. After prewarming the
plasma at 37°C, the coagulation cascade was triggered with a start mix
containing recombinant TF (Dade Innovin; DADE-Behring), synthetic
phospholipid vesicles (DOPS/DOPC/DOPE [1,2-dioleoyl-sn-glycero-3phosphoserine/1,2-dioeoyl-sn-glycero-3-phosphocholine/1,2-dioleoyl-snglycero-3-phosphoethanolamine], 20/60/20, mol/mol/mol), and CaCl2 (final concentrations in plasma: approximately 6.4 ng/mL TF [approximately
136 pM], 15 ␮M phospholipids, and 16 mM CaCl2), in the absence or in the
presence of APC (0.4 nM). At regular time points samples were drawn from
the plasma mixture and diluted in a “quenching” buffer (50 mM Tris
[tris(hydroxymethyl)aminomethane], pH 7.9, 175 mM NaCl, 20 mM EDTA
[ethylenediaminetetraacetic acid], 0.5 mg/mL ovalbumin for samples taken
for thrombin and FXa quantification; 25 mM HEPES [N-2-hydroxyethylpiperazine-N⬘-2-ethanesulfonic acid, pH 7.7], 175 mM NaCl, 5 mg/mL
bovine serum albumin [BSA], 3 mM CaCl2, and 1 ␮M Pefabloc TH for the
sample taken for FVa quantification). The latter quenching buffer contained
the reversible thrombin inhibitor Pefabloc TH (Pentapharm, Basel, Switzerland) to prevent further activation of FV. Thrombin concentration was
measured directly using the chromogenic substrate S2238. FXa and FVa
concentrations were quantified via prothrombinase-based assays using
purified proteins and synthetic phospholipid vesicles. Reaction conditions
were as follows: 40 ␮M phospholipid vesicles (DOPS/DOPC, 10/90,
mol/mol), 5 nM bovine FVa, 0.5 ␮M human prothrombin, and approximately 3 mM CaCl2 for the FXa assay; 40 ␮M phospholipid vesicles
(DOPS/DOPC, 10/90, mol/mol), 0.52 nM bovine FXa, 0.5 ␮M human
prothrombin, approximately 3 mM CaCl2, and 1 ␮M Pefabloc TH for the
FVa assay. Pefabloc TH was added to the FVa assay mixture to prevent
further activation of FV by the thrombin generated in the assay. From the
amount of thrombin formed (quantified with the chromogenic substrate
S2238), the FXa or FVa concentration was calculated using a calibration
curve made with known amounts of FXa or FVa. The reaction mixtures
used for the construction of the calibration curve for FVa contained 1 ␮M
Pefabloc TH, thereby automatically correcting for the (small) effects of
Pefabloc TH on the activity of the prothrombinase complex and on the
quantification of thrombin with S2238. Thrombin, FXa, and FVa generation
Table 1. Demographic and clinical characteristics of the FVII Lazio-homozygous patients
Patient
code, no.
Age, y
Sex
FVII:Ag, %
FVII:C, %
Clinical phenotype
FV R506Q;
FV Leiden
1
49
M
—
⬍1
Conjunctival bleeding, hemorrhagic pleuritis, hemarthrosis (elbow)
RR
2
38*
F
1
2
Epistaxis, metrorrhagia, melena
RR
3
42
M
1
1
Epistaxis, gum bleeding, hemarthrosis
RR
4
38
F
1
Menorrhagia, hemarthrosis (elbow and ankle)
RR
Hemarthrosis (hip), melena
RR
Menorrhagia at menarche, then asymptomatic
RQ
Hemarthrosis
RR
—
5
57*
M
1
1
6
37
F
⬍1
⬍2
7
54
M
1
1
— indicates not done.
*Deceased.
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4016
BLOOD, 1 DECEMBER 2003 䡠 VOLUME 102, NUMBER 12
CASTOLDI et al
curves were constructed by plotting concentration as a function of
subsampling time. Each time course was measured at least in duplicate.
Thrombin generation curves were corrected for ␣2M-thrombin according to
Hemker et al,28 and the area under the curve (the endogenous thrombin
potential, ETP)29 was calculated as a measure of the total amount of
thrombin generated in plasma.
Study of the effect of FV Leiden on thrombin generation in a
plasma model of FVII deficiency
Preparation of FV-FVII doubly deficient plasma. Congenitally FVIIdeficient plasma (FVII:C ⬍ 1%) was purchased from George King
Bio-Medical (Overland Park, KS), and 15 ␮g/mL corn trypsin inhibitor
(Kordia, Leiden, The Netherlands) was added to prevent contact activation.
FV was removed from the plasma by immunodepletion using a monoclonal
antibody (RU-FV3B1) directed against the heavy chain of FVa30 coupled to
Sepharose beads (CNBr-activated Sepharose 4B; Amersham Pharmacia
Biotech, Uppsala, Sweden). After 4 passages over the RU-FV3B1 column,
the residual FV concentration in the plasma was 5% of normal, as
determined by a prothrombinase-based FV assay.31 Measurement of FVIII
levels before and after FV-immunodepletion showed that the plasma had
not been significantly diluted during the procedure. Freshly prepared
FV-depleted FVII-deficient plasma (referred to as FV-FVII doubly deficient
plasma from here onward) was aliquoted, snap-frozen, and stored at ⫺80°C
until use.
Reconstitution of the FV-FVII doubly deficient plasma with purified
FV and variation of the FVII concentration. To mimic the plasma of the
FVII Lazio-homozygous patients, FV-FVII doubly deficient plasma was
supplemented with 0.2% FVII (in the form of normal plasma) and
reconstituted with purified normal FV or FV Leiden,32,33 or with a 1:1
mixture of normal FV and FV Leiden, to a final FV concentration of 23 nM.
Coagulation was triggered with approximately 6.4 ng/mL TF in the
presence and absence of 20 nM APC.
In order to vary the FVII concentration, the FV-FVII doubly deficient
plasma was mixed with a congenitally FV-deficient plasma (George King
Bio-Medical) in various proportions, ranging from 0% to 10% FV-deficient
plasma (ie, 0%-10% FVII). Plasma mixtures were then reconstituted with
purified normal FV or FV Leiden. In order to obtain measurable thrombin
generation curves at all FVII concentrations both in the absence and
presence of APC, the TF concentration was set at approximately 1.9 ng/mL,
while the APC concentration in the reaction mixture was 10 nM.
Measurement of thrombin generation using a fluorogenic substrate.
Thrombin generation in the reconstituted plasma was measured continuously using a low-affinity fluorogenic substrate for thrombin (Z-Gly-GlyArg-AMC; BACHEM, Bubendorf, Switzerland). Plasma was mixed with
the fluorogenic substrate (300 ␮M final concentration) in the well of a
microtiter plate and prewarmed at 37°C for 3 minutes. Coagulation was
triggered with a mixture containing TF, phospholipids, and CaCl2 in the
presence or absence of APC (as described in “Measurement of thrombin,
FXa, and FVa generation in extrinsically triggered plasma”) and thrombin
generation was followed in time in a fluorometer (SPECTRAmax GEMINI
XS; Molecular Devices, Sunnyvale, CA). Raw data from the fluorometer
(expressed as relative fluorescence units, RFUs) were corrected for
inner-filter effects and substrate consumption and subsequently converted
to thrombin concentrations using a thrombin standard kindly provided by
Prof H. C. Hemker. The contribution of ␣2M-thrombin to the measured
signal was calculated and subtracted as described by Hemker et al.34
Corrected data were exported to a Microsoft Excel sheet (Microsoft,
Seattle, WA) and averaged with the “moving average” function, after which
the first derivative of the data was calculated to obtain the thrombin
generation curve.
Results
Selection of patients and genetic analysis
Of all patients with severe FVII deficiency referred to our lab for
mutation screening by 2 hemophilia centers in central Italy, 7 were
found to be homozygous for the FVII Lazio mutation (Table 1). Of
the patients, 2 (nos. 3-4) were brother and sister, whereas the others
were apparently unrelated. All patients had extremely low FVII
levels (FVII:Ag ⱕ 1%, FVII:C ⱕ 2%), and all but one (no. 6) were
severe bleeders. FVII deficiency in patient no. 6 was diagnosed at
menarche, when she presented with menorrhagia. Since then she
has been asymptomatic and had 2 uncomplicated pregnancies
without need for treatment.
To verify whether the difference in clinical phenotype between
patient no. 6 and the other patients could possibly be attributed to
intragenic polymorphisms that modulate FVII levels, a multipoint
FVII gene haplotype analysis was carried out in all patients. The
following polymorphisms were tested: 2 single-nucleotide polymorphisms10 and a decanucleotide insertion (5⬘F7) in the promoter
region, a minisatellite in intron 7 (IVS7 VNTR),7 and a missense
mutation in exon 8 (R353Q).8 All FVII Lazio alleles under study
turned out to be identical at each polymorphic position (Table 2), in
accordance with the founder effect hypothesis for this mutation.16
Since the FX gene is contiguous to the FVII gene, the haplotype
analysis was also extended to a hexanucleotide insertion in the FX
gene promoter (5⬘F10).24 Again, all patients were found to be
homozygous for the same allele at this polymorphic site (Table 2).
Screening of the FVII-deficient patients for common thrombophilic mutations, namely FV R506Q (FV Leiden), FV H1299R (FV
R2), and PT 20210G⬎A, revealed that patient no. 6 was heterozygous for FV Leiden, whereas all other patients did not carry any of
the mutations.
Generation of FXa, FVa, and thrombin in the patients’ plasma
In order to gain an insight into the molecular mechanism underlying the marked difference in clinical phenotype among patients,
plasma was collected from the FV Leiden carrier (patient no. 6) and
from a noncarrier (patient no. 3). Coagulation was initiated with TF
(approximately 6.4 ng/mL), phospholipids, and CaCl2, and the
generation of FXa, FVa, and thrombin was followed in time by
quantifying the amounts of FXa, FVa, and thrombin in samples
taken from the plasma mixture at regular time points. To test the
influence of FV Leiden, the experiments were performed both in
the absence and in the presence of APC (0.4 nM). A normal plasma
was included in the analysis as a control.
In the normal control (Figure 1), fast coagulation factor
activation was observed. The FXa generation curve reached a peak
after 1.5 minutes, and FV was quantitatively activated within 1
minute. This resulted in the rapid generation of a large amount of
thrombin. The addition of APC considerably reduced the generation of FVa, but it had no effect on FXa and thrombin generation at
the TF concentration used in our experimental set-up.
Table 2. FVII-FX gene haplotype analysis in the FVII Lazio-homozygous patients
FVII gene
Alleles in the population
FVII Lazio haplotype
FX gene
ⴚ402G > A
ⴚ401G > T
5ⴕ F7
IVS7 VNTR
R353Q
5ⴕ F10
G/A
G/T
A1/A2
a/b/c/d
R/Q
I/D
G
G
A1
b
R
I
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BLOOD, 1 DECEMBER 2003 䡠 VOLUME 102, NUMBER 12
PROTECTION BY FV LEIDEN IN SEVERE FVII DEFICIENCY
4017
Figure 1. Time courses of FXa, FVa, and thrombin generation in normal plasma. Plasma was triggered with approximately 6.4 ng/mL TF in the absence (䉫) and in the
presence (䉬) of 0.4 nM APC, and the generation of FXa (A), FVa (B), and thrombin (C) was followed in time with the subsampling method. Concentrations of activated
coagulation factors refer to the reaction mixture.
In the FVII-deficient patients (Figure 2), the activation of all
coagulation factors was significantly delayed compared with the
normal control. The FXa, FVa, and thrombin generation curves
showed a lag phase of several minutes, and considerably lower
amounts of FXa and thrombin were formed. FXa formation was
affected the most by the lack of FVII. At the peak of the FXa
generation curve, about 60 pM FXa (⬍ 1% of the normal
control) was formed in both patients. In the absence of APC,
more FXa was formed in the plasma of patient no. 6 than in the
plasma of patient no. 3 (Figure 2A). The addition of APC
(Figure 2D) resulted in further down-regulation of FXa generation in the plasma of patient no. 3, but not in the plasma of
patient no. 6, who carried the FV Leiden mutation. FVa
generation was quantitatively not affected by the deficiency of
FVII. In the absence of APC (Figure 2B) the time courses of FVa
generation obtained in the 2 patients were superimposable,
whereas in the presence of APC (Figure 2E) more FVa was
formed in the plasma of patient no. 6 than in that of patient no. 3,
which is in line with their respective FV genotypes. As a
consequence of the sharp reduction in FXa generation, thrombin
formation was also decreased in the FVII-deficient patients
compared with the normal control. However, thrombin generation was less dramatically affected than FXa generation. In the
absence of APC, the thrombin peak was about 25% (patient no.
3) to 28% (patient no. 6) of that measured in normal plasma (as
well as considerably delayed), while the ETP was about 70% of
that of normal plasma. Between the 2 patients there was hardly
any difference in the amount of thrombin formed in the absence
of APC (Figure 2C). However, in the presence of APC (Figure
2F) thrombin generation in patient no. 3 showed a slightly
longer lag phase and the total amount of thrombin generated was
about half (56%) of that of patient no. 6.
Effect of FV Leiden on thrombin generation in a plasma
model of FVII deficiency
Since the difference in thrombin generation between the 2 patient
plasmas might theoretically be due to other plasma variables in
addition to the FV, we decided to investigate the effect of the FV
Leiden mutation on thrombin generation in a model system of FVII
Figure 2. Time courses of FXa, FVa, and thrombin generation in the plasmas of the FVII Lazio-homozygous patients. The plasmas of patient no. 3 (open symbols) and
patient no. 6 (closed symbols) were triggered with approximately 6.4 ng/mL TF in the absence (A-C) and in the presence (D-F) of 0.4 nM APC, and the generation of FXa, FVa,
and thrombin was followed in time by the subsampling method. Each curve represents the average of at least 2 measurements of the whole time course. Concentrations of
activated coagulation factors refer to the reaction mixture.
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4018
CASTOLDI et al
deficiency, that is, FV-FVII doubly deficient plasma supplemented
with a variable amount of FVII and reconstituted with purified
normal FV or FV Leiden.
To mimic FVII Lazio-homozygous patients, FV-FVII doubly
deficient plasma was supplemented with 0.2% FVII17 and reconstituted with normal FV, FV Leiden, or, to simulate heterozygosity for
FV Leiden, a 1:1 mixture of normal FV and FV Leiden. Reconstituted plasmas were triggered with TF (approximately 6.4 ng/mL),
phospholipids, and CaCl2 in the presence and absence of 20 nM
APC, and thrombin generation was followed with a thrombinspecific fluorogenic substrate. As shown in Figure 3A, the 3
plasmas yielded superimposable thrombin generation curves in the
absence of APC. However, in the presence of APC (Figure 3B),
considerably less thrombin (approximately 64%) was formed in the
plasma containing normal FV than in the plasmas containing FV
Leiden. A normal plasma measured under the same experimental
conditions is shown for comparison in Figure 3C.
Comparison of Figure 3 with Figures 1-2 shows that thrombin
generation curves obtained with the fluorogenic method are about
2.4 times higher than the corresponding curves measured with the
subsampling method. This is due to the fact that the fluorogenic
substrate present in the reaction mixture competes with the
physiologic substrates and inhibitors of thrombin in plasma. The
predominant effect of this interference is the protection of thrombin
from inhibition by antithrombin and other plasma inhibitors, which
likely explains the increased levels of thrombin measured in
reaction mixtures containing fluorogenic substrate.
To further characterize the interaction between FVII deficiency
and FV Leiden, we also investigated the effect of varying FVII
levels on thrombin generation in FV-FVII doubly deficient plasma
reconstituted with either normal FV or FV Leiden and triggered
with approximately 1.9 ng/mL TF (Figure 4). Both in the absence
and in the presence of APC (10 nM), thrombin generation
progressively increased with increasing FVII concentration. In the
absence of APC, thrombin generation curves obtained at each
particular FVII concentration were very similar between plasma
mixtures reconstituted with normal FV or FV Leiden (data not
shown). However, in the presence of APC (Figure 4), plasma
reconstituted with FV Leiden (closed symbols) yielded clearly
higher and faster thrombin generation than the corresponding
plasma reconstituted with normal FV (open symbols). Interestingly, the difference in lag time and peak height of thrombin
generation between plasmas containing normal FV and FV Leiden
decreased at increasing FVII concentration and became negligible
at FVII levels 10% or higher (Figure 4). The same trend was
observed for the total amount of thrombin formed, calculated from
BLOOD, 1 DECEMBER 2003 䡠 VOLUME 102, NUMBER 12
Figure 4. FVII-titration of thrombin generation in plasma containing either
normal FV or FV Leiden. FV-FVII doubly deficient plasma was supplemented with
increasing amounts of FVII and reconstituted with either normal FV (open symbols
and dashed lines) or FV Leiden (closed symbols and solid lines). Plasma was
triggered with approximately 1.9 ng/mL TF in the presence of 10 nM APC and
thrombin generation was followed continuously with a fluorogenic substrate. FVII
concentrations were as follows: 0% (dashes), 0.1% (diamonds), 0.2% (squares),
0.5% (triangles), 1% (circles), and 10% (crosses/stars).
the area under the thrombin generation curve. The amount of
thrombin generated in plasma reconstituted with normal FV was
approximately 40% of that formed in plasma reconstituted with FV
Leiden at very low FVII (ⱕ 0.1%), and gradually increased to 86%
at 10% FVII. At 50% FVII (data not shown) the total amount of
thrombin generated in plasma reconstituted with normal FV was
almost the same (95%) as that generated in plasma reconstituted
with FV Leiden.
Discussion
The variable penetrance of genetic defects predicting severe
coagulation factor deficiencies indicates that additional genetic
and/or environmental variability modulates the clinical expression
of these defects. The search for such modifiers has been mainly
targeted to hemophilic disorders, due to their relatively high
prevalence in the population. In the case of hemophilia A, evidence
has been provided that coinheritance of the FV Leiden19,20 or PT
20210G⬎A mutations22 can ameliorate the clinical phenotype,19,20
and that FV Leiden increases thrombin generation as measured in
vitro.35 In addition, the onset of symptoms in children with
hemophilia A was found to be significantly delayed in carriers of
thrombophilic defects such as FV Leiden, PT 20210G⬎A, and
protein C deficiency.21 More recently, a case of severe hemophilia
B with mild clinical expression attributable to carriership of FV
Leiden has also been reported.23
Figure 3. Time course of thrombin generation in simulated patients’ plasma and in normal plasma. (A-B) FV-FVII doubly deficient plasma, to which 0.2% normal plasma
had been added, was reconstituted with purified normal FV (E), 50% normal FV and 50% FV Leiden (F), and FV Leiden (F). Reconstituted plasmas and normal plasma were
triggered with approximately 6.4 ng/mL TF in the absence (A) and in the presence (B) of 20 nM APC, and thrombin generation was followed continuously with a fluorogenic
substrate. (C) Normal plasma triggered in the absence (䉫) and in the presence (䉬) of APC.
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BLOOD, 1 DECEMBER 2003 䡠 VOLUME 102, NUMBER 12
In contrast to hemophilic disorders, which affect the propagation phase of thrombin generation, FVII deficiency impairs the
initiation phase.36 Therefore it is questionable whether thrombophilic mutations that enhance downstream procoagulant reactions
can compensate for the inefficient initiation of coagulation due to a
FVII defect. The FVII Lazio mutation offers a unique opportunity
to investigate the potentially protective role of common thrombophilic mutations in patients with severe FVII deficiency, since (1)
its prevalence in the Italian population is compatible with the
recruitment of homozygous patients; (2) the very low FVII levels
associated with the homozygous state cause a severe but not lethal
bleeding diathesis, which favors the detection of other inherited or
acquired conditions ameliorating the clinical phenotype; and (3)
being a splicing mutation, it results in a very low amount of normal
FVII molecules, a condition that enables “mimicking” in a model
system. The design of a parallel model system would have been
complicated in the case of other mutations causing FVII deficiency
through poor secretion of altered molecules or normal amounts of
dysfunctional molecules.
Our study was based on the comparison of 7 FVII Lazio
homozygous patients who had equally low FVII levels but different
clinical outcomes (Table 1). While 6 of them were severely
affected, the only FV Leiden carrier of the group had been
asymptomatic since the age of 14 years, in spite of repeated
challenges of the hemostatic system. This striking difference in
clinical presentation could not be attributed to possible upregulation of FVII levels by other intragenic functional polymorphisms, because an extended haplotype analysis showed that all
patients had inherited 2 identical copies of the same FVII allele
(Table 2).
The efficiency of the coagulation cascade in the plasma of 2
patients, 1 of whom was the FV Leiden carrier, was evaluated at 3
different levels (FXa, FVa, and thrombin generation) after extrinsic
activation of plasma in the absence and presence of APC. All
activation steps were significantly delayed in the plasma of the 2
FVII-deficient patients, and considerably less FXa and thrombin
were formed than in a normal plasma. Comparing the 2 patients,
coagulation factor activation was more efficient in the plasma of
the FV Leiden carrier, particularly in the presence of APC. Better
discrimination in the presence of APC provides evidence that the
observed differences between the 2 patients are indeed attributable
to FV Leiden. Moreover, comparison between the thrombin
generation curves obtained in the plasmas of the 2 patients
(Figure 2C,F) indicates that relatively small differences in the
amount of thrombin formed may underlie major differences in
clinical phenotype.
The amount of FXa generated in the plasmas of the FVIIdeficient patients appeared to be 2 orders of magnitude lower than
that of FVa on a molar basis, which indicates that FXa is the
limiting component in thrombin formation. The ability of APC to
down-regulate FXa generation in the plasma of the FVII-deficient
patient without the FV Leiden mutation (Figure 2A,D) was an
intriguing finding. A possible explanation for this phenomenon is
PROTECTION BY FV LEIDEN IN SEVERE FVII DEFICIENCY
4019
that FIXa, formed either by the TF/FVIIa complex37 or by the
thrombin feedback on FXI,38 contributes to FXa generation via the
intrinsic coagulation pathway. A contribution of the intrinsic
FX-activating complex would make FXa generation partially
dependent on FVIIIa, which is inactivated by APC, leading to
reduced FXa generation in the presence of APC. Since FV
stimulates APC-mediated FVIIIa inactivation39 and FV Leiden is a
poor APC cofactor,40,41 this hypothesis would also explain the
relative inability of APC to decrease FXa formation in the FV
Leiden carrier. The fact that more FXa is formed in the plasma of
the FV Leiden carrier than in the noncarrier even in the absence of
added APC is probably due to the endogenous APC formed by
activation of plasma protein C.
In a study on hemophilia A it was observed that not all patients
carrying FV Leiden were characterized by a mild clinical course,42
suggesting that FV Leiden alone might not be sufficient to confer
protection against bleeding. In addition, FV Leiden and other
thrombophilic defects were found to be rare or absent in a group of
severe hemophiliacs with a mild bleeding diathesis,43 indicating
that the mild phenotype in these patients was due to other
(unknown) mitigating factors. To demonstrate that FV Leiden
contributes to the increased thrombin generation in patient no. 6,
we performed experiments in FV-FVII doubly deficient plasma
reconstituted with either normal FV or FV Leiden. Since in this
model the FV used for reconstitution is the only variable between
the plasma samples, the experiment presented in Figure 3 clearly
demonstrates that the presence of FV Leiden can account for the
observed differences in thrombin generation in severely FVIIdeficient plasmas, at least in the presence of APC. Moreover,
variation of the FVII concentration in the reconstituted FV-FVII
doubly deficient plasma indicated that the effect of FV Leiden on
thrombin generation is most pronounced at extremely low FVII
levels and gradually disappears as FVII levels increase (Figure 4).
This finding further suggests that the protective effect of FV Leiden
in severe FVII deficiency might be restricted to FVII mutations that
predict unmeasurable FVII levels. Mutation-specific protection
against bleeding by FV Leiden has already been proposed for
hemophilia A.42
Overall, our findings indicate that coinheritance of FV Leiden
leads to enhanced thrombin generation and can mitigate the
bleeding phenotype in patients with severe FVII deficiency due to
homozygosity for the Lazio mutation. In addition, our plasma
model of FVII deficiency shows that the ability of FV Leiden to
increase thrombin generation is maximal at the lowest FVII
concentrations and is lost at higher FVII levels.
Acknowledgments
The authors wish to thank Prof H. C. Hemker and Dr S. Be´ guin
(Synapse b.v., Maastricht, the Netherlands) for helpful suggestions,
as well as Dr T. Lindhout for assistance in the use of the
fluorometer.
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