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Blood, Vol. 91 No. 4 (February 15), 1998:
pp. 1140-1144
Factor V Cambridge: A New Mutation (Arg306 Thr)
Associated With Resistance to Activated Protein C
By
David Williamson,
Karen Brown,
Roger Luddington,
Caroline Baglin, and
Trevor Baglin
From the Department of Haematology, Addenbrooke's NHS Trust,
Cambridge, UK.
 |
ABSTRACT |
A new factor V mutation associated with resistance to activated
protein C and thrombosis (factor V Cambridge, Arg306Thr)
was found in one patient from a carefully selected group of 17 patients
with venous thrombosis and confirmed APC resistance in the absence of
the common Gln506 mutation. The Arg306 mutation
was also present in a first degree relative who also had APC
resistance. Other potential causes of APC resistance, such as a
mutation at the Arg679 site and the factor V HR2 haplotype,
were excluded. Subsequent screening of 585 patients with venous
thromboembolism and 226 blood donors did not show any other individual
with this mutation. Factor VThr306 is the first description
of a mutation affecting the Arg306 APC cleavage site and is
the only mutation, other than factor V Leiden
(Arg506Gln), that has been found in association with APC
resistance. This finding confirms the physiologic importance of the
Arg306 APC-cleavage site in the regulation of the
prothrombinase complex. It also supports the concept that APC
resistance and venous thrombosis can result from a variety of genetic
mutations affecting critical sites in the factor V cofactor.
| |
INTRODUCTION |
IN 1993, A POOR ANTICOAGULANT response to
activated protein C (APC) was reported as a common cause of familial
thrombophilia.1-3 APC resistance is present in 3% to 5%
of asymptomatic Caucasians and is found in approximately 20% of
unselected patients with venous thrombosis.3,4 In at least
95% of cases, resistance to APC is caused by a single point mutation
in the factor V gene.5 A transition (G to A) at nucleotide
1691 in exon 10 results in the synthesis of a variant factor V molecule
(factor V Leiden) with the substitution Arg Gln at amino acid
position 506.6-8 Factor V is converted to the active
cofactor, Va, during assembly of the prothrombinase complex. Thrombin
generation is limited by cleavage of factor Va by APC at
Arg506, followed by cleavage at Arg306 and
Arg679. In vitro experiments have shown that cleavage at
Arg506 has no effect on cofactor activity but is necessary
for exposure of the inactivating cleavage sites, primarily at
Arg306.9-12 The rate of inactivation of factor
VaGln506 is slower than that of factor
VaArg506, resulting in an increased thrombin potential in
vitro13 and an increased risk of thrombosis in
vivo.14
In the 5% to 10% of patients with deep vein thrombosis and APC
resistance without the FVGln506 mutation, APC resistance
can be due to pregnancy,15 lupus anticoagulant activity,16 or high factor VIII levels.17
Patients with the FVGln506 mutation in trans with the HR2
haplotype may have lower APC sensitivity ratios,18 but no
mutation, other than FVGln506, has been identified as a
cause of an APC-resistant factor V protein. The aim of this study was
to identify mutations in the factor V gene sequence encoding the
primary inactivation site at Arg306 in patients with venous
thromboembolism and APC resistance. Patients were selected from a large
cohort with venous thromboembolism on the basis of having confirmed APC
resistance in the absence of the FVGln506 mutation.
| |
MATERIALS AND METHODS |
Patients.
APC sensitivity ratios were measured on plasma samples from 602 patients consecutively investigated after a diagnosis of deep vein
thrombosis or pulmonary embolus. The patients were not taking warfarin
at the time of blood sampling. Deep vein thrombosis was diagnosed by
ultrasound and venography and pulmonary embolus by ventilation-perfusion lung scanning. Four hundred twenty-four patients
had a diagnosis of deep vein thrombosis only and 178 had symptomatic
pulmonary embolism.
Standard APC sensitivity ratio.
Samples were centrifuged twice at 2,500g for 10 minutes and
aliquots of platelet-poor plasma frozen at  80°C until assay. The APC sensitivity ratios were determined after filtration of plasma
through a 0.2-µm syringe filter (Gelman Sciences,
Northampton, UK) with Coatest APC Resistance-C kit
(Chromogenix, Molndal, Sweden). Plasma was incubated with
an equal volume of activated partial thromboplastin time
(APTT) reagent for 5 minutes. Clotting was initiated by
the addition of CaCl2. Clotting times were expressed as a
ratio of the clotting time in the presence of APC divided by the
clotting time in the absence of APC. The plasma filtration method
greatly reduces resistance to APC as a result of platelet phospholipid
contamination and increases the specificity of the assay for true APC
resistance from 32% to 98%.19 In healthy controls, APC
ratios determined by this method are greater than 2.2.19
Modified APC resistance assay.
APC resistance in the presence of factor V-depleted plasma was assessed
using the Coatest APC Resistance-C kit and factor V-depleted plasma
(Chromogenix).20 Plasma was prediluted 1 in 5 with factor
V-depleted plasma and APC sensitivity ratios were determined as in the
standard assay. Modified APC sensitivity ratios were less than 2.0 in
40 patients with the Gln506 mutation who were tested
(range, 1.29 to 1.96) and greater than 2.2 in 40 unselected patients
without the mutation (range, 2.23 to 4.64).
Extended APC resistance assay.
An extended APC resistance assay was performed as initially described
by Dahlback et al.1 Platelet-poor plasma was incubated with
an equal volume of APTT reagent for 5 minutes. Clotting times were
recorded after the addition of CaCl2 supplemented with APC over a final concentration range of 0 to 100 nmol/L (Diagnostica Stago,
Asnières, France).
Natural anticoagulants, lupus anticoagulant activity, and the
FII20210 mutation.
Levels of natural anticoagulants and lupus anticoagulant activity were
measured as previously described, with normal ranges as previously
determined.21 Restriction enzyme analysis with HindIII after DNA amplification using a mutagenic primer was
used to screen samples for the recently reported G to A transition at
position 20210 in the 3 -untranslated region of the prothrombin gene.22,23
Plasma factor V coagulant activity.
Plasma factor V coagulant activity was measured in a
one-stage APTT assay on an MDA180 coagulometer (Organon
Teknika, Cambridge, UK) using factor V-deficient
plasma and Platelin LS phospholipid substitute (Organon
Teknika). In healthy controls, the normal range is 70% to 170%.
Factor VGln506.
Restriction enzyme analysis for detection of the FVGln506
mutation (factor V Leiden) was performed as previously
described.24 A 147-bp fragment encoding the APC cleavage
site was amplified by polymerase chain reaction (PCR), and the DNA
product was digested with Mnl I and analyzed by agarose gel
electrophoresis.
Factor V exon 7 amplification.
A 228-bp DNA fragment containing exon 7 of the factor V gene was
amplified from genomic DNA by PCR. The amplification primers were
synthesized corresponding to intronic sequences upstream (5-TGTCTTTCTGTCCTAAC-3) and downstream
(5-TCTTGAACCTTTGCCCA-3) of the exon 7 sequence. Each
amplification reaction (50 µL) contained 0.5 µg of genomic DNA, 200 µmol/L of each deoxynucleotide triphosphate, 25 pmol of each
amplification primer, and 1.25 U of AmpliTaq DNA polymerase (Perkin
Elmer Cetus, Norwalk, CT) in 10 mmol/L Tris-HCl, 50 mmol/L KCl, pH 8.3, and 1.5 mmol/L MgCl2. The amplification reactions were
performed in a Perkin Elmer Cetus DNA thermal cycler with an initial
denaturation step for 10 minutes at 94°C, followed by 35 cycles
including denaturation at 94°C for 20 seconds, annealing at
42°C for 20 seconds, and extension at 74°C for 20 seconds, with
a final extension for 10 minutes at 74°C. At the end of the reaction, 5 µL of the reaction mixture was analyzed by
electrophoresis on a 1% agarose gel.
DNA sequencing.
The amplified FV exon 7 DNA was directly sequenced by dideoxy
sequencing using the ThermoSequenase radiolabeled terminator cycle
sequencing kit and 33P-labeled dideoxynucleotide
terminators (Amersham Life Science, Bucks, UK). The amplified DNA (5 µL) was pretreated by incubation at 37°C for 15 minutes with 10 U
of exonuclease I and 2 U of shrimp alkaline phosphatase (Amersham Life
Science) before sequencing according to the kit manufacturer's
protocol. A nested sequencing primer was used, corresponding to the
noncoding strand sequence 5-TGGTATGAACCCCAACAA-3.
Restriction enzyme analysis for the factor V Cambridge
(ArgThr) mutation.
Restriction enzyme digestion of the PCR products was performed by
incubating an aliquot of the amplified DNA overnight at 60°C with
10 U of the enzyme BstNI (New England Biolabs [UK] Ltd, Herts, UK) in the presence of 100 µg/mL bovine serum
albumin. The digestion products were analyzed by
electrophoresis on a 2% agarose gel. The normal exon 7 amplified
product has one BstNI restriction site that produces fragments
of 66 and 162 bp after digestion. The factor V Thr306
mutation results in the loss of this site, which results in an uncut
228-bp fragment after digestion.
Factor V exon 13 amplification and DNA sequencing.
A 400-bp fragment of the 3 end of exon 13 encoding the
Arg679 site was amplified with an intronic upstream primer
(5-TGCCACAATGGATTATTGTG-3) and a downstream exonic primer
(5-AGAAACGAATTCAGTGCCAT-3). With the exception of an
annealing temperature of 50°C, PCR conditions were the same as for
exon 7 amplification. A nested sequencing primer, corresponding to the
noncoding strand sequence 5-TAGGGCAGTAAGATTGAACT-3, was
used to sequence codons 631 through 727.
Factor V HR1/HR2 haplotype analysis.
A 420-bp fragment was amplified using exon 13 exonic primers
5-CAGACCTCAGCCATACAA-3 and
5-CTGACTGAGTTTCTGGAGA-3. Each amplification reaction (50 µL) contained 0.5 µg genomic DNA. A total of 200 µmol/L of each
deoxynucleotide triphosphate, 50 pmol of each amplification primer, and
1.25 U AmpliTaq in 10 mmol/L Tris-HCl, 50 mmol/L KCl, pH 8.3, and 1.5 mmol/L MgCl2. Amplification was performed with an initial
denaturation step at 94°C for 10 minutes, followed by 35 cycles of
94°C for 20 seconds, 50°C for 20 seconds, and 74°C for 20 seconds followed by a final extension step at 74°C for 10 minutes.
After amplification, 5 µL of the reaction mixture was analyzed by
electrophoresis in a 1% agarose gel.
Restriction enzyme digestion of the PCR product was performed by
overnight incubation at 37°C with 10 U of Rsa I (New
England Biolabs [UK] Ltd). The digestion products were analyzed by
electrophoresis in a 2% agarose gel. The HR1 haplotype
(His1299) is represented by an undigested fragment of 420 bp. The HR2 haplotype (Arg1299) is represented by fragments
of 121 and 299 bp representing the presence of an Rsa I site.
| |
RESULTS |
Determination of APC resistance and selection of patients.
Samples for sequencing of the factor VArg306 APC-cleavage
site were selected from 602 patients with venous thromboembolism. Four of these patients were homozygous for the Gln506 mutation
and had APC sensitivity ratios between 1.2 and 1.9. A total of 112 patients were heterozygous with APC sensitivity ratios from 1.5 to 3.3 (median, 2.2). Four hundred eighty-six patients had a normal genotype
and ratios of 1.3 to 6.0 (median, 3.4), including 22 who had
sensitivity ratios of less than 2.2. These 22 patients were
investigated further.
Three of the 22 patients had detectable lupus anticoagulant activity
and free protein S levels were low in 2 others (9% and 25%; normal,
>55%). The remaining 17 patients had normal levels of antithrombin,
protein C, and free protein S; no detectable lupus anticoagulant
activity; and the prothrombin gene 20210G/A mutation was not present.
Plasma from these patients was therefore subjected to the modified APC
resistance assay after dilution in factor V-deficient plasma and exon 7 of the factor V gene sequenced to identify any mutations affecting the
Arg306 APC-cleavage site.
Of the 17 selected patients, 6 had a modified APC sensitivity ratio of
less than 2.0, despite the absence of the Gln506 mutation.
DNA sequencing and restriction enzyme analysis.
One of the 17 selected patients was found to be heterozygous for a
mutation (G to C) changing the codon for amino acid 306 from AGG
(arginine) to ACG (threonine) (Fig 1).
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| Fig 1.
Sequencing of amplified factor V exon 7 DNA from the
patient and a normal control. The mutation (G to C) is present at codon 306, resulting in an arginine306threonine
substitution in the factor V protein (factor V Cambridge).
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The new G to C mutation at codon 306 removes a recognition site for the
restriction enzyme BstNI. The 228-bp amplified fragment from a
normal FV gene has a single cutting site, resulting in fragments of 162 and 66 bp after enzyme digestion. As expected, digestion of exon 7 DNA
from the patient with the Thr306 mutation showed bands of
228, 162, and 66 bp arising from the products of the mutant and normal
alleles (patient II.1 in Fig 2).
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| Fig 2.
Family study of factor V Cambridge pedigree. The standard
and modified APC sensitivity ratios and BstNI restriction
enzyme digestion of exon 7 DNA are shown. Lane 1, DNA size markers.
Lanes 2 through 5, BstNI digestion of DNA from family members.
Lane 6, undigested exon 7 DNA fragment.
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Restriction enzyme analysis using BstNI was performed on the
remaining patient population. A normal digest pattern was detected in
all 601 patients, including the 16 selected patients with APC resistance in the absence of the Gln506 mutation. In
addition, BstNI digestion of DNA from 226 anonymous blood
donors was normal.
Family study.
The index patient (II.1, Fig 2) with the factor V Cambridge mutation
(Arg306Thr) is a 49-year-old man who developed a
spontaneous proximal deep vein thrombosis at the age of 47 years. He
was treated with unfractionated heparin for 5 days and warfarin (target
INR 2.5) for 6 months. He has now been off anticoagulant therapy
without recurrence of venous thromboembolism for 2 years. His only
living relatives are a 44-year-old sister and his mother and father, aged 73 and 74 years, respectively, none of whom have suffered a
thrombotic event. Blood samples were obtained from the family members
for DNA analysis and phenotypic study.
The patient's mother was also found to have the
Arg306Thr mutation (I.2, Fig 2). His father and
sister did not have the mutation. The Arg679 site was
sequenced in all four family members and was normal. Plasma factor V
levels were normal in all four (94% to 134%) and all were homozygous
for the HR1 haplotype.
The patient and his affected sister had APC resistance in both standard
and modified APC resistance assays, whereas the unaffected father and
sister were not APC resistant (Fig 2). The APC resistance phenotype of
the patient and his affected mother was the same as that found in
patients with the Gln506 mutation
(Fig 3).
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| Fig 3.
(Top panel) The effect of added APC on the APTT of plasma
from the index patient with factor V Cambridge (Thr306;
 ) compared with patients heterozygous ( ) and homozygous ( ) for
the Gln506 mutation and a normal control
(Arg506;  ). (Bottom panel) APC resistance of family
members with (I.2 [] and II.1 [ ]) and without (I.1 []
and II.2 [ ]) the Thr306 mutation.
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The patient's mother has not been exposed to any high-risk situtation
for thrombosis other than two pregnancies.
| |
DISCUSSION |
We have described a new factor V mutant (factor V Cambridge,
Arg306Thr) associated with APC resistance and
thrombosis. It is, to date, the only mutation other than factor V
Leiden associated with this phenomenon and the first description of a
mutation affecting the Arg306 APC cleavage site. The degree
of APC resistance associated with Thr306 is the same as
that due to the Gln506 mutation.
Thrombin generation is dependent on assembly of the prothrombinase
complex on a phospholipid surface. The activated form of factor V
(factor Va) is a component of the complex and enhances the rate of
formation of thrombin several thousand fold.25 Cleavage of
factor Va by activated protein C limits prothrombinase activity and
hence thrombin generation. Inactivation of factor Va occurs via
sequential cleavage by APC at Arg506, followed by
Arg306 and Arg679 with loss of cofactor
activity. Cleavage at Arg306 is responsible for the loss of
approximately 70% of this activity, whereas cleavage at
Arg679 is responsible for the loss of approximately
30%.9,10,12,26 In vitro, cleavage at Arg506
has no direct effect on cofactor activity, but cleavage at this site is
necessary for exposure of the inactivating cleavage sites at
Arg306 and Arg679. The rate of inactivation of
factor VaGln506 (factor V Leiden) is slower than that of
normal factor VaArg506. On incubation with APC, factor
VaArg506 is completely inactivated after 5 minutes, whereas
factor VaGln506 retains approximately 50% of its initial
activity.11 At low concentrations, the difference in APC
inactivation of the mutant and wild-type proteins is even more
pronounced.27 The mutant protein is associated with an
increased thrombin potential in vitro,13 an eightfold
increased risk of deep vein thrombosis in heterozygotes, and a 50- to
100-fold increased risk in homozygotes.14
Egan et al28 have recently studied factor V inactivation
using site-directed mutagenesis to produce mutant recombinant factor V
proteins. They have confirmed the importance of the Arg306
site by demonstrating that a recombinant factor V molecule with a
mutated 306 site (replacement of arginine by alanine or glutamine) is
inactivated at a slower rate than plasma or recombinant wild-type factor V and at a similar rate to recombinant
FVaGln506.28 However, APC cleavage patterns of
factor V with a threonine substitution at the 306 site have not yet
been reported. Despite the presumed physiologic importance of cleavage
at Arg306, no mutation affecting this site has been
identified until now. We used a rigorous screening strategy to identify
patients with venous thromboembolism who showed APC resistance in the
absence of the factor V Leiden mutation. In a large cohort of patients, we used an optimized standard APC sensitivity assay and a ratio of less
than 2.2 as a high predictive value for the Gln506
mutation.19 Patients with this degree of APC resistance in the absence of the Leiden mutation were therefore identified as candidates for other mutations. Because APC resistance has been demonstrated in the presence of lupus anticoagulant activity and thrombotic events may have been due to other forms of thrombophilia, only patients with isolated APC resistance were investigated further. Sequencing exon 7 of the factor V gene showed a normal sequence in all
but one of the selected patients. This patient was found to have a G to
C mutation altering the amino acid codon 306 from AGG (arginine) to ACG
(threonine). This mutation would remove the primary APC
cleavage-inactivation site from the mutant protein. Other potential
causes of APC resistance, such as a mutation at the Arg679
site and the factor V HR2 haplotype, were excluded. APC resistance was
also present in the modified APC resistance assay after dilution in
factor V-deficient plasma, confirming that the phenotype is due to a
defect in factor V and not in another protein such as factor VIII.
Conclusive evidence of the relationship between the Thr306
mutation and APC resistance will require purification of the mutant
protein and analysis of the APC cleavage pattern. Nevertheless, the
presence of this mutation in a family with APC resistance is further
evidence of the physiologic importance of the Arg306
cleavage site. It seems likely in the absence of any other form of
thrombophilia or predisposing high-risk situation that the mutation and
the associated APC resistance was the cause of deep vein thrombosis in
the index patient.
In selecting patients, the APC sensitivity ratio of 2.2 was chosen for
its high specificity for the Gln506 mutation, but the
sensitivity associated with this cut-off is only 50%. It is possible
that some patients with a ratio greater than 2.2, who were not selected
by our screening strategy, could have the Thr306 mutation
and it is possible that patients with the Gln506 mutation
could also have the Thr306 mutation, particularly those
with very low APC sensitivity ratios. However, we screened the whole
patient cohort and did not find any other affected patient. Similarly,
the mutation was absent in 226 blood donors. Therefore, the
Thr306 mutation is not a common polymorphism and it is a
rare cause of APC resistance. However, the finding confirms the
physiologic importance of the Arg306 APC-cleavage site in
the regulation of the prothrombinase complex. It also supports the
concept that APC resistance and venous thrombosis can result from a
variety of genetic mutations affecting critical sites in the factor V
cofactor.
| |
FOOTNOTES |
Submitted October 30, 1997;
accepted November 21, 1997.
Address reprint requests to Trevor Baglin, FRCP,
Department of Haematology, Addenbrooke's NHS Trust, Cambridge CB2 2QQ,
UK.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. section 1734 solely
to indicate this fact.
| |
ACKNOWLEDGMENT |
The authors are grateful to Prof Robin Carrell for review of this
manuscript.
| |
REFERENCES |
1.
Dahlback B,
Carlsson M,
Svensson P:
Familial thrombophilia due to a previously unrecognised mechanism characterised by poor anticoagulant response to activated protein C: Prediction of a cofactor to activated protein C.
Proc Natl Acad Sci USA
90:1004,
1993[Abstract/Free Full Text]
2.
Griffin J,
Evatt B,
Wideman C,
Fernandez J:
Anticoagulant protein C pathway is defective in majority of thrombophilic patients.
Blood
82:1989,
1993[Abstract/Free Full Text]
3.
Koster T,
Rosendaal F,
de Ronde H,
Briet E,
Vandenbrouke J,
Bertina R:
Venous thrombosis due to poor anticoagulant response to activated protein C: Leiden Thrombophilia Study.
Lancet
342:1503,
1993[Medline]
[Order article via Infotrieve]
4.
Svensson P,
Dahlback B:
Resistance to activated proteion C as a basis for venous thrombosis.
N Engl J Med
330:517,
1994[Abstract/Free Full Text]
5.
Zoller B,
Svensson P,
He X,
Dahlback B:
Identification of the same factor V gene mutation in 47 out of 50 thrombosis-prone families with inherited resistance to activated protein C.
J Clin Invest
94:2521,
1994
6.
Bertina R,
Koeleman B,
Koster T,
Rosendaal F,
Dirven R,
de Ronde H,
van der Velden P,
Reitsma P:
Mutation in blood coagulation factor V associated with resistance to activated protein C.
Nature
369:64,
1994[Medline]
[Order article via Infotrieve]
7.
Voorberg J,
Roelse J,
Koopman R,
Buller H,
Berends F,
Ten Cate J,
Mertens K,
Van-Mourik J:
Association of idiopathic venous thromboembolism with single point-mutation at Arg506 of factor V.
Lancet
343:1535,
1994[Medline]
[Order article via Infotrieve]
8.
Greengard J,
Sun X,
Xu X,
Fernandez J,
Griffin J,
Evatt B:
Activated protein C resistance caused by Arg506Gln mutation in factor Va.
Lancet
343:1361,
1994[Medline]
[Order article via Infotrieve]
9.
Kalafatis M,
Rand M,
Mann K:
The mechanism of inactivation of human factor V and human factor Va by activated protein C.
J Biol Chem
269:31869,
1994[Abstract/Free Full Text]
10.
Kalafatis M,
Bertina R,
Rand M,
Mann K:
Characterisation of the molecular defect in factor V R506Q.
J Biol Chem
270:4053,
1995[Abstract/Free Full Text]
11.
Heeb M,
Kojima Y,
Greengard J,
Griffin J:
Activated protein C resistance: Molecular mechanisms based on studies using purified Gln506-factor V.
Blood
85:3405,
1995[Abstract/Free Full Text]
12.
Griffin J,
Heeb J,
Kojima Y,
Fernandez J,
Kojima K,
Hackeng T,
Greengard J:
Activated protein C resistance: Molecular mechanisms.
Thromb Haemost
74:444,
1995[Medline]
[Order article via Infotrieve]
13.
Nicolaes G,
Thomassen M,
Tans G,
Rosing J,
Hemker H:
Effect of activated protein C on thrombin generation and on the thrombin potential in plasma of normal and APC-resistant individuals.
Blood Coagul Fibrinol
8:28,
1997[Medline]
[Order article via Infotrieve]
14.
Rosendaal F,
Koster T,
Vandenbrouck J,
Reitsma P:
High risk of thrombosis in patients homozygous for factor V Leiden (activated protein C resistance).
Blood
85:1504,
1995[Abstract/Free Full Text]
15.
Cumming A,
Tait R,
Fildes I,
Yoong A,
Keeney S,
Hay C:
Development of resistance to activated protein C during pregnancy.
Br J Haematol
90:725,
1995[Medline]
[Order article via Infotrieve]
16.
Ehrenforth S,
Radtke K,
Scharrer I:
Acquired activated protein C-resistance in patients with lupus anticoagulant.
Thromb Haemost
74:797,
1995[Medline]
[Order article via Infotrieve]
17.
Henkens C,
Bom V,
Van der Meer J:
Lowered APC-sensitivity ratio related to increased factor-VIII clotting activity.
Thromb Haemost
74:1198,
1995[Medline]
[Order article via Infotrieve]
18.
Bernardi F,
Faioni E,
Castoldi E,
Lunghi B,
Castaman G,
Sacchi E,
Mannucci P:
A factor V genetic component differing from factor V R506Q contributes to the activated protein C resistance phenotype.
Blood
90:1552,
1997[Abstract/Free Full Text]
19.
Luddington R,
Brown K,
Baglin T:
Effect of platelet phospholipid exposure on activated protein C resistance: Implications for thrombophilia screening.
Br J Haematol
92:744,
1996[Medline]
[Order article via Infotrieve]
20.
Jorquera J,
Montoro J,
Fernandez M,
Aznar JA,
Aznar J:
Modified test for activated protein C resistance.
Lancet
344:1162,
1994[Medline]
[Order article via Infotrieve]
21.
Lee L,
Jennings I,
Luddington R,
Baglin T:
Markers of thrombin and plasmin generation in patients with inherited thrombophilia.
J Clin Pathol
47:631,
1994[Abstract/Free Full Text]
22.
Poort S,
Rosendaal F,
Reitsma P,
Bertina R:
A common genetic variation in the 3-untranslated region of the prothrombin gene is associated with elevated prothrombin levels and an increase in venous thrombosis.
Blood
88:3698,
1996[Abstract/Free Full Text]
23.
Brown K,
Williamson D,
Luddington R,
Baker P,
Baglin T:
Risk of venous thromboembolism associated with a G to A transition at position 20210 in the 3-untranslated region of the prothrombin gene.
Br J Haematol
98:907,
1997[Medline]
[Order article via Infotrieve]
24.
Beauchamp N,
Daly M,
Hampton K,
Cooper P,
Preston F,
Peake I:
High prevalence of a mutation in the factor V gene within the UK population: Relationship to activated protein C resistance and familial thrombosis.
Br J Haematol
88:219,
1994[Medline]
[Order article via Infotrieve]
25.
Rosing J,
Tans G,
Govers Riemslag J,
Zwaal R,
Hemker H:
The role of phospholipids and factor Va in the prothrombinase complex.
J Biol Chem
255:274,
1980[Abstract/Free Full Text]
26.
Aparicio C,
Dahlback B:
Molecular mechanisms of activated protein C resistance.
Biochem J
313:467,
1996
27.
Nicolaes G,
Tans G,
Thomassen C,
Hemker H,
Pabinger I,
Varadi K,
Schwarz H,
Rosing J:
Peptide bond cleavages and loss of functional activity during inactivation of factor Va and factor VaR506Q by activated protein C.
J Biol Chem
270:21158,
1995[Abstract/Free Full Text]
28.
Egan J,
Kalafatis M,
Mann K:
The effect of Arg306Ala and ARg506Gln substitutions in the inactivation of recombinant human factor Va by activated protein C and protein S.
Prot Sci
6:2016,
1997[Abstract]
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2546 - 2553.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
K. R. Viel, D. K. Machiah, D. M. Warren, M. Khachidze, A. Buil, K. Fernstrom, J. C. Souto, J. M. Peralta, T. Smith, J. Blangero, et al.
A sequence variation scan of the coagulation factor VIII (FVIII) structural gene and associations with plasma FVIII activity levels
Blood,
May 1, 2007;
109(9):
3713 - 3724.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
E. A. Norstrom, S. Tran, M. Steen, and B. Dahlback
Effects of Factor Xa and Protein S on the Individual Activated Protein C-mediated Cleavages of Coagulation Factor Va
J. Biol. Chem.,
October 20, 2006;
281(42):
31486 - 31494.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
R. L. Bick
Hereditary and Acquired Thrombophilic Disorders
Clinical and Applied Thrombosis/Hemostasis,
April 1, 2006;
12(2):
125 - 135.
[PDF]
|
|
|
|
|
|
|
B. S. Donahue
The Response to Activated Protein C After Cardiopulmonary Bypass: Impact of Factor V Leiden
Anesth. Analg.,
December 1, 2004;
99(6):
1598 - 1603.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
N. Sayinalp, I. C. Haznedaroglu, S. Aksu, Y. Buyukasik, H. Goker, H. Parlak, O. I. Ozcebe, S. Kirazli, S. V. Dundar, and A. Gurgey
The Predictability of Factor V Leiden (FV:Q506) Gene Mutation via Clotting-Based Diagnosis of Activated Protein C Resistance
Clinical and Applied Thrombosis/Hemostasis,
July 1, 2004;
10(3):
265 - 270.
[Abstract]
[PDF]
|
|
|
|
|
|
|
B. S. Donahue
Factor V Leiden and Perioperative Risk
Anesth. Analg.,
June 1, 2004;
98(6):
1623 - 1634.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
E. Castoldi, J. M. Brugge, G. A. F. Nicolaes, D. Girelli, G. Tans, and J. Rosing
Impaired APC cofactor activity of factor V plays a major role in the APC resistance associated with the factor V Leiden (R506Q) and R2 (H1299R) mutations
Blood,
June 1, 2004;
103(11):
4173 - 4179.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Steen, E. A. Norstrom, A.-L. Tholander, P. H. B. Bolton-Maggs, A. Mumford, J. H. McVey, E. G. D. Tuddenham, and B. Dahlback
Functional characterization of factor V-Ile359Thr: a novel mutation associated with thrombosis
Blood,
May 1, 2004;
103(9):
3381 - 3387.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. van der Neut Kolfschoten, R. J. Dirven, H. L. Vos, G. Tans, J. Rosing, and R. M. Bertina
Factor Va Is Inactivated by Activated Protein C in the Absence of Cleavage Sites at Arg-306, Arg-506, and Arg-679
J. Biol. Chem.,
February 20, 2004;
279(8):
6567 - 6575.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
D. Scanavini, D. Girelli, B. Lunghi, N. Martinelli, C. Legnani, M. Pinotti, G. Palareti, and F. Bernardi
Modulation of Factor V Levels in Plasma by Polymorphisms in the C2 Domain
Arterioscler. Thromb. Vasc. Biol.,
January 1, 2004;
24(1):
200 - 206.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
B. A. Kerlin, S. B. Yan, B. H. Isermann, J. T. Brandt, R. Sood, B. R. Basson, D. E. Joyce, H. Weiler, and J.-F. Dhainaut
Survival advantage associated with heterozygous factor V Leiden mutation in patients with severe sepsis and in mouse endotoxemia
Blood,
November 1, 2003;
102(9):
3085 - 3092.
[Abstract]
[Full Text]
[PDF]
|
|
|
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Abstract
Introduction
Abstract