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INTRODUCTION |
Protein C is a multi domain vitamin K-dependent plasma
serine protease zymogen, which upon activation by the
thrombin·TM1 complex,
down-regulates the blood coagulation cascade by selectively inactivating factors Va and VIIIa (1-3). Monovalent and divalent cations regulate the protein C anticoagulant pathway at the level of
both the protein C zymogen activation and the catalytic function of
activated protein C (APC). In the zymogen activation process, binding
of Ca2+ to the N-terminal Gla domain and the first
epidermal growth factor like domain-1 is required for efficient
assembly of the protein into the activation complexes on cell surfaces
(4, 5). In addition to the Gla domain, Ca2+ binding to a
high affinity site in the catalytic domain induces a conformational
change in the activation peptide of protein C that is required for
rapid activation by the thrombin·TM complex (6). Following the
activation, the non-catalytic domains of APC remain covalently linked
to the serine protease domain to mediate the Ca2+- and
Gla-dependent binding of APC to its specific cofactors, protein S or the endothelial cell protein C receptor (7-9). Although several previous studies have demonstrated that Ca2+ and
other divalent cations stimulate the amidolytic and esterolytic activity of APC (10, 11), it is not known if occupancy of the
Ca2+-binding site in the catalytic domain of APC is also
required for its physiological function.
Similar to Ca2+, the monovalent cation Na+
regulates the protein C anticoagulant pathway at the level of both
zymogen activation and APC function. In this case, it is believed that
Na+ allosterically regulates the activity of thrombin and
that protein C is activated preferentially by the Na+-free
form of thrombin, which is referred to as the slow form (12, 13). The
Na+-bound or fast form of thrombin is believed to function
in the procoagulant pathway by specifically activating fibrinogen (12, 13). A dissociation constant of 30-100 mM for
Na+ binding to thrombin has been reported (12, 14). In
contrast to protein C activation, however, it has been demonstrated
that bovine APC displays a strict requirement for Na+ (or
other monovalent cations) for expression of its amidolytic activity
toward tri-peptide chromogenic substrates (15). In previous studies an
apparent dissociation constant of 87-129 mM for
Na+ binding to bovine APC and activated Gla domainless
protein C (aGDPC) has been reported (15-17). The exact role of
Na+ in the activity of APC toward its physiological
substrates, factors Va and VIIIa, is not known.
In contrast to the divalent cation-binding sites of APC, the monovalent
cation-binding site(s) of APC has not been identified. In the case of
thrombin, a single metal-binding site for Na+ has been
localized to a conserved loop consisting of residues 221-225 in the
chymotrypsin numbering system (18, 19). The predicted consensus
Na+-binding sequence in this loop is conserved in APC but
not in trypsin (19). To determine whether this loop in APC binds
Na+, we prepared a mutant lacking the Gla domain (GDPC) in
which the 221-225 loop of GDPC (Gly221-Tyr225)
was replaced with the corresponding residues of trypsin
(Ala221-Pro225). Both the wild type and GDPC
mutant (GDPC tryp/loop) were expressed in mammalian cells, and
sufficient quantities of both derivatives were isolated for activation
and characterization. In addition to this mutant, we recently prepared
and characterized another GDPC mutant in which Glu80 in the
Ca2+ binding loop of the molecule was replaced with Lys
(E80K) (6). In this previous study, we demonstrated that the 70-80
loop in this mutant was stabilized in the Ca2+ conformer
possibly by Lys80 forming a salt bridge with
Glu70 (6). In the current study, the catalytic activities
of the activated wild type, tryp/loop, and E80K GDPC (aGDPC)
derivatives were monitored by their ability to hydrolyze several
chromogenic substrates in both the absence and presence of
Na+ and Ca2+. We found that unlike the wild
type protease, the aGDPC tryp/loop mutant lost its ability to bind
Na+ and no longer discriminated between various monovalent
cations. The affinity of the mutant protease for Ca2+ was
also impaired ~16-fold. Interestingly, further study suggested that
the affinity of the wild type aGDPC for Na+ in the presence
Ca2+ was improved ~20-fold, and the binding of aGDPC E80K
to Na+ was of the high affinity type independent of
Ca2+. These results suggest that the 221-225 loop is a
Na+-binding site in APC, which is allosterically linked to
the divalent cation-binding loop of the protease. The allosteric
coupling of the two metal ion-binding loops in APC has likely played a
role in the divergent evolution of this highly specific anticoagulant enzyme.
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EXPERIMENTAL PROCEDURES |
Mutagenesis, Expression, and Purification--
The expression of
Gla domainless protein C (GDPC) and GDPC E80K by the RSV-PL4
expression/purification vector system in human 293 cells has been
previously described (6, 20). The 221-225 loop
(Gly221-Tyr225) of GDPC was replaced with the
corresponding sequence of trypsin (GDPC tryp/loop) by the polymerase
chain reaction mutagenesis approach, and the mutant molecule was
expressed in the same vector system as described previously (20, 21).
Accuracy of the mutations was confirmed by sequencing prior to
expression. The wild type and mutant zymogens were purified from the
cell culture supernatants as described previously (20).
Human plasma protein C (22), human plasma thrombin (23), human factor
Va (24), bovine antithrombin (25), recombinant human prethrombin-1
(26), and recombinant human thrombomodulin fragment 4-6 (TM4-6, the
minimum fragment of TM required as a cofactor for thrombin activation
of protein C) (20) were prepared by the cited methods. The chromogenic
substrate Spectrozyme PCa (SpPCa) was purchased from American
Diagnostica (Greenwich, CT), and S2266 and S2238 were purchased from
Kabi Pharmacia/Chromogenix (Franklin, OH).
Activation of GDPC and GDPC Tryp/Loop by the Thrombin·TM4-6
Complex--
The initial rate of protein C activation by the
thrombin·TM4-6 complex (1 nM thrombin in complex with
100 nM TM) was measured as a function of different
concentrations of GDPC or GDPC tryp/loop in 0.1 M NaCl,
0.02 M Tris-HCl, pH 7.5 (TBS), containing 0.1% polyethylene glycol 8000 (PEG 8000), and 2.5 mM
Ca2+ at room temperature. After inhibition of thrombin
activity by antithrombin, the initial rates of activation were measured
from the rate of activated protein C generation in an amidolytic
activity assay using 1 mM SpPCa in TBS buffer containing
0.1% PEG 8000 and 2.5 mM Ca2+. The rate of
hydrolysis was measured at 405 nm at room temperature in a
Vmax kinetic plate reader (Molecular Devices,
Menlo Park, CA). Fixed reaction times of 5 min were employed for each
GDPC concentration. Over this time only the initial rate of activation was measured and less than 1% substrate was activated. The
concentration of active protein C derivatives in reaction mixtures was
determined by reference to a standard curve, which was prepared by
total activation of GDPC at the time of each experiment. This was
accomplished by activation of 1 µM each protein C
derivative with 10 nM thrombin in complex with 100 nM TM4-6 and 2.5 mM Ca2+ for 90 min at 37 °C. Under these experimental conditions, all protein C
zymogens were completely activated in less than 30 min. The
Km and kcat values of protein
C activation were calculated from the Michaelis-Menten equation.
Protein C activation was also carried out on thrombin·TM complex
assembled on Affi-Gel 10 (Bio-Rad) as described previously (22). The
active site concentrations of the aGDPC derivatives were determined by
an active site-specific immunoassay using BioCap-FPR-ck
(biotinyl-
-aminocaproyl-D-phenylalanine prolylarginine chloromethyl ketone) (Hematologic Technologies Inc.,
Essex Junction, VT) as described previously (27). The aGDPC derivatives
were desalted by passing through PD-10 (Amersham Pharmacia Biotech) gel
filtration columns equilibrated with Chelex-treated (Bio-Rad) 5 mM Tris-HCl, pH 8.0, buffer containing 0.1% PEG 8000 and
stored at
80 °C.
Steady-state Kinetics--
The steady-state kinetic parameters
of wild type and mutant enzyme toward S2266
(D-Val-Leu-Arg-p-nitroanilide) was performed at room
temperature in 5 mM Tris-HCl, pH 8.0, containing 0.1% PEG
8000, 2.5 mM CaCl2, and 0.2 M of
different monovalent chloride salts (LiCl, NaCl, KCl, and choline
chloride). The rate of hydrolysis was measured at 405 nm at room
temperature in a Vmax kinetic plate reader as
described above. The apparent Km and
kcat values for substrate hydrolysis were
calculated from the Michaelis-Menten equation. The specificity constant
for each chloride salt was expressed as the ratio of
kcat/Km. The specificity
constant for the bulky monovalent cation choline (Ch+) was
used as a reference to determine the monovalent cation specificity as
described by Dang and Di Cera (19). The concentration of S2266 ranged
from 20 µM to 15 mM depending on the
Km values, and the concentration of enzymes ranged
from 10 to 100 nM depending on the
kcat values.
Dissociation Constant (Kd(app)) for
Na+--
The values for
Kd(app) of Na+ binding to
each protease was determined from the effect of varying concentrations
of Na+ on the activity of the protease toward three
synthetic substrates SpPCa, S2266, and S2238 in both the absence
(Chelex-treated buffer) and presence of 2.5 mM
Ca2+. In all experiments a constant ionic strength of 0.2 M was maintained by addition of choline chloride. This
procedure has been commonly used in the past to study the effect of
monovalent cations on the catalytic function of various enzymes (12,
19). The values for Kd(app) were
calculated from the hyperbolic increase in the rate of substrate
hydrolysis as a function of increasing Na+ concentrations.
Factor Va Inactivation--
The time course of human factor Va
inactivation by activated GDPC was measured by a two-stage assay. In
the first stage, factor Va (50 nM) was incubated with aGDPC
(5 nM) at room temperature in 0.02 M Tris-HCl,
pH 7.5, containing 0.15 M NaCl or 0.15 M KCl and 0.1% PEG 8000. This stage of the assay was carried out in both the
absence and presence of 2.5 mM Ca2+. For this
purpose, aGDPC and factor Va (in 5 mM Ca2+)
were passed through two separate PD-10 gel filtration columns equilibrated with Chelex-treated 0.02 M Tris-HCl, 0.1% PEG
8000 and used immediately in kinetic experiments. In the second stage, at different time intervals (0-12.5 min) the remaining activity of
factor Va was determined by measuring its ability to accelerate factor
Xa activation of recombinant human prethrombin-1 as described previously (28).
Data Analysis--
The apparent Km and
kcat values for substrate hydrolysis were
calculated from the Michaelis-Menten equation, and the affinity of
Na+ for each aGDPC derivative
(Kd(app)) was determined by nonlinear
regression fits of data to a rectangular hyperbola using ENZFITTER
(R. J. Leatherbarrow, Elsevier, Biosoft). All values are the
average of at least 3-5 independent measurements ±S.D.
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RESULTS |
Expression and Purification of Recombinant
Proteins--
Recombinant wild type and mutant GDPC derivatives were
expressed in 293 cells and isolated as described under "Experimental Procedures". SDS-PAGE analysis (Fig. 1)
indicated that both derivatives expressed as two subforms with
identical apparent molecular weights that correspond to
and
protein C that are glycosylation variants observed previously with this
protein (20). Under reducing conditions, a light chain was also
observed with both derivatives. With both proteins a fraction of the
protein samples remained non-reducible suggesting that the GDPC
derivatives were expressed as a mixture of single and two-chain
proteins. This property has been also observed for both recombinant and
plasma-derived human protein C in the past (29, 30). We have previously
demonstrated that both single and double-chain APC derivatives have
identical catalytic activities (31). These results suggest that the
mutation does not alter the post-translational modifications or the
processing of the protein. With the mutant GDPC, however, a minor band
migrating at ~35 kDa was also observed (Fig. 1). The nature of this
band was not characterized.

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Fig. 1.
SDS-PAGE analysis of recombinant GDPC and
GDPC tryp/loop. Under reducing conditions: lane M,
molecular mass standards (in kDa); lane 1, GDPC; lane
2, GDPC tryp/loop. Under non-reducing conditions: lane
3, GDPC; lane 4, GDPC tryp/loop. HC, heavy
chain; LC, light chain.
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Comparison of the initial rate of protein C activation by the
thrombin·TM4-6 complex as a function of different zymogen
concentrations suggested that both GDPC derivatives were activated at a
similar rate (Fig. 2). SDS-PAGE analysis
of the activated products indicated that both zymogens were completely
converted to activated forms (data not shown). This was consistent with
the observation that the concentrations of enzymes as determined by the
active site titration were similar (within 80%) with the values
calculated based on the absorbance at 280 nm.

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Fig. 2.
Kinetic analysis of GDPC and GDPC tryp/loop
activation by the thrombin·TM complex. The initial rates of
activation were determined as a function of various GDPC ( ) or GDPC
tryp/loop ( ) concentrations with 1 nM thrombin and 100 nM TM4-6 in TBS buffer containing 0.1% PEG 8000 and 2.5 mM Ca2+ at room temperature as described under
"Experimental Procedures." The kinetic constants,
Km and kcat, were determined
from nonlinear regression fit of the data using the Michaelis-Menten
equation (solid lines).
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To determine whether the mutant aGDPC can bind Na+, the
effect of increasing concentration of Na+ on the activity
of aGDPC derivatives toward the chromogenic substrates S2266, S2238,
and SpPCa was studied. Since the catalytic domain of APC contains a
Ca2+-binding site (6), these studies were carried out both
in the absence and presence of Ca2+. In the absence of
Ca2+, the amidolytic activity of aGDPC was strongly
dependent on the presence of Na+ in the reaction buffer
(Fig. 3A). The amidolytic
activity of aGDPC was enhanced with increasing concentrations of
Na+ and reached saturation with a
Kd(app) of 44.1 ± 8.6 mM. In a previous study, the interaction of Na+
with bovine aGDPC was shown to be cooperative with a Hill coefficient of 1.5 (17). A similar cooperativity for Na+ binding to
human aGDPC was also observed in this study in the absence of
Ca2+ (data not shown). However, when the ionic strength of
medium was adjusted to 0.2 M with choline chloride, no
significant cooperativity was observed, and the nonlinear regression
fits of data to a rectangular hyperbola was found to be suitable for
obtaining the Kd(app) values (Fig.
3A). In the presence of Ca2+, no cooperativity
for Na+ binding to aGDPC was observed irrespective of
whether the ionic strength of medium was adjusted to 0.2 M
with choline chloride. In this case an ~5-6-fold stimulation of the
amidolytic activity of aGDPC was observed at saturating Na+
concentrations (Fig. 3A). Interestingly, the affinity of
Na+ for the protease was also improved ~20-fold in the
presence of Ca2+ (Kd(app) = 2.3 ± 0.3 mM), and at higher concentrations of
Na+ (>60 mM) the activity of aGDPC was
slightly diminished. A similar improvement in the affinity of
Na+ for the protease in the presence of Ca2+
was observed at physiological temperature, although the
Kd(app) values were slightly elevated
(141.0 ± 20.9 and 5.3 ± 0.3 mM in the absence
and presence of 2.5 mM Ca2+, respectively). To
ensure that this effect of Ca2+ on the Na+
binding properties of APC was not a phenomenon related to the Gla
domainless form of APC or an effect related to a particular substrate,
the amidolytic activity of plasma-derived APC was monitored as a
function of Na+ in both the absence and presence of
Ca2+ with three different chromogenic substrates (S2266,
S2238, and SpPCa). In all cases, similar results were obtained (data
not shown). Only the results with SpPCa hydrolysis for aGDPC at room temperature are presented in these figures.

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Fig. 3.
Na+ dependence of the
amidolytic activity of activated GDPC and GDPC tryp/loop in the absence
or presence of Ca2+. A, the initial rate of
hydrolysis of SpPCa (200 µM) by aGDPC (5 nM) in the
absence ( ) or presence ( ) of 2.5 mM Ca2+
was measured as described under "Experimental Procedures."
Solid lines are nonlinear regression fits to a rectangular
hyperbola. In all measurements total ionic strength was adjusted to 0.2 M with choline chloride. B, the same as above
except that the initial rate of hydrolysis of SpPCa (500 µM) by aGDPC tryp/loop (100 nM) was measured
in the absence ( ) or presence ( ) of 2.5 mM
Ca2+.
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It is worth noting that in previous studies, the amidolytic activity of
bovine APC displayed a strict requirement for Na+ (16). In
the current study, we noticed some base-line amidolytic activity for
human aGDPC in the absence of Na+ and Ca2+
(Fig. 3A). However, we believe that our results are
consistent with the literature since a similar base-line activity was
also observed in previous studies (15, 16). It was previously suggested that the base-line amidolytic activity of APC in the absence of Na+ and Ca2+ may be due to the presence of
other monovalent cations such as Tris+ and/or choline
(Ch+) in the reaction buffer (15, 16).
The Na+ concentration dependence of the amidolytic activity
of aGDPC tryp/loop mutant was studied in a similar fashion. In the
absence of Ca2+, no Kd(app)
could be estimated for the Na+ interaction with the mutant
since no saturation of Na+ binding to the mutant was
observed up to 400 mM (data are presented for up to 200 mM NaCl in Fig. 3B). No attempt was made to
increase the concentration of Na+ above 400 mM
since the effect of high ionic strength on the structure of the enzyme
is not known. However, in the presence of Ca2+, the
amidolytic activity of the mutant was insensitive to the absence or
presence of Na+ (Fig. 3B). These results clearly
suggest that the mutant has lost its ability to bind Na+
and further suggest that Ca2+ binding to aGDPC has a
profound effect on the ability of the protease to interact with
Na+.
There are two known Ca2+-binding sites on aGDPC that can
influence the Na+ binding properties of the protease. The
first Ca2+-binding site resides in the epidermal growth
factor like domain-1 of the light chain and the other was localized to
the C-terminal catalytic domain of APC (6, 32). The
Ca2+-binding site in the catalytic domain is located on a
loop between residues Glu70 and Glu80,
analogous to the Ca2+ binding loop in trypsin (33).
Previously, we prepared and characterized a GDPC derivative in which
Glu80 was replaced with Lys (E80K) (6). In that study, we
demonstrated that the catalytic domain of the mutant was stabilized in
the Ca2+ conformer possibly as a result of
Lys80 in the mutant forming a salt bridge with
Glu70 (6). To characterize further the
Na+-binding site of APC and determine the
Ca2+-binding site responsible for altering the
Na+ binding properties of the protease, the amidolytic
activity of aGDPC E80K toward SpPCa was studied as a function of
different concentrations of Na+. Interestingly, the
Kd(app) values for Na+
binding to this mutant was of high affinity and insensitive to the
absence or presence of Ca2+ (5.1 ± 0.7 mM
in the absence of Ca2+ and 5.0 ± 0.6 mM
in the presence of Ca2+) (Fig.
4). The
Kd(app) of Na+ for the
mutant did not change even if the amidolytic activity of the mutant was
monitored in the presence of 0.1 mM EDTA or EGTA to chelate
divalent metal ions.

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Fig. 4.
Na+ dependence of the amidolytic
activity of activated GDPC E80K in the absence or presence of
Ca2+. The initial rate of hydrolysis of SpPCa (200 µM) by aGDPC E80K (5 nM) in the absence ( )
or presence ( ) of 2.5 mM Ca2+ was measured
as described under "Experimental Procedures." Solid
lines are nonlinear regression fits to a rectangular hyperbola. In
all measurements a constant ionic strength of 0.2 M was
maintained by addition of choline chloride.
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It is known that there is an ~20% Ca2+ stimulation of
the amidolytic activity of aGDPC toward chromogenic substrates with a Kd(app) of ~50 µM (6).
To determine whether mutagenesis of the 221-225 loop influences the
affinity of the 70-80 loop for binding to Ca2+, the
amidolytic activity of aGDPC tryp/loop was monitored as a function of
increasing Ca2+ concentrations. Two interesting
observations emerged. First, unlike the small effect of
Ca2+ on the amidolytic activity of aGDPC, Ca2+
stimulated the amidolytic activity of the mutant toward the chromogenic substrates 8-10-fold (data not shown). Second, the
Kd(app) for Ca2+ binding to
the aGDPC tryp/loop mutant increased to ~800 µM,
representing about 16-fold weaker interaction than that observed for
Ca2+ binding to aGDPC. Taken together, these results
suggest that occupancy of one metal-binding site of aGDPC with its
specific metal cation influences the affinity of the other site for its specific metal ligand. We conclude that the 70-80 and 221-225 loops
of aGDPC are allosterically linked.
It is known that other monovalent alkali cations can substitute for
Na+ in stimulation of the amidolytic activity of bovine APC
and that the activity increases in parallel with increasing cation
radius (16, 17). To determine whether the mutant can discriminate among
various alkali cations, the kinetic constants were determined for
hydrolysis of S2266 and SpPCa by the wild type and mutant aGDPC.
Although similar results were obtained with both chromogenic substrates, the Km value of the mutant for the
substrates in the presence of all cations was elevated to 1.5-3
mM. Due to poor solubility of SpPCa at concentrations above
~3 mM, kinetic analysis was only carried out for S2266.
As shown in Fig. 5, and consistent with
previous findings (16), the order of monovalent cation preference for
the optimal catalytic function of aGDPC was K+ (ionic
radius = 1.52 Å) > Na+ (ionic radius = 1.16 Å) > Li+ (ionic radius = 0.90 Å) (34). In these
studies, the specificity constant
(kcat/Km) for the bulky
monovalent cation choline (Ch+, ionic radius = 3.5 Å)
was set as a reference, and data were analyzed as described by Dang and
Di Cera (19). As shown in Fig. 5, unlike aGDPC, the mutant has lost its
ability to discriminate among Li+, Na+, and
Ch+. However, similar to aGDPC, the mutant displayed
~2-3-fold improved specificity constant with K+. These
results may suggest that the binding site for K+ on APC is
distinct from that of Na+. However, this notion was not
supported by the observation that Na+ competitively
inhibited the binding of K+ to aGDPC (data not shown).
Furthermore, similar to the case with Na+, the
Kd(app) for K+ binding to
aGDPC was lower affinity (74.3 ± 2.1 mM) in the
absence of Ca2+ and higher affinity (5.4 ± 1.1 mM) in the presence of 2.5 mM Ca2+.
These results strongly suggest that both Na+ and
K+ bind to the same site on the protease and that
Ca2+ binding to the 70-80 loop allosterically modulates
the structure of the monovalent cation binding loop of APC.

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Fig. 5.
Comparison of the relative specificity of
activated wild type and GDPC tryp/loop toward hydrolysis of the
chromogenic substrate S2266. The kinetic constants,
kcat and Km, for the
hydrolysis of S2266 in the presence of various monovalent cations (0.2 M) in 5 mM Tris-HCl, pH 8.0, 2.5 mM
Ca2+, and 0.1% PEG 8000 were calculated as described under
"Experimental Procedures." The concentration of enzymes ranged from
10 to 100 nM, and the concentration of substrate ranged
from 20 µM to 15 mM. The specificity constant
(kcat/Km) for each cation was
calculated and expressed as relative specificity using the specificity
constant for choline chloride as a reference as described by Dang and
Di Cera (19).
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Next, we compared the effect of Na+ and K+ on
the activity of aGDPC toward its natural substrate factor Va in both
the absence and presence of Ca2+. The assay system required
to study this question is not straightforward because factor Va is made
up of two subunits, held together by Ca2+ (35, 36), and
both subunits of factor Va are required for its function. However, it
has been reported that the affinity of factor Va for Ca2+
is high and that subunit dissociation requires EDTA or denaturation of
the cofactor (36, 37). Therefore, we desalted human factor Va (in 5 mM Ca2+) by passing it through a PD-10 gel
filtration column that was equilibrated with Chelex-treated 20 mM Tris-HCl, pH 7.5, buffer containing 0.1% PEG 8000. Desalted factor Va was used immediately in the kinetic experiments
before subunit dissociation and concomitant factor Va inactivation
could occur. Previously, we employed this strategy to study the
Ca2+ dependence of prothrombin activation by factor Xa in
the presence of factor Va (38). Again, the aGDPC inactivation rate of
factor Va in the presence of K+ was ~2-fold more
efficient than Na+, and Ca2+ was required for
the optimal activity of the protease with both monovalent cations (Fig.
6). The catalytic activity of the mutant toward Va inactivation in the presence of Ca2+ ion was
impaired more than 30-fold and again the mutant did not discriminate
between either Na+ or Ch+ ions (Fig. 6).

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Fig. 6.
The time course of factor Va inactivation by
activated GDPC in the presence of Na+ or
K+. Human factor Va (50 nM) was incubated
with activated GDPC (5 nM) in 20 mM Tris-HCl,
pH 7.5, 0.1% PEG 8000 and either 150 mM Na+
( , ) or 150 mM K+ ( , ) at room
temperature. At the indicated time intervals the remaining factor Va
activity was determined from the rate of thrombin generation from
recombinant prethrombin-1 as described under "Experimental
Procedures." The open symbols represent factor Va
degradation in the absence of Ca2+, and the closed
symbols represent factor Va degradation in the presence of 2.5 mM Ca2+. Factor Va degradation by aGDPC
tryp/loop ( ) was carried out in the presence of Ca2+ and
either Na+ or Ch+ under the same conditions
mentioned above except that the concentration of the mutant enzyme was
increased to 75 nM. Identical results were obtained with
both monovalent cations.
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DISCUSSION |
The effect of Na+ and other monovalent cations on the
catalytic activity of bovine APC has been studied in the past. It has been demonstrated that the binding of Na+ or another alkali
metal to bovine APC is required for expression of the amidolytic
activity of the protease toward tri-peptide chromogenic substrates (15,
17). In the current study, we have identified the 221-225 loop as the
monovalent cation-binding site in human APC. Our strategy for the
mutagenesis was based on 1) the observation that trypsin does not
require Na+ for its catalytic activity, and 2) the report
that the 221-225 loop is a Na+-binding site in thrombin
(18, 19). Based on studies with thrombin, Dang and Di Cera (19)
identified residue 225 as a key residue that may determine whether or
not a serine protease can bind Na+. Serine proteases with a
Pro at this position, like trypsin, are unable to bind Na+,
but plasma serine proteases with a Tyr at this position, like APC and
thrombin, are expected to bind Na+. We initially generated
a GDPC mutant in which Tyr225 was replaced with Pro.
However, we could not isolate sufficient quantity of this mutant for
complete characterization. This was due to lack of expression of this
mutant as an intact molecule. SDS-PAGE analysis of the
Tyr225
Pro mutant indicated that the majority of the
expressed protein is proteolytically cleaved at two or more sites (data
not shown). As an alternative, we replaced all 5 variant residues of
the loop (Gly221-Pro225) with the corresponding
residues of trypsin. We isolated sufficient quantity of this mutant to
homogeneity and demonstrated that, similar to thrombin, this loop is a
Na+-binding site in APC. We should also add that our
qualitative results suggest that the Try225
Pro mutant
also lost its ability to bind Na+, in agreement with the
proposal that Pro225 does not support Na+
binding to the 221-225 loop of serine proteases.
In the previous Na+ binding studies with APC, a
Kd(app) of 87-129 mM for
Na+ binding to bovine APC or aGDPC was reported (15-17).
In these studies, positive cooperativity (Hill coefficient = 1.5)
for Na+ binding to bovine aGDPC was observed (17). In the
current study, a similar result for the recombinant human aGDPC was
observed only if choline chloride was not included in the reaction
buffer to compensate for the ionic strength during Na+
titrations. However, minimal cooperativity for Na+ binding
to aGDPC was observed when the total ionic strength in all measurements
was adjusted to 0.2 M with choline chloride. Under these
conditions, the data fit well to a hyperbolic binding equation, and a
Kd(app) of ~44 mM for
Na+ binding to aGDPC was obtained. In addition to
monovalent cations, previous studies by Hill and Castellino (11,
39-41) has established that divalent cations also stimulate the
amidolytic activity of APC and aGDPC and that separate binding sites
for these cations exist on each enzyme. Interestingly, when we included
Ca2+ in the reaction buffer, the
Kd(app) for Na+ binding to
aGDPC was improved ~20-fold suggesting that the
Ca2+-binding site of APC allosterically modulates the
Na+ binding loop of the molecule. Further support for an
allosteric link between the two metal-binding sites was provided by the
observation that the affinity of the aGDPC tryp/loop mutant for
Ca2+ decreased ~16-fold. These results indicate that the
conformation of the Na+- and Ca2+-binding sites
are interdependent and that the two metal ions allosterically regulate
the structure and function of this anticoagulant enzyme.
Previously, Hill and Castellino (11, 39, 40) reported that APC contains
a single divalent cation-binding site outside of the Gla domain that is
critical for the optimal expression of its amidolytic activity.
Recently, we demonstrated that this divalent cation-binding site is
located in the catalytic domain of APC in a loop between the residues
Glu70-Glu80, which is also known as the
Ca2+ binding loop in trypsin (6, 33). The binding of
Ca2+ to this site is essential for rapid activation of
protein C by the thrombin·TM complex (6). In thrombin, which does not
bind Ca2+, Glu70 is not conserved, but it is
replaced with Lys. In the crystal structure of thrombin, an internal
salt bridge between Lys70 and Glu80 stabilizes
this loop (42). Previously, we prepared and characterized a GDPC mutant
in which Glu80 was replaced with Lys (E80K) so that the
loop was stabilized in the Ca2+ conformer, possibly by
Lys80 in the mutant forming a salt bridge with
Glu70 (6). Interestingly, the affinity of this mutant for
Na+ was of high affinity (5 mM) and was not
sensitive to the absence or presence of Ca2+ in the
reaction buffer. Results obtained with this mutant support the
hypothesis that the conformation of the monovalent and divalent cation-binding sites are allosterically linked. Furthermore, no cooperativity for Na+ binding to this mutant was observed
in either absence (10 µM EDTA) or presence of 2.5 mM Ca2+. It is possible that the cooperativity
observed for the Na+ binding to aGDPC in the absence of
Ca2+ is due to binding of Na+ to the 70-80
loop and partially substituting for Ca2+. In support of
this hypothesis, cooperativity is observed only for Na+
(ionic radius 1.16 Å) which has similar ionic radius as
Ca2+ (1.14 Å), but not with any other alkali cation.
Several previous studies by Di Cera and co-workers (12, 18) suggest
that Na+ binding to the 221-225 loop of thrombin modulates
the activity and specificity of this enzyme in an allosteric fashion.
These authors believe that the Na+-bound form (fast form)
has improved catalytic activity toward chromogenic substrates and
specifically clots fibrinogen, whereas the Na+-free form
(slow form) specifically activates protein C (12). It is interesting to
note that the catalytic activity of the loop mutant of aGDPC was
impaired but that the activation of the zymogen form of the mutant by
the thrombin·TM complex was not impaired and rather improved
slightly. These results suggest that Na+ binding to either
thrombin or protein C is not required for recognition and rapid
activation by the thrombin·TM complex. It is not known if APC,
similar to thrombin, can exist in two distinct slow and fast forms in
plasma. The concentration of Na+ in blood is very close to
the Kd(app) of this cation for thrombin,
so that the two forms of thrombin may exist in equilibrium under
physiological conditions (12, 14). In the case of APC, however, the
Kd(app) for Na+ is
~25-30-fold lower than the concentration of this cation in blood,
which suggests that APC may predominantly exist in the fast form under
physiological conditions.
The other interesting observation of this study is that the catalytic
activity of aGDPC in the presence of K+ was 2-3-fold more
efficient than Na+. This was true whether the activity was
monitored by hydrolysis of chromogenic substrates or by the
inactivation of the natural substrate factor Va. Both thrombin and
factor Xa, unlike APC, function more efficiently in the presence of
Na+. The molecular basis for the improved catalytic
property of APC in the presence of K+ was not understood.
Since K+ is primarily an interacellular metal ion, it is
not known if it can play a role in up-regulation of the catalytic
activity of APC in plasma. However, it is worth noting that similar to Na+, the affinity of K+ for binding to aGDPC
was improved dramatically in the presence of Ca2+. The
local concentration of K+ at the site of clot formation
and/or inflammation due to K+ release from aggregated
platelets and/or lysed cells may increase to a level that could
specifically activate the APC anticoagulant pathway. Further study will
be required to determine whether there is validity for this hypothesis.
Finally, the Ca2+ binding loop on the N-terminal
-barrel
and the Na+ binding loop on the C-terminal
-barrel of
APC are located 15-20 Å away from the catalytic triad and ~30 Å apart from each other (Fig. 7). The
observation that stabilization of these loops by various metal ions
allosterically modulates the catalytic function of APC provides further
insight into how coagulation proteases and other more specialized
serine proteases may have evolved from a common ancestral fold into
highly specific enzymes with very diverse functions. Whereas the key
catalytic residues are conserved in all serine proteases, other
residues at the vicinity of the primary specificity pocket (S1) or
remote from this pocket have diversified. There is overwhelming
evidence in the literature that variant residues restrict the
specificity of coagulation proteases by enabling them to specifically
interact with additional residues surrounding the P1 site of various
peptide substrates. In recent years, homology modeling based on the
known crystal structures and subsequent mutagenesis studies have
identified a number of variant residues critical for determination of
the P3-P3' specificity of coagulation proteases (27, 43-47). It is also becoming clear that the binding of specific cofactors or substrates to variant residues and loops remote from the catalytic pocket allosterically regulates the specificity and function of coagulation serine proteases (48-51). However, except for a few studies with small synthetic substrates and inhibitors, the molecular basis of the coagulation protease specificity with the natural macromolecular substrates have remained largely elusive. The
results presented in this study suggest that this may partly be due to evolution of complex allosteric links among different surface loops in
coagulation serine proteases that are not easily identified by homology
modeling based on the crystal structures.

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Fig. 7.
MOLSCRIPT plot of the catalytic domain of
activated protein C derived from the crystal structure of the activated
GDPC-PPACK complex (52). The non-catalytic epidermal growth
factor-like domains-1 and -2 and the inhibitor PPACK were removed. The
70-80 Ca2+ loop is located on the N-terminal six-stranded
-barrel with a Ca2+ ion positioned at the geometric
center of the loop (shown in red). The putative
Na+ binding loop (from
Cys220-Gly226) is located on the C-terminal
six-stranded -barrel with a Na+ ion positioned at the
geometric center of the loop (shown in yellow). The
catalytic triad residues His57, Asp102, and
Ser195 are shown in red at the interface of two
-barrels.
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