From the
Department of Chemistry, Cleveland State
University, Cleveland, Ohio 44115 and the
Department of Molecular Cardiology, Lerner
Research Institute, Cleveland Clinic Foundation, Cleveland, Ohio 44195
Received for publication, January 9, 2003 , and in revised form, May 6, 2003.
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ABSTRACT |
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INTRODUCTION |
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Human factor V circulates in plasma as a single chain glycoprotein of
Mr 330,000 consisting of multiple domains
A1-A2-B-A3-C1-C2, at a concentration of 20 nM
(1518).
The factor V gene is 80 kb in length and contains 24 introns, whereas the mRNA
is 6.9 kb long (19). Single
chain factor V does not appear to interact with factor Xa and to have cofactor
activity. Factor V is cleaved sequentially by
-thrombin at
Arg709, Arg1018, and Arg1545 to produce the
active cofactor, factor Va, that consists of a heavy chain
(Mr 105,000) and a light chain (Mr
74,000). The heavy chain derives from the NH2-terminal part of
factor V (A1-A2 domains, residues 1709), whereas the light chain
represents the COOH-terminal end of the procofactor (A3-C1-C2 domains,
residues 15462196)
(17). The heavy and light
chains are noncovalently associated via divalent metal ions
(20). Activation of the
procofactor by
-thrombin is required for expression of its cofactor
activity (i.e. interaction with factor Xa and prothrombin). Both
chains of the cofactor are required for the high affinity interaction with
factor Xa
(2123).
The data accumulated thus far indicate that the binding of factor Va and/or
factor Xa to the lipid bilayer most likely results in conformational changes
in one or both proteins that contribute to aspects of the factor Va-factor Xa
interaction. Factor Va and factor Xa interact stoichiometrically in the
presence and absence of phospholipids. In the absence of phospholipids, the
Kd for the interaction is 0.8 µM
and is dependent upon the presence of Ca2+ ions
(24). Upon binding of both
proteins to lipids, the Kd of the interaction
decreases by
1000-fold to 1 nM
(4,
5), suggesting that multiple
points of contact between the two proteins are exposed, resulting in the
tighter interaction. Because of the physiological concentration of the two
proteins in plasma, the only physiologically relevant
Kd for the interaction between the two proteins
is the Kd observed in the presence of a membrane
surface. Factor Va is inactivated by activated protein C
(APC),1 following
cleavage at Arg506, Arg306, and Arg679 only
in the presence of a membrane surface
(25,
26). Cleavage of factor Va by
APC at Arg506 results in a 10-fold decrease in the affinity of the
molecule for factor Xa and is required for efficient cleavage at
Arg306
(2527).
Subsequent cleavage at Arg306, which is lipid-dependent and is the
inactivating cleavage site, completely abolishes the ability of the cofactor
to interact with factor Xa because of the dissociation of the heavy chain
fragments from the light chain, hence eliminating the factor Vafactor Xa
interaction (27). Thus,
whereas activation of factor V by
-thrombin allows for proper
interaction of factor Va with factor Xa, APC inhibits this interaction by
eliminating the binding site(s) of the cofactor for the enzyme. These combined
data suggest that the regulation of normal hemostasis and control of
thrombosis is exerted following the activation and inactivation processes of
the cofactor, which ultimately and explicitly control its interaction with
factor Xa. As a consequence, a complete understanding of prothrombinase
assembly and function, which is a prototype for most membrane-bound complexes
contributing to the blood clotting processes, necessitates the identification
of the distinct amino acids from each protein responsible for their
interaction as well as a complete understanding of the macromolecular
interactions that occur between them.
We have shown that a synthetic peptide containing a sequence of amino acids from the middle portion of factor Va heavy chain (amino acid residues 307348; N42R) (every peptide is identified by the first and last amino acids, with the number of residues composing the peptide in the middle) inhibits factor Va cofactor activity (28). We have recently demonstrated, using overlapping peptides, that a nonapeptide from N42R containing amino acid sequence 323331 of factor Va (AP4'), is the active inhibitory portion of N42R and represents a direct binding site for factor Xa (29). Both peptides, N42R and AP4', also produced a "cofactor" effect on factor Xa, increasing the catalytic efficiency of the enzyme by 21- and 4-fold, respectively, as compared with factor Xa alone (29). The present study was undertaken to identify the amino acid residues within AP4' that are responsible for its biological activities.
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EXPERIMENTAL PROCEDURES |
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Assay Measuring Thrombin FormationThe formation of thrombin
was analyzed using the fluorescent thrombin inhibitor DAPA as described
(25,
26,
29,
39) using a PerkinElmer LS-50B
Luminescence Spectrometer with ex = 280 nm,
em = 550 nm, and a 500-nm-long pass filter in the emission
beam (Schott KV-500). The buffer used in all cases was composed of 20
mM Hepes, 0.15 M NaCl, 5 mM CaCl2,
pH 7.4 (HBS(Ca2+), "assay buffer"). The
final concentration of factor Va in the mixture was 4 nM, with
factor Xa at 10 nM. Under these conditions, the rate of thrombin
formation is linearly related to the amount of active cofactor (factor Va).
The initial rate of thrombin formation (nM IIa/min) was calculated
as described (25,
26,
29). The concentration of
peptide given in each figure is the final concentration of the peptide in the
assay mixture. All data were initially analyzed and stored using the software
FL WinLab (PerkinElmer Life Sciences) provided by the manufacturer and further
analyzed and plotted with the software Prizm (GraphPad, San Diego, CA).
Fluorescence Anisotropy MeasurementsFluorescence anisotropy
of [OG488]-EGR-hXa was measured using a PerkinElmer LS-50B
Luminescence Spectrometer in L format as recently described
(29). Anisotropy measurements
were performed in a quartz cuvette under constant stirring (low) with
ex = 490 nm and
em = 520 nm with a long
pass filter (Schott KV-520) in the emission beam. At each addition, anisotropy
was measured for 20 s and averaging eight successive readings. In all cases,
the total addition of peptide did not exceed 10% of the volume of the
reaction. The concentration of peptide given in each graph is the final
concentration of the peptide in the assay mixture. All data were initially
analyzed and stored using the software FL WinLab (PerkinElmer Life Sciences)
and further analyzed and plotted with the software Prizm (GraphPad). Some of
the data were also plotted using DeltaGraph (DeltaPoint, Monterey, CA).
Mutagenesis and Transient Expression of Recombinant Factor V
MoleculesThe complete 6.909-kb-long cDNA sequence for factor V
containing a leader sequence (from 91 to 174 bp) that encodes the signal
peptide was inserted into the mammalian expression vector pMT2 at
SalI sites. Mutant molecules were designed by keeping in mind that in
order to positively identify the amino acids responsible for the effector and
receptor properties of factor Va, we have to express recombinant factor V
molecules with these specific amino acids mutated in such a manner that the
entire conformation of the recombinant factor V molecules possesses a tertiary
conformation as close as possible to the wild type factor Va molecule. For
these reasons, we have made three conservative and one nonconservative
mutation. As conservative substitutions, phenylalanine was substituted for a
tyrosine in order to test the function of the hydroxyl present on the later
amino acid. Methionine has a similar core structure as glutamic acid; however,
the carboxyl group of the latter is replaced by a thioether group in the
former. Although valine and isoleucine are both hydrophobic in nature, the
structure of the side chain of the latter is significantly different from the
structure of the side chain of the former. Finally, we have previously
observed that the presence of a glutamic acid at position 323 was a
prerequisite for optimum inhibitory potential of AP4'
(29). We have thus performed a
nonconservative mutation by replacing the carboxyl group of glutamic acid with
a phenyl group found in phenylalanine to eliminate any potential contribution
of this hydrophilic amino acid to the interaction with factor Xa. PCR-based
site-directed mutagenesis was used for constructing the factor V mutants
(i.e. pMT2-FV (Glu323 Phe/Tyr324
Phe, factor VFF), pMT2-FV (Glu330
Met/Val331
Ile, factor VMI), and pMT2-FV
(Glu323
Phe, Tyr324
Phe, Glu330
Met, Val331
Ile, factor ValFF/MI)). The
mutagenic primers for the mutant Glu323
Phe/Tyr324
Phe were
5'-GAAGAGGTGGTTCTTCTTCATTGCTGC-3' (sense)
and 5'-GCAGCAATGAAGAAGGAACCACCTCTTC-3'
(antisense), whereas for the mutant Glu330
Met/Val331
Ile the primers were
5'-CATTGCTGCAGAGATGATCATTTGGGACTATGC-3' (sense) and
5'-GCATAGTCCCAAATGATCATCTCTGCAGCAATG-3' (antisense)
(underlined letters indicate the mismatch). The outer PCR primers were
5'-ACATCCACTACCGCAATATGAC-3' (sense) and
5'-CCTCAGGCAGGAACAACACCATGA-3' (antisense), corresponding to
nucleotide positions 964979 (upstream of a XcmI restriction
site) and 14811504 (downstream of a Bsu361 site), respectively
(according to Jenny et al.
(17); GenBankTM accession
number M16967
[GenBank]
). Each site-directed mutagenesis was carried out by three-step
PCRs. The first two PCRs were performed using the sense outer primer and
antisense mutagenic primers or antisense outer primer and sense mutagenic
primers. The complete DNA fragment removed from the pTM2-FV wild type sequence
by digesting with SalI was used as a template for the PCR. The third
reaction was performed using outer sense and antisense primers and the
purified PCR products of the first two reactions as templates. All PCRs were
performed with high fidelity Taq polymerase. PCRs were programmed as
follows: denaturation at 94 °C for 15 s, annealing at 55 °C for 30 s,
and extension at 68 °C for 1 min/kb in a GeneAmp PCR system 9700 DNA
thermal cycler (PerkinElmer Life Sciences). The amplicons of each third
reaction were ligated into the cloning plasmid vector pGEM-T, and the
integrity of the constructs was verified following sequencing using an ABI
Prism automatic DNA sequencer and the universal primers T7 and SP6 as well as
other factor V sequence-specific primers as needed at the sequencing facility
at the Cleveland Clinic Foundation. The quadruple mutant pMT2-FV
(E323F/Y324F/E330M/V331I) was made using the pGEM-T plasmid vector containing
the DNA insert of factor VFF as template and the mutagenic primers
used for factor VMI. The inserts (nucleotides 9641504) were
removed from the pGEMT-FV plasmid constructs (mutant fragments) following
digestion with XcmI and Bsu361 restriction enzymes.
Following purification of the inserts from the agarose gel, the factor V
inserts that possess the mutations (nucleotide positions 9641504) were
religated into pMT2-FV, in which the DNA fragment between the XcmI
and Bsu361 restriction sites was removed. The ligated plasmids were
transformed into JM109 bacterial competent cells. Positive
ampicillin-resistant clones for pMT2-FV mutants were selected. The correct
sequences and orientations of the inserts were established by DNA sequence
analysis with factor V-specific primers. The wild-type pMT2-FV and mutant
pMT2-FV plasmids were isolated from the bacterial culture by the QIAfilter
plasmid Midi kit (Qiagen Inc., Valencia, CA).
Recombinant Factor V-Wild Type and Factor V Mutant Transfection into COS-7 CellsCOS-7 cells (ATTC) or COS-7L, (Invitrogen) were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 2 mM L-glutamine, and antibiotics (100 µg/ml streptomycin and 100 IU/ml penicillin) in a humidified atmosphere of 5% CO2 and 95% air at 37 °C. The purified plasmids pMT2-FV wild type, pMT2-FV(E323F/Y324F), pMT2-FV(E330M/V331I), and pMT2-FV(E323F/Y324F/E330M/V331I) were used to transfect into COS-7/COS-7L cells as follows. Each plasmid (12 µg) was transfected into 5080% confluent COS-7/COS-7L cells in a 100-mm culture plate using lipid-based transfection reagent FUGENE 6 (Roche Diagnostics) according to the manufacturer's instructions. Following 48 h of transfection, the medium was removed. Cells were washed twice with serum-free medium, and 610 ml of conditioned medium VP-SFM supplemented with 4 mM of L-glutamine was added. After growing the cells in the conditioned medium for 24 h, medium was harvested, and 610 ml of fresh condition medium was added. After growing the cells for another 24 h, the media were harvested again. The harvested medium containing recombinant factor V was briefly centrifuged at 4,500 rpm at 4 °C to remove insoluble particles. All control media and solutions containing the recombinant factor V molecules were concentrated using centrifugal ultrafiltration (Centricon YM 30,000; Millipore Corp.), washed with 20 mM Tris, 0.15 M NaCl, 5 mM CaCl2, pH 7.4, following repeated concentration steps, and stored on ice at 4 °C. The activity and the integrity of the molecules were verified before and after thrombin activation by clotting assays using factor V-deficient plasma and by Western blotting using the appropriate monoclonal and polyclonal antibodies. The recombinant molecules were stable under these conditions for at least 1 week. The concentrations of the recombinant molecules were determined by an ELISA developed in our laboratory and described below. The values found in our ELISA were similar to the concentration of recombinant factor V determined using the commercially available factor V ELISA kit (Affinity Biologicals, Hamilton, Ontario, Canada). It is noteworthy that in all experiments performed herein, before concentration of the recombinant molecules, the double mutant (factor VFF/MI) was found to be consistently secreted in lower concentrations than the wild type procofactor molecule or the single mutants (i.e. the mutants with only two amino acids mutated at the time, factor VFF or factor VMI). However, its concentration was normalized and adjusted in all prothrombinase assays. Thus, all recombinant molecules were assayed under similar experimental conditions.
Determination of the Concentration of the Recombinant Factor V
MoleculesThe concentration of factor V was determined with an
ELISA. 20 µl of polyclonal anti-human factor V (10 mg/ml) was diluted into
20 ml of coating buffer (0.077 M NaHCO3, 0.007
M Na2CO3, pH 9.5). A 96-well microtiter plate
(Costar, Corning Glass) was loaded with 200 µl/well of the polyclonal
antibody mixture and incubated overnight at 4 °C. The final concentration
of the antibody in each well was 10 µg/ml. The plate was washed three times
with 25 mM Tris, 0.15 M NaCl, pH 7.4, 0.05% Tween 20
(TBS-Tween, washing buffer), following by incubation with 100 µl/well of 5%
nonfat dry milk in TBS-Tween at 37 °C for 1 h. Following extensive
washing, the plate was loaded (100 µl/well) with either serial dilutions of
purified plasma factor V in VP-SFM medium with 4 mM L-glutamine
within the range of 20 µg to 5 ng of factor V in triplicate or with various
volumes of recombinant factor V solutions in triplicate. Normal plasma factor
V was diluted in the medium in order to rule out the possibility that the cell
culture media would interfere with the ELISA assay. Following incubation at 37
°C for 1 h, the plate was washed three times followed by incubation with
HFVHC 17 at 5 µg/ml final concentration (per well) for 1
h at 37 °C. Following extensive washing, a 1:2000 dilution of goat
anti-mouse antibody coupled to peroxidase was loaded on the wells (100
µl/well), and incubation was allowed to proceed at 37 °C for an
additional 1 h. The plates were developed with OPD tablets (5 mg), which were
dissolved in 0.1 M citric acid, 0.1 M
Na2HPO4 (12 ml). Immediately prior to use, 12 µl of
30% H2O2 was added to the OPD solution (developing
solution), and 100 µl was applied per well. Following incubation at room
temperature, the reaction was stopped with 150 µl of 1.5 M
H2SO4. The absorbance at 490 nm was monitored using an
Amersham Biosciences THERMOMAX microplate reader. Because of slight
differences in time of incubation with the substrate, in every experiment a
plasma factor V standard (serial dilutions of purified plasma factor V) was
run, and all values obtained with the recombinant molecules were compared with
the plasma factor V standard values within the same 96-well plate. No
comparison in concentration was made between recombinant molecules from one
plate and the other. The determination of the concentration of the recombinant
molecules was performed by averaging the value found for each sample run in
triplicate.
Measurement of Rates of -Thrombin Formation in a
Prothrombinase AssaySince even after concentration, the
recombinant molecules resulting from the transient expression were obtained in
limited amounts, cofactor activity measurements using DAPA were not possible.
The activity of all recombinant factor V molecules was thus measured in a
discontinuous assay as follows. All factor V species were activated with 10
nM thrombin for 20 min followed by the addition of DFP (5
mM). The factor Va solution was then incubated for an additional 30
min on ice. Control experiments demonstrated that under these conditions, no
interference of the DFP with the assay could be observed, since DFP is readily
hydrolyzed in aqueous solution. Assay mixtures contained PCPS vesicles (20
µM), DAPA (3 µM), various recombinant factor Va
species (0.5 nM), and prothrombin (1.4 µM) in 20
mM HEPES, 0.15 M NaCl, 5 mM CaCl2,
pH 7.4. The mixture was allowed to incubate at ambient temperature for 5 min,
and the reaction was initiated by the addition of factor Xa (5 nM).
Aliquots of the reaction mixture were removed at various time intervals, as
indicated in the figures, and diluted 3-fold in 20 mM HEPES, 0.15
M NaCl, 50 mM EDTA, 0.1% polyethylene glycol 8000, pH
7.4, in a 96-well microtiter plate (Costar, Corning Glass) to quench the
reaction. The formation of
-thrombin in each sample was monitored using
the chromogenic substrate Spectrozyme TH (0.4 mM, final
concentration). The change in the absorbance at 405 nm was monitored using the
Amersham Biosciences THERMOMAX microplate reader. The initial rate of
-thrombin generation under these conditions is linear, and in all
experiments no more than 10% of prothrombin was consumed during the initial
course of the assay. An excess of the specific
-thrombin inhibitor DAPA
was included in all experiments to prevent potential feedback reactions
catalyzed by
-thrombin that is generated during the assay. All data
were analyzed with the software Prizm (GraphPad). Some of the data were also
plotted using DeltaGraph (DeltaPoint).
Gel Electrophoresis and Western BlottingSDS-PAGE analyses were performed using 515% gradient gels according to the method of Laemmli (40). When necessary, proteins were visualized after staining with Coomassie Brilliant Blue. In several experiments, proteins were transferred to polyvinylidene difluoride (PVDF) membranes (Millipore Corp.) according to the method described by Towbin et al. (41). After transfer to nitrocellulose, factor V heavy and light chain(s) were detected using the appropriate monoclonal and polyclonal antibodies (3134). Immunoreactive fragments were visualized with chemiluminescence.
Analysis of the Inactivation of Membrane-bound Factor Va (with or
without EGR-hXa)The inactivation of factor Va by APC was studied
in the presence or absence of synthetic peptides. EGR-hXa was preincubated
with peptides and added to purified membrane-bound factor Va. The final
concentrations of all reagents were as follows: factor Va, 20 nM;
EGR-hXa, 50 nM; PCPS vesicles, 5 µM; pentapeptides,
200 µM; AP4', 8 µM; A9A, 8 µM.
The reaction was initiated by the addition of APC (5 nM). Samples
of the mixture were withdrawn at selected time intervals (indicated in the
legends to the figures), mixed with 2% SDS, and heated for 5 min at 90 °C
prior to analysis by gel electrophoresis. Following transfer to PVDF
membranes, factor Va-immunoreactive fragments were visualized with monoclonal
antibody HVaHC 17 and chemiluminescence.
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RESULTS |
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These peptides were first assayed for inhibition of prothrombinase function. Under the conditions employed, all three peptides showed various degrees of inhibition, with E5A being more potent at low concentrations, whereas A5V was more potent at high concentrations of peptide (Fig. 2, dark filled bars). Thus, as previously suggested, the pentapeptides contain amino acids that all appear to contribute to the inhibitory potential of AP4' (29). Experiments using the control peptides demonstrated that replacing Glu323 and Tyr324 by 2 phenylalanines resulted in diminished inhibitory potential of F5A (Fig. 2, hatched bars, 100 and 300 µM), whereas replacing Glu330 and Val331 by methionine and isoleucine, respectively, resulted in a diminished inhibitory potential of A5I (Fig. 2, hatched bar, 500 µM). However, both control peptides had an inhibitory effect on prothrombinase, albeit less potent than their parent peptides. The open bars in Fig. 2 depict the positive and negative control peptides AP4' and A9A previously characterized (29).
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We have shown that AP4' interacts with [OG488]-EGR-hXa
with half-maximal saturation of 65 µM
(29). We have thus tested the
three pentapeptides for their ability to interfere with the fluorescence
anisotropy of membrane-bound [OG488]-EGR-hXa in the absence of
factor Va (Fig. 3). The data
show that increasing concentrations of A5V produce a significant decrease
(quench) in the fluorescent anisotropy of [OG488]-EGR-hXa
(Fig. 3, filled inverted
triangles), demonstrating direct interaction between the peptide and
[OG488]-EGR-hXa. Half-maximal saturation was reached at
150
µM peptide. A control pentadecapeptide (P15H) previously shown
to have no effect on prothrombinase assembly and function
(29) produced a
20%
decrease in the anisotropy of [OG488]-EGR-hXa when used at 300
µM (not shown). Peptide E5A had a similar effect on the
anisotropy of [OG488]-EGR-hXa (
22% decrease in the anisotropy,
assuming that the decrease observed in the presence of A5V is the maximum
possible effect) (Fig. 3,
filled diamonds). Under similar experimental conditions, and in the
presence of 400 µM peptide F5E, there was a 42% decrease in the
anisotropy of [OG488]-EGR-hXa
(Fig. 3, filled
triangles). This effect of F5E remained constant even at concentrations
as high as 700 µM peptide. The data indicate that A5V alone
appears to contain most of the amino acids that are responsible for the direct
interaction between [OG488]-EGR-hXa and AP4'. However, an
additional contribution of 3 amino acids common to F5E and A5V is also
apparent. No significant effect of the control peptide A5I was observed on the
fluorescent anisotropy of [OG488]-EGR-hXa (the decrease in the
anisotropy observed was similar to that of a control peptide inducing
20%
change in the fluorescent anisotropy) (Fig.
3, filled squares). Overall, these data point to two
amino acids, Glu330 and Val331, within the sequence of
AP4' that appear to be largely responsible for the decrease of the
anisotropy of [OG488]-EGR-hXa when incubated with the peptide. It
is noteworthy that since all three peptides inhibit prothrombinase, they most
likely interact with factor Xa; however, only A5V appears to contain amino
acids capable of producing a significant decrease of the fluorescent
anisotropy of [OG488]-EGR-hXa.
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High concentrations of AP4' and N42R were shown to have a cofactor
effect on factor Xa (29). To
understand which amino acids from AP4' are responsible for this effect,
we assayed the pentapeptides for their ability to increase the catalytic
efficiency of factor Xa during activation of prothrombin in the absence of
factor Va. Incubation of E5A (100 µM) with factor Xa resulted in
a modest but significant and reproducible increase of the activity of factor
Xa with respect to prothrombin activation (150%) when compared with the
activity of factor Xa alone (not shown). No increase in the activity of factor
Xa was observed in the presence of any of the other peptides shown in
Fig. 1. Thus, amino acids
Glu323 and Tyr324 may be responsible for the cofactor
effect of AP4' on factor Xa
(29). Overall, these data
demonstrate that Glu323, Tyr324, Glu330, and
Val331, which represent amino acids located at the extremities of
AP4', are crucial for the inhibitory potential of the peptide on both
prothrombinase assembly and function. These amino acids may be thus required
for expression of factor Va cofactor function.
Elimination of the Protective Effect of Factor Xa during Membrane-dependent Inactivation of Factor Va by APCIt has been established that factor Xa protects factor Va from inactivation by APC (4244). APC inactivation of factor Va following cleavage of the membrane-bound cofactor at Arg506/Arg306 and generation of the Mr 30,000 fragment (containing amino acid residues 307506) is fast and occurs within 1 min (Fig. 4A) (26). Following preincubation of the cofactor with EGR-hXa, the appearance of the Mr 30,000 fragment of factor Va is considerably delayed (Fig. 5B, lanes 28), and an increase in the concentration of an Mr 60,000/54,000 doublet is observed (Fig. 4B, lanes 26). This doublet represents initial cleavage of the cofactor at Arg306 (26, 31). Our data are in agreement with a previous report showing that upon incubation of the membrane-bound cofactor with EGR-hXa, the rate of cleavage at Arg506 is considerably impaired (44). Altogether, these data suggest that upon interaction with the membrane-bound cofactor, the EGR-hXa molecule impairs cleavage by APC at Arg506 by hindering access of APC to specific amino acids that are required for its interaction with factor Va and subsequent catalysis. Thus, by preincubating the cofactor with EGR-hXa, we are creating an artificial and transient factor VLEIDEN molecule (31). However, since the factor Va molecule still possesses an arginine at position 506, inactivation of the molecule will still proceed but with the order of cleavages reversed; the first cleavage at Arg306 will be followed by a slow cleavage at Arg506 and generation of the Mr 30,000 fragment. As a consequence, the appearance of the Mr 30,000 will be delayed, whereas the Mr 60/54,000 doublet will be more pronounced compared with the cleavage of factor Va in the absence of EGR-hXa (Fig. 4B, lanes 28). These data verify the crucial importance of prior cleavage at Arg506 for cleavage at Arg306 and rapid inactivation of the cofactor as previously suggested (26, 31, 32). These data also demonstrate that we can distinguish between the binding of EGR-hXa to the membrane-bound cofactor or not by the absence or presence, respectively, of the Mr 30,000 fragment (at the beginning of the reaction). Our previous (29) and present data show that a factor Xa binding site is contained within amino acid residues 323331 of factor Va heavy chain. Thus, we can formulate the hypothesis that upon preincubation of EGR-hXa with a peptide from factor Va that contains a binding site for factor Xa, binding to the cofactor will be prevented, resulting in the elimination of the protective effect of EGR-hXa from APC inactivation. As a consequence, membrane-bound factor Va will be rapidly cleaved by APC with the appearance of the Mr 30,000 fragment. The data resulting from such an experiment are illustrated in Fig. 5. To simplify the figure and for the easy analysis of the data, we have focused on the portion of the gels around the region showing the Mr 30,000 fragment resulting from cleavage of factor Va heavy chain at Arg506/Arg306. The data only show the progress of the reaction through the first 10 min.
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Under the conditions employed, upon preincubation of EGR-hXa with AP4', inactivation of membrane-bound factor Va by APC occurred normally with rapid cleavages at Arg506/Arg306 and appearance of the Mr 30,000 fragment (Fig. 5A). The appearance of the Mr 30,000 fragment was not observed when EGR-hXa was preincubated with A9A, a peptide containing a scrambled version of AP4' (29) (Fig. 5B), demonstrating that 1) AP4' inhibits the interaction of EGR-hXa with membrane-bound factor Va, and thus, allows APC cleavage of the cofactor, and 2) A9A, which does not interact with factor Xa, allows protection of the membrane-bound cofactor by EGR-hXa from cleavage by APC. Preincubation of EGR-hXa with either E5A or A5V results in the appearance of the Mr 30,000 fragment following incubation of the membrane-bound cofactor with APC (Fig. 5, C and E). These data strongly suggest that the two pentapeptides contain amino acids capable of interfering with the binding of EGR-hXa to membrane-bound factor Va, thus inhibiting the protective effect of the cofactor by EGR-hXa and allowing APC cleavage of the heavy chain at Arg506/Arg306, resulting in formation of the Mr 30,000 fragment. Consistent with the findings shown in Figs. 2 and 3, the control peptides, A5I (Fig. 5F) and F5A (Fig. 5G), did not interfere with the ability of EGR-hXa to protect membrane-bound factor Va from inactivation by APC. Interestingly, pentapeptide F5E, which inhibited prothrombinase function and induced a modest decrease in the fluorescent anisotropy of [OG488]-EGR-hXa, was unable to inhibit EGR-hXa protection of factor Va under the conditions employed (Fig. 5D). Overall, these data confirm our previous findings and demonstrate that peptides E5A and A5V contain specific amino acids that are involved in their direct interaction with factor Xa. It is noteworthy that in control experiments, a delay in factor Va inactivation (a decrease in the rate of appearance of the Mr 30,000 fragment) was observed when APC was preincubated with high concentrations of AP4' prior to the addition to the purified membrane-bound factor Va molecule in the absence of EGR-hXa. The bulk of data thus far accumulated demonstrate that amino acids Glu323, Tyr324, Glu330, and Val331 of factor Va heavy chain, which are located at the extremities of AP4', are crucial for the interaction of the cofactor with factor Xa.
Expression and Activation of Recombinant Human Factor VaIn
view of the data shown above, we used recombinant technology to assess the
effect of these 4 amino acids residues on factor Va cofactor function. Three
recombinant factor V molecules were made: factor VFF and factor V
with the mutations Glu323 Phe/Tyr324
Phe,
factor VMI and factor V with the mutations Glu330
Met/Val331
Ile, and factor VFF/MI and factor V
with four substitutions Glu323
Phe, Tyr324
Phe, Glu330
Met, and Val331
Ile. All
recombinant molecules were expressed in COS-7L cells. The concentration of
each molecule prior to all assays was calculated using an ELISA recently
developed in our laboratory. The capture antibody was a polyclonal antibody
(sheep anti-factor V), whereas the detecting antibody was the monoclonal
antibody
HFVHC 17
(3134).
For comparison, we have also used a commercially available kit containing two
polyclonal antibodies, previously employed by other investigators, to assess
the concentration of recombinant factor V in conditioned media
(4548).
A linear correlation between the absorbance at 490 nm and increasing
concentrations of purified plasma factor V was observed between 10 ng and 10
µg of plasma factor V in both our assay and the commercial ELISA kit (not
shown). Further, all plasma and recombinant molecules gave similar results in
the two ELISA (i.e. similar absorbance values at 490 nm were observed
for a given volume containing a recombinant molecule); thus, all molecules
were recognized with similar affinities by the detecting antibodies. The
concentrations of the recombinant molecules in the media before concentration,
using centrifugal ultrafiltration, varied from 300 ng/ml to 3.2 µg/ml.
Fig. 6 shows an SDS-PAGE of the
secreted molecules used in our experiments following concentration and
incubation with
-thrombin. Fig.
6, lane 1, is a control sample representing the heavy and
light chains of plasma factor Va and demonstrates that all heavy and light
chains of the recombinant factor Va molecules (wild type or mutated)
(Fig. 6, lanes
36) have similar molecular weights, which do not appear to be
significantly different from the molecular weight of the heavy and light
chain(s) of the plasma-derived factor Va molecule
(Fig. 6, lane 1).
Thus, introduction of the mutations in factor Va heavy chain does not alter
the ability of the recombinant molecules to be recognized by the monoclonal
antibodies; nor does it alter their mobility on an SDS-PAGE. In
Fig. 6, lane 2, a
control sample is shown where all steps were carried out with cells that do
not contain the cDNA for factor V (mock transfection). The open arrow
identifies a Mr 50,000 fragment that is recognized by the
monoclonal antibody to the light chain of the cofactor. This band is present
in the plasma-derived cofactor molecule (visible upon prolonged exposure of
the autoradiogram) and is also recognized by a polyclonal antibody to human
factor V. Overall, the data demonstrate that the quality of the recombinant
molecules is similar to the quality of plasma-derived factor Va.
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Functional Characterization of the Recombinant Molecules
The recombinant molecules were first screened for clotting activity in a one-
and two-stage clotting assay
(35). Whereas factor
VFF and factor VMI had 10-fold less clotting
activity than the wild type recombinant factor Va molecule, the double mutant
(factor VaFF/MI) was unable to promote clotting under the
conditions employed in a one- or two-stage clotting assay using factor
V-deficient plasma (i.e. the clotting activity of the double mutant
was similar to the clotting activity observed when using the concentrated
media obtained from mock-transfected COS-7L cells; not shown). Thus, whereas
mutation of 2 amino acids at the time results in a cofactor with impaired
clotting activity, substitution of all 4 amino acid residues resulted in the
elimination of factor Va clotting activity. These data suggest that these 4
amino acids are of crucial importance for the expression of factor Va clotting
activity.
We next investigated the ability of the thrombin-activated recombinant
molecules (at 0.5 nM) to interact with factor Xa (5 nM)
and be assembled into prothrombinase using an assay employing purified
reagents and a chromogenic substrate. Since the assay is conducted with
limiting cofactor concentrations, any dearth in cofactor activity may be
explained by the inability of the recombinant molecules to act as a cofactor
for factor Xa. The data demonstrated that under the conditions employed wild
type recombinant factor Va (Fig.
7, filled circles) displayed similar cofactor activity as
plasma-derived factor Va (Fig.
7, open circles), 2550 and 2100 mOD/min, respectively.
Under similar experimental conditions, factor VaFF
(Fig. 7, filled inverted
triangles) and factor VaMI
(Fig. 7, filled
triangles) showed decreased cofactor activities, of 450 and 150 mOD/min,
respectively. Thus, the activity of the two mutants is 5.7- and 17-fold less
compared with wild-type factor Va. However, the activity of factor
VaMI was 3-fold lower than the activity of factor
VaFF and was always consistently lower than factor VaFF
in several other experiments using various concentrations of factor Xa.
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In preliminary experiments, using several preparations of the double mutant
(factor VaFF/MI), we have observed that the rate of thrombin
generation by this mutant, under the conditions employed above (0.5
nM factor Va with 5 nM factor Xa), could not be
distinguished from the rate of thrombin generation by factor Xa alone
(Fig. 7, open
diamonds, 32 mOD/min) or from the rate of thrombin generation by
factor Xa in the presence of the media collected from mock-transfected cells
(Fig. 7, stars, 36
mOD/min). We have thus decided to measure thrombin generation by
prothrombinase in the presence of increasing concentrations of factor
VaFF/MI (Fig. 7, 1
nM (open squares), 2 nM (open
triangles), and 5 nM (open inverted triangles) with
factor Xa constant at 5 nM. The data obtained showed a small but
significant increase in the catalytic efficiency of factor Xa in the presence
of increasing concentrations of the double mutant, up to 90 mOD/s in the
presence of 5 nM factor VaFF/MI. A direct comparison
between wild type factor Va and the double mutant revealed a 284-fold decrease
in cofactor activity of factor VaFF/MI (5100
mOD·min1·nM1
for the wild type and 18
mOD·min1·nM1
for factor VaFF/MI). These data demonstrate that even in the
presence of stoichiometric concentrations
(Fig. 7, open inverted
triangles) factor VaFF/MI is a poor cofactor for factor
Xa.
It has been established that binding sites from both chains of the cofactor contribute to the interaction of the cofactor with factor Xa (11, 2124). In the experiment shown in Fig. 7, we have assessed the contribution of factor Va to prothrombinase at limited cofactor concentration. The experimental conditions were thus designed to make prothrombinase assembly sensitive to any alterations in the factor Va molecule. However, if the binding site from the light chain depends on the binding of the heavy chain, by increasing the concentration of the cofactor molecule (with factor Xa kept constant at 5 nM) we do not expect to observe an increase in the rate of thrombin generation when using the mutant molecules. In contrast, if the binding site on the light chain can increase the catalytic efficiency of factor Xa, independently of the binding site located on the heavy chain, by increasing the concentration of the cofactor, an increase in the catalytic efficiency of all mutants will be observed.
A direct comparison between factor VaFF and factor
VaMI (1 nM mutant cofactor with 5 nM factor
Xa) demonstrated that the activity of factor VaFF was 780 mOD/min,
whereas the activity of factor VaMI was 200 mOD/min
(Fig. 8, filled
circles and filled diamonds, respectively). These data, together
with the data shown in Fig. 7,
verify our previous conclusion that under similar experimental conditions, the
absence of amino acids Glu330 and Val331 has a more
profound effect on cofactor function than the absence of amino acids
Glu323 and Tyr324. In the presence of 10 nM
factor VaFF/MI, the rate of thrombin formation was 180 mOD/min
(Fig. 8, filled
triangles); this rate is 25-fold smaller than the rate of thrombin
generation in the presence of wild type recombinant factor Va (
4500
mOD/min, Fig. 8, filled
squares). However, when comparing prothrombinase activity per
nM enzyme used, and assuming a 1:1 stoichiometry between factor Va
and factor Xa, the activity of the double mutant when used at 10 nM
(5 nM prothrombinase) was 36
mOD·min1·nM1,
whereas the activity of the wild type molecule was 4500
mOD·min1·nM1
(1 nM prothrombinase). These data show a 125-fold decrease in
activity of the double mutant when used at saturating concentrations compared
with the wild type molecule. Altogether, our data suggest that whereas the
remaining binding site for factor Xa on the light chain of the cofactor may be
able to partially compensate for the absence of residues Glu323 and
Tyr324, the absence of amino acids Glu330 and
Val331 from the heavy chain of the recombinant molecule appears
more difficult to overcome. However, absence of all 4 amino cannot be
efficiently overcome by the binding site(s) on the light chain, resulting in a
factor Va molecule with severely impaired cofactor activity. Thus, the binding
site(s) from the light chain of the molecule can increase factor Xa activity
independently of the binding site from the heavy chain as previously suggested
(23).
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The fact that an increase in the concentration of factor VaFF/MI is required to obtain a weak cofactor effect demonstrates 1) a weakened interaction of the mutant cofactor with factor Xa and 2) that discrete amino acids from both chains of factor Va must interact with factor Xa in order to achieve the dramatic increase in the catalytic efficiency of prothrombinase when compared with factor Xa alone; replacement of the specific amino acids with residues containing a different functional group results in the elimination of the contribution of those amino acids to the activity of prothrombinase. It is noteworthy that our data do not exclude the contribution of another portion of the heavy chain to the interaction with factor Xa as suggested earlier (49, 50). However, amino acids Glu323, Tyr324, Glu330, and Val331 from the heavy chain of factor Va appear to be critical for the interaction of the cofactor with the enzyme and contribute a major binding site for factor Xa.
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DISCUSSION |
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Factor Xa alone possesses the catalytic machinery for cleavage and
activation of prothrombin; however, its interaction with factor Va and a
membrane surface are required to obtain a physiologically relevant rate for
catalysis. Membrane-bound factor Xa alone activates prothrombin following two
sequential cleavages: Arg271 followed by cleavage at
Arg320
(5456).
These cleavages proceed through the intermediate prethrombin 2 and produce
-thrombin and the activation fragment, fragment 1·2. The
interaction of factor Va with factor Xa and its incorporation into the
prothrombinase complex alters the order and the rate of peptide bond
cleavages, resulting in the formation of an active intermediate, meizothrombin
(first cleavage at Arg320), followed by the formation of fragment
1·2 and
-thrombin (cleavage at Arg271)
(7,
5456).
The factor Va effect on factor Xa, besides reversing the order of cleavages
during prothrombin activation, is also manifested by the increase in the
overall kcat of the prothrombin, activating reaction by
3,000-fold. The dramatic effect of factor Va on the catalytic machinery of
factor Xa is yet to be understood at the molecular level. A recent study using
recombinant mutant prothrombin molecules has shown that whereas the catalytic
efficiency of factor Xa for cleavage at Arg320 is increased by
20,000-fold in the presence of factor Va, the catalytic efficiency of the
enzyme for cleavage at the Arg271 is only increased by 453-fold
following binding to the cofactor
(57). However, the overall
catalytic efficiency of the assembled prothrombinase for cleavage at each site
separately was similar. Thus, cleavage at Arg320 largely benefits
from the interaction of the cofactor with factor Xa when compared with
cleavage at Arg271
(57). As a consequence, it
appears that incorporation of factor Va into prothrombinase results in the
rearrangement of the prothrombin molecule in such a manner that cleavages at
Arg320 and Arg271 by the factor Va-factor Xa complex
independently become more favorable for catalysis by prothrombinase than by
membrane-bound factor Xa. This rearrangement of the prothrombin molecule upon
its incorporation into the complex and its interaction with factor Va and
factor Xa was previously suggested following the determination of the x-ray
crystal structure of prethrombin 2
(58).
The binding of factor Va to factor Xa involves both chains of the cofactor (11, 2224). Whereas the binding site on the light chain of factor Va remains to be identified, the binding site on the heavy chain of the cofactor has been the object of intense investigation. Because APC and plasmin inactivate the cofactor following limited proteolysis of the heavy chain and dissociation of a portion of the molecule (2528), several studies have attempted to identify amino acid regions from the cofactor that are important for its interaction with factor Xa. The regions studied were usually located around the inactivating cleavage sites of the cofactor or comprised an entire region located between two cleavage sites. A binding site for factor Xa was reported within amino acid residues 493506 of the heavy chain (49, 50). We have previously suggested that a binding site for factor Xa is located within amino acid region 307348 of the heavy chain of factor Va (28). We have next focused on this region of the cofactor, because our findings suggested that whereas cleavage of the heavy chain by plasmin at Arg348 is of no consequence for the activity of the cofactor, cleavage by APC at Arg306 or by plasmin at Lys309, Lys310, and Arg313, which are all membrane-dependent, resulted in rapid and complete inactivation of factor Va (26, 28). Thus, this region of factor Va, which most likely undergoes conformational rearrangement(s) upon binding of the cofactor to a cell surface at the place of vascular injury, contains several amino acids that are important for its incorporation into prothrombinase. Using overlapping peptides from the region 307351, we have recently identified a nonapeptide containing amino acid residues 323331 (AP4') that represents a binding site for factor Xa. We have further shown that the peptide interferes with the binding of factor Va to [OG488]-EGR-hXa and can induce a "cofactor" effect in the absence of factor Va, increasing the catalytic efficiency of the enzyme by 4-fold when used at high concentrations (29). In the present study, the use of overlapping pentapeptides from AP4' led to the identification of 4 amino acids located at its extremities that are required for its overall activity. Recombinant molecules mutated at the specific sites confirmed our findings.
The measure of the factor Va cofactor effect on factor Xa when using the
recombinant factor Va molecules and an assay measuring -thrombin
generation is usually determined by the extent of the increase in the rate of
-thrombin formation. Thus, the experiments using recombinant molecules
cannot distinguish between binding and effector function of factor Va. The
identification of the 4 amino acids described herein and their putative
function within the factor Va molecule was based purely on the experiments
using synthetic peptides. The experiments using recombinant molecules were
only confirmatory and can only attest to their importance within the overall
structure of the cofactor. Our data show that whereas peptide A5V was able to
interfere with the fluorescent anisotropy of [OG488]-EGR-hXa, E5A
had no significant effect on the fluorescent anisotropy of the enzyme. In
contrast, whereas E5A alone was able to increase the catalytic efficiency of
the enzyme toward prothrombin, peptide A5V was unable to produce a similar
effect. Since a perturbation of the signal of membrane-bound
[OG488]-EGR-hXa (positive or negative) is usually correlated with
direct binding (4,
5,
29,
30), overall the data suggest
that whereas amino acids Glu323 and Tyr324 alone may
contribute to the cofactor effect of factor Va (i.e. increase in the
catalytic efficiency of factor Xa), residues Glu330 and
Val331 may be responsible for the high affinity interaction between
the cofactor and the enzyme on the membrane surface.
The reporter group Oregon Green 488 is attached to a chloromethyl ketone, which is covalently linked to the histidine located in the active site of the enzyme (59, 60). Its intensity is only perturbed in the presence of peptide A5V, which, when tested alone, does not have any effect on the catalytic efficiency of the enzyme. In contrast, peptide E5A, which increases the activity of factor Xa toward prothrombin, does not have any significant effect on the fluorescent anisotropy of the probe attached to the active site of the enzyme. Thus, the changes in the structure of factor Xa induced by E5A are different from the changes induced on the molecule by A5V. Using recombinant molecules, we have validated the data obtained with synthetic peptides, and we have shown that under similar experimental conditions, factor VaFF had consistently higher cofactor activity than factor VaMI, but both recombinant molecules had impaired cofactor activity when compared with the wild type cofactor. Thus, under any conditions employed, the binding site on the light chain together with any remaining binding site from the heavy chain is more efficient in increasing factor Xa catalytic activity in the presence of amino acid residues Glu330 and Val331 than in the presence of amino acids Glu323 and Tyr324. However, lack of all 4 amino acids results in a factor Va molecule (factor VaFF/MI) that is severely deficient in its cofactor activity.
In the activity assays described here, the exact concentration of each recombinant molecule used was of crucial importance for the validation of our experiments, since impaired activity of the recombinant molecules could be explained by the wrong concentration used in the assay. An alternative explanation for the decreased activities of the mutant factor Va molecules could also be the fact that the recombinant molecules have altered conformation, due to the change in the amino acid side chain, which will ultimately impair their cofactor function. Finally, the reason for impaired cofactor activity could be the lack of the critical amino acids required for proper cofactor function. In order to eliminate the first two possibilities, since the latter explanation is the ultimate desired effect of our experiments, the concentration of each molecule prior to all assays was calculated using an ELISA recently developed in our laboratory. All molecules were recognized with similar affinities by the detecting antibodies, and SDS-PAGE analysis did not reveal any electrophoretic abnormalities between normal plasma factor Va and the recombinant factor Va molecules. Further, we have made three conservative and one nonconservative mutation in such a manner that the entire conformation of the recombinant factor V molecules possesses a tertiary conformation as close as possible to the wild type factor Va molecule. Since the cofactor activity of the recombinant wild type factor Va molecule was similar to the activity of the plasma-derived cofactor, we must conclude that 1) the concentrations of the recombinant factor Va molecules obtained from the ELISA were accurate, and 2) the recombinant molecules were properly folded. As a conclusion, impaired cofactor effect of the mutant molecules is the consequence of the mutations. All of these findings can be offered as arguments against generalized conformational change phenomena resulting in impaired cofactor activity. Further, several studies using recombinant factor V and factor V mutants have been performed by various investigators using methodologies similar to ours (4548). No problems with the conformation of the recombinant proteins have yet been reported. Thus, any difference observed in the cofactor activities of the recombinant mutant molecules is a direct consequence of the specific mutations. Altogether, our data would suggest that amino acids Glu323, Tyr324, Glu330, and Val331 are critical for the high affinity interaction of factor Va with factor Xa on the membrane surface and provide a reasonable starting point for a systematic investigation of the amino acids that play crucial role for the expression of cofactor activity. Finally, it is important to note that the possibility that the mutant molecules have a weakened chain-chain interaction because of the mutations resulting in a weaker interaction between factor Va and factor Xa could not be excluded by our studies.
As stated earlier, our data do not exclude the presence of another binding site for factor Xa on the heavy chain of the cofactor. The existence of a binding site for the enzyme was suggested between amino acid residues 493 and 506 (49, 50). A peptide containing these amino acids was found to inhibit prothrombinase activity in a system using purified reagents with an IC50 of 3 µM. Kinetic analyses of the inhibition pattern suggested a mixed type of inhibition. The inhibitory peptide also interfered with the amidolytic activity of the enzyme toward a chromogenic substrate when used at high concentrations and was also found to interact with prothrombin. Thus, it was not clear if the inhibitory peptide inhibited factor Xa or prothrombin interaction with factor Va or if the peptide interfered with the binding of the cofactor to the phospholipid bilayer. Further, when the inhibitory peptide was amidated at the COOH terminus or when a glutamine was substituted for an arginine at the carboxyl terminus, its inhibitory activity was lost (49, 50). It is thus possible that Arg506 from factor Va or its surrounding amino acids may also provide for an interactive site with factor Xa, albeit this interaction with the enzyme may be secondary to the interaction of factor Xa with the peptide containing amino acid residues 323331.
In conclusion, our data identify amino acid residues Glu323,
Tyr324, Glu330, and Val331 from the factor Va
heavy chain to be essential for expression of optimum factor Va cofactor
function. A synthetic peptide containing these amino acids that will inhibit
cofactor activity will have the same effect as inhibiting factor Xa and/or
-thrombin directly and may thus be a potent anticoagulant with few or
no side effects, since it is unlikely that this peptide will interfere with
the functions of any other protein of the blood clotting cascade.
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FOOTNOTES |
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¶ To whom all correspondence should be addressed: Chemistry Dept., Cleveland State University, Science Bldg., 2351 Euclid Ave., Cleveland, OH 44115. Tel.: 216-687-2460; Fax: 216-687-9298; E-mail: m.kalafatis{at}csuohio.edu.
1 The abbreviations used are: APC, activated protein C; DFP, diisopropyl
fluorophosphate; OPD, o-phenylenediamine dihydrochloride; PS,
L--phosphatidylserine; PC,
L-
-phosphatidylcholine; PCPS, small unilamellar
phospholipids vesicles composed of 75% PC and 25% PS (w/w); DAPA,
dansylarginine-N-(3-ethyl-1,5-pentanediyl)amide; EGR-hXa,
glutamylglycinylarginyl chloromethyl ketone active site-blocked human factor
Xa; [OG488]-EGR-hXa, human factor Xa labeled in the active site
with Oregon Green 488; PVDF, polyvinylidene difluoride; ELISA, enzyme-linked
immunosorbent assay(s); factor VaFF, recombinant human factor Va
with the mutations Glu323
Phe/Tyr324
Phe;
factor VaMI, recombinant human factor Va with the mutations
Glu330
Met/Val331
Ile; factor
VaFF/MI, quadruple mutant, recombinant human factor Va with the
mutations Glu323
Phe/Tyr324
Phe and
Glu330
Met/Val331
I; mOD,
103 OD.
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ACKNOWLEDGMENTS |
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REFERENCES |
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