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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 91 No. 12 (June 15), 1998:
pp. 4593-4599
The Structure and Function of Murine Factor V and Its Inactivation by
Protein C
By
Tony L. Yang,
Jisong Cui,
Alnawaz Rehumtulla,
Angela Yang,
Micheline Moussalli,
Randal J. Kaufman, and
David Ginsburg
From the Departments of Human Genetics, Radiation Oncology,
Biological Chemistry, and Internal Medicine, University of Michigan
Medical School, Ann Arbor, MI; and the Howard Hughes Medical Institute,
University of Michigan, Ann Arbor, MI.
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ABSTRACT |
Factor V (FV) is a central regulator of hemostasis, serving both as
a critical cofactor for the prothrombinase activity of factor Xa and
the target for proteolytic inactivation by the anticoagulant, activated
protein C (APC). To examine the evolutionary conservation of FV
procoagulant activity and functional inactivation by APC, we cloned and
sequenced the coding region of murine FV cDNA and generated recombinant
wild-type and mutant murine FV proteins. The murine FV cDNA encodes a
2,183-amino acid protein. Sequence comparison shows that the A1-A3 and
C1-C2 domains of FV are highly conserved, demonstrating greater than
84% sequence identity between murine and human, and 60% overall amino
acid identity among human, bovine, and murine FV sequences. In
contrast, only 35% identity among all three species is observed for
the poorly conserved B domain. The arginines at all thrombin cleavage
sites and the R305 and R504 APC cleavage sites (corresponding to amino
acid residues R306 and R506 in human FV) are invariant in all three
species. Point mutants were generated to substitute glutamine at R305, R504, or both (R305/R504). Wild-type and all three mutant FV
recombinant proteins show equivalent FV procoagulant activity. Single
mutations at R305 or R504 result in partial resistance of FV to APC
inactivation, whereas recombinant murine FV carrying both mutations
(R305Q/R504Q) is nearly completely APC resistant. Thus, the structure
and function of FV and its interaction with APC are highly conserved
across mammalian species.
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INTRODUCTION |
HUMAN FACTOR V (FV) is synthesized as a
single-chain precursor glycoprotein of 2,224 amino acids (aa),
consisting of a 28-aa N-terminal signal peptide, followed by the
2,196-aa mature protein.1,2 Mature FV is composed of
internally repeated homologous domains A (A1, A2, A3) and C (C1, C2),
and a nonrepeated B domain, organized in the order A1, A2, B, A3, C1,
and C2.1-3 FV is activated to FVa by thrombin cleavage at
three residues (R709, R1018, and R1545), removing most of the B
domain.4-7 The resulting noncovalent heterodimer is
composed of a heavy-chain (residues 1-709) and a light-chain (1546-2196) held together by divalent cation-dependent
interactions.1,5,8-11 The function of the B domain remains
unclear, although it may be involved with facilitating FV activation by
thrombin.12
FVa is a critical protein in the coagulation cascade. It is an
essential cofactor for factor Xa (FXa), together forming the prothrombinase complex which, in the presence of calcium and a phospholipid surface, efficiently converts prothrombin to active thrombin.13 FVa is also a proteolytic target for activated
protein C (APC).14,15 APC exerts its anticoagulant function
through its inactivation of FVa and factor VIIIa
(FVIIIa).16-20 In humans, APC inactivates FVa to FVi via
proteolytic cleavage at R306, R506, and R679 in the FVa heavy
chain.14,15,21,22 An initial rapid cleavage at R506
facilitates the otherwise slow cleavage reaction at R306. Cleavage at
R306 is associated with the complete loss of FVa
activity.23 Although cleavage at R506 does not result in
complete loss of FVa activity, the 10-fold reduction in APC inactivation of the mutant form of FVa, R506Q, results in a higher risk
for thrombotic disorders.24-26
APC resistance is a very frequent finding in patients with thrombotic
disorders.27-29 The R506Q mutation in human FV accounts for
nearly all individuals with APC resistance, with an allele frequency of
2% to 7% in European populations,30 making it one of the
most common genetic risk factors for thrombosis.25,31 Cosegregation of the R506Q mutation increases the penetrance of thrombosis in protein C-deficient32 and protein
S-deficient33 patients.
Analysis of human1 and bovine34 FV cDNAs shows
a high degree of sequence conservation. Sequence data has not yet been reported for any other mammalian species. To develop an animal model
for the study of FV function in vivo and to examine the evolutionary
conservation of FV procoagulant activity and functional inactivation by
APC, we have cloned and sequenced the coding region of the murine FV
cDNA and analyzed FV mutations at the putative APC cleavage sites. The
murine FV cDNA shows a high degree of evolutionary conservation.
Recombinant murine FV proteins carrying one or both of two mutations at
the APC cleavage sites (R305Q and R504Q, homologous to R306Q and R506Q
in human FV) maintain normal procoagulant function. However, mutant
murine FV proteins carrying either R305Q or R504Q are partially
resistant to APC inactivation, whereas the double-mutant is markedly
resistant to APC.
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MATERIALS AND METHODS |
Chemicals and reagents.
The mouse C57BL/6J bone marrow (BM) cDNA library was a gift of J. Lowe
(University of Michigan, Ann Arbor) and the mouse Sv129 genomic library
was purchased from Stratagene (La Jolla, CA). The Muta-Gene in vivo
mutagenesis kit and Affi-Gel 10 affinity matrix were purchased from
Bio-Rad (Hercules, CA). OPTI MEM I media and Trizol for total RNA
isolation were from GIBCO-BRL (Gaithersburg, MD). Normal and
FV-deficient human plasmas were purchased from George King Bio-Medical,
Inc (Overland Park, KS). Thromboplastin (with 25 mmol/L calcium) was
obtained from Sigma (St Louis, MO). Human thrombin was purchased from
Calbiochem (San Diego, CA). APC was from Enzyme Research Labs Inc
(South Bend, IN), and phospholipid vesicles (Inosithin) were a gift of
P.J. Fay (University of Rochester, Rochester, MD). FV dilution buffer
is from Pharmacia Heper (Franklin, OH). Precharged Ni2+
affinity purification matrix was bought from Invitrogen (Carlsbad, CA),
as ProBond. Nitrocellulose membrane (BA85) was purchased from
Schleicher & Schuell (Keene, NH).
Cloning of murine FV genomic DNA.
Two  phage spanning exons 7 to 13 were cloned as described
previously.35 The intron/exon junctions were determined by
DNA sequencing using primers based on the murine FV cDNA sequence. Introns were amplified by polymerase chain reaction (PCR) and their
size determined by comparison to a commercial 1-kb DNA ladder (GIBCO-BRL) separated by agarose gel electrophoresis. A 790-bp fragment
of the murine FV cDNA containing the 3 end of the coding sequence and
including 13 bp beyond the termination codon was used as a probe to
screen the same mouse genomic library. Two additional phage were
isolated, and a portion of their sequence beyond the FV stop codon was
determined (sequence identical for both clones).
Cloning of the murine FV cDNA.
A total of 5 × 106 clones from a C57BL/6J BM cDNA
library, in plasmid pCDM8, was screened by standard
methods36 using the human FV cDNA as probe. Seven unique
cDNA clones were identified (mFV1-7, Fig
1). Additional sequence corresponding to
the B domain was obtained from a 4.7-kb genomic Nco I fragment
(designated as "exon 13" in Fig 1). An additional 275 bp from the
A2 domain was obtained by reverse transcriptase (RT)-PCR amplification
of BM and liver RNA templates obtained from a C57BL/6J X DBA mouse. Total RNA was isolated from mouse liver and BM using Trizol, according to manufacturer's instructions. First-strand cDNA synthesis was performed with avian myeloblastosis virus (AMV) RT and a
primer complementary to sequence in exon 13 (5-CTGGAGAAAGGGACACC-3) at 42°C for 1 hour, followed by PCR amplification using primers in
exons 7 (5-TCATAGCCGCAGAGGAGGTCA-3) and 12 (5-ATGGGGAACAGGGTCAAGGTG-3). BM and liver experiments were performed
independently; two BM- and three liver-derived clones were sequenced
and found to be identical (clone labeled "PCR," Fig 1). The
overlapping clones are shown in Fig 1. The entire cDNA sequence was
determined on both strands.
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| Fig 1.
Schematic of murine FV cDNA and protein structure. The
top bar represents the murine FV protein with the domain boundaries numbered above. The two putative APC cleavage sites at R305 and R504 in
murine FV are indicated by arrows. FV domains are labeled as A1-A3, B,
and C1-C2. The FV coding sequence (lower bar) was cloned from a
C57BL/6J BM cDNA library using the human FV cDNA as probe. The seven
unique cDNAs identified are shown as single lines and labeled mFV1-7.
The clone labeled "PCR" indicates the DNA fragment obtained
through RT-PCR, and "exon 13" is the segment obtained from a
genomic library. The major restriction enzyme sites used in the
assembly are labeled on the top of the bar [Sac I (781),
Apa I (1258), Pst I (1834), Nde I (2073),
Kpn I (3976)]. Cla I and Sal I cut the vector
sequences adjacent to the FV cDNA.
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Site-directed mutagenesis.
A Sac I-Pst I fragment (nucleotides 781-1834, Fig 1) of
the murine FV cDNA, containing both putative APC cleavage sites (R305 and R504), was cloned into pSELECT-1 (Promega, WI) and the mutations R305Q, R504Q, or both mutations in cis were introduced by
site-directed mutagenesis following the manufacturer's instructions.
The mutagenesis oligonucleotides were
5-CCAAAGAAAACGCA(AG)GAGCCCCAAGACC-3 (R305Q) and
5-CCTGGACCAGCA(AG)GGGTGTACAG-3 (R504Q), in which the
underlined nucleotides represent the mutations, with the native
sequence in parentheses. Presence of appropriate mutations was
confirmed by DNA sequencing.
Assembly of the wild-type and mutant FV cDNAs.
The full-length FV cDNA was assembled in an eight-step procedure from
five different clones into the expression vector pCMV537 as
a Cla I-Sal I fragment (Fig 1). The plasmid vector
pCMV5 contains the cytomegalovirus promoter, and the human growth
hormone (hGH) polyadenylation signal. The 5 end of the FV cDNA to the
Apa I site (1-1263) was derived from clone mFV1; the second
fragment, Apa I-Pst I (1263-1839), is from clone
"PCR"; the third fragment, Pst I-Nde I
(1839-2078), is from clone mFV2; the fourth fragment, Nde
I-Kpn I (2078-3981), is from the exon 13 genomic clone; and the
last fragment, from Kpn I to the 3 end (3981-6585), is taken from clone mFV3 (Fig 1). The mutations were first introduced into the
Sac I-Pst I fragment (786-1839, Fig 1), and both
wild-type and mutant cDNAs were then assembled in parallel. The
integrity of junction regions in each construct was confirmed by DNA
sequencing.
Transient transfection of COS-1 cells.
COS-1 cells grown in a 100-mm plate were transfected with 10 µg of
the appropriate wild-type or mutant construct by calcium phosphate
precipitation,38 and grown for 24 hours before 3 mL serum-free OPTI-MEM I media were added and then grown for another 48 hours before harvesting.
FV clotting assay.
Conditioned media were procured, diluted in dilution buffer, and
assayed for FV activity. Samples (50 µL) were mixed 1:1 with human
FV-deficient plasma, and warmed at 37°C for 3 minutes. Prewarmed thromboplastin with 25 mmol/L CaCl2 (100 µL) was then
added, and the time to clot formation was measured in a Medical
Laboratory Automation (Pleasantville, NY) Electra 750 coagulation timer. A standard curve was generated using
dilutions of pooled normal human plasma.
APC resistance assay.
Wild-type and mutant FV conditioned media were obtained as described
above, and concentrated threefold to fivefold using Centricon-30 concentrators (Amicon, Beverly, MA) to yield preparations with 500 to
1,000 mU/mL FV activity and activated using thrombin (1 U/mL). Complete
activation was usually obtained within 5 minutes, at which time
(designated t = 0) 0.1 µg/mL human APC, 100 µg/mL phospholipid
vesicles, and 5 mmol/L CaCl2 were added to initiate FVa
inactivation by APC. Samples were taken at various time points, diluted
50-fold in 50 mmol/L Tris (pH = 7.3), 0.2% bovine serum albumin, and
immediately assayed for FV activity. FV activity was determined as
described above except that 100 µL of the sample was incubated with
100 µL of human FV-deficient plasma and 100 µL thromboplastin for 3 minutes, then 100 µL of 25 mmol/L CaCl2 was added and the
time to form a clot was determined.
Generation of polyclonal rabbit antimurine FV heavy-chain
antibodies.
Murine FV cDNA encoding amino acids  27 to 662 (includes the signal
peptide and heavy chain) was amplified by PCR and cloned into the
vector pGEX-KG (kindly provided by J.E. Dixon, University of Michigan,
Ann Arbor).39 The corresponding Escherichia
coli-expressed insoluble GST-fusion protein (GST-mFV-H) was first
washed in 2 mol/L guanidine-HCl, 50 mmol/L Tris-Cl, pH 6.8, 5 mmol/L
EDTA, 1 mmol/L phenylmethylsulfonyl fluoride, and solubilized in the same solution containing 3 mol/L guanidine-HCl. GST-mFV-H was then
precipitated by dilution to 0.3 mol/L guanidine-HCl, washed twice, and
separated by electrophoresis on a 3%/8% sodium dodecyl sulfate
(SDS)-polyacrylamide gel.40 The portion of gel containing GST-mFV-H was excised, lyophilized, and used as the immunogen for the
generation of rabbit antisera by a commercial supplier (Rockland,
Gilbertsville, PA). A second murine FV heavy-chain fusion protein
(Thx-mFV-H) containing amino acids 12 to 662 was expressed in the
vector pET-32a (Novagen, Madison, WI), solubilized in 8 mol/L urea, 500 mmol/L NaCl, 20 mmol/L Tris-Cl, pH 7.9, purified on Ni2+
affinity matrix, and coupled to Affi-Gel 10 affinity purification matrix according to the manufacturer's instructions. Crude rabbit antiserum was affinity purified, eluted with 20 mmol/L citrate buffer,
pH 2.5, and neutralized by NaOH.
Analysis of APC cleavage of murine FV.
Serum-free conditioned media from COS-1 cells transfected with
wild-type or mutant murine FV expression plasmids were procured and
treated first with thrombin (1 U/mL) for 5 minutes at 37°C, and then
with APC (0.1 µg/mL) plus phospholipid vesicles (100 µg/mL) and 5 mmol/L CaCl2 for 25 minutes at 37°C. Samples were separated by electrophoresis on an 8% SDS-polyacrylamide gel. Western
blotting was performed as previously described41 with the
affinity-purified polyclonal antimurine FV heavy-chain antibody. Mouse
anti-rabbit IgG conjugated to horseradish peroxidase was purchased from
Accurate (Westbury, NY), and the ECL chemiluminescence kit from
Amersham (Arlington Heights, IL).
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RESULTS AND DISCUSSION |
The intron-exon junction sequences in the murine FV gene.
The genomic structure of murine FV exons 7-13 was determined from two
previously identified SV129 genomic clones.35 The intron-exon junction sequences have been deposited in GenBank (GenBank
Accession No. AF040572-AF040577). Complete conservation of exon
structure is observed in this region, compared with the human
gene.42 As in the human, the entire mouse FV B domain is
contained within a single exon (exon 13).
Analysis of the murine FV cDNA coding sequence.
The complete cDNA sequence for the murine FV coding region (6,552 bp)
was determined on both strands (GenBank Accession No. U52925). The
sequence in the vicinity of the initiation codon fits the consensus
described by Kozak.43 A consensus polyadenylation signal
(AATAAA) is found 39 bp following the termination codon (determined
from the genomic sequence, and confirmed by RNA PCR).
The murine FV cDNA encodes a 2,183-aa protein, which includes a 28-aa
signal peptide, followed by the mature protein of 2,155 aa. Alignment
of the predicted aa sequence for murine FV with the human1
and bovine34 sequences shows that the functionally important A1-A3 and C1-C2 domains are highly conserved (>84%
sequence identity between murine and human, and 60% overall amino acid identity among the human, bovine, and murine FV sequences) (Fig 2). In contrast, only 35% identity among
all three species is observed for the poorly conserved B domain. A
similar high degree of interspecies divergence has been noted for the
FVIII B domain, which is also poorly conserved in homology comparison
between FV and FVIII. These data have suggested that the B domain may serve primarily a spacer function, with little selective pressure to
conserve a specific aa sequence. Consistent with this hypothesis, human
FV and FVIII lacking B domain sequences retain procoagulation activity.44,45
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| Fig 2.
FV amino acid sequence comparison. The human FV amino
acid sequence ("Human") and the bovine FV amino acid sequences
("Bovine") were compared pairwise against the murine FV sequence.
A multiple comparison of all three sequences simultaneously was also
done ("Mult"). All comparisons were performed by the MegAlign
computer program (DNASTAR, Madison, WI), using the Clustal
method. The numbers on the top of the bar indicate the
domain boundaries in murine FV, deduced from sequence comparison with
the human and bovine proteins. Values for each domain are the
percentage amino acid identity, with the percentage amino acid
similarity in parentheses.
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The murine FV protein is 41 aa shorter than human FV. This difference
is due primarily to a relative deletion within the murine B domain. The
human FV B domain contains two tandem repeats of a 17-aa sequence and
31 tandem repeats of a 9-aa motif. The murine sequence has only one
17-aa repeat, and is missing 9 aa corresponding to parts of the 26th
and 27th 9-aa repeat, as well as 11 aa immediately preceding the first
repeat. Differences in these repeat sequences account for nearly all of
the variation in length (37 of 41 aa) between human and murine FV.
Bovine FV contains only one 17-aa repeat and 29 copies of the 9-aa
motif.34
A number of distinct structural features noted in the human FV B
domain1 are also conserved in the mouse. Human FV contains 37 potential N-linked glycosylation sites, with 25 located in the B
domain. By comparison, murine FV contains 27 potential N-glycosylation sites, with 17 located in the B domain similar to that of bovine FV (28 total and 18 in the B domain). Interspecies variation among the B
domain 9-aa repeat motifs accounts for the greater number of potential
N-linked glycosylation sites in human FV relative to mouse or bovine
FV. Sixteen of the potential N-linked glycosylation sites in murine FV
(9 in the B domain) are conserved in both human and bovine sequences.
Thus, the B domain appears to be a consistent site for extensive
N-linked glycosylation, a processing step that may be required for
efficient secretion of FV.46
All cysteine residues predicted to form disulfide linkages in bovine FV
are conserved in murine FV as well as in human FV, as are the three
predicted free cysteines located one each in the A2, C1, and C2
domains.47,48 Murine FV, like bovine FV, lacks the single
free cysteine in the human FV B domain.1 Based on the
conservation between the species, this would predict disulfide linkages
in murine FV between Cys138-Cys164, Cys219-Cys300, Cys470-Cys496, Cys573-Cys654, Cys1657-Cys1683, Cys1838-Cys1992, and Cys1997-Cys2152. Residues Cys537, Cys583, and Cys1919 are predicted to be free cysteines.
The arginine residues at all thrombin and APC cleavage sites are
conserved between human, murine, and bovine FV, except at the R679 APC
cleavage site (Fig 3). Bovine FV is cleaved
instead at R662,49 where human FV has a proline at the
corresponding position. Interestingly, murine FV has arginine at both
positions (R656, corresponding to bovine R662, and two consecutive
arginines, R678 and R679, corresponding to human R679). From our
present data we are unable to precisely determine at which homologous position APC cleaves murine FV. The sequences surrounding the cleavage
sites are also highly conserved between species. In addition, the
sequence in the vicinity of the R506 APC cleavage site is well
conserved in comparison between the homologous segments of FV and
FVIII.
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| Fig 3.
Cross species alignment of FV sequences in the vicinity
of (A) human APC cleavage sites R306 and R506; (B) thrombin cleavage sites; and (C) the acidic region at the C-terminus of the heavy chain,
including the human APC cleavage site R679. Cleavage sites are numbered
according to either the human (hFV) or bovine (bFV) protein, as
indicated in parentheses. The " " indicates the cleavage site
in both (A) and (B) at the conserved underlined arginine residue. The
third APC cleavage site (C) varies between the human and bovine
proteins, as indicated. Only the sequence surrounding the FV APC
cleavage site R506 is conserved between FV and FVIII; all other APC and
thrombin cleavage sites fail to show significant homology across these
two proteins (not shown).
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Three of the seven tyrosine sulfation sites predicted in human
FV50 are conserved in murine FV. These are Tyr664, Tyr697,
and Tyr1470, corresponding to Tyr665, Tyr698, and Tyr1510 of human
FV.1 The same three tyrosine residues and surrounding
acidic residues are also conserved in bovine FV.34 Tyrosine
sulfation of FV appears to be required for efficient activation by
thrombin.51 The serine phosphorylation site corresponding
to Ser690 of bovine FV is not present in murine FV.52
Procoagulant activity of the wild-type and mutant FV proteins.
Plasmid expression vectors carrying wild-type murine FV cDNA, and
mutants at the R305 and R504 APC cleavage sites (R305Q and R504Q) as
well as the double mutant (R305Q/R504Q), were transiently transfected
into COS-1 cells, and the FV procoagulant activity in the conditioned
media measured by reconstitution of human FV-deficient plasma. Using
normal human plasma as standard, the conditioned media from wild-type
and all mutants contained similar FV clotting activities (ranging from
450 mU/mL to 650 mU/mL). Thus, introduction of these mutations does not
appear to affect FV procoagulant function, consistent with observations
for the corresponding human native and recombinant FV
proteins.21,53-55 This observation also shows that murine
FV efficiently complements human FV function in plasma.
APC resistance of wild-type and mutant FV.
The recombinant murine FV proteins were assayed for susceptibility to
APC inactivation using standard methods (see Materials and Methods).
Conditioned media were concentrated and then activated by thrombin
before addition of APC. Wild-type recombinant murine FV was completely
inactivated by t = 5 minutes (Fig 4). In
contrast, introduction of a single mutation at R305Q or R504Q results
in partial resistance to APC. The double mutant (R305Q/R504Q) is markedly resistant to APC inactivation, retaining ~70% of its initial peak activity at t = 25 minutes (Fig 4). The observed ~30%
decrease in activity in the R305Q/R504Q double-mutant could be
explained by either cleavage at the COOH terminus of the FVa heavy
chain similar to the R679 cleavage in humans22,23,53-56 or
by inhibition of FV activation due to binding of APC to the APC-resistant mutant.57
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| Fig 4.
APC resistance of recombinant wild-type and mutant murine
FV. Each point represents the average of the activity measured in conditioned media from three independent transfections. APC resistance assays were performed as described in the experimental procedures. APC
was added to the reactions at t = 0. ( ), Wild-type FV; ( ), FV
R305Q; ( ), FV R504Q; ( ), FV R305Q/R504Q.
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Because murine FV was not recognized by available anti-human FV
antibody reagents (data not shown), we generated an affinity-purified rabbit polyclonal anti-murine FV heavy-chain antibody (see Materials and Methods). This antibody detects intact single-chain mouse FV, and
FV heavy-chain in both conditioned media from transfected COS-1 cells
and mouse plasma by Western blot analysis, but does not recognize the
light chain (Fig 5). To assess the
fragments resulting from APC cleavage of murine FV, conditioned media
were reacted with human thrombin and APC (for 25 minutes) and the
products were analyzed by Western blot (Fig 5). Intact single-chain FV (~300 kD) is evident in unreacted conditioned media for wild-type and
all the mutant FVs. After the addition of thrombin, identical ~110-kD
heavy-chain fragments are generated from the wild type and all three
mutants. After treatment with APC, an ~50-kD band is seen in both
wild-type and the R504Q mutant, consistent with the expected N-terminal
fragment resulting from cleavage at R305, and associated with complete
loss of FV cofactor activity (Fig 4). The antibody appears to recognize
epitopes restricted to the N-terminal segment (residues 1-305) of the
FV heavy chain, explaining the failure to visualize the C-terminal
cleavage fragments. The ~70-kD fragment generated by APC cleavage of
the R305Q mutant is consistent with cleavage at R504 and the complete
loss of the second N-terminal cleavage as a result of the R305Q
mutation. Cleavage by APC of the R305Q/R504Q mutant generates a large
~105-kD fragment, consistent with the loss of both heavy-chain
cleavages. The doublet of this large fragment is consistent with slow
cleavage at the third APC cleavage site (within residues 657-709 comprising the acidic domain). This cleavage could be responsible for
the observed ~30% loss of FV cofactor activity (Fig 4).
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| Fig 5.
APC cleavage of recombinant wild-type and mutant murine
FV. Western blot analysis was performed using a rabbit anti-murine FV
antibody which recognizes an epitope(s) on the N-terminal half of the
FV heavy chain (residues 1-305). Lanes A through E are untreated
conditioned media, lanes F through J are following activation with
thrombin, and lanes K through O are following thrombin activation and
subsequent treatment with APC. Lanes A, F, and O are conditioned media
from mock-transfected cells; lanes B, G, and K, from cells transfected
with wild-type FV; lanes C, H, and L, from cells transfected with R305Q
mutant FV; lanes D, I, and M, from cells transfected with R504Q mutant
FV; lanes E, J, and N, from cells transfected with R305Q/R504Q mutant
FV.
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APC resistance and FV evolution.
Interest in FV function has increased recently as a result of the
identification of a common sequence variation in the human FV gene,
which appears to be a major risk factor for thrombosis. Substitution of
glutamine for arginine 506, resulting in a mutant FV that is partially
resistant to APC inactivation, has been identified in as many as 7% of
normal individuals in some populations (FV Leiden).30 This
common mutation is associated with a marked increase in the risk of
thrombosis.25,31,58 Despite this frequent variation in
humans, our studies confirm the maintenance of very similar FV
structure and function across species. Although previous studies have
suggested fundamental differences in the interaction of human APC with
protein S and FV from other species including rats,59 our
data are consistent with conservation of the FV/APC interaction among
mammalian species. Variation in carbohydrate structure, which has
recently been shown to modulate FV inactivation by APC,60
may also explain some previously observed differences in the
interaction between FV and APC across species. The location of APC
cleavage sites is conserved, and resistance to APC similar to that
observed in human FV Leiden is obtained with a mutation at the
homologous position in murine FV. In addition, the functional assays
performed in human plasma indicate that FV procoagulant functions is
also highly conserved.
Taking all of these observations together, it is perhaps surprising
that mutation at the critical R506 APC cleavage site should be
tolerated at the high population frequency observed for the human R506Q
mutation, given the negative selection that would be anticipated. These
observations suggest that a significant recent change in hemostatic
balance may have decreased dependence on APC function in humans
compared with other mammals. Alternatively, a recent, as yet
unidentified positive selection in humans for the mutant R506Q allele
may counteract the strong negative selection that would appear to have
been present previously in evolution. Recent genetic evidence of a
founder effect for this mutation further supports this latter
hypothesis.61 The high degree of structural and functional
conservation between human and murine FV in both procoagulant and APC
anticoagulant function suggests that the mouse should be an appropriate
model in which to study FV function in vivo. The mouse may also provide
the necessary in vivo tools to identify the positive selection
responsible for the high population frequency of the human FV R506Q
mutation.
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FOOTNOTES |
Submitted August 27, 1997;
accepted February 9, 1998.
Supported by National Institutes of Health Grants No. 1P01HL57346-01A1
(D.G.), HL39639 (D.G.), HL52173 (R.J.K.), and HL53777 (R.J.K.).
Address reprint requests to David Ginsburg, MD, The University of
Michigan Medical School, 4520 MSRB I, 1150 W Medical Center Dr, Ann
Arbor, MI 48109-0650.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
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Abstract
Introduction
Abstract