From the Departments of Molecular and Experimental Medicine and of
Vascular Biology, The Scripps Research Institute,
La Jolla, California 92037
Factor Va inactivation by activated protein C is
associated with cleavages at Arg306,
Arg506, and Arg679 with Arg306
cleavage causing the major activity loss. To study functional roles of
the Arg306 region, overlapping 15-mer peptides representing
the sequence of factor Va residues 271-345 were synthesized and
screened for anticoagulant activities. The peptide containing residues
311-325 (VP311) noncompetitively inhibited prothrombin activation by
factor Xa, but only in the presence of factor Va. Fluorescence studies showed that VP311 bound to fluorescence-labeled
5-dimethylaminonaphthalene-1-sulfonyl-Glu-Gly-Arg factor Xa in solution
with a Kd of 70 µM.
Diisopropylphosphoryl factor Xa and factor Xa but not factor VII/VIIa
or prothrombin bound to immobilized VP311 with relatively high
affinity. These results support the hypothesis that residues 311-325,
which are positioned between the A1 and A2 domains of factor Va and
likely exposed to solvent, contribute to the binding of factor Xa by factor Va. Based on this hypothesis, it is suggested that cleavage by
activated protein C at Arg306 in factor Va not only severs
the covalent connection between the A1 and A2 domains but also disrupts
the environment and structure of residues 311-325, thereby
down-regulating the binding of factor Xa to factor Va.
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INTRODUCTION |
Blood coagulation factor Va
(FVa)1 is the essential
cofactor for the prothrombinase complex that consists of factor Xa
(FXa), phospholipids, calcium ions, and FVa and that is responsible for conversion of prothrombin to thrombin (1-6). FVa generated by limited
proteolysis of FV is usually composed of a heavy chain containing the
A1-A2 domains in amino acid residues 1-709 and a light chain
containing the A3-C1-C2 domains in residues 1546-2196. These two
chains are noncovalently associated in the presence of divalent metal
ions (3, 7). Protein C is a vitamin K-dependent plasma
protein zymogen that is cleaved by thrombin to yield the active serine
protease, activated protein C (APC). APC down-regulates blood
coagulation by proteolytic inactivation of the cofactors factor Va and
factor VIIIa (8, 9). Irreversible proteolytic inactivation of FVa by
APC is reported to be associated with three cleavages at
Arg306, Arg506, and Arg679 in the
FVa heavy chain, whereas cleavage at only Arg306 in FV
causes full loss of its activity (10). The importance of specific
cleavages has been studied using purified Gln506-FVa that
lacks the Arg506 cleavage site (11-14). Inactivation of
FVa by APC proceeds via a biphasic reaction that consists of a rapid
and a slow phase. The rapid phase is associated with an initial
cleavage at Arg506 and partial loss of activity (~30%),
whereas extensive or complete inactivation of FVa requires cleavage at
Arg306. The contribution of cleavage at Arg679
to FVa inactivation is presently unclear. All published results suggest
that cleavage at Arg306 plays the most important role for
inactivation of FVa as well as for FV. Inactivation of FVa by APC is
associated with loss of the ability of FVa to bind FXa and with
dissociation of the A2 domain of FVa from the rest of the cleaved FVa
(15, 16). To help clarify why cleavage at Arg306
inactivates FV and FVa, overlapping 15-mer peptides representing FVa
heavy chain residues 271-345 were synthesized and screened for their
ability to inhibit prothrombin activation using purified prothrombinase
components. The results presented here suggest that the region between
the A1 and A2 domains of FVa involving residues 311-325 of FVa
provides a binding site for FXa and implies that APC cleavage at
Arg306 down-regulates FVa activity, at least in part, by
disrupting the immediate environment and/or structure of this
FXa-binding site.
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EXPERIMENTAL PROCEDURES |
Peptide Synthesis--
Peptides with amino-terminal
-amino
groups and carboxyl-terminal carboxamide moieties were prepared under
the supervision of Dr. Richard Houghten of the Torrey Pines Institute
for Molecular Studies using the simultaneous multiple synthesis method
(17) and were analyzed by reverse-phase high pressure liquid
chromatography and mass spectral analysis to verify purity and
composition (17, 18). Alternatively, some peptides were synthesized by
and purchased from the Peptide Synthesis Group (Beckman Center,
Stanford University, Palo Alto CA).
Proteins--
Human FVa, prothrombin, and phospholipid vesicles
(20% phosphatidylserine, 80% phosphatidylcholine) were prepared as
described (18-20). Human FXa was purchased from Enzyme Research Labs
(South Bend, IN). Diisopropylphosphoryl (DIP)-FXa (
99% inactivated) was prepared by incubation of FXa at 1 mg/ml with 2 mM
diisopropyl fluorophosphate (Sigma) on ice for 2 h followed by
prolonged dialysis at 4 °C against Tris-buffered saline (0.05 M Tris-HCl, 0.1 M NaCl, 0.02%
NaN3, pH 7.4). Human 1,5-dansyl-Glu-Gly-Arg-factor Xa
(DEGR-Xa) was purchased from Hematologic Technologies, Inc. (Essex
Junction, VT). FVII/VIIa and rabbit anti-FVII were purchased from
Celsus (Cincinnati OH), and monoclonal antibody against prothrombin was from Biodesign (Kennebunk, ME).
Prothrombinase Assay--
Prothrombinase assays were performed
at room temperature as described elsewhere (19) and employed 20 pM FVa, 1 nM FXa, 25 µM or 50 µM phospholipid vesicles, 5 mM
CaCl2, and 0.3 µM prothrombin unless
otherwise indicated in buffer containing 0.05 M Hepes, 0.1 M NaCl, 5 mM CaCl2, 0.1 mM MnCl2, 0.02% NaN3, and 0.5%
bovine serum albumin. The rate of prothrombin activation was assessed using the chromogenic substrate
H-D-cyclohexylglycyl-L-
-aminobutyryl-L-arginine-p-nitroanilide (final concentration, 0.2 mM) (American Bioproducts,
Parsippany, NJ) in an EL312 microplate reader using Kineti-calc
software (Biotek, Winooski, VT). It should be noted that this
amidolytic assay cannot distinguish formation of
-thrombin from
meizo-thrombin.
Fluorescence Titrations--
Fluorescence titrations were
performed using an SLM Aminco Bowman Series 2 Luminescence Spectrometer
(Spectronic Instruments, Inc., Rochester, NY) following the procedures
of Krishnaswamy et al. (21) with some modifications. For
these experiments the excitation wavelength was 340 nm (band pass, 4 nm) and the emission wavelength was 545 nm (band pass, 16 nm). A
408-nm-long pass filter (KV-408) was used in the emission path to
minimize scattered light artifacts. All buffers were filtered with
0.2-µm filters, and protein solutions were centrifuged to remove
particulate matter. The sample compartment was maintained at 25 °C
with a circulating water bath. Microliter additions of a 1 mM stock solution of peptide or buffer alone were added to
a square 5-mm path length cuvette containing 300 µl of reaction
mixture of DEGR-Xa at 200 nM in 50 mM Hepes, pH
7.4, 0.15 M NaCl, 5 mM CaCl2, and
fluorescence intensity measurements were made 1 min after each
addition. Three 5-s readings were made and averaged to determine the
final value. Three titrations were done to allow for correction of
fluorescence intensity values because of light scattering or any other
artifacts. Titration A involved additions of peptide to DEGR-Xa.
Titration B involved additions of control buffer to DEGR-Xa. Titration
C involved additions of peptide to buffer alone. The corrected
fluorescence change was then calculated according to the expression
|
(Eq. 1)
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where FA, FB, and
FC are the fluorescent intensities from the
above titration mixtures and FC' is
the intensity recorded for control buffer alone in the absence of added
peptide. The net fluorescence intensity change
(F/Fo) was converted to percent, and nonlinear
least squares regression was used to fit the data to the single ligand
binding equation
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(Eq. 2)
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where [P] is the peptide concentration. The
Kd and
Fmax were derived
from data fitted using this equation.
Plate Binding Assays--
Binding assays were performed as
described (19). Peptides at 20 µM were coated on the
wells of Xenobind microtiter plates (Xenopore, Saddle Brook, NJ)
according to manufacturer's instructions and then blocked with 3%
hydrolyzed fish skin gelatin (Sigma) in Tris-buffered saline. After
washing the plate with Tris-buffered saline, various concentrations of
DIP-FXa or FXa in binding buffer consisting of 0.05 M Tris,
0.2 M NaCl, 5 mM CaCl2, 0.1 mM MnCl2, 0.02% NaN3, and 0.5%
porcine skin gelatin (Sigma) were incubated in plate wells for 1 h
at room temperature. Following washings, bound DIP-Xa was detected
using a monoclonal antibody to FX (purified IgG from Biodesign), which
was quantitated with biotin-secondary antibody, streptavidin-alkaline
phosphatase, and phosphatase substrate as described (19). Detection of
bound factor VII/VIIa and prothrombin was similarly made using
appropriate antibodies. Detection of bound FXa was made using a
chromogenic substrate
N-
-benzyloxycarbonyl-D-arginyl-L-glycyl-L-arginine-p-nitroanilide (Chromogenix, Franklin, OH). The absorbance values observed for duplicate noncoated wells lacking peptides served as nonspecific controls for binding and were subtracted from absorbance values for
corresponding duplicate peptide-coated wells. Nonspecific binding
ranged from 5 to 30% of total observed binding in various experiments.
 |
RESULTS |
To clarify potential functional roles of the region around the APC
cleavage site at Arg306 in the FVa heavy chain, seven
overlapping 15-mer synthetic peptides representing FVa sequences from
residues 271-345 (Table I) were tested
for their ability to inhibit prothrombinase assays in the presence and
absence of FVa (Fig. 1). At 100 µM, peptide VP311 strongly inhibited prothrombinase
activity in the presence of FVa, whereas peptide VP321 had a moderate
inhibitory effect on prothrombinase activity. In the absence of FVa,
VP311 did not inhibit prothrombin activation; however, it modestly and
reproducibly enhanced prothrombinase activity by 50% (Fig. 1). To
define prothrombinase inhibition by peptides, various concentrations of
peptides were preincubated with FXa, FVa, or prothrombin, followed by
addition of other prothrombinase components for activity assays (Fig.
2). VP311 inhibited prothrombinase
activity only in the presence of FVa (Fig. 2B). In the
absence of FVa, VP311 at 100-200 µM reproducibly modestly enhanced prothrombinase activity by approximately 50% (Figs.
2B and 3B). Peptide
VP321 showed only moderate inhibition in the presence of FVa, whereas
at 200 µM it also modestly enhanced prothrombinase
activity in the absence of FVa (Fig. 2C). Peptide VP301,
which contains Arg306 and peptide VP331, like VP271, VP281,
and VP291 (Fig. 1 and data not shown), had no effect on prothrombinase
activity under any preincubation conditions (Fig. 2, A and
D).

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Fig. 1.
Inhibition of prothrombinase by synthetic
overlapping 15-mer peptides representing residues 271-345 of factor
V. Peptides at a final concentration of 100 µM were
preincubated with 1 nM (final concentration) FXa for 30 min
at room temperature. Phospholipid vesicles, FVa, and prothrombin were
added as described under "Experimental Procedures." Aliquots from
the reaction mixture were removed every 30 s into amidolytic assay
buffer containing 10 mM EDTA to stop the prothrombinase
reaction. The rate of appearance of thrombin amidolytic activity was
determined. Stipled and striped bars show
prothrombinase activity in the presence or absence of FVa,
respectively. The percentage of activity was calculated by defining the
activity of control reaction mixtures in the absence of peptide as
100%. Peptide abbreviations and sequences are given in Table I.
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Fig. 2.
Inhibition of prothrombinase by synthetic
15-mer peptides representing residues 301-345 of factor V. Peptides at various concentrations (0-200 µM) were
preincubated with (preinc. w.) 0.3 µM
prothrombin (FII) ( ), 1 nM FXa ( ), 20 pM FVa (×), or 0.3 µM prothrombin without
subsequent FVa addition ( ) for 15 min at room temperature. Then the
other prothrombinase components were added to initiate thrombin
formation, except where the absence of FVa is indicated. The synthetic
peptides (Table I) were VP301 (A), VP311 (B),
VP321 (C), and VP331 (D). The percentage of
prothrombinase activity (rate of thrombin formation) without peptide
was defined as 100%. Each point is the average of duplicate
determinations.
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Fig. 3.
Effect of peptides VP311 and VP311reverse on
prothrombinase in the presence and absence of factor Va. Peptide
VP311 (closed symbols and solid line) or a
control peptide, VP311 reverse, that contained the reverse amino acid
sequence (open symbols and dashed line) were
preincubated at concentrations indicated for 30 s with FXa
(circles) or with FVa (triangles) and
phospholipids for 30 s prior to addition of other prothrombinase
components to initiate thrombin formation. Prothrombinase assays were
performed as described under "Experimental Procedures" except that
the buffer contained 0.05 M NaCl. In panel B,
FVa was omitted. Symbols represent the mean of two
(panel A) or three (panel B) separate
experiments.
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A control peptide with the reverse sequence of amino acids of VP311,
designated VP311reverse, was synthesized and tested in parallel with
VP311 for inhibition of prothrombinase. Fig. 3A shows that
under conditions where VP311 inhibited prothrombinase by up to 90%,
peptide VP311reverse inhibited prothrombinase only slightly. In the
absence of FVa (Fig. 3B) where VP311 at 100-200 µM stimulated prothrombinase activity by 80%,
VP311reverse in contrast slightly inhibited prothrombinase activity
just as it did in the presence of FVa. Moreover, the inhibition of
prothrombinase by VP311 cannot be simply due to a net high positive
charge effect or an effect due to adjacent basic residues because
VP301, which also contains a high net positive charge and two sets of
adjacent basic residues, did not inhibit prothrombinase activity
(Fig. 1). These results suggest that residues 311-335 in the FVa heavy chain may contribute to FXa-FVa and/or prothrombin-FVa
interactions.
A series of Lineweaver-Burk plots for prothrombinase activity at
varying prothrombin concentrations is seen in Fig.
4 for various concentrations of VP311.
Peptide VP311 inhibited prothrombinase activity with a pattern of
noncompetitive inhibition, and the apparent Ki under
these experimental conditions was 140 µM. This suggests
that the effect of VP311 is not explained by competition for binding of
the substrate, prothrombin, to FVa.

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Fig. 4.
Lineweaver-Burk plots for prothrombin
activation at varying concentrations of VP311. FXa (final
concentration, 1 nM) was preincubated with 0, 50, 100, or
200 µM of VP311 in duplicate for 15 min. FVa and
phospholipids were then added, and various concentrations of
prothrombin (0.07 to 0.67 µM) were added to initiate
prothrombin activation that was quantitated as described under
"Experimental Procedures."
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The specific binding of peptide VP311 to FXa was measured to test the
hypothesis that the sequence of VP311 represents a FXa-binding site in
FVa. Because we found that the addition of VP311 to DEGR-Xa quenched
the dansyl fluorescence of the labeled protein, binding of VP311 to the
protein in solution was monitored by fluorescence intensity changes of
the dansyl group in DEGR-Xa (Fig. 5). The apparent Kd of peptide VP311 for DEGR-Xa was
determined, based on the average value from three experiments, to be
71 ± 9 µM with a
Fmax of
39%. This agrees reasonably well with the concentration of peptide
VP311 required for 50% inhibition of the prothrombinase assays,
i.e. 40-140 µM (Figs. 2B,
3A, and 4). The VP311-dependent decrease in
dansyl fluorescence of DEGR-FXa (Fig. 5) was specific because the
control peptide VP311reverse at 0-100 µM did not cause a
significant change (<4%) in dansyl fluorescence (data not shown).
Moreover, peptide VP301 that has a high positive charge because of its
Arg/Lys content and that contains Arg306, which presents
the peptide bond cleaved during inactivation of FVa by activated
protein C, did not cause a significant change in the fluorescence of
DEGR-Xa. These data support the hypothesis that FVa residues 311-335
provide a binding site for FXa.

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Fig. 5.
Binding of Peptide VP311 to DEGR-Xa.
Peptide VP311 (+) or VP301 ( ) was titrated into a 200 nM
solution of DEGR-Xa in HBS containing 5 mM
CaCl2. The dansyl fluorescence intensity of DEGR-Xa was
monitored as described under "Experimental Procedures." The
percentage of fluorescence is relative to the original fluorescence
intensity of DEGR-Xa without peptide additions defined as 100%. The
line for VP311 was generated according to a single ligand binding
equation using nonlinear regression, with Kd = 71 µM and Fmax = 39%.
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An additional approach was used to assess the binding of VP311 to FXa
in which FXa was bound to peptides immobilized on microtiter plates. In
this type of solid phase binding assay that involves a small
surface-bound peptide, apparent Kd values may be
significantly lower than apparent Kd values
determined in fluid phase binding assays, possibly because proteins
have an abnormally low off-rate constant once bound near a surface that
is multivalent because it is coated with ligand and possibly because
the hydrophobic surface itself may contribute to protein binding.
Furthermore, immobilized peptides have reduced degrees of freedom.
Thus, apparent binding constants determined by solid phase assays
cannot be considered to be real binding constants and cannot be
compared with fluid phase real binding constants. Nevertheless, these
types of assays can be useful to compare relative binding affinities
for similar ligands. DIP-FXa and FXa bound tightly to immobilized VP311
(Fig. 6). Apparent Kd
values calculated by Scatchard analysis using Enzfitter software
averaged 10 nM (n = 4 experiments) for
DIP-FXa and 46 nM (n = 2 experiments) for
FXa. This demonstrated that both normal FXa and FXa with a modified
active site bound to VP311. As controls, factor VII/VIIa showed no
binding to VP311 and prothrombin showed only weak binding (apparent
Kd > 400 nM) (Fig. 6). Moreover, FXa
did not bind to the immobilized basic peptide VP301. These results
further support the hypothesis that FVa residues 311-335 provide a
FXa-binding site.

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Fig. 6.
Binding of DIP-FXa and FXa to immobilized
peptide VP311. Various concentrations of DIP-FXa ( ), FXa ( ),
factor VII/VIIa ( ), or prothrombin ( ) were incubated in
microtiter plate wells that had been coated with peptide VP311, and
then bound proteins were detected as described under "Experimental
Procedures."
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DISCUSSION |
Synthetic peptides that inhibit multicomponent enzyme complexes
can provide useful information about protein-protein interactions. To
identify potential roles in the prothrombinase complex of FVa heavy
chain residues near the APC cleavage site at Arg306, seven
15-mer peptides representing FVa residues 271-345 were studied, and
peptide VP311 (residues 311-325) was found to inhibit prothrombinase
activity but only in the presence of FVa. The sequence of peptide VP311
represents a major part of the connecting region between the A1
(residues 1-301) and A2 (residues 320-656) domains of the heavy chain
(residues 1-709) of FVa (6, 22). Inhibition of prothrombinase activity
by VP311 only in the presence of FVa suggests that this connecting
region of FVa containing residues 311-325 might contribute to FXa-FVa
and/or prothrombin-FVa interactions. Alternatively or additionally,
VP311 could inhibit prothrombinase activity by disrupting important FVa
intramolecular interactions. Kinetic data showed prothrombinase
inhibition by VP311 to be noncompetitive with respect to prothrombin,
suggesting that VP311 is not simply competing for prothrombin binding
to the prothrombinase complex.
Human FVa heavy chain has 84% overall homology with bovine FVa heavy
chain, whereas peptide VP311 has 12 of 15 residues identical in human
and bovine FV. Protein regions with a high percentage of homology
between different species are often functionally important. Peptide
VP311 also has a sequence motif that is present in peptides representing sequences in APC and human group II secretory
phospholipase A2 that have been implicated in
prothrombinase inhibition. This motif (KRXXKR) is present in
the inhibitory peptide 142-155, including residues 146-151 of APC
(KRMEKK), which was shown to inhibit FXa coagulant activity in the
presence of FVa (23). A similar motif is present in phospholipase
A2 from residues 52-57 (KRLEKR). A peptide from residues
51-74 of phospholipase A2 was found to bind specifically
to FXa (24). This motif in peptide VP311 involving residues 315-320
(RRHMKR) may be responsible for binding to a specific FVa-binding
exosite on FXa. Based on these three peptides, each of which inhibit
prothrombinase only in the presence of FVa, the putative FXa-binding
motif is (K/R)RXYK(R/K) where there may be a preference for
E at residue Y and for a bulky hydrophobic or neutral side
chain at residue X. The residues preceding the basic
hexapeptide motif in the proteins include Trp, Tyr, and Gln and may
indicate a requirement for a large side chain capable of H-bonding.
To test the hypothesis that VP311 disrupts FXa-FVa interactions by
binding to FXa, both solution phase and solid phase binding studies
were performed. In solution, peptide VP311 bound to DEGR-Xa with a
Kd of 71 µM, whereas peptides
VP311reverse and VP301 did not significantly bind to DEGR-Xa. The
Kd of 71 µM based on fluorescence
titrations is similar to the VP311 concentration 40-140
µM required for 50% inhibition of prothrombinase activity. The interactions of bovine factor Va and bovine DEGR-Xa have
been studied (25, 26). Upon binding to DEGR-Xa factor Va causes an
increase in the fluorescence intensity of the dansyl reporter group in
DEGR-Xa. In the presence of phospholipid vesicles the calculated
Kd of bovine factor Va for DEGR-Xa was 1 nM. Unlike these results peptide VP311 caused a quenching
of dansyl fluorescence intensity rather than an increase. It is not entirely clear why the direction of the effect would be opposite of
that for factor Va. However, because fluorescence intensity is
dependent on a variety of factors, including protein conformation and
solvent exposure, it should not be unexpected that two molecules of
such drastically different size might have different effects on the
fluorescent intensity of the dansyl group.
Although protein binding studies using immobilized peptides do not
yield real equilibrium binding constant values and cannot be compared
with fluid phase binding constants, such studies can provide useful
qualitative descriptions of binding and may allow comparisons of
relative affinities for different ligands or peptides. Binding assays
using immobilized peptides showed that DIP-FXa and FXa do bind to VP311
with relatively high affinity, whereas two homologous vitamin
K-dependent proteins, factor VII and prothrombin, do not
bind to immobilized VP311 with comparable measurable affinity. Thus,
the fluid phase and the solid phase binding studies combined with the
prothrombinase inhibition data support the hypothesis that FVa residues
311-325 contain a binding site for FXa.
In the absence of FVa, peptide VP311 at 200 µM
reproducibly mildly enhanced rather than inhibited FXa activity,
possibly mimicking in some way the cofactor effect of FVa on FXa. This
effect in the absence of FVa is consistent with the concept that the
sequence of VP311 binds to FXa. The control peptide, VP311reverse, did not stimulate FXa activity, showing specificity for the normal 311-325
sequence. In parallel to the ability of VP311 to stimulate FXa activity
in the absence of FVa, it was recently reported that a peptide
corresponding to FVIII residues 698-712 enhances FIXa activity in the
absence of FVIIIa, whereas the same FVIII peptide inhibits FIXa
activity in the presence of FVIIIa (27). Thus, each respective peptide
may represent a protease-binding site on the respective cofactors, and
binding of each peptide may induce a conformational change in its
respective coagulation protease, producing a mild enhancement of the
protease activity that is much less effective than that of the intact
cofactor.
FV and FVIII possess a common domain structure, A1-A2-B-A3-C1-C2 (6,
22, 28). There is approximately 40% amino acid sequence identity
between FV and FVIII in the amino-terminal heavy chain regions (A1-A2),
and the three A domains of FV and FVIII show a minimum of 30% identity
with any other A domain (28). In addition, schematic models of the
structures of FVa and FVIIIa based on electron micrographs show certain
similarities (29-31). The three A domains of FV and FVIII resemble the
three A domains of human ceruloplasmin whose three-dimensional
structure was solved using x-ray crystallography (32). Ceruloplasmin is
a six-domain structure comprising a heterotrimer of heterodimers, each
dimer containing two
-barrel structures homologous to plastocyanin (32, 33). A homology model of the three A domains of FVIII based on
this ceruloplasmin structure has recently been published (34), and
another FVIII homology model based on nitrite reductase has appeared
(35). The FVIII homology models propose that the A1-A2-A3 domains of
FVIIIa form a trimer of heterodimers, with each domain containing two
similar but distinct
-barrel plastocyanin-like structures (34).
Based on the homologies of FV, FVIII, and ceruloplasmin, some
reasonable though speculative insights about FVa structure may be drawn
from inspection of the FVIII homology model and the ceruloplasmin x-ray
crystallographic structure. The APC cleavage site at Arg306
in the FVa heavy chain is in the solvent-exposed sequence (residues 302-319) connecting the A1 and A2 domains, and VP311 contains much of
this sequence that is easily accessible to FXa and/or APC. Binding of
FXa to this connecting region could block the accessibility of
Arg306 to APC, thereby causing the known protective effect
of FXa against FVa cleavage by APC (36-40). Furthermore, the APC
cleavage site at Arg506 in FVa is situated between the two
plastocyanin-like
-barrels of the A2 domain and is exposed to
solvent, homologous to Arg562 in FVIIIa (35).
In the prothrombinase complex, FVa and FXa interact stoichiometrically
and FVa has an extended binding site for FXa with contributions from
both the heavy and light chains (2, 41-43). Included in this extended
binding interaction are residues 311-325, as shown here, and residues
493-506, which were previously shown to interact with FXa (44, 45). In
the human ceruloplasmin x-ray crystallographic structure the sequences
homologous to residues 493-506 and 311-325 of FVa are adjacent on the
protein surface and are generally within 9-20 Å of one another (32).
Inspection of the FVIIIa homology model structure of Pemberton et
al. (34) indicates that the peptides homologous to these two
sequences of FVa are directly adjacent to one another on the surface of
the "bottom" of the protein. The distance in the FVIIIa model
between the
-carbons of FVIII residues 562 and 385 (corresponding to
FV residues 506 and 325) is 15.1 Å, and the
-carbons of FVIII
residues 561 and 382 (corresponding to FV residues 505 and 322) are 9.2 Å apart. Because cleavage at Arg306 in FV or FVa causes
loss of most or all FVa activity, whereas cleavage at only
Arg506 ablates approximately 30% of FVa activity (10, 14),
the structural integrity of the region around Arg306 is
apparently more important than that of Arg506 for the
structure and function of FVa. The Arg306 cleavage may be
lethal because of loss of the FXa-binding site, destabilization of the
trimeric A1-A2-A3 structure of FVa because of loss of the covalent link
between the A1 and A2 domains potentially with dissociation of the A2
domain (16), or an overlapping combination of these effects.
In conclusion, our data suggest that residues 311-325 in FVa provide a
FXa-binding site that may be essential for prothrombinase activity.
Cleavage of FVa at Arg306 by APC severs the covalent
linkage between the A1 and A2 domains and likely alters FVa tertiary
structure, especially of the FXa-binding site involving residues
311-325, such that FXa binding is ablated or greatly diminished and
FVa is irreversibly inactivated.
We thank Yolanda Montejano and Marissa
Maley for assistance in purification of FV and prothrombin, Dr.
Stephen Kent for mass spectral analyses of peptides, and Dr. Richard
Houghten and James Winkle for peptide synthesis.