Proexosite-1 on Prothrombin Is a Factor Va-dependent Recognition Site for the Prothrombinase Complex*
Lin Chen,
Likui Yang and
Alireza R. Rezaie
From the
Edward A. Doisy Department of Biochemistry and Molecular Biology, St.
Louis University School of Medicine, St. Louis, Missouri 63104
Received for publication, March 17, 2003
, and in revised form, May 12, 2003.
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ABSTRACT
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Although the contribution of basic residues of exosite-1 to the catalytic
function of thrombin has been studied extensively, their role in the
specificity of prothrombin recognition by factor Xa in the prothrombinase
complex (factor Xa, factor Va, phosphatidylcholine/phosphatidylserine
vesicles, and Ca2+) has not been examined. In this
study, we prepared several mutants of prethrombin-1 (prothrombin lacking Gla
and Kringle-1 domains) in which basic residues of this site (Arg35,
Lys36, Arg67, Lys70, Arg73,
Arg75, and Arg77 in chymotrypsinogen numbering) were
individually substituted with a Glu. Following expression in mammalian cells
and purification to homogeneity, these mutants were characterized with respect
to their ability to function as zymogens for both factor Xa and the
prothrombinase complex. Factor Xa by itself exhibited similar catalytic
activity toward both the wild type and mutant substrates; however, its
activity in the prothrombinase complex toward most of mutants was severely
impaired. Further kinetic studies in the presence of Tyr63-sulfated
hirudin-(5465) peptide suggested that although the peptide inhibits the
prothrombinase activation of the wild type zymogen with a
KD of 0.50.7 µM, it is
ineffective in inhibiting the activation of mutant zymogens
(KD = 230 µM). These results
suggest that basic residues of proexosite-1 on prothrombin are factor
Va-dependent recognition sites for factor Xa in the prothrombinase
complex.
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INTRODUCTION
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Prothrombin is a vitamin K-dependent serine protease zymogen that is
proteolytically converted to thrombin by the prothrombinase complex (factor
Xa, cofactor Va, negatively charged phospholipid vesicles, and
Ca2+) in the final step of the blood clotting cascade
(16).
Factor Xa specifically catalyzes the cleavage of two peptide bonds after the
two basic residues, Arg273 and Arg322, to convert
prothrombin to thrombin (1).
Although factor Xa by itself can catalyze the cleavage of both peptide bonds
on the substrate, its catalytic efficiency is improved by greater than 5
orders of magnitude when it is assembled into the prothrombinase complex
(1,
3). Results of several kinetic
studies have indicated that such a dramatic improvement in the rate of
prothrombin activation by the prothrombinase complex is derived from an
100-fold decrease in the apparent Km and a
greater than 1000-fold enhancement in the kcat of the
activation reaction (3,
7). The improvement in the
apparent Km of the activation reaction is
mediated through the Ca2+-dependent assembly of both
prothrombin and factor Xa on negatively charged membrane surfaces via their
N-terminal Gla domains (3,
7). However, the improvement in
the kcat of the activation reaction is believed to arise
from the factor Va-mediated protein-protein interaction between factor Xa and
prothrombin in the prothrombinase complex
(7,
8).
The mechanism of the factor Va-mediated protein-protein interaction that
improves the catalytic efficiency of factor Xa in the prothrombinase complex
is under intensive investigation. Based on recent kinetic data, it has been
hypothesized that factor Va binding to factor Xa allosterically exposes a
secondary binding site"exosite"remote from the catalytic pocket on
the protease that is a specific recognition site for interaction with the
prothrombin lacking the Gla and both Kringle-1 and -2 domain
(prethrombin-2)1
portions of the substrate (9).
In support of this hypothesis, it has been demonstrated that factor Va
enhances the kcat of both prothrombin and prethrombin-2
activation by factor Xa to a similar extent
(9,
10). The putative
cofactor-mediated interaction sites, on either the protease or the substrate,
have not been identified. However, an active site inhibited thrombin and a
C-terminal fragment derived from the cleavage of thrombin by chymotrypsin at
Trp148 have been shown to inhibit competitively the activation of
prethrombin-2 by factor Xa in the prothrombinase complex
(9). Based on such results, it
has been hypothesized that the factor Va-mediated factor Xa interactive site
on prothrombin is located on the protease domain that does not include either
the fibrinogen recognition (exosite-1) or the heparin-binding site (exosite-2)
(9).
Other studies (11,
12) have demonstrated recently
that the heavy chain of factor Va contains a binding site for exosite-1 of
thrombin and that this site is also present in a low affinity precursor state
"proexosite-1" on prothrombin. Thus, an alternative hypothesis for
the mechanism of the cofactor function of factor Va is that the cofactor in
the prothrombinase complex may provide a binding site for direct interaction
with the proexosite-1 of prothrombin
(13,
14). In support of this
hypothesis, it has been demonstrated that the factor Va-mediated acceleration
of prothrombin activation by the prothrombinase complex can be specifically
inhibited by an exosite-1-specific peptide ligand derived from the C-terminal
domain of the leech inhibitor, hirudin
(14).
The exosite-1 of thrombin plays a pivotal role in the catalytic function of
thrombin. This site has several basic residues that can interact with nearly
all natural substrates, inhibitors, and cofactors of thrombin including
fibrinogen, factors V and VIII, PAR-1, heparin cofactor II, and thrombomodulin
(TM)
(1519).
Previous mutagenesis of basic residues of exosite-1 has been demonstrated to
dramatically impair the reactivity of thrombin with all of these
macromolecules
(2023).
Despite an extensive characterization of basic residues of exosite-1 in
thrombin, the contribution of these residues to the specificity of prothrombin
recognition by factor Xa in the prothrombinase complex has not been studied.
In this study, we substituted all basic residues of this site including
Arg35, Lys36, Arg67, Lys70,
Arg73, Arg75, and Arg77 (chymotrypsinogen
numbering (24)) with Glu in
individual constructs in prethrombin-1 (prothrombin lacking both the Gla and
Kringle-1 domains) and expressed the mutant proteins in mammalian cells as
described (25). Following
purification to homogeneity, the properties of mutant proteins were analyzed
with respect to their ability to function as zymogens for factor Xa in both
the absence and presence of factor Va on phospholipid vesicles. It was
discovered that whereas factor Xa activates the wild type and prethrombin-1
mutant zymogens with a comparable rate in the absence of factor Va, the
protease exhibits a dramatic catalytic defect toward mutant substrates in the
presence of the cofactor. Further kinetic studies in the presence of the
proexosite-specific peptide, Tyr63-sulfated hirudin-(5465)
(Hir-(5465)(
), suggested that
the hirudin peptide inhibits the prothrombinase activation of prethrombin-1
with a KD of 0.7 µM. However, the
competitive inhibitory effect of the hirudin peptide in the prothrombinase
activation of mutants was impaired at varying degrees, which correlated well
with the extent of impairments observed in the activation of mutant zymogens.
These results suggest that basic residues of proexosite-1 are specific
recognition sites for factor Xa in the prothrombinase complex. Interestingly,
we also discovered that the epidermal growth factor-like domains 46 of
TM (TM46) can inhibit the prothrombinase activation of prethrombin-1
with a KD of 0.5 µM. The possible
physiological significance of this finding is discussed.
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EXPERIMENTAL PROCEDURES
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Construction and Expression of Mutant ProteinsThe
expression of wild type prethrombin-1 (prothrombin lacking both Gla and
Kringle-1 domains) and prethrombin-2 (prothrombin lacking Gla, Kringle-1, and
Kringle-2 domains) by the pNUT-PL2 expression/purification vector system in
baby hamster kidney cells has been described previously
(10,
26). Prethrombin-1 mutants in
the chymotrypsinogen numbering system
(24): Arg35
Glu and Ala (R35E and R35A), Lys36
Glu (K36E),
Arg67
Glu (R67E), Lys70
Glu (K70E),
Arg73
Glu (R73E), Arg75
Glu (R75E), and
Arg77
Glu (R77E) were prepared by PCR mutagenesis methods as
described (26). Following
confirmation of accuracy of mutations by DNA sequencing, the mutant constructs
were expressed in baby hamster kidney cells using the same
expression/purification vector system described above. All derivatives were
purified to homogeneity by immunoaffinity chromatography using the
Ca2+-dependent monoclonal antibody, HPC4, as described
(26). TM46 was
expressed in HEK293 cells and purified to homogeneity as described
(27).
Human plasma proteins, antithrombin, and factors Va and Xa were purchased
from Hematologic Technologies Inc. (Essex Junction, VT). Phospholipid vesicles
containing 80% phosphatidylcholine and 20% phosphatidylserine (PC/PS) were
prepared as described (28).
The chromogenic substrate S2238 was purchased from Kabi Pharmacia/Chromogenix
(Franklin, OH). The chromogenic substrate
N-p-tosyl-Gly-Pro-Arg-p-nitroanilide
(GPR-pNA) and Tyr63-sulfated hirudin-(5465)
(Hir-(5465)(
) were purchased
from Sigma.
Prethrombin-1 ActivationThe initial rate of prethrombin-1
activation by factor Xa was studied in both the absence and presence of factor
Va on PC/PS vesicles. In the absence factor Va, the time course of activation
of each prethrombin-1 derivative (2 µM) by factor Xa (5
nM) was measured at room temperature in 0.1 M NaCl, 0.02
M Tris-HCl (pH 7.5) containing 0.1 mg/ml bovine serum albumin, 0.1%
polyethylene glycol 8000, and 2.5 mM CaCl2
(TBS/Ca2+). At different time intervals, small aliquots
of activation reactions were transferred to wells of a 96-well assay plate
containing 20 mM EDTA, and the rate of thrombin generation was
determined from the cleavage of S2238 (100 µM) at 405 nm by a
Vmax Kinetic Microplate Reader (Molecular Devices, Menlo
Park, CA). The concentration of thrombin generated was determined from
standard curves prepared from the cleavage rate of S2238 (100
µM) by known concentrations of wild type and mutant thrombins.
In the presence of factor Va, the concentration dependence of prethrombin-1
(0.320 µM) activation by human factor Xa (0.051
nM) in complex with a saturating concentration of human factor Va
(30 nM) was measured on PC/PS vesicles (35 µM) in
TBS/Ca2+. After 130 min of incubation at room
temperature, the reactions were terminated by addition of EDTA, and the
initial rate of thrombin generation was measured from the cleavage rate of
S2238 as described above. The apparent Km and
kcat values for prethrombin-1 activation were calculated
from the Michaelis-Menten equation. It was ensured that the factor Va
concentration (30 nM) was in excess in all activation reactions.
Thus, the factor Va (0.110 nM) concentration dependence of
activation reactions by factor Xa (0.2 nM) were carried out on
PC/PS vesicles (35 µM) in TBS/Ca2+ using 1
µM wild type or mutant prethrombin-1. The
Kd(app) values were calculated from hyperbolic
dependence of activation rates on the concentrations of factor Va as described
(29). In all reactions, it was
ensured that less than 10% of prethrombin-1 was activated at all
concentrations of the substrates.
Prethrombin-1 Activation in the Presence of
Hir-(5465)-(
)The
inhibitory effect of the hirudin peptide on the kinetics of prethrombin-1 and
prethrombin-2 activation by both factor Xa and prothrombinase was studied.
Thus, the rate of activation of each prethrombin-1 derivative (1
µM) by factor Xa alone (5 nM) or factor Xa
(0.051 nM) in complex with factor Va (30 nM) on
PC/PS vesicles was monitored in the presence of increasing concentrations of
the hirudin peptide in the same TBS/Ca2+ buffer system.
The concentration of thrombin generated in each reaction was calculated from
standard curves as described above except that GPR-pNA was used as
the chromogenic substrate because the cleavage rate of this substrate has been
reported not to be affected by the occupancy of exosite-1 by the hirudin
peptide (18). To simplify
comparisons of the hirudin peptide dependence of the activation reactions, the
data for all activation reactions were normalized to maximal thrombin
generation in the absence of the peptide. The dissociation constants
(KD) for the interaction of the hirudin peptide
with prethrombin-1 was calculated from Equations
1 and
2 as described
(14).
 | (Eq. 1) |
 | (Eq. 2) |
Vobs is the observed initial rate of prethrombin-1
(Pre-1) activation; Vlim is the limiting rate at a
saturating hirudin peptide (Hir) concentration;
Vo is the initial rate of activation in the
absence of the hirudin peptide; KD is the
dissociation constant for the hirudin peptide binding to prethrombin-1; and
[Pre-1·Hir] represents the prethrombin-1-hirudin peptide complex
concentration. The quadratic binding equation assumes that the concentration
of prethrombin-1 in complex with either factor Xa or factor Va is small enough
to be neglected under experimental conditions where
[Pre-1]o is in excess of the initial concentrations of
both the enzyme and the cofactor
(14).
Cleavage of Chromogenic SubstratesApproximately 1 mg of
each prethrombin-1 derivative was activated to completion, and thrombin
mutants were purified by cation exchange chromatography on a Mono S column
using a linear gradient of 0.10.6 M NaCl as described
(25). The concentrations of
thrombin mutants were determined by their absorbance at 280 nm, assuming a
molecular mass of 36.6 kDa and an extinction coefficient
(
) of 17.1 and by
stoichiometric titrations with a known concentration of antithrombin as
described (25). The
steady-state kinetics of hydrolysis of S2238 (1.5100 µM)
by thrombin derivatives (0.5 nM) were studied in
TBS/Ca2+ as described
(25). The rate of hydrolysis
was measured at 405 nm at room temperature in a Vmax
Kinetic Microplate Reader as described above. The
Km and kcat values for the
chromogenic substrate hydrolysis were calculated from the Michaelis-Menten
equation, and the catalytic efficiencies were expressed as the ratios of
kcat/Km.
 |
RESULTS
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Expression and Purification of Recombinant ProteinsWild
type and mutant prethrombin-1 derivatives were expressed in baby hamster
kidney cells using the pNUT-PL2 expression/purification vector system as
described previously (26). All
recombinant proteins were purified to homogeneity by an immunoaffinity
chromatography using the Ca2+-dependent monoclonal
antibody HPC4 as described
(26). Except for elevated
Km(app) values, factor Xa in the prothrombinase
complex activates both prethrombin-1 and prethrombin-2 with similar and normal
Vmax values
(10,
30). Thus, these truncated
substrates are ideal reagents for probing the extent that protein-protein
interactions in the prothrombinase complex contribute to the high specificity
of the catalytic reaction.
Amidolytic ActivityBecause the zymogenic properties of
prethrombin-1 mutants by the prothrombinase complex are studied from the
initial rate of thrombin generation in an amidolytic activity assay, it was
essential to determine the kinetic parameters for the cleavage of the
chromogenic substrate S2238 by mutant thrombins. Thus, following activation
and purification on a Mono S column, the concentration of the active site of
mutants was determined by stoichiometric titration with antithrombin as
described (25). The
concentration of active enzymes correlated well with the concentration of
substrates determined based on their absorbance at 280 nm (within
90100%). Kinetic parameters for the hydrolysis of S2238 are presented
in Table I. With the exception
of the K70E mutant, which exhibited a dramatically impaired
Km value, all other mutants cleaved S2238 with
kinetic constants that were similar to those observed for wild type thrombin.
These results strongly suggest that with the exception of K70E, the mutations
did not adversely affect the folding, charge stabilizing system, or the
reactivity of the catalytic pockets of mutant enzymes. The interaction of
p-aminobenzamidine with the K70E mutant was also impaired
3-fold
(Ki = 38 and 107 µM for wild type
and mutant thrombin, respectively). Thus, the conformation of the P3-P1
binding pocket of the K70E mutant must have been altered.
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TABLE I Kinetic constants for the cleavage of the chromogenic substrate S2238
by thrombin derivatives
The kinetic constants were calculated from the cleavage rate of increasing
concentrations of S2238 (1100 µM) by each thrombin
derivative (0.5 nM) in TBS/Ca2+. Kinetic values are the
average of three measurements ± S.E.
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Prethrombin-1 Activation by Factor Xa and the Prothrombinase
ComplexThe initial rate of activation of prethrombin-1 derivatives
by factor Xa was studied both in the absence and presence of factor Va on
PC/PS vesicles. As shown in Fig.
1A, factor Xa exhibited similar activity toward both the
wild type and mutant zymogens in the absence of factor Va. However, the
activation of all mutants by factor Xa in the prothrombinase complex was
markedly impaired (Fig.
1B). The concentration dependence of zymogen activation
indicated that the prothrombinase complex activated wild type prethrombin-1
with apparent Km and kcat
values of 9.1 ± 0.7 µM and 1529 ± 56 mol/min/mol,
respectively. In the case of mutants, these values could only be determined
for the R75E mutant (17.1 ± 2.8 µM and 913 ± 87
mol/min/mol) because the rate of thrombin generation was dramatically impaired
and remained linear for up to 20 µM substrate (the highest
concentration available) for all other mutants
(Fig. 1B).

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FIG. 1. Activation of prethrombin-1 derivatives by factor Xa and the
prothrombinase complex. A, in the absence of factor Va, factor Xa
(5 nM) and PC/PS vesicles (35 µM) were incubated with
prethrombin-1 derivatives: wild type ( ), R35E (), K36E ( ),
R67E ( ), K70E ( ), R73E ( ), R75E ( ), and R77E
( ) (2 µM each) at room temperature in
TBS/Ca2+. At different time intervals, small aliquots of
the activation reactions were transferred to wells of a 96-well assay plate
containing 20 mM EDTA, and the rate of thrombin generation was
measured from the cleavage rate of S2238 as described under
"Experimental Procedures." B, factor Xa (0.051
nM) in complex with factor Va (30 nM) and PC/PS vesicles
(35 µM) was incubated with different concentrations of
prethrombin-1 derivatives: wild type ( ), R35E (), K36E ( ),
R67E ( ), K70E ( ), R73E ( ), R75E ( ), and R77E ( )
at room temperature in TBS/Ca2+. After 130 min of
incubation at room temperature, the reactions were terminated by addition of
EDTA, and the initial rate of thrombin generation was measured as described
above. Solid lines in both wild type and R75E thrombins are nonlinear
regression fits of kinetic data to the Michaelis-Menten equation, and all
others are fits to a linear equation.
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Because the saturation of the initial rate of activation was not feasible
with mutant substrates for the kinetic analysis, the overall extent of
impairment with each proexosite-1 mutant residue was estimated from the
initial rate of activation of a limiting concentration of each mutant
substrate (at least 10-fold below Km values) in
the presence of increasing concentrations of factor Va on PC/PS vesicles. A
saturable dependence of thrombin generation on factor Va concentrations was
observed yielding both Kd(app) values for the
factor Xa-factor Va interaction and the maximum rate of thrombin generation
with all derivatives. The results presented in
Table II suggested that
although the Kd(app) for the enzyme-cofactor
interaction was independent of the substrate, the second-order rate of
thrombin generation was impaired at varying degrees with all mutant
substrates. The most impairment (75150-fold) was observed with R67E and
K70E mutants. The activation of all other mutants was impaired 418-fold
(Table II). These results
clearly suggest that none of the mutant residues under study interact with
factor Xa in the absence of factor Va; however, they are critical recognition
sites for the cofactor and/or the protease in the prothrombinase complex.
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TABLE II Kinetic constants for interaction of factor Xa with factor Va
(Kd(app)), maximum rate of thrombin generation (V), and
dissociation constants (KD) for binding of
Hir-(5465)-(SO3-) to prethromnin-1
derivatives
The apparent Kd values for the factor Xa-factor Va
interaction and the maximal rate of thrombin generation were determined from
the saturable cofactor-dependent increase in the initial rate of prethrombin-1
(1 µM) activation on PC/PS vesicles in TBS/Ca2+ as
described under "Experimental Procedures." The
KD values for binding of
Hir-(5465)-(SO3-) to prethrombin-1 derivatives
were determined from nonlinear regression analysis of inhibition kinetic data
(shown in Fig. 2) according to
Equations 1 and
2 as described in the text. ND,
not determinable. All values are the average of at least two measurements
± S.E.
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FIG. 2. Loss of factor Va-dependent inhibitory effect of
Hir-(5465)( ) on factor Xa
activation of prethrombin-1 derivatives. A, the inhibitory effect
of the hirudin peptide was monitored by incubating each prethrombin-1
derivative (1 µM) with factor Xa (0.051 nM) in
complex with a saturating concentration of factor Va (30 nM) on
PC/PS vesicles (35 µM) in the presence of increasing
concentrations of the hirudin peptide shown on the x axis. The
initial rate of thrombin generation was measured by an amidolytic activity
assay using GPR-pNA, and the data were normalized to % of activity at
each concentration of the inhibitor (100% in the absence of the inhibitor) as
described under "Experimental Procedures." The symbols are as
follows: , wild type prethrombin-1; , R35E; , K36E; ,
R67E; , K70E; , R73E; , R75E; and , R77E. Solid
lines are best fit of data according to Equations
1 and
2. The
KD values determined from the slope of these
curves are presented in Table
II. B, the same as A except that prethrombin-1
() or prethrombin-2 ( ) in the presence of factor Va and
prethrombin-1 ( ) in the absence of factor Va was used in the activation
reactions.
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Inhibition of Activation by
Hir-(5465)-(
)The
Tyr63-sulfated hirudin peptide is proven to be a useful probe to
analyze the interaction of different ligands with exosite-1 of thrombin
(15,
31). It was demonstrated
recently (14) that the hirudin
peptide can also interact with the proexosite-1 of the substrate prothrombin.
Thus, to determine whether the loss of specific interactions of proexosite-1
residues with prothrombinase accounts for the defective catalytic reactions
with different prethrombin-1 derivatives, the initial rates of activation of
mutant substrates were measured in the presence of increasing concentrations
of the hirudin peptide. The results presented in
Fig. 2A and
Table II indicated that the
hirudin peptide inhibits the initial rate of wild type prethrombin-1
activation with a KD of 0.7 µM.
However, the ability of the peptide to inhibit activation of prethrombin-1
mutants was impaired at varying degrees. Interestingly, the degree of
impairment in KD values correlated well with the
degree of impairment in the observed activation rates of mutant substrates.
Thus, the hirudin peptide exhibited no inhibitory effect toward the
prothrombinase activation of either R67E or K70E mutants which were also
ineffective substrates for the activation complex. Parallel with impairment in
the activation rates, the KD values were elevated
340-fold with all other mutants
(Table II). It should be noted
that the highest concentration of the hirudin peptide in the prothrombinase
assays was 40 µM (shown only up to 30 µM in
Fig. 2); thus a larger
uncertainty was associated with the KD values for
R73E and K36E, and the activation of both mutants (with the exception of R67E
and K70E) was impaired the most. It should be noted that the inhibitory
property of the peptide was specific for factor Xa in the presence of factor
Va because in the absence of the cofactor no inhibition of thrombin generation
was observed (Fig.
2B). It is also known that the fragment-2 domain of
prethrombin-1 can enhance the rate of substrate activation by factor Xa in the
presence of factor Va (32).
This raises the possibility that the hirudin peptide directly or indirectly
interferes with the effect of fragment-2 in the activation reaction. However,
as shown in Fig. 2B,
the inhibitory effect was independent of the fragment-2 because a similar
KD of 0.6 ± 0.1 µM was
obtained when prethrombin-2 rather than prethrombin-1 was used in the
prothrombinase inhibition reaction.
The binding of epidermal growth factor-like domains 46 of
thrombomodulin (TM46) to exosite-1 of thrombin changes the
macromolecular substrate specificity of thrombin from a fibrinogen clotting
procoagulant enzyme to a protein C-activating anticoagulant one
(17,
33,
34). It is known that
TM46 binds to exosite-1 of thrombin with a
KD of
5 nM
(35,
36). TM46 is a highly
specific ligand for the exosite-1 of thrombin, and unlike the hirudin peptide,
it is not known if TM46 can also interact with proexosite-1 of the
substrate. This question was investigated by examining the ability of the TM
fragment to inhibit the activation of prethrombin-1 by the prothrombinase
complex. As shown in Fig. 3,
TM46 effectively inhibited the rate of substrate activation by factor
Xa specifically in the presence of factor Va with a
KD of 0.5 ± 0.1 µM that is
slightly better than the corresponding value observed with the hirudin
peptide, but
100-fold weaker than its interaction with exosite-1 of
thrombin. Taken together, these results confirm the proposal that the
interaction of proexosite-1 with the cofactor/enzyme of the prothrombinase
complex is required for normal prothrombin activation and that the loss of the
zymogenic properties of mutants can be unequivocally attributed to the loss of
this interaction.

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FIG. 3. The TM46 concentration dependence of prethrombin-1 inhibition by
factor Xa in the absence and presence of factor Va. The inhibitory effect
of TM46 was monitored by incubating prethrombin-1 (1 µM)
with 5 nM factor Xa in the absence ( ) or 0.1 nM
factor Xa in the presence () of a saturating concentration of factor Va
(30 nM) on PC/PS vesicles (35 µM) in the presence of
increasing concentrations of TM46 shown on the x axis. The
initial rate of thrombin generation was measured by an amidolytic activity
assay using GPR-pNA, and the data were analyzed as described under
the legend of Fig. 2. The
nonlinear regression of data according to Equations
1 and
2 yielded a
KD of 0.5 ± 0.1 µM for the
TM46 inhibition of the activation reaction.
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DISCUSSION
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Because of its pivotal role in numerous physiological processes, the
structure and function of thrombin are being extensively studied by many
laboratories. It has been well established that in addition to its catalytic
pocket with a trypsin-like S1 specificity, two basic regions remote from the
catalytic pocket of thrombin (exosites-1 and 2) play essential roles in
determining the macromolecular substrate and inhibitor specificity of thrombin
(15,
37). Structural and
mutagenesis data have indicated that the occupancy of both exosites by various
ligands can regulate the proteolytic activity of thrombin both in the
procoagulant and anticoagulant pathways. Thus, the interaction of fibrinogen
and PAR-1 with exosite-1 of thrombin is required for the protease to function
in the procoagulant pathway
(15,
18,
20). Similarly, the
interaction of both cofactors V and VIII with this site is a prerequisite for
their activation by thrombin and subsequent amplification of the blood
coagulation cascade (22,
23,
38). On the other hand, the
binding of TM46 to exosite-1 changes the specificity of thrombin from a
procoagulant to an anticoagulant enzyme by enabling the protease to rapidly
activate the precursor protein of the anticoagulant pathway, protein C
(17,
33,
34). Despite a wealth of
knowledge about the role of exosite-1 in the catalytic function of thrombin,
much less is known about the role of this site in the zymogenic properties of
the precursor prothrombin in the prothrombinase complex. The current study was
undertaken to address this question by a mutagenesis approach.
Factor Xa activated all prethrombin-1 mutants at comparable rates in the
absence of factor Va, suggesting that proexosite-1 of prothrombin does not
interact with the protease in the absence of the cofactor in a detectable
manner. On the other hand, the catalytic efficiency of factor Xa toward most
mutants was severely impaired in the presence of factor Va. The greatest
degree of impairment (
75150-fold) was observed during the
prothrombinase activation of R67E and K70E mutant zymogens. With other
mutants, the impairments ranged from
4-fold for R75E,
7-fold for
R35E,
9-fold for R73E, and
1520-fold for K36E and R77E. These
results suggest that all basic residues of proexosite-1 are important
recognition sites on the substrate for interaction with factor Va/factor Xa in
the prothrombinase complex. It is not known if the degree of impairment in the
activation rates directly reflects the order of importance of the mutant
residues in their interaction with the activation complex. This is because the
proper folding of all mutant zymogens cannot be ascertained. In particular,
the x-ray crystal structure of thrombin suggests that both Arg67
and Lys70 make intramolecular salt bridge/hydrogen bond contacts
with Glu80 (37).
The observations that the Km value for the
hydrolysis of S2238 with K70E thrombin was impaired and the
Ki value for interaction with
p-aminobenzamidine was also elevated suggest that the conformation of
the P3-P1 binding pocket of the mutant enzyme has been altered. These results,
together with the previous observation that the active site pocket and
exosite-1 of thrombin are allosterically linked
(39,
40), strongly suggest that the
mutagenesis of Lys70 disrupts the integrity of exosite-1 in the
mutant thrombin. If the same ionic interactions also exist between
Lys70 and Glu80 in the precursor protein, it is possible
that mutagenesis of Lys70 has also disrupted the integrity of
proexosite-1 in the mutant zymogen. However, R67E thrombin exhibited normal
amidolytic activity; thus mutagenesis of this residue has likely no adverse
effect on the active site pocket and possibly also not on the integrity of
exosite-1 in the mutant thrombin. Accordingly, the mutagenesis of
Arg67 may have no significant effect on the structure of
proexosite-1 in the mutant zymogen. All other mutants are expected to fold
properly because they are not known to be involved in intramolecular salt
bridges in thrombin and presumably also not in prothrombin. This is consistent
with the observation that their amidolytic activity has not been adversely
affected. Thus, the degree of impairments in the substrate properties of these
mutants likely reflects their order of importance for their recognition
specificity of the prothrombinase complex. This proposal is also consistent
with the observation that the competitive effect of the hirudin peptide was
impaired with the proexosite-1 mutants of the substrate and that the degree of
impairment correlated well with losses in the zymogenic activities of mutants.
Taken together, these results firmly establish that basic residues of
proexosite-1 on prothrombin are factor Va-dependent recognition sites for the
prothrombinase complex.
The mechanism by which factor Va improves the catalytic efficiency of
factor Xa in the prothrombinase complex is largely unknown. An attractive
hypothesis is that the binding of factor Va to factor Xa allosterically
exposes a cryptic substrate recognition exosite remote from the catalytic
pocket of the protease. In support of this hypothesis, it has been
demonstrated that the active site inhibited thrombin and a specific
chymotrypsin fragment of thrombin can competitively inhibit the activation of
prothrombin by factor Xa in the prothrombinase complex
(9). Based on the sequence of
the inhibitory chymotrypsin fragment, the putative site is believed to include
neither the fibrinogen nor the heparin-binding exosites
(9). Thus, the underlying cause
of the defective zymogenic properties of proexosite-1 mutants cannot be
attributed to the loss or weakening of an interactive site on mutants for the
proposed factor Va-mediated exosite on factor Xa. On the other hand, in a
different study, an important role for a direct interaction between factor Va
and the proexosite-1 of the substrate has been proposed
(14). This latter hypothesis
is based on the observation that the exosite1-specific peptide ligand derived
from the C-terminal domain of hirudin could competitively inhibit the
activation of prothrombin and prethrombin-1 by factor Xa in the presence but
not in the absence of factor Va
(14). In agreement with the
latter hypothesis, the results of this study suggest that a direct interaction
between basic residues of proexosite-1 and an acidic region of factor Va may
account for the loss of the zymogenic activities of mutant proteins. Several
lines of evidence suggest that the complementary proexosite-1 interactive site
on factor Va may be located on the C-terminal end of the factor Va heavy
chain, possibly involving residues within 659698
(4144).
This region of factor Va has a high homology for the hirudin peptide and
contains several functionally important sulfated Tyr residues that are
critical for both the activation of pro-cofactor V by thrombin and the
cofactor activity of factor Va in the prothrombinase complex
(4143).
It should be emphasized that our results do not exclude the possibility that
other functionally important interactive sites exist on prothrombin that can
directly or indirectly interact with factor Xa in the prothrombinase
complex.
Finally, the observation that TM46 can also effectively inhibit the
activation of prethrombin-1 by the prothrombinase complex suggests that TM can
also bind to proexosite-1 of prothrombin. It is not known whether or not this
property of TM can play a significant role in the modulation of the
prothrombinase activity under physiological conditions. In particular the
KD for this interaction (
500 nM)
is relatively high, and the binding of prothrombin to membrane surfaces has
been reported to counteract the interaction of the hirudin peptide with
proexosite-1 of the substrate
(14). However, the negative
effect of the membrane interaction is reported to be dependent on the
composition of the phospholipid vesicles
(14). Noting the plasma
concentration of prothrombin (
1.5 µM) and the high
concentration of TM in small vessels (
500 nM)
(45,
46), a significant portion of
prothrombin is expected to exist in dissociable complex with TM in
microcirculation. Based on our results, following conversion of prothrombin to
thrombin, the affinity of exosite-1 for TM is enhanced
100-fold. Thus it
is conceivable that the TM interaction with proexosite-1 can regulate the
prothrombinase activity under certain physiological conditions. More
importantly, when the TM-associated prothrombin is activated to thrombin, the
protease cannot dissociate from the cofactor, ensuring that both procoagulant
and anticoagulant thrombins are concurrently generated. This may be highly
critical for the regulation of the blood coagulation cascade.
 |
FOOTNOTES
|
---|
* This work was supported by NHLBI Grant HL68571 (to A. R. R.) from the
National Institutes of Health. 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 Biochemistry and
Molecular Biology, St. Louis University School of Medicine, 1402 S. Grand
Blvd., St. Louis, MO 63104. Tel.: 314-577-8130; Fax: 314-577-8156; E-mail:
rezaiear{at}slu.edu.
1 The abbreviations used are: prethrombin-2, prothrombin lacking the Gla and
both Kringle-1 and -2 domains; prethrombin-1, prothrombin mutant in which the
Gla and Kringle-1 domains have been deleted by recombinant DNA methods; TM,
thrombomodulin; TM46, TM fragment containing the epidermal growth
factor-like domains 4, 5, and 6; GPR-pNA,
N-p-tosyl-Gly-Pro-Arg-p-nitroanilide; TBS,
Tris-buffered saline; PC, phosphatidylcholine; PS, phosphatidylserine; Hir,
hirudin. 
 |
ACKNOWLEDGMENTS
|
---|
We thank Audrey Rezaie for proofreading the manuscript.
 |
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