Originally published In Press as doi:10.1074/jbc.M303153200 on June 4, 2003
J. Biol. Chem., Vol. 278, Issue 35, 33550-33561, August 29, 2003
Structural Requirements for Expression of Factor Va Activity*
Michael Kalafatis 
¶,
Daniel O. Beck and
Kenneth G. Mann ||
From the
Department of Chemistry, Cleveland State
University, Cleveland, Ohio 44115, the
Department of Molecular Cardiology, The Lerner
Research Institute. The Cleveland Clinic Foundation, Cleveland, Ohio 44195,
and the ||Department of Biochemistry, University of
Vermont, College of Medicine, Burlington, Vermont 05405
Received for publication, March 27, 2003
, and in revised form, June 4, 2003.
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ABSTRACT
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Thrombin activated factor Va (factor VIIa, residues 1–709
and 1546–2196) has an apparent dissociation constant
(Kd,app) for factor Xa within prothrombinase of
0.5 nM. A protease (NN) purified from the venom of the snake
Naja nigricollis nigricollis, cleaves human factor V at
Asp697, Asp1509, and Asp1514 to produce a
molecule (factor VNN) that is composed of a Mr
100,000 heavy chain (amino acid residues 1–696) and a
Mr 80,000 light chain (amino acid residues
1509/1514–2196). Factor VNN, has a
Kd,app for factor Xa of 4 nM and
reduced clotting activity. Cleavage of factor VIIa by NN at
Asp697 results in a cofactor that loses 60–80% of its
clotting activity. An enzyme from Russell's viper venom (RVV) cleaves human
factor V at Arg1018 and Arg1545 to produce a
Mr 150,000 heavy chain and Mr 74,000
light chain (factor VRVV, residues 1–1018 and
1546–2196). The RVV species has affinity for factor Xa and clotting
activity similar to the thrombin-activated factor Va. Cleavage of factor
VNN at Arg1545 by 
-thrombin (factor
VNN/IIa) or RVV (factor VNN/RVV) leads to enhanced
affinity of the cofactor for factor Xa (Kd,app
0.5 nM). A synthetic peptide containing the last 13 residues
from the heavy chain of factor Va (amino acid sequence 697–709, D13R)
was found to be a competitive inhibitor of prothrombinase with respect to
prothrombin. The peptide was also found to specifically interact with
thrombin-agarose. These data demonstrate that 1) cleavage at
Arg1545 and formation of the light chain of factor VIIa
is essential for high affinity binding and function of factor Xa within
prothrombinase and 2) a binding site for prothrombin is contributed by amino
acid residues 697–709 of the heavy chain of the cofactor.
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INTRODUCTION
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The prothrombinase complex responsible for the generation of
-thrombin in the hemostatic process is composed of factor Va and factor
Xa associated on a phospholipid membrane in the presence of
Ca2+ (1,
2). Although factor Xa alone
can convert prothrombin to -thrombin, the prothrombinase complex has a
catalytic efficiency five orders of magnitude greater than factor Xa acting
alone (3). Plasma factor V
circulates as a large single chain protein of Mr 330,000
(4–6).
The cDNA sequences for human, murine, porcine, and bovine factor V have been
reported previously
(7–11).
The factor V molecule is composed of triplicated "A" domains,
duplicated "C" domains, and a "B" region. Human factor
V is cleaved by -thrombin at Arg709, Arg1018, and
Arg1545 and generates the active cofactor factor Va, which is
composed of a heavy chain (A1-A2 domains, Mr 105,000,
amino acid residues 1–709) non-covalently associated with the light
chain (A3-C1-C2 domains, Mr 74,000, amino acid residues
1546–2196). The interaction between the two chains is promoted by
divalent cations (12,
13).
Activation of factor V by -thrombin is required for the interaction
of the cofactor with factor Xa and prothrombin. Factor Va and factor Xa
interact stoichiometrically in the absence of phospholipids with a
Kd of 0.8 µM, and the interaction is
dependent upon the presence of Ca2+
(14). The
Kd for the factor Va-prothrombin interaction is 1
µM and appears to be independent of the presence of
Ca2+
(15). In the presence of a
membrane surface and Ca2+, the Kd of
the factor Va-factor Xa association is 1 nM
(3,
16). Both chains of the
cofactor are required for the interaction with factor Xa
(17–20).
The factor Va-prothrombin interaction is promoted by the heavy chain of the
molecule (15,
18). The factor Va-membrane
interaction, governed by a Kd value of
3nM, occurs at diffusionally limited rates, involves both
hydrophobic and Ca2+-dependent electrostatic
interactions, and results in penetration of a portion of the light chain into
the membrane bilayer
(21–25).
Two sites on the light chain of the cofactor appear to be responsible for the
interactions of factor Va with the membrane surface. The factor Va-membrane
complex is a "true receptor" for factor Xa on the platelet surface
(26,
27).
Although proteolytic cleavage of factor Va by
APC1 at
Arg506/Arg679 results in a 10-fold decrease in the
affinity of the molecule for factor Xa, the same cleavages result in the
elimination of its interaction with prothrombin
(15,
18,
28,
29). It has been shown that
prothrombin and thrombin have two separate electropositive exosites (anion
binding exosite I, ABE-I, and anion binding exosite II, ABE-II) that are
responsible for the majority of the functions of the molecules
(30–39).
Whereas ABE-I has been involved in the binding to thrombomodulin
(40), fibrinogen
(41), PAR1
(42), the COOH-terminal
hirudin peptides (43), and
heparin cofactor II (44) among
others, ABE-II was found to be involved in the interaction with protease nexin
(45) and antithrombin III
(44). Data from separate
laboratories have demonstrated that both exosites bind factors V and VIII
(33,
34,
35). Interestingly, proexosite
I of prothrombin, which is present at a low affinity state on the molecule,
and its affinity for its ligands increases by 100-fold following
activation and formation of thrombin
(33,
39), was found to be directly
involved in the productive interaction with factor Va within prothrombinase
(32,
33).
Thrombin, factor Xa, and the RVV-factor V activator can produce factor Va
by discrete proteolytic cleavages (for details see reviews
(1,
2)). It has also been reported
that a protease from the venom of Naja naja oxiana produces a factor
Va-like molecule lacking a major portion of the carboxyl terminal domain of
the heavy chain, with diminished cofactor activity
(46). The reasons for this
diminished cofactor activity, which is mostly observed at low factor Xa
concentrations, have yet to be elucidated. The present study was undertaken to
understand the role of the last 13 amino acids from the COOH-terminal portion
of the heavy chain of the cofactor. Thus, our present study adds to the
knowledge regarding the structure/function relationships required for an
active factor Va molecule.
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EXPERIMENTAL PROCEDURES
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Materials and Reagents—Hepes, Sepharose CL-4B,
1-palmitoyl-2-oleoylphosphatidylserine (PS) from bovine brain, and
1-palmitoyl-2-oleoylphosphatidylcholine (PC) from hen egg were purchased from
Sigma (St. Louis, MO). The thromboplastin reagent used in the clotting assays
was purchased from Organon Teknika Corp. (Durham, NC).
Dansylarginine-N-(3-ethyl-1,5-pentanediyl)amide (DAPA),
glutamylglycinylarginyl chloromethyl ketone (EGR), prothrombin-Sepharose,
thrombin coupled to agarose through the active site as described
(30), human APC, human factor
Xa, and factor Xa labeled in the active site with Oregon Green 488
([OG488]-EGR-hXa) as described
(47,
48), were from Hematologic
Technologies Inc. (Essex Junction, VT). Human prothrombin, -thrombin,
and human factor V were purified as previously reported
(49–51).
Human factor Va was prepared and purified as recently described using a
heparin column (48). The
enzyme from the snake venom N. nigricollis nigricollis (NN) was
prepared as described (52).
Bovine factor X was isolated as described
(53,
54), activated to factor Xa
using the activator from Russell's viper venom, and purified using
benzamidine-Sepharose (Amersham Biosciences) as described
(55). The synthetic peptide
representing amino acid residues 697–709 of factor Va heavy chain
(DYQNRLAAALGIR, D13R) was purchased from Peninsula Laboratories Europe Ltd.
(Mereyside, UK). The synthetic pentadecapeptide representing factor V residues
337–351 from factor Va heavy chain previously shown to have no effect on
prothrombinase activity (PVIPANMDKKYRSQH, P15H) as well as the peptide shown
to contain a binding site for factor Xa (EYFIAAEEV, AP4') were
synthesized, in the Biotechnology Core of the Cleveland Clinic Foundation
(Cleveland, OH), purified by high-performance liquid chromatography (HPLC),
and characterized by mass spectrometry as described
(48). Phospholipid vesicles
composed of 75% PC and 25% PS were prepared as previously described
(56). The concentrations of
the phospholipid vesicles were determined by phosphorous assay
(57).
Generation of Various Factor V Species—The various factor V
species were generated by incubation of single chain human plasma factor V
with the purified enzymes. Phospholipid vesicles slightly increase the rate of
cleavage of factor V by NN, whereas PCPS vesicles diminish the rate of
activation of factor V by -thrombin
(58). Thus, the experiments
studying the effect of -thrombin on factor VNN were
conducted in the absence of PCPS vesicles. The nomenclature for the
proteolyzed factor V products is as follows: factor VIIa: human
factor V incubated with -thrombin (1 unit/ml, 10 nM) at
37 °C for 5 min; factor VNN: human factor V (220 nM)
incubated with NN venom enzyme (2.4 nM) at 37 °C for 3 h;
factor VIIa/NN: factor VNN incubated with
-thrombin (1 unit/ml) at 37 °C for 10 min. In all cases, 20
nM hirudin was added following proteolytic treatment. To generate
factor VRVV, 212 nM human factor V was incubated at 37
°C with 4.2 nM RVV-V activator for 2 h. In some experiments
following full activation to factor VRVV, 2.1 nM APC and
50 µM PCPS vesicles were added and the reaction was allowed to
proceed for 1 h. Aliquots were taken at the time points indicated in the
figure legends and analyzed by SDS-PAGE.
Cleavage of Factor VIIa by NN Protease and Inactivation by
APC— -Thrombin-cleaved human factor VIIa (212
nM) was incubated at 37 °C with 50 µM PCPS
vesicles and 2.1 nM NN protease. After 3 h, 2.1 nM APC
was added and the reaction was allowed to proceed for 1 h. At selected time
intervals prior to and subsequent to addition of APC, aliquots of the mixture
were withdrawn and assayed for cofactor activity in a clotting assay using
factor V-deficient plasma. At the same intervals, aliquots were prepared for
SDS-polyacrylamide gel electrophoresis analyses.
Determination of Factor Va Cofactor Activity—Cofactor
activity was measured at selected time intervals by clotting assay using
factor V-deficient plasma and standardized to the percentage of control
(50). In a typical assay 50
µl of factor V-deficient plasma was mixed with an equal volume of the
sample to be analyzed. To start the assay 100 µl of the PT reagent
(Simplastin Excel) was added while rocking the tube at 37 °C. The assay
end point was determined by visualization of the fibrin clot. Control
experiments using a solution of RVV-V activator or NN alone (in the absence of
factor V) in the clotting assay, at the same concentrations that are used to
cleave factor V, gave no interference with the clotting process (with or
without normal plasma). Thus, trace amounts of snake venom enzymes when
introduced in plasma during the clotting assay (or in the prothrombinase
assay) did not interfere with the results obtained. The assay was initiated
with the addition of the factor Va followed by the addition of
thromboplastin.
Measurements of Rates of Thrombin Formation using
DAPA—Functionally defined apparent dissociation constants
(Kd,app) for factor Xa binding to factor
Va-PCPS were inferred from an assay measuring -thrombin generation at
various factor Xa concentrations
(59,
60). These studies were
performed using a PerkinElmer Life Sciences MPF-44A fluorescence
spectrophotometer with a 
ex 280 nm (slit at 8 nm),
em 550 nm (slit at 16 nm), and a 500-nm long pass filter in
the emission beam. In a typical experiment a mixture (80 ml) composed of
prothrombin (1.4 µM), PCPS vesicles (20 µM), and
DAPA (3 µM) was incubated in the dark for 20 min in 20
mM HEPES, 0.15 M NaCl, 5 mM CaCl2,
pH 7.4, buffer. An aliquot of the mixture (1800 µl) was added to a cuvette
containing the indicated factor V species (0.5 nM), and the base
line fluorescence was monitored for 15–20 s at ambient temperature.
Factor Xa (100 pM to 10 nM) was then added to initiate
the reaction. -Thrombin generation was continuously monitored by the
change in the fluorescence intensity of DAPA present in the reaction mixture.
The use of DAPA in the reaction mixture prevents feedback reactions catalyzed
by the newly formed thrombin.
Determination of the Apparent Binding Constants for Factor
Xa—To determine the apparent dissociation constant
(Kd,app) of factor Xa for the various factor Va
species, a titration with factor Xa measuring prothrombin activation (1.4
µM) was performed using concentrations ranging from 100
pM to 10 nM.
The Kd,app for the factor Va-factor Xa
interaction is given by Equation
1,
 | (Eq. 1) |
where [Va]F and [Xa]F are the concentrations of free
factor Va and factor Xa; the final concentration of factor Va within the
mixture was 0.5 nM. The initial rate of thrombin formation for each
factor Xa concentration was plotted as a function of the factor Xa
concentration (using the software DeltaGraph PRO, 3.5, Delta Point Inc.,
Monterey, CA) using the quadratic equation that is the solution for
Equation 1
(16,
61).
 | (Eq. 2) |
Assuming that n = moles of factor Xa bound/mole of factor Va at
saturation; throughout this study n = 1; the stoichiometry of the
factor Va-factor Xa interaction was fixed at 1.
obs = V0 +
[Xabound·V
], where obs is the rate at
XaT = 0 and V is the rate at infinite factor Xa
concentrations (i.e. when the fixed concentration of factor Va is
saturated with factor Xa). Prior to each experiment, the maximum amount of
complex formed under the above conditions was verified by replacing
[Va]F and [Xa]F in
Equation 1 by,
 | (Eq. 3) |
 | (Eq. 4) |
where [Va]T and [Xa]T are the total concentrations of
factor Va and factor Xa.
Inhibition of Thrombin Formation—The formation of thrombin
was also analyzed using the fluorescent thrombin inhibitor DAPA as described
(48) using a PerkinElmer Life
Sciences LS-50B luminescence spectrometer (PerkinElmer Life Sciences LLC,
Norwalk, CT) 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, prothrombin at 350 nM, PCPS vesicles at 10
µM, in the presence of 2 mM CaCl2. Factor
Va was incubated with the peptides (at concentrations ranging from 0.5
nM to 100 µM) for 10 min and added to the assay. The
initial rate of thrombin formation (nM
IIa·min–1) was calculated as described
(48). 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) and further analyzed and plotted with
the software Prizm (GraphPad, San Diego, CA).
Fluorescence Anisotropy Measurements—Fluorescence anisotropy
of [OG488]-EGR-hXa was measured using a PerkinElmer Life Sciences
LS-50B luminescence spectrometer in L-format as recently described
(48). Anisotropy measurements
were performed in a quartz cuvette under constant stirring (low) with
ex = 490 nm, 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 eight successive readings were averaged. In all
cases, the total addition of peptide did not exceed 10% of the volume of the
reaction. 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).
Direct Binding of the Peptides to Thrombin—Thrombin
immobilized to agarose through the active site was used
(30). Peptide solutions of
D13R and P15H (48) were
dissolved in water to a given concentration. In control experiments it was
determined that the maximum amount of peptide retained by the thrombin-agarose
column was approximately 400 µg. The peptides were thus diluted in 20
mM Hepes, 0.1 M NaCl, pH 7.4, in a manner so that
400 µg was contained in each of the starting solutions. The two
peptides used in this study contained aromatic amino acid residues; thus,
their presence in the flowthrough or the eluate of the column was ascertained
by the optical density. Position identification of bound material was verified
by LC/MS as detailed below in the analytical facility of Dr. David Anderson at
Cleveland State University.
Mass Spectrometry Instruments and Conditions—Eluate was
separated from the salt components using an HPLC system (HP 1100, HPLC
gradient system, Agilent Technologies, Palo Alto, CA) with a C18 column (1 mm
x 15 cm, GraceVydac, Hesperia, CA) and buffers A (0.3% acetic acid in
water) and B (0.3% acetic acid in acetonitrile), and the eluted material was
monitored using a Micromass Quatro II ESI-Triple quadrupole mass spectrometer
(Waters, Milford, MA). The data were collected using a Compaq Professional
Workstation (AP200, Hewlett-Packard, Palo Alto, CA) and analyzed by MassLynx
version 3.3 (Waters).
Gel Electrophoresis—SDS-PAGE analyses were performed using
5–15% and 4–12% gradient gels according to the method of Laemmli
(62). Proteins were visualized
after staining with Coomassie Brilliant Blue in 50% methanol, 10% acetic acid
followed by destaining by diffusion using a solution of 50% methanol and 10%
acetic acid.
Amino Acid Sequence Analyses—Human factor V (630
nM) was incubated with PCPS vesicles (200 µM) and the
purified NN protease for 3 h at 37 °C. The reaction was stopped by the
addition of 2% SDS, 2% 
-mercaptoethanol, and the mixture was heated for
5 min at 90 °C. Approximately 40 µg of total factor V digest was
analyzed on an 8–18% linear gradient SDS-PAGE gel. Similar experiments
were performed with the RVV-cleaved cofactor. Following electrophoretic
separation, proteins were transferred to a PVDF membrane, and the
NH2-terminal sequences were determined using automatic Edman
degradation on an Applied Biosystems 475A protein sequencing system as
described (60) in the
laboratory of Dr. Alex Kurosky (University of Texas, Medical Branch at
Galveston).
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RESULTS
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Limited Proteolysis of Factor V by N. nigricollis nigricollis (NN)
Protease—Factor V prior to the activation by -thrombin
typically displays 13- to 15-fold lower clotting activity than the active
cofactor factor Va obtained following activation by -thrombin
(50). The apparent activity of
single chain factor V is a consequence of a limited amount of cleavage, which
produces the active cofactor during the initial phase of the assay
(63,
64). In experiments using
purified factor Xa, prothrombin, and phospholipid in the presence of the
fluorescent thrombin inhibitor DAPA, which prevents feedback activation of
single chain factor V, measurements of factor V activity give results that are
400-fold lower than for thrombin-activated factor V
(3). Thus, the single-stage
clotting assay overestimates factor V cofactor activity, because the clotting
end-point occurs when 1% of the total circulating procofactor is
activated and relies upon the adventitious activation of the procofactor by
the initial thrombin produced during the assay. For isolated human factor V
the single stage assay yields a value of 54 units/mg
(Fig. 1, lane 1),
which increases 15-fold to 740 units/mg upon activation with -thrombin
(factor VIIa, Fig.
1, lane 12). Treatment of factor V with the NN protease
results in the progressive development of a species (factor VNN,
Fig. 1, lanes
2–11) with a specific activity lower than factor V (21 units/mg,
Fig. 1, lane 11). NN
cleaved factor V (factor VNN, lane 11) exhibits a heavy
chain of Mr 100,000 and a light chain doublet of
Mr 80,000, compared with a heavy chain of
Mr 105,000 and light chain doublet of
Mr 74,000/72,000 for thrombin-activated factor V (factor
VIIa, lane 12). These data suggest that the impaired
cofactor activity of factor VNN is due either to the shorter heavy
chain or the longer light chain or both.
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FIG. 1.
Cleavage of factor V by the enzyme contained in the snake venom of
N. nigricollis nigricollis. Factor V was incubated with the NN
protease as described under "Experimental Procedures." At selected
time intervals aliquots of the mixture were withdrawn and assayed for cofactor
activity in a clotting assay using factor V-deficient plasma (the activity of
each sample is shown at the bottom of the figure in units/mg). Fragments were
visualized following Coomassie Blue staining on a 4–12% (linear
gradient) SDS-PAGE. Lane 1, factor V, control; lanes
2–11, factor V at 1, 3, 5, 10, 20, 30, 60, 90, 120, and 180 min
following the addition of the enzyme; lane 12, factor Va control
activated with -thrombin. Molecular weight markers are shown in
lane 13.
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NH2-terminal amino acid sequence analyses of the fragments
resulting from incubation of factor V with NN is shown in
Fig. 2. The data show
preferential cleavage by NN between the X and Asp residues of
selected X-Asp-Z bonds (where X is any amino acid
and Z is typically tyrosine), consistently producing fragments with a
D-Y at the NH2 terminus (Fig.
2B). Cleavage by NN results in new NH2 termini
at Asp697, Asp1509, and Asp1514. These
products correspond to the fragments with Mr 100,000
(residues 1–696, fragment a,
Fig. 2A),
Mr 80,000 (residues 1509-/1514–2196, fragments
b/c, Fig. 2A),
and a fragment of Mr 65,000 (fragment d). The
latter fragment exhibited an NH2-terminal sequence that matches a
region of factor V starting at amino acid residue 697
(Fig. 2B). The
arrowhead in Fig.
2B, column d, identifies the Arg709
thrombin cleavage site illustrating that the heavy chain of factor
VNN is 13 residues shorter than for factor VIIa. The
enzyme also cleaves at another site on the B region of the molecule, giving
rise to fragment d (Fig.
2A) and another fragment (arrowhead below
fragment d, Fig.
2A), which remains to be explicitly identified. A high
background complicated the sequencing of this fragment; however, in one
experiment an NH2-terminal sequence starting with D-F-S was found
for the fragment depicted by the arrowhead in
Fig. 2A. In the B
domain of factor V there are several repeats of 9 amino acids and two of these
repeats start with the sequence D-F-S (residues 1260 and 1305)
(8). Thus, a cleavage site in
the B region of factor V is suggested at either Asp1260 or
Asp1305. The high levels of carbohydrate present in this region may
be responsible for the difficulties in obtaining an extended sequence
(8). Overall, NN cleavage of
factor V produces a factor "Va-like" molecule, factor
VNN, composed of a heavy chain (residues 1–696) and a light
chain (residues 1509-/1514–2196). The reduced activity of factor
VNN in the clotting assay (<3% activity of factor
VIIa) thus must be the consequence of either a shorter heavy chain
or a longer light chain or both.
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FIG. 2.
Identification of the proteolytic cleavages of the factor V molecule by
the enzyme contained in the snake venom of N. nigricollis
nigricollis. Human factor V was incubated with PCPS vesicles and the
purified NN protease as described under "Experimental Procedures."
After electrophoresis, the proteins were transferred to a PVDF membrane.
A, one of the PVDF membranes that was used for the
NH2-terminal sequence analysis stained with Coomassie Blue and
represents the fragments derived from human factor V following cleavage by NN.
The letters a–d depict fragments of human factor V. The
arrowhead underneath fragment d denotes a fragment that is
derived from the B region of factor V and does not stain with Coomassie Blue.
The NH2-terminal sequence of this fragment could not be determined
positively. B, NH2-terminal sequence of the fragments
a–d shown in A. The numbers in
parentheses indicate picomoles of amino acid at the given cycle.
Fragment b was found to contain two amino acid sequences. According
to the amino acid sequence derived from the cDNA of the human cofactor
(8) these sequences are
identified to represent cleavage by NN at Asp1509 and
Asp1514. The arrowhead at the right of the
sequence shown for fragment d depicts Arg709, which is a
specific cleavage site by -thrombin for activation of human factor V
(8).
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The Importance of the COOH Terminus of the Heavy Chain—To
examine the factor V proteolytic product with a full-length light chain and
truncated heavy chain, factor VIIa was further subjected to
proteolysis by NN. Using the plasma clotting assay, the cofactor activity of
factor VIIa (210 nM, 750 units/mg) was followed during
treatment with NN protease (2.1 nM), with parallel analysis by
SDS-PAGE. After 10-min incubation with NN, factor VIIa lost
40% of its initial clotting activity
(Fig. 3A); after 1 h,
20% clotting activity remains. No further loss in clotting activity was
observed (up to 2 h, Fig.
3A). The cleavage of factor VIIa at
Asp697 of the heavy chain is correlated with the loss of 80% of the
clotting activity of factor VIIa (factor VNN/IIa,
Fig. 3A, 180
min). The remaining cofactor activity of factor VIIa/NN can be
eliminated by cleavage of the heavy chain by APC and phospholipids, at
Arg506, Arg306, and Arg679
(Fig. 3A, lanes
13–18, open circles), yielding a fragmentation pattern similar to
that observed for APC inactivation of factor VIIa
(65). The data demonstrate
that removal of the last 13 residues (amino acid region 697–709) from
the COOH terminus of the heavy chain of factor VIIa results in a
molecule with greatly diminished activity when measured in a clotting
assay.
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FIG. 3.
Loss of factor VIIa clotting activity as a consequence of
cleavage of the cofactor by the enzyme contained in the snake venom of N.
nigricollis nigricollis. A, human factor VIIa was
incubated with PCPS vesicles and NN protease, and the activity of the molecule
was measured as described under "Experimental Procedures." After 3
h APC was added, and the reaction was allowed to proceed for 1 h. B,
5–15% linear gradient SDS-PAGE. Fragments were visualized by Coomassie
Blue staining: Lane 1, factor VIIa, control, no enzyme;
lanes 2–12, factor VIIa, PCPS vesicles and NN at 1,
3, 5, 7, 10, 20, 30, 60, 90, 120, and 180 min; lanes 13–18,
factor VNN, PCPS, and APC at 1, 3, 5, 10, 30, and 60 min. The time
of incubation of factor VIIa with NN or of factor
VIIa/NN with APC is shown on the top of the gel. The
vertical arrows in A and B indicate the addition of
APC to the reaction mixture. The horizontal arrow in B
identifies a fragment from factor Va heavy chain (amino acid residues
1–506) that co-migrates with the light chain of the cofactor
(65).
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Activation of Factor V by Russell's Viper Venom Factor V Activator and
the Importance of the Light Chain—In contrast to the NN protease,
RVV-V activator is an efficient activator of factor V. Although several
laboratories have used the RVV-V activator to activate factor V
(12,
66,
67) no explicit description of
the activation/cleavage sites has been reported. Popular consensus is that the
RVV-V protease activates factor V to produce a species with only a light
chain, equivalent (by electrophoretic migration) and with similar clotting
activity to factor VIIa
(12,
66,
67). We have reported that
APC-inactivated membrane-bound factor V (cleaved at Arg306,
Arg506, Arg679, and Lys994) is further
cleaved by the RVV-V activator at Arg1018 and Arg1545
(65). However, additional
RVV-V cleavages within the region 1–1018 could not be ruled out, because
those experiments were complicated by further APC fragmentation of the
products (65).
Fig. 4 shows that incubation of
factor V with catalytic amounts of the RVV-V protease results in a species,
factor VRVV, which possesses cofactor activity in the plasma
clotting assay similar to the -thrombin-activated cofactor, factor
VIIa (approaching 700 units/mg,
Fig. 4A). The
activation of factor V by the RVV-V activator is a slower process than
-thrombin activation at similar catalyst and substrate concentrations.
In addition to the light chain (Mr 74,000), factor
VRVV displays a Mr 150,000 heavy chain
(Fig. 4B).
NH2-terminal amino acid sequence analyses of the fragments obtained
following cleavage of factor V by the RVV-V activator (not shown) confirm our
previous findings with the APC-treated factor V
(65) consistent with the
conclusion that RVV-V activator cleaves the procofactor at Arg1018
and Arg1545.
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FIG. 4.
Activation of human factor V by RVV-V activator and inactivation by
APC. A, human factor V was incubated at 37 °C with RVV-V
activator for 2 h. After full activation to factor VRVV, APC and
PCPS vesicles were added, and the reaction was allowed to proceed for 1 h.
Factor V clotting activity was measured as described under "Experimental
Procedures." B, 4–12% linear gradient SDS-PAGE. Fragments
were visualized by Coomassie Blue staining: Lane 1, factor V,
control; lanes 2–10, factor V and 4.2 nM RVV at 1,
3, 5, 10, 15, 30, 60, 90, and 120 min; lanes 11–19, factor
VRVV, PCPS and APC at 1, 3, 5, 7, 10, 15, 30, and 60 min.
Vertical, arrows indicate the addition of APC to the reaction
mixture. The position of the heavy chain of factor VRVV is
indicated at left.
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The subunit compositions for each factor V molecule and their clotting
activities are illustrated in Fig.
5, and their characteristics are reported in
Table I. Lane 1 shows
thrombin-activated factor V, followed by factor VRVV, which
possesses a normal light chain and a heavy chain that is longer than that of
factor VIIa. Treatment of factor V with NN produces factor
VNN (Fig. 5) with a
shorter heavy chain and longer light chain than factor VIIa. Factor
VNN possesses minimum clotting activity. Factor VNN can
be further cleaved by -thrombin at Arg1545 to reduce the
light chain to normal size (factor VNN/IIa). Coincidentally, the
clotting activity of the molecule increases to 50% of that of factor
VIIa. Cleavage of factor VRVV with -thrombin
produces a species indistinguishable from factor VIIa (factor
VRVV/IIa), whereas factor VNN/RVV appears with a normal
light chain and truncated heavy chain (identical to factor
VNN/IIa).
Functional Activity of the Factor V Species Derived from NN and
Thrombin—Cleavage of factor V by NN produces a molecule exhibiting
impaired cofactor activity either because of impaired factor Xa receptor
capability, diminished catalytic effector properties, or both. To compare the
receptor capacities of the factor VIIa, factor VNN,
factor VRVV, factor VNN/IIa, and factor
VNN/RVV, thrombin generation was measured in the DAPA assay as a
function of added factor Xa (Fig.
6). The use of DAPA in the reaction also inhibited feedback
cleavage by thrombin of the factor VNN and factor VRVV
species. The curves in Fig. 6 represent best fits to Equation 2
(see "Experimental Procedures"), and each yields a
Kd,app describing on a functional basis the
affinity between factor Xa and the factor V species
(16,
61). These dissociation
constants and corresponding activities in the clotting assay are listed in
Table I. Fits were obtained
from similar data compiled for the other factor V species, and the results of
these are also given in Table
I. Compared with factor VIIa
(Fig. 6A, filled
circles), factor VNN (Fig.
6A, open circles) exhibits both reduced factor
Xa affinity and diminished clotting activity at limiting concentrations of
factor Xa (Table I). The
Kd,app for factor VNN with human
factor Xa was 4nM (with a 1:1 stoichiometry). The effector
efficiency of VNN was not affected at saturating concentrations of
factor Xa. Upon treatment with -thrombin or RVV, factor VNN
improves its affinity for factor Xa and its ability to serve optimally as
receptor for factor Xa within prothrombinase
(Fig. 6A, open
squares); however, only partial recovery of the clotting activity was
observed (Table I). Similar
results were observed when factor VRVV was treated with NN
(Kd,app for factor VRVV/NN of
0.5 nM). Because both factor VIIa and factor
VIIa/NN possess a fully formed light chain, resulting from cleavage
at Arg1545 (Fig.
6B, lanes 1 and 3), it appears that
cleavage at Arg1545 is enough to restore optimal binding of the
cofactor for factor Xa. However, the clotting activities of factors
VNN/IIa and VNN/RVV still remained impaired as compared
with factor VIIa (Table
I and Fig. 5).
These data suggest that cleavage of the heavy chain at Asp697
results in a cofactor with reduced capabilities within prothrombinase that are
related to functions of the molecule other than the binding to factor Xa;
i.e. the COOH-terminal residues of the heavy chain of the cofactor do
not appear to be involved in the factor Va-factor Xa interaction or the factor
Va-membrane interaction. The latter was demonstrated by Bakker et al.
(46) using a factor Va
molecule lacking the Asp683-Arg709 domain of the heavy
chain. Collectively the data demonstrate that at saturating concentrations of
factor Xa (
10 nM, Fig.
6 and Table I)
cleavage at Arg1545 is not required for full expression of factor
Xa cofactor activity, however, this cleavage is necessary for full expression
of factor Va cofactor activity at limiting amounts of factor Xa, as those
available in the clotting assay. Thus, although cleavage at Arg1545
is not a prerequisite for expression of maximum factor Va cofactor activity,
it is required for optimal factor Xa binding. Overall, the data also indicate
that elimination of 13 amino acids from the COOH-terminal portion of the heavy
chain of factor VIIa results in partial loss in cofactor activity
with no diminution in the factor Xa binding capability.
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FIG. 6.
Requirement for cleavage at Arg1545 in factor V for high
affinity binding of factor Xa to factor Va and optimum function within
prothrombinase. A, each of the factor Va species were assayed for
activity at a fixed concentration of 0.5 nM and various factor Xa
concentrations in a purified prothrombinase assay as described under
"Experimental Procedures." The graphs represent: factor
VIIa, filled circles; factor VNN, open
circles; factor VNN/IIa, open squares. The reaction
was initiated by the addition of varying amounts of factor Xa (100
pM to 10 nM). -Thrombin generation was
continuously monitored by the change in fluorescence intensity of DAPA. The
slope of the graph of prothrombin generation was calculated separately and
plotted as a function of the concentration of factor Xa used. The solid
lines represent hyperbolas that were obtained by fitting the data to
Equation 2 shown under
"Experimental Procedures" using non-linear least squares analysis
with n fixed as a locked parameter at 1 (i.e. the factor
Va:factor Xa stoichiometry was fixed to 1). The initial estimates were a
Kd of 1 nM and a fixed stoichiometry of 1
(n = 1). B, the identities of the cofactor molecules assayed
in A are shown. The symbols at the bottom of the
gel represent the cofactor species used in A. The star
identifies an intermediate fragment deriving from cleavage of factor V by the
NN protease. Lane 1, factor VIIa, lane 2, factor
VNN, lane 3, factor VNN/IIa. Positions of the
heavy and light chains of the various species are shown on the side of the
gel.
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Inhibition of Prothrombinase Activity by a Synthetic Peptide Containing
the 13 Amino Acids from the COOH-terminal Portion of the Heavy Chain of Factor
Va (D13R)—To ascertain the importance of the region 697–709
for cofactor activity, we obtained a 13-amino acid peptide encompassing this
region (D13R). This peptide was tested for inhibition on factor Va cofactor
activity in a prothrombinase assay using purified reagents. In the
prothrombinase assay purified human factor Va was preincubated with the
synthetic peptide and added to a cuvette containing PCPS, prothrombin, DAPA,
and factor Xa. D13R showed complete inhibition of factor Va cofactor activity
(Fig. 7A, filled
squares) with an IC50 of 12 µM. Complete
inhibition occurred at 100 µM peptide. The residual
6–7% prothrombinase activity observed at this peptide concentration is
due to the activity of factor Xa alone. Under similar experimental condition a
control pentadecapeptide from the middle portion of factor Va (P15H
(48)) did not show any
inhibition of prothrombinase function (Fig.
7A, filled triangles). The pentadecapeptide has
7 amino acids in common with D13R (in a different order), contains a tyrosine
and an aspartic acid, derives from the factor Va sequence, and overall possess
a similar charge as D13R. P15H is thus an adequate control for D13R. Overall
data demonstrate that D13R inhibits prothrombinase by impairing the
interaction of the cofactor with one of the components of prothrombinase. It
is noteworthy that D13R was previously found to be non-inhibitory in a
clotting assay using factor V-deficient plasma
(68). These discrepancies may
result from the way the assays are conducted, and from the definition of
factor Va activity per se in each assay. In the prothrombinase assay,
the peptide is preincubated with factor Va and the end-point of the assay is
measured by the rate of thrombin formation as measured by its complexation
with DAPA. In the clotting assay, the end-point of the assay is the formation
of a fibrin clot. We must thus conclude that the nature of an assay is
important for the determination of the inhibitory potential of a peptide.
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FIG. 7.
Function of a peptide containing amino acid residues 697–709
(D13R). A, prothrombinase function. Increasing concentrations of
D13R were incubated with factor Va. Factor Va cofactor activity was assayed in
the presence of increasing concentrations of D13R as described under
"Experimental Procedures" in a prothrombinase assay using purified
reagents (filled squares). The control peptide P15H previously shown
to have no effect on factor Va cofactor activity is shown by the filled
triangles (48). The data
with D13R represent the average value found in three independent experiments,
whereas the results with P15H represent the average value found in two
independent experiments. B, prothrombinase assembly. Reaction
mixtures containing a [OG488]-EGR-hXa at 10 nM and 10
µM PCPS were titrated with factor Va to saturation (25
nM (48)). The
preformed complexes were titrated with increasing concentrations of AP4'
(filled triangles), P15H (filled inverse triangles), and
D13R (filled squares). Delta r was obtained by subtracting
the value of the anisotropy of [OG488]-EGR-hXa alone (average of
eight readings) in the absence of factor Va from the anisotropy observed in
the presence of a given concentration of factor Va (average of eight readings)
as described (48). The
graph represents two different things: Three titrations of
membrane-bound [OG488]-EGR-hXa with factor Va (filled
circles, in nM), followed by the displacement of factor Va
from [OG488]-EGR-hXa by the synthetic peptides (in
µM). C, kinetic analyses of prothrombinase inhibition
by D13R. The data are plotted as V0 (initial velocity, in
arbitrary units) as a function of increasing prothrombin concentration in the
presence of increasing concentrations of inhibitory peptide (D13R). The
lines drawn represent the best fit through the points with an
R2 of at least 0.99. The concentrations of D13R used in
the experiments are as follows: control no peptide (filled squares),
2 µM peptide (filled triangles), 3 µM
peptide (filled circles), 4 µM peptide (filled
diamonds), and 5 µM (filled inverted triangles).
The data represent the average of the results found in three independent
experiments. The apparent inhibition constant (Ki)
reported in the text is the value derived from the formula: IC50 =
Ki(1 + S/Km)
(69), where
Km is the Michaelis-Menten constant of the reaction in the
absence of inhibitor.
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To verify if the peptide inhibits prothrombinase activity by impairing the
direct interaction of the cofactor with factor Xa, we have employed an assay
using a fluorescent derivative of the enzyme as recently described
(48):
[OG488]-EGR-hXa. Titration of increasing concentrations of D13R
into a preformed complex of membrane-bound [OG488]-EGR-hXa-human
factor Va (10 nM [OG488]-EGR-hXa and 25 nM
factor Va) did not result in the decrease of the factor Va-dependent
anisotropy of [OG488]-EGR-hXa even in the presence of 100
µM D13R (Fig.
7B, filled squares). Under similar experimental
conditions P15H didn't have any effect on the factor
Va-[OG488]-EGR-hXa interaction, whereas a previously described
nonapeptide from the heavy chain of factor Va (AP4') showed complete
inhibition of the binary interaction with an IC50 of 8
µM (Fig.
7B, filled triangles)
(48). These data are
consistent with the data of Fig.
6, which indicated that deletion of 13 amino acids from the
COOH-terminal part of the heavy chain of factor Va does not interfere with the
binding of factor Xa to the cofactor (Fig.
6, factor VaNN/IIa). Altogether these data suggest that
D13R inhibits prothrombinase, because it interferes with other interactions in
prothrombinase.
The mechanism of D13R inhibition of prothrombinase was addressed by
investigating the effect of the peptide on the kinetic parameters of
prothrombinase (Km and Vmax) in the
presence of varying concentrations of inhibitor (2–5 µM,
Fig. 7C). Under the
conditions employed and in the presence of increasing concentrations of
peptide, the saturation curves were sigmoidal
(Fig. 7C). The curves
tended toward a similar asymptote, which is the Vmax of
prothrombinase (525 ± 17 nM IIa/min), whereas the apparent
K0.5 of the enzymatic reaction increased with increasing
concentration of inhibitor (Fig.
7C, 2 µM (filled triangles), 3
µM (filled circles), 4 µM (filled
diamonds), and 5 µM (filled inverted triangles)).
These data represent an unusual competitive inhibition mechanism were only
free substrate (prothrombin) can produce thrombin in the presence of
prothrombinase (69). According
to this model D13R binds prothrombin in competition with the binding of
prothrombin to prothrombinase (factor Va-factor Xa)
(69). This unusual competitive
mechanism was previously described for the inhibition of prothrombin
activation by the sulfated version of Hir54–65 peptide, which inhibited
prothrombinase with a Ki of 3 µM
(33). The inhibition constant
(Ki) of prothrombinase by D13R calculated from the
IC50 (Fig.
7A) using the Km obtained from
Fig. 7C (0.4
µM ± 0.03, filled squares) was found to be 6.4
µM. These values are in complete agreement and strongly suggest
that the hirudin-like peptide D13R is an unusual competitive inhibitor of
prothrombinase, because it inhibits prothrombin activation by interfering with
the interaction of the substrate (prothrombin) with the enzyme
(prothrombinase) rather than interfering with the active site of the enzyme
like a classical competitive-type inhibitor
(69).
Direct Binding of D13R to Thrombin and Prothrombin—All the
data presented thus far suggest that a binding site for prothrombin is located
within D13R, which represents the last 13 amino acids from the heavy chain of
factor Va. To ascertain that the peptide interacts directly with prothrombin
and/or thrombin, we have used a prothrombin-Sepharose column and a
thrombin-agarose column. Attempts to study the binding of D13R to
prothrombin-Sepharose failed, because D13R was consistently found in the
flowthrough of the column. Two facts may account for this result: 1) the
peptide has low affinity for prothrombin-Sepharose and our experimental
conditions did not allow for the binary interaction to occur and/or 2) ABE-I
of prothrombin, which has been reported to interact with hirudin-like peptides
and to interfere with prothrombin activation
(33), may have been blocked
during the coupling procedure. However, although it has been previously
demonstrated that factor Va interacts with prothrombin and thrombin via ABE-I,
it has been also shown that the affinity of ABE-I for its ligands is increased
following activation and thrombin formation
(33,
39). We have thus used a
thrombin-agarose column where thrombin was coupled to the agarose beads via
the active site as described
(30). Because D13R and the
control peptide P15H both contain tyrosine residues, we can measure their
optical density, thus we can identify the presence of the peptides in the
collected fractions. The thrombin-agarose column was loaded with the peptides
and washed extensively. Subsequently, elution was performed with 2 M NaCl. The
results of these experiments are shown in
Fig. 8. The control peptide,
P15H (filled squares), which has 7 amino acids identical to D13R, was
detected in the flowthrough of the thrombin-agarose column. In contrast, D13R
bound to the thrombin-agarose column and eluted with the high salt buffer
(Fig. 8, arrow, filled
circles, and filled triangles). The identity of D13R in the
elution fractions from both runs was verified by LC/MS (not shown). The data
demonstrate that D13R contains a binding site for thrombin. A direct
comparison, of the amino acid sequence of D13R with the amino acid sequence of
several hirudin-like peptides that interact with thrombin, suggests that the
hirudin-like motif DYQ
(30–33,
70–72)
contained within D13R may be responsible for its interaction with the
thrombin-agarose column.
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FIG. 8.
Direct interaction of the hirudin-like peptide from the COOH terminus of
factor Va heavy chain with thrombin-agarose. Chromatography of control and
acidic peptides was performed on a thrombin-agarose column as described
(30). Each run represents 400
µg of peptide. This amount of peptide was determined to be saturable for
the thrombin-agarose column used. Elution was performed with 2 M
NaCl and was started at the point indicated by the arrow. The
presence of the peptides in the corresponding fractions was monitored by
absorbance at 280 nm. Identification of D13R in the elution fractions was
performed by LC/MS as described under "Experimental Procedures."
Results show P15H (filled squares), D13R (first run, filled
circles), and D13R (second run, filled triangles).
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Taken together the data indicate that prothrombinase assembly can occur
following cleavages at Asp697, Asp1509, and
Asp1514, whereas optimum function requires cleavage at
Arg1545 and generation of the light chain of factor Va. This
cleavage appears to be required for optimum binding of factor Xa, which in
turn is a prerequisite for subsequent expression of efficient catalytic
effector function by the cofactor at low physiological concentrations of
factor Xa. Thus, although deletion of residues 697–709 in the heavy
chain of factor Va species did not affect factor Xa binding, the clotting
function of factor Va was significantly affected by truncation at the COOH
terminus. Thus, the COOH-terminal portion of the heavy chain of factor Va
appears to contribute to the prothrombin binding site of prothrombinase and is
required for expression of optimum clotting activity.
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DISCUSSION
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Fig. 9 is a schematic
representation of the factor V products derived following NN
(Fig. 9A), RVV-V
activator (Fig. 9B),
and -thrombin (Fig.
9C) treatment. NN cleaves factor V at Asp697
and Asp1509/1514 to produce a molecule that has less clotting
activity than unactivated factor V (factor VNN,
Fig. 9A). The present
work demonstrates that formation of the light chain of factor Va (following
cleavage at Arg1545 of factor V) is a common feature of the cleaved
products, which exhibit optimum association for factor Xa in the assembly of
prothrombinase (Fig. 9). Thus,
factor VIIa/NN, factor VRVV/NN, and factor
VIIa display similar affinities for factor Xa
(Kd of 0.5, 0.57, and 0.3, respectively,
Table I). Each of these three
forms of factor V has a fully formed light chain (1546–2196) and differs
only in the COOH terminus of the heavy chain. With an extended light chain, as
in factor VNN (where the NH2 terminus has the additional
residues 1509-/1514–1545), a weaker factor VNN-factor Xa
interaction was observed (Kd of 4.1 nM).
Thrombin cleavage at Arg1545 restored the affinity of factor
VNN for factor Xa to levels found for factor VIIa
(Kd of 0.5 nM,
Fig. 9D). The current
study indicates that formation of the light chain (1546–2196) is
required to capture factor Xa on the lipid surface and that the addition of as
few as 30 additional residues in the light chain reduces the cofactor and
clotting activity by an order of magnitude or more. This correlates with
observations made during tissue factor-initiated coagulation, in which the
cleavage of factor V at Arg1545 coincides with explosive activation
of prothrombin (73,
74). It is noteworthy that a
similar situation is observed for factor VIII where cleavage at
Arg1689 by -thrombin in the light chain of the cofactor is
required for activation of the cofactor and optimum activity within the
intrinsic tenase complex
(75).
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FIG. 9.
Schematic representation of various factor V species. The domain
structure of the human procofactor (composed of 2196 amino acids) is as
follows (8): the heavy chain
region (residues 1–709) contains two A domains (A1-A2), which
are associated through a region (amino acids 304–316) containing mostly
basic amino acid residues. The COOH-terminal portion of the heavy chain
(657–709) contains a cluster of acidic amino acids, three potential
sulfation sites, and Ser692, which is phosphorylated by a
platelet-derived casein kinase II-like kinase
(76,
77). The light chain of factor
Va (amino acids 1546–2196) includes one A and two C domains
(A3-C1-C2). Both chains are connected in the factor V molecule
through the highly glycosylated B region. The binding site for factor Xa
(AP4') recently described is illustrated as part of the A2 domain
(48), whereas peptide D13R
identified in this study is shown as part of the COOH-terminal portion of the
heavy chain of the cofactor. A, cleavage of factor V by NN results in
a heavy chain containing amino acid residues 1–696 and a
light-chain-spanning amino acid region 1509/1514–2196. It is noteworthy
that all NN cleavage sites described herein are found near the
location of potential sulfation sites
(84). The prothrombin binding
site located on D13R is released from the heavy chain of factor Va and is part
of the B region. B, our sequencing data demonstrate that the
RVV-factor V activator activates factor V following two cleavages only: at
Arg1018 and Arg1545 to produce a heavy chain (amino acid
region 1–1018) and a light chain (amino acid region 1546–2196). It
is important to note that the consequence of cleavage at Arg1545
and Arg1018 of factor V by RVV-V activator is the production of two
fragments of Mr 150,000 (amino acid residues 1–1018
for one and 1019–1545 for the other, the latter being highly
glycosylated). Distinction between these two fragments on a SDS-PAGE gel
following staining with Coomassie Blue or silver nitrate is sometimes impaired
because: 1) the two fragments comigrate and 2) the highly glycosylated
fragment (amino acid residues 1019–1545) does not always stain with
either Coomassie Blue or silver nitrate. C, -thrombin produces
an active cofactor, factor Va, following cleavage of factor V at
Arg709, Arg1018, and Arg1545. These cleavages
release a heavy chain of Mr 105,000 (amino acid region
1–709) and a light chain of Mr 74,000 (amino acid
region 1546–2196). The two subunits of the molecule are associated
through Ca2+ ions. D, -thrombin and
RVV-V activator cleave factor VNN at Arg1545. This
cleavage releases a light chain (amino acid region 1546–2196) and
restores binding to factor Xa. However, the cofactor with the shorter heavy
chain still has impaired clotting activity because of impaired interaction
with prothrombin.
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Although the species factors VIIa/NN, VRVV/NN, and
VIIa exhibited similar affinities for factor Xa, factors
VIIa/NN and VRVV/NN had lower and more variable activity
(20–40%) in clotting assays using factor V-deficient plasma
(Fig. 9D). However,
for prothrombin activation at high (i.e. saturating) concentrations
of factor Xa, the catalytic efficiency of the factor VIIa/NN-factor
Xa complex was as high as that observed for the factor VIIa-factor
Xa complex. Furthermore, we have found that the missing amino acid sequence
from the heavy chain of factor VNN represents a binding site for
prothrombin and that the clotting activity of factor VIIa/NN is
considerably impaired by removal of residues 607–709 from the
COOH-terminal region of the heavy chain. Thus, this region of the molecule is
crucial for cofactor activity under low factor Xa concentrations
(physiological conditions).
Bakker et al.
(46), using a protease
purified from the venom of the snake N. naja oxiana, prepared a
species of factor Va missing an even larger portion (residues 683–709)
of the heavy chain. These investigators concluded that the region
683–709 is required for the binding of factor Xa and/or prothrombin to
factor Va. Thus, whereas amino acid region 697–709 contains a binding
site for prothrombin and appears to be required for prothrombin interaction
and expression of factor Va clotting activity under conditions of limiting
factor Xa concentrations (physiological conditions), it is also possible that
the additional stretch from amino acid residues 683–696 may be also
required for factor Xa and/or prothrombin binding. Thus, this highly acidic
COOH-terminal segment of the heavy chain, which has also been found to be
phosphorylated by a platelet-derived casein kinase II
(76,
77), appears to play a
critical regulatory role for factor Va cofactor function under limiting
concentrations of factor Xa.
We have demonstrated an interaction between D13R, a hirudin-like peptide,
and thrombin-agarose. We have also shown that D13R is a competitive inhibitor
of prothrombinase function. We can thus conclude that D13R contains at least a
portion of the binding site of factor Va for prothrombin. Several studies have
suggested a contribution from the heavy chain of factor Va to the interaction
of prothrombin with prothrombinase. Our findings are in complete agreement
with the data from Dharmawardana and Bock
(30) and Anderson et
al. (33) demonstrating a
productive interaction between factor Va and ABE-I of thrombin and prothrombin
and suggesting that this interaction involves hirudin-like motifs from the
COOH terminus of the factor Va heavy chain
(33). The only hirudin-like
motif of D13R is the sequence Asp-Tyr-Gln
(70). Thus, our findings
together with those on record provide for a new binding site for thrombin and
prothrombin on the COOH terminus of the heavy chain of factor Va to which the
amino acid residues Asp697-Gln699 contribute.
Factor V requires proteolytic processing for expression of full cofactor
activity (3,
4–6,
12,
66,
67). The activation of factor
V by -thrombin, factor Xa
(78,
79), RVV-factor V activator
(80,
81), plasmin
(68,
82), cathepsin G
(83), human neutrophil
elastase (83), and other
enzymes has been the subject of investigation the past decade and has produced
some controversy. Early data using bovine factor V suggested that generation
of the heavy chain of the cofactor following cleavage at Arg713 by
-thrombin (the equivalent of Arg709 in the human molecule)
is sufficient for expression of maximum cofactor activity in an assay
measuring -thrombin generation and employing purified reagents and
saturating concentrations of factor Xa
(4). Using a similar assay and
human plasma factor V, it was reported that cleavage of human plasma factor V
at Arg1018 by factor Xa and generation of a heavy chain portion of
the cofactor (amino acid residues 1–1018) and of a
Mr 220,000 fragment (amino acid residues 1019–2196,
factor VXa) was sufficient to promote maximum cofactor activity
(79). No increase in cofactor
activity was found when factor VXa was treated with
-thrombin (factor VXa/IIa), and no differences in activities
were observed between VXa and factor VIIa. In contrast,
using a clotting assay (5) or
an assay that measures -thrombin formation and employs limiting factor
Xa concentrations (67), it was
reported that cleavage of factor V at Arg1545 and generation of the
light chain alone was sufficient for optimum factor Xa activity within
prothrombinase. The data presented here suggest that at saturating
concentrations of factor Xa, cleavage at Arg709 is sufficient for
optimum factor Va cofactor expression, whereas under conditions of limiting
factor Xa, cleavages at both Arg1545 and Arg709 are
required to promote maximum cofactor activity. Hence, results obtained in a
clotting assay or using factor Xa concentrations below the
Kd for the factor Xa-factor Va bimolecular interaction
within prothrombinase cannot and should not be compared with the data obtained
when using saturating amounts of factor Xa to study the effect of various
factor Va species on prothrombinase assembly and function. Thus, differences
between different groups concerning the "active species of factor
Va" are most likely related to the assay conditions used rather than to
specific proteolytic cleavages of the factor V molecule.
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FOOTNOTES
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* This work was supported by Established Investigator Award 0040100N from the
American Heart Association (to M. K.) and by Merit Award R37 HL34575 from the
National Institutes of Health (to K. G. M.). The costs of publication of this
article were defrayed in part by the payment of page charges. This article
must therefore be hereby marked "advertisement" in
accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 
¶
To whom correspondence should be addressed: Dept. of Chemistry, Cleveland
State University, 2121 Euclid Ave., Science Bldg. SR370, 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; NN, protease from the
venom of N. nigricollis nigricollis; 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; LC/MS, liquid
chromatography/mass Spectrometry, D13R, synthetic peptide containing the last
13 amino acids from the factor Va heavy chain; factor VNN, factor V
cleaved by the protease from N. nigricollis nigricollis; factor
VIIa, factor V cleaved by -thrombin; factor
VRVV-V, factor V cleaved by the RVV-V activator; factor V
NN/IIa, factor V cleaved by the protease from N. naja
nigricollis then cleaved by thrombin; factor V NN/RVV, factor
V cleaved by the protease from N. nigricollis nigricollis then
treated with RVV-V activator; ABE-I, anion binding exosite I; ABE-II, anion
binding exosite II; RVV-V, Russell's viper venom factor V activator; HPLC,
high-performance liquid chromatography.
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ACKNOWLEDGMENTS
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We thank Dr. Alex Kurosky and Steve Smith from the University of Texas,
Medical Branch at Galveston, for sequencing from polyvinylidene difluoride
membranes, Dr. Satya Yadav, from the Cleveland Clinic Foundation for peptide
synthesis, and Drs. David Anderson and Xiang Zhou from the Cleveland Mass
Spectrometry Facility at Cleveland State University for LC/MS analysis of the
peptides used in this study.
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REFERENCES
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