From the Division of Hematology, Stanford University School of Medicine, Stanford, California 94305
Received for publication, December 15, 2000, and in revised form, March 5, 2001
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ABSTRACT |
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The interaction interface between human
thrombin and human factor V (FV), necessary for complex formation and
cleavage to generate factor Va, was investigated using a site-directed
mutagenesis strategy. Fifty-three recombinant thrombins, with a
total of 78 solvent-exposed basic and polar residues substituted with
alanine, were used in a two-stage clotting assay with human FV.
Seventeen mutants with less than 50% of wild-type (WT) thrombin FV
activation were identified and mapped to anion-binding exosite I
(ABE-I), anion-binding exosite II (ABE-II), the
Leu45-Asn57 insertion loop, and the
Na+ binding loop of thrombin. Three ABE-I mutants (R68A,
R70A, and Y71A) and the ABE-II mutant R98A had less than 30% of WT
activity. The thrombin Na+ binding loop mutants, E229A and
R233A, and the Leu45-Asn57 insertion loop
mutant, W50A, had a major effect on FV activation with 5, 15, and 29%
of WT activity, respectively. The K52A mutant, which maps to the S'
specificity pocket, had 29% of WT activity. SDS-polyacrylamide gel
electrophoresis analysis of cleavage reactions using the
thrombin ABE mutants R68A, Y71A, and R98A, the Na+ binding
loop mutant E229A, and the Leu45-Asn57
insertion loop mutant W50A showed a requirement for both ABEs and the
Na+-bound form of thrombin for efficient cleavage at the FV
residue Arg709. Several basic residues in both ABEs have
moderate decreases in FV activation (40-60% of WT activity),
indicating a role for the positive electrostatic fields generated by
both ABEs in enhancing complex formation with complementary negative
electrostatic fields generated by FV. The data show that thrombin
activation of FV requires an extensive interaction interface with
thrombin. Both ABE-I and ABE-II and the S' subsite are required for
optimal cleavage, and the Na+-bound form of thrombin is
important for its procoagulant activity.
Thrombin is a serine protease that interacts with a large number
of macromolecular substrate receptors, cofactors, and inhibitors that
have both procoagulant and anticoagulant properties. This functional
versatility of thrombin is revealed by its structure (1). Thrombin has
the characteristic serine protease fold but differs in having a deep
narrow active site cleft occluded by the
Leu45-Asn57 (60 insertion loop) and
Leu144-Gly155 (149 insertion loop) surface
loops. Unique to thrombin are two cationic surface domains termed
anion-binding exosite I
(ABE-I)1 and II (ABE-II) that
are important for the binding of ligands to overcome the steric
hindrance of the occluded active site cleft. ABE-I is important for the
binding of fibrinogen (2-4), fibrin (5), heparin cofactor II (6, 7),
PAR1 (8, 9), thrombomodulin (2, 10-12), and hirudin (13-15). ABE-II
is important for the binding of platelet glycoprotein Ib (16)
and the glycosaminoglycan-bound serpins antithrombin III (6), heparin
cofactor II (6), and protease nexin I (17, 18).
Both exosites are involved in the binding of coagulation factors V and
VIII, which is important for the amplification of the coagulation
cascade (19). Studies with the ABE-I-specific inhibitor hirugen (19,
20) have implicated the thrombin ABE-I in cleavage of factor V, whereas
studies using the ABE-II triple mutant thrombin RA
(R89A/R93A/R98A) have implicated ABE-II (19). The specific proteolytic cleavage of human factor V (FV) by the serine protease thrombin generates factor Va (FVa), which acts as a cofactor with factor Xa, prothrombin, and Ca2+ ions to form the
prothrombinase complex on the surface of anionic phospholipids. This
leads to the amplification of the coagulation pathway at the sites of
vascular injury. FV, a single chain 330,000-kDa cofactor protein, is
activated to FVa with the release of the B domain activation products
(E fragment and C1 fragment) by cleavage at the residues
Arg709, Arg1018, and Arg1545 (21).
FVa is composed of the 105-kDa heavy chain (A1-A2 domain) and the
74-kDa light chain (A3-C1-C2 domains) held together by a calcium ion
(22, 23). The crystal structure of the light chain C2 domain implies a
Ca2+-independent mode of binding to the phospholipid
membrane via immersion of hydrophobic residues into the laminar
membrane core, with direct interactions between the basic C2 domain
residues and the negatively charged phosphatidylserine head groups and generalized complementary electrostatic interactions (24). The heavy
chain interacts with prothrombin, whereas both chains interact with
factor Xa. As part of the prothrombinase complex, factor Xa has a
300,000-fold increase in catalytic efficiency in thrombin generation
from prothrombin (25). Specific cleavage of FV occurs preferentially
first at Arg709, giving rise to the heavy chain, followed
by cleavage at Arg1018 and then by the rate-limiting
cleavage at Arg1545, which gives rise to the light chain.
Cleavage of both Arg709 and Arg1545 is
important for the full cofactor activity of factor Va, and the cleavage
of Arg1018 enhances the rate of cleavage of
Arg1545 (26).
In this study, we used 53 mutant thrombins in which solvent-accessible
polar and charged residues were substituted with alanine to fully
define thrombin residues important in the recognition and cleavage of
FV. This provided insights into the roles of the anion-binding exosites
for specificity toward FV and of residues involved in substrate
recognition and cleavage within the active site cleft.
Materials--
Purified human FV and FVa were purchased from
Hematologic Technologies Inc. Factor V-deficient plasma and
thromboplastin were purchased from Sigma. The expression and
purification of wild-type (WT) and alanine-substituted mutant thrombins
from Chinese hamster ovary cells and of the mutant R62Q from
insect cells have been described in detail previously (27, 28). The
concentration of active thrombin molecules was determined by titration
with D-Phe-Pro-Arg-chloromethyl ketone using the
chromogenic substrate H-D-Val-Leu-Arg-p-nitroanilide (S-2266,
Chromogenix, Sweden). The catalytic activity of the purified
recombinant WT and mutant thrombins toward
H-D-Phe-Pip-Arg-p-nitroanilide (S-2238,
Chromogenix, Sweden), fibrinogen, protein C, and thrombin-activatable
fibrinolysis inhibitor (TAFI) has been described (27).
Activation of Factor V by WT and Mutant Thrombins--
Cleavage
assays were performed in 50-µl volumes containing 300 nM
human FV and 100 pM thrombin in assay buffer (10 mM Hepes, pH 7.4, 150 mM NaCl, 5 mM
CaCl2, and 0.1% polyethylene glycol 6000) at 37 °C for
30 min, and the reactions were then placed on ice. The
concentration of FVa generated in the cleavage assay was determined by
a one-stage clotting assay. Cleavage reactions were diluted 1000-fold
in assay buffer on ice, and 50 µl was then added to 50 µl of
FV-deficient plasma and left at room temperature for 10 min. The
clotting assay was started with 100 µl of thromboplastin (containing
5 mM CaCl2 and equilibrated at room
temperature), and the clotting reaction was monitored over time at 600 nm in a SoftmaxTM plate reader (Molecular Dynamics) at room temperature until maximum clotting was achieved (no further change in absorbance at
600 nm). The t1/2 value was recorded as the time at
which 50% clot formation occurred. The effective concentration of
diluted human factor Va from the cleavage reaction in the clotting
assay was determined from a calibration curve using various
concentrations of purified human FVa versus the t1/2 value for clotting. For mutants showing
decreased clotting ability, dose-response curves were constructed over
a range of thrombin concentrations using the above protocol.
SDS-PAGE Analysis of Cleavage Reactions--
Cleavage reactions
containing 300 nM human FV and 0.5 nM thrombin
in assay buffer were incubated from 1 to 120 min before being
terminated by the addition of SDS loading buffer and boiling for 5 min.
Cleavage products were resolved by electrophoresis on 5-18% gradient
SDS-polyacrylamide gels (Bio-Rad) and then stained with biosafe
Coomassie Blue (Bio-Rad). The intensity of Coomassie Blue-stained bands
was determined by direct scanning of stained SDS-PAGE gels using the
UMAX Astra 4000U scanner and Umax VistaScan 3.5.2 software. Scanned
images were saved as TIFF files at a resolution of 600 dots per
inch. Pixel densities were calculated for each band from TIFF
files imported into Scion Image 1.62c.
Molecular Modeling of Thrombin--
The x-ray crystal structure
of thrombin (EC 3.4.21.5) bound with the active site inhibitor
D-Phe-Pro-Arg-chloromethyl ketone was obtained from the
Protein Data Bank (entry 1PPB). Thrombin is depicted as a
space-filling (CPK) model with solvent removed using the RasMol
V2.5 software package.
Screening for Thrombin Mutants Defective in Human FV
Activation--
A site-directed mutagenesis strategy was used to
determine residues on thrombin important for the interaction between
thrombin and human FV necessary for activation of FV to FVa.
Fifty-three thrombin mutants (Table I),
where solvent-exposed polar and charged residues were mutated to
alanine (27), were used in a two-stage clotting assay using purified
human FV to assay FV activation. Seventeen thrombin mutants with
~50% or less FV activation compared with WT thrombin were identified
and mapped to ABE-I, ABE-II, the Leu45-Asn57
insertion loop, and the Na+ binding loop (Fig.
1, Table
II). ABE-I had six residues (K21A, K65A,
H66A, R70A, R73A, K77A) with less than 50% of WT activity. Two mutant
thrombins showed less than 15% of WT activity (R68A, Y71A) (Fig. 1,
Table II). The double mutant K106A/K107A showed only 24% of the WT
type activity, indicating that the effects of the single mutations
(K106A, 62%; K107A, 52%) were additive for the double mutant.
Interestingly, substitution of Arg62 with Gln gave a
greater decrease in clotting activity (23% of WT activity) compared
with the alanine-substituted form of Arg62 (65% of WT
activity). ABE-II showed one mutant (R98A) with greatly reduced FV
activation (27% of WT activity), whereas two triple mutants (with a
total of six residues substituted) showed less than 50% of WT
activation (R89A/R93A/E94A and R245A/K248A/Q251A). Both the ABE-I and
ABE-II mutants have normal
kcat/Km values toward the
chromogenic substrate S-2238, suggesting that the reduced activity is
due to impaired binding to the exosites rather than to an effect on
catalysis (Table I). The residues Glu229,
Arg233, and Asp234 form part of a
surface-exposed Na+ binding loop that modulates the
activity of thrombin (29). Mutation of each residue resulted in 5, 15, and 32% of WT activity, respectively. Interestingly, the E229A mutant
shows a 13-fold reduction in the
kcat/Km for S-2238
(Km = 36.3 µM;
kcat = 33.2 s Dose-response Curves for Mutants with Diminished Human FV
Activation--
For mutants with a marked decreased in FV activation
(less than 20% of WT activity), dose-response curves were constructed, because these responses in the screening assay may not be in the linear
range for cleavage (Fig. 2). The mutants
R68A and R233A had 12.5 and 15.4% of WT activity in the initial
screen, each requiring 6-fold more thrombin to generate the same amount
of human FVa generated by cleavage with 100 pM WT thrombin.
The 6-fold more thrombin required for cleavage by the mutants
corresponded well with the initial screen of the clotting assays.
Consistent with this, the 5.2% of WT activity of E229A in the initial
screen also corresponded well in requiring 12-fold more thrombin to
generate the same amount of FVa obtained by 50 pM WT
thrombin. The mutant W50A required 4-fold more thrombin to generate the
same amount of FVa compared with 100 pM WT thrombin. Of all
the mutants tested, Y71A appears to have had the greatest effect on
cleavage of FV. Even at a concentration of 600 pM, only 25 nmol of FVa were produced in 30 min by Y71A, which is equivalent to a
30-fold decrease in FV activation by Y71A as compared with WT
thrombin.
SDS-PAGE Analysis of Cleavage Reactions--
SDS-PAGE analysis of
cleavage reactions on FV by representative thrombin mutants of the
major interaction interfaces (ABE-I, ABE-II, the Na+
binding loop, and the Leu45-Asn57 loop) was
employed to assess their role in the differential cleavage of the three
thrombin cleavage sites Arg709, Arg1018, and
Arg1545 (Fig. 3, A
and B). Cleavage of FV at Arg709 by WT thrombin
occurred within 1 min, with the appearance of the heavy chain (VaH).
The 75-kDa band appearing within 2-5 min represents both the light
chain (VaL) and the E fragment (generated by cleavage at
Arg709 and Arg1018), which are difficult to
resolve under these conditions. Within 5 min the majority of the
330-kDa FV was converted into VaH and other intermediate protein
species. Within 30 min there was almost complete conversion to the VaL
and VaH species. For the mutant thrombins W50A, R68A, and R98A, the VaH
appeared to be delayed by 1-2 min compared with WT thrombin, with the
intact protein persisting for 10 min, suggesting that cleavage at
Arg709 is delayed. Generation of the VaH protein species in
cleavage reactions for the mutants Y71A and E229A was further delayed, requiring 5-10 min before detection of the heavy chain. The appearance of the VaH chain by specific cleavage at Arg709 between WT
and mutant thrombins was assessed by running cleavage products from
1-min and 5-min reactions on the same gel. This was performed to avoid
making comparisons between several gels showing differing degrees of
staining. The amount of VaH generated was quantified by scanning
stained gels directly, the mean pixel density for each band was
determined, and the results are presented as a percentage of the VaH
band intensity generated by cleavage with WT thrombin (Fig.
4). The effect of the Y71A and E229A
substitutions on cleavage at Arg709 was most prominent,
with almost undetectable levels of VaH at 1 min compared with WT
thrombin (0.2 ± 0.5 and 2.4 ± 2.4% band intensity,
respectively). After 5 min, the Y71A and E229A mutants generated only
5.1 ± 1.1 and 24.9 ± 3.0% of the VaH compared with WT
thrombin. The effects of the R68A, W50A, and R98A substitutions were
less severe, showing 26.0 ± 2.8, 34.8 ± 2.1, and 43.8 ± 2.7%, respectively, of the VaH band intensity respective to WT
thrombin within 1 min. Cleavage reactions with the mutant thrombin W50A showed persistence of the E-C1-VaL protein species over the
entire time course, suggesting impairment of cleavage at
Arg1018. To confirm this, cleavage reactions of WT and W50A
were run on the same gel (Fig. 3C), which clearly shows the
persistence of the E-C1-VaL fragment over 120 min.
The appearance of the VaL species for the cleavage reactions by all the
mutant thrombins was difficult to interpret, because cleavages at
Arg709 and Arg1018 generate the E fragment,
which comigrates with the VaL chain. In addition, cleavage at
Arg1018 enhances cleavage at Arg1545 (32).
Hence, it is difficult to assess the effect of the substitutions on
cleavage at Arg1545.
Using a total of 53 mutant thrombins, which have a total of 78 alanine substitutions, we were able to map the interaction interface
between thrombin and FV using a two-stage clotting assay. This revealed
an extensive binding surface that utilizes ABE-I, ABE-II, the
Na+ binding loop, and the
Leu45-Asn57 insertion loop (Fig.
5).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1)
compared with WT thrombin (Km = 4.7 µM; kcat = 55.9 s
1), whereas the remaining two mutants have
normal catalysis toward S-2238, suggesting that substitution of
Glu229 affects substrate binding within the active site
cleft. Two residues, situated on the
Leu45-Asn57 loop, also have reduced FV
activation. Trp50 forms part of a hydrophobic "YPPW
lid" that restricts the specificity of thrombin by occluding the
active site, and Lys52 protrudes and contributes to the
specificity of the S' subsite (30, 31). Alanine substitution at these
residues showed FV activation of 29%. The mutant thrombin W50A has a
5-fold reduction in kcat/Km
for S-2238, reflected by a 6-fold increase in Km,
whereas K52A has normal catalysis toward S-2238. Mutation of
Trp50, like Glu229, appears to alter the active
site cleft, affecting substrate binding.
Thrombin nomenclature and S-2238 amidolytic activities of purified
thrombin mutants
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Fig. 1.
Human FV activation by thrombin mutants.
The ability of purified WT and mutant thrombins to activate human FV
was determined using a two-stage clotting assay as described under
"Experimental Procedures." The effect of alanine substitutions on
FV activation is reported relative to 100% WT activity. The error bars
represent the standard deviation of at least two separate independent
experiments (carried out in duplicate).
Mutant thrombins with less than 50% of human factor V activation
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Fig. 2.
Dose dependence of thrombin cleavage of human
FV. FV (390 nM) was incubated with several
concentrations of WT and mutant thrombins ranging from 50 to 600 pM at 37 °C for 30 min. The concentration of human FVa
generated after the 30-min cleavage reaction was determined using a
two-stage clotting assay as outlined under "Experimental
Procedures." A shows the dose dependence curves for WT
thrombin (squares) and the ABE-I mutants R68A
(circles) and Y71A (triangles). B
shows the dose dependence curves for WT thrombin (squares)
and the Na+ binding loop mutants E229A (circles)
and R233A (triangles) and the
Leu45-Asn57 loop mutant W50A
(diamonds).
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Fig. 3.
SDS-PAGE analysis of cleavage reactions for
ABE-I, ABE-II, and Na+ binding and
Leu45-Asn57 loop mutants. Cleavage
reactions containing 390 nM human FV and 0.5 nM
thrombin were performed at 37 °C at several time points ranging from
1 to 120 min. Cleavage products were resolved by SDS-PAGE on a 5-18%
gradient gel. A shows the expected sizes of cleavage
products. B shows gels for WT thrombin, W50A, R68A, Y71A,
R98A, and E229A. C shows time course experiments for the
cleavage of WT and W50A thrombins over 0, 1, 5, 10, and 60 min.
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Fig. 4.
Comparison of human FV heavy chain release by
WT and mutant thrombins. The intensity of Coomassie Blue-stained
FV heavy chain bands (VaH) generated from cleavage reactions at 1 and 5 min for WT and thrombin mutants (W50A, R68A, Y71A, R98A, and E229A) was
determined by direct scanning of stained SDS-PAGE gels as described
under "Experimental Procedures." The band intensity for each mutant
is presented on the bar graph as the percent VaH band intensity with
respect to WT thrombin.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 5.
Space-filling model of thrombin
residues with decreased human FV activation. Thrombin is depicted
as a space-filling model using the RasMol V2.5 software package showing
residues, when substituted with alanine, with less than 50% of WT
human FV activation. Mutants with 15% of WT activity are depicted in
a darker color. Residues of interest are labeled using the
single amino acid codes. The top panel shows the classic
front view of thrombin (31) with the active site cleft running
horizontally left to right, with the
Leu45-Asn57 insertion loop on the
top of the cleft occluding the active site
Ser205 (red). The active site is shown with the
bound active site inhibitor D-Phe-Pro-Arg-chloromethyl
ketone as a brown stick model. The
Leu45-Asn57 insertion loop residues are colored
magenta, and the Na+ binding loop residues are
green. ABE-I runs to the right of the active site
cleft, and ABE-II is on the opposite side of the molecule. The
middle and bottom panels show the same molecule
but rotated 90° to the left and right,
respectively, to show ABE-I and ABE-II. ABE-I residues are
blue, and ABE-II residues are depicted in
yellow.
The ABE-I of thrombin is a surface patch with a high density of surface-exposed basic amino acids. This region extends away from the active site cleft and is essential for the binding of fibrinogen (2-4), heparin cofactor II (5, 6), PAR1 (8, 9), thrombomodulin (2, 10-12), and the inhibitor hirudin (13-15). Binding to ABE-I is important to overcome steric hindrance to the occluded active site. Basic residues in ABE-I have a dual role in binding its many ligands. Studies with PAR1 (33) and hirudin (34) show that the ABE-I residues contribute to a positive electrostatic field that extends into the solvent. This is important for enhancing the rate of complex formation with complementary electrostatic fields generated by its ligands (electrostatic steering). Secondly, basic residues are used for direct interactions in complex formation (ionic tethering), although which residue is involved differs between the various ligands (33, 34). The ABE-I also has a number of hydrophobic residues important for hydrophobic interactions with PAR1 (9), thrombomodulin (35), and hirudin (14, 15). Alanine substitution of the ABE-I residues Arg68, His66, Arg70, and Tyr71 significantly reduced FV activation, which was due to impaired binding within ABE-I rather than to a direct effect on catalysis. The mutations either affect direct interactions with FV or indirectly affect local protein structure. Both Arg68 and Arg70 have been shown to form direct interactions with ligands in the crystal structure of thrombin bound to either a peptide based on the N-terminal domain of the PAR1 receptor (Arg68) (9) or the C-terminal tail of hirudin (Arg68 and Arg70) (14, 15). Hence, it is possible that these residues may also be in direct interactions with FV. Likewise, the residue Tyr71 is important for hydrophobic interactions with the C-terminal tail of hirudin (14, 15) and the thrombomodulin 4-5-6 epidermal growth factor-like domain (35). The substitution of residue His66 has a large effect on FV activation; however, this residue does not make any direct interactions as seen in the published crystal structures of thrombin bound with various ligands. Furthermore, the H66A mutation has a large effect on the thrombomodulin-dependent activation of thrombin-activatable fibrinolysis inhibitor (TAFI) and protein C (27); however, the crystal structure of thrombin bound with the 4-5-6 epidermal growth factor-like domains of thrombomodulin showed that this residue makes no direct major interactions (35). Together with the observation of normal catalysis toward S-2238, would suggest that the H66A substitution has a local effect on the binding in ABE-I. Interestingly, the effect of substitution of Arg62 with Gln is more severe than that seen with Ala. The effect of the R62Q mutation is similar to that of the dysthrombin Quick I (R62C), where the cysteine substitution appears to distort the 60-70 autolysis loop, affecting the binding of hirudin (36), fibrinogen (37), and heparin cofactor II (38) within ABE-I. Hence, it appears that alanine substitution does not affect loop structure to the same degree as glutamine or cysteine substitutions. The role of ABE-I in the recognition and cleavage at the thrombin cleavage sites within FV was difficult to determine because of the complexity of cleavage patterns on SDS-PAGE. What is clear from cleavage studies using the ABE-I mutants of Arg68 and Tyr71 is the importance of ABE-I in the recognition and cleavage of FV at Arg709. These studies suggest a major interaction with a hirudin-like domain N-terminal to the Arg709 cleavage site (21) and are consistent with equilibrium binding studies by Bock and co-workers (39).
The ABE-II, which is located on the opposite side of the molecule, is involved in the binding of glycosaminoglycan-bound serpins antithrombin III (6), protease nexin I (17, 18), heparin cofactor II (6, 7, 28), bovine FV, and recombinant human FVIII (19), as well as in the binding of prothrombin fragment F2 (40) and platelet membrane glycoprotein Ib (16). The mutant thrombins R98A and R89/R93/E94A have an effect on the activation of FV (27-40% of WT activity), suggesting an important role for the interaction of ABE-II with human FV. The importance of these three residues has been shown by Esmon and Lollar (19) using the triple thrombin mutant R89A/R93A/R98A, which had a greatly reduced ability to activate bovine FV. The residues Arg89 and Arg98 lie in close proximity (4 Å), whereas Arg93 is over 12 Å away from these residues (Fig. 5). Mutation of these residues impairs heparin binding and heparin-ATIII-accelerated inhibition of thrombin (41) and chondroitin sulfate-enhanced affinity of thrombomodulin to ABE-II (41). The R98A substitution has a major effect on cleavage of Arg709 on FV, and this effect compares well with cleavage studies of bovine FV using the thrombin triple mutant RA (R89A/R93A/R98A), suggesting a role of ABE-II for cleavage of human FV at Arg709 (19).
It is less clear whether both ABE-I and ABE-II are important for cleavage at Arg1018 and Arg1545. Studies by Thorelli et al. (26, 32) show that cleavage at Arg1018 facilitates cleavage at Arg1545. Therefore, the late appearance of the VaL could be a direct consequence of poor cleavage of Arg1018 delaying the appearance of the C1-VaL species and subsequent cleavage at Arg1545, or the mutations may directly affect recognition and cleavage at the individual cleavage sites. The effect of the ABE-I and ABE-II mutations on cleavage of Arg1018 and Arg1545 was difficult to determine because of comigration of the E-fragment and VaL on SDS-PAGE.
Several residues in both ABE-I and ABE-II have reduced FV activation ranging from 52% (K107A) to 65% (R62A). However, the magnitude in reduction suggests that these residues are unlikely to participate in major interactions. Mutagenesis studies support the hypothesis that ABE-I basic residues, not involved in direct interactions with hirudin (33) or PAR1 (34), make small but collectively important contributions to the localized positive electrostatic field generated by ABE-I. Hence, it is likely that both ABE-I and ABE-II positive electrostatic fields are important in enhancing complex formation through the interaction of complementary electrostatic fields with FV (42, 43). Both Arg709 and Arg1545 have sequences N-terminal to the cleavage site with a high density of acidic residues, which, if exposed to the solvent, could contribute to a negative electrostatic potential, allowing rate enhancement by electrostatic steering with complementary electrostatic fields of thrombin ABE-I and -II.
The residues Glu229 and Arg233 make ion pair interactions with residues Lys236 and Asp146, respectively, which are important for maintaining the structural integrity of the Na+ binding loop (44, 45). Mutation of either residue leads to a conformational change of the Na+ binding loop, favoring the anticoagulant form of thrombin (46). Consistent with this, E229A and R233A showed markedly reduced FV activation, suggesting that efficient recognition and cleavage of FV requires the Na+ form of thrombin. The thrombin substitution W50A appears to have an effect on recognition and cleavage at Arg709, but the persistence of the E-C1-Val species shows that recognition and cleavage at Arg1018 is also impaired. Trp50 is spatially well separated (17 Å) from the Na+-binding site; however, mutation of Trp50 may have an indirect effect on the Na+ binding loop. Substitution of Trp50 with serine appears to affect Na+ binding and favors the anticoagulant form of thrombin with enhanced protein C specificity (45). Likewise, it is possible that alanine substitution of Trp50 could favor the anticoagulant form of thrombin with reduced ability to activate FV.
Specificity of the thrombin S1' substrate binding site is restricted to small polar P1' residues, due in part to occlusion by the Lys52 side chain (1). Alanine substitution of this residue resulted in a decrease in FV activation, suggesting a role for this residue and the S1' subsite in defining the specificity of thrombin toward FV. Alanine substitution of this residue has also been shown to be important in defining the specificity of thrombin toward antithrombin III (31) and fibrinogen (12, 47).
In summary, thrombin activation of human FV requires an extensive
interaction interface involving ABE-I, ABE-II, the
Leu45-Asn57 loop, and the Na+
binding form of thrombin. It will be interesting to analyze and compare
the structural requirements for the activation of human factor VIII,
which is homologous to human FV and important for the assembly of the
intrinsic tenase enzyme complex.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant R01 HL57530 and the Cheong Har Family Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence may be addressed: Division of Hematology,
Stanford University School of Medicine, CCSR (Rm. 1155), Stanford, CA 94305-5156. Tel.: 650-725-4043; Fax: 650-736-0974; E-mail: lawrence.leung@stanford.edu or tmyles{at}stanford.edu.
Published, JBC Papers in Press, April 18, 2001, DOI 10.1074/jbc.M011324200
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ABBREVIATIONS |
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The abbreviations used are: ABE, anion-binding exosite; PAR1, protease-activated receptor 1; FV, factor V; WT, wild-type; SDS-PAGE, SDS-polyacrylamide gel electrophoresis; VaH, factor Va heavy chain; VaL, factor Va light chain; serpin, serine protease inhibitor.
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