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J Biol Chem, Vol. 274, Issue 36, 25510-25516, September 3, 1999
From the A collection of 56 purified thrombin mutants, in
which 76 charged or polar surface residues on thrombin were mutated to
alanine, was used to identify key residues mediating the interactions
of thrombin with thrombomodulin (TM), protein C, and
thrombin-activatable fibrinolysis inhibitor (TAFI). Comparison of
protein C activation in the presence and absence of TM identified 11 residues mediating the thrombin-TM interaction
(Lys21, Gln24, Arg62,
Lys65, His66, Arg68,
Thr69, Tyr71, Arg73,
Lys77, Lys106). Three mutants (E25A, D51A,
R89A/R93A/E94A) were found to have decreased ability to activate TAFI
yet retained normal protein C activation, whereas three other mutants
(R178A/R180A/D183A, E229A, R233A) had decreased ability to activate
protein C but maintained normal TAFI activation. One mutant (W50A)
displayed decreased activation of both substrates. Mapping of these
functional residues on thrombin revealed that the 11 residues mediating
the thrombin-TM interaction are all located in exosite I. Residues important in TAFI activation are located above the active-site cleft,
whereas residues involved in protein C are located below the
active-site cleft. In contrast to the extensive overlap of residues
mediating TM binding and fibrinogen clotting, these data show that
distinct domains in thrombin mediate its interactions with TM, protein
C, and TAFI. These studies demonstrate that selective enzymatic
properties of thrombin can be dissociated by site-directed mutagenesis.
Thrombin is unique among the clotting cascade serine proteases in
its ability to interact with a large number of diverse substrates. These interactions can paradoxically lead to either procoagulant or
anticoagulant effects. Procoagulant properties of thrombin include
cleavage of fibrinogen to form a fibrin clot (1), activation of
platelets through cleavage of the thrombin receptor(s) (2), feedback
activation of factors V, VIII, and XI (3, 4), and activation of the
transglutaminase, factor XIII (5). Binding of thrombin to the
endothelial cell surface protein thrombomodulin (TM)1 alters the substrate
specificities of the enzyme, leading to loss of its procoagulant
activities. The thrombin-TM complex acts as an anticoagulant through
activation of protein C (PC), which attenuates the clotting cascade by
inactivation of the activated cofactors V and VIII (6). Additionally,
thrombin activity can be regulated by several serine protease
inhibitors, including antithrombin III, heparin cofactor II, and
protease nexin I (7-9).
Recently, a new substrate for the thrombin-TM complex has been
identified termed plasma procarboxypeptidase B or thrombin-activatable fibrinolysis inhibitor (TAFI) (10). Activated TAFI functions as a
fibrinolysis inhibitor by cleavage of C-terminal lysines from partially
degraded fibrin, diminishing the activation of plasminogen, leading to
delayed clot lysis (11-13). Like PC, there is a 1,000-fold increase in
the catalytic efficiency of TM-bound thrombin for TAFI activation
compared with free thrombin. Unlike PC, TAFI is a metalloproteinase,
has a short half-life, and has no identified specific plasma inhibitor,
and the thrombin cleavage site in TAFI shares no homology to the site
in PC. The thrombin domains involved in TAFI activation have not yet
been identified.
Insights into the diverse specificities of thrombin have come from
structural analysis of the crystallized molecule bound to either
specific inhibitors (PPACK, hirudin) or substrate peptides (fibrinopeptide A, hirulog) (reviewed in Ref. 14). The catalytic triad
(His43, Asp99,
Ser205)2 is
located within a canyon-like cleft flanked by two major insertion loops, Leu46-Asn57 (the Trp50 loop)
and Leu144-Gly155 (the autolysis loop), that
sterically restrict access to the active site. There are two patches of
positively charged residues located on opposite sides of the active
site termed exosite I and exosite II, and it is primarily these two
domains that interact with the majority of substrates of thrombin and
contribute to its exquisite specificity. Thrombin also contains a
sodium-binding site, which is intimately involved in mediating
allosteric changes within the molecule (15). Sodium is coordinated by
the carbonyl oxygen atoms of Arg233 and Lys236,
and the entire binding environment involves a charge-stabilizing system
that includes Glu229, Glu146,
Arg197, Asp234, and Tyr237.
Functional studies into the contribution of these structural domains to
the activities of thrombin have come from a variety of methodologies
including the use of proteolyzed thrombin preparations (16, 17),
specific antibodies (18-20), peptide probes (2, 21, 23-25),
deletional or substitutional mutagenesis (26-30), naturally occurring
thrombin mutants (31-34), and ion substitution (35-37). We have
previously described the approach of alanine-scanning mutagenesis, in
which 79 highly exposed charged or polar residues on thrombin were
individually mutated to alanine, and this library of mutants was
screened for clotting activity, PC activation, and inhibition by
antithrombin III (38, 39). These studies demonstrated that all of these
functional residues were located on one hemisphere of the molecule,
which included the active site cleft and exosites I. Residues in which
mutations caused changes in the procoagulant and anticoagulant
activities of thrombin were not spatially distinct, but specific
mutations that dissociated the clotting activity of thrombin from its
PC anticoagulant activity were identified. This work led to the
development of a novel class of anticoagulants, termed protein C
activators, which have demonstrated in vivo anticoagulant
(40, 41) and antithrombotic activity (42).
To identify key residues in thrombin involved in TAFI activation, we
decided to use the library of thrombin alanine mutants to compare the
activation of the TM-dependent substrates PC and TAFI.
Previous studies with the thrombin mutants used the strategy of
transient expression of mutant prothrombin, activation by Echis carinatus venom, and thrombin quantitation by enzyme-linked
immunosorbent assay. Although allowing for rapid screening, this
strategy is limited by a number of factors, including obtaining
limiting amounts of the mutants, the presence of venom in the media
that can interfere in assays of the activities of thrombin, and
accuracy/reproducibility of the quantitation of enzyme concentration.
To circumvent these problems, we have expressed these mutants in stable
cell lines and purified them to homogeneity to have a collection of
readily usable reagents that can broadly be used for
structural-functional analyses of thrombin-substrate interactions. In
the present study, we used this purified collection to identify
thrombin residues involved in the activation of PC and TAFI. By using
two TM-dependent substrates, we were able to distinguish
residues important in the thrombin-TM interaction from those involved
with the individual substrates. The results show that distinct domains
in thrombin are involved in PC, TAFI, and TM binding and that PC and
TAFI activation can be dissociated from each other.
Proteins and Reagents--
Purified plasma protein C was from
Hematologic Technologies (Burlington, VT). Purified fibrinogen was from
Alexis Corp. (San Diego, CA). S-2238
(Phe-Pip-Arg-p-nitroanilide) and S-2366
(Glu-Pro-Arg-p-nitroanilide) were purchased from
Chromogenix. 3-(2-Furyl)acryloyl-Ala-Arg-OH was from Bachem (Torrance,
CA). Recombinant soluble TM (Solulin) and recombinant TAFI (43) were
from Berlex Biosciences (Richmond, CA). Q-Sepharose Fast Flow was from
Amersham Pharmacia Biotech, and Amberlite CG50 cation-exchange resin
was from ICN Biomedicals. The detergent CHAPS, E. carinatus
snake venom, QAE-Sephadex A-50, polyethylene glycol-8000, and
N-glycanase were from Sigma. PPACK was from Calbiochem.
Construction and Expression of Mutant Prothrombins--
The
full-length human prothrombin gene was inserted into the expression
vector pRc/CMV (Invitrogen), and codons for 76 polar and charged
surface residues were mutated to alanine by oligonucleotide-directed mutagenesis as described previously (38). 56 purified plasmid DNAs
(QIAGEN Maxi kit) encoding pRc/CMV-hPT mutants were linearized with
ScaI, and 10 µg of each was used to transfect 1 × 106 CHO AA8 cells grown in a 35-mm well by the calcium
phosphate method. CHO cells were grown and passaged in CHO S-serum-free medium (Life Technologies, Inc.) containing 10% fetal bovine serum and
100 units/ml penicillin and 100 µg/ml streptomycin. Stable cells
lines were obtained by limiting dilution and selection in G-418 (Life
Technologies, Inc.). 12 colonies were randomly selected for each
mutant, expanded to confluence in a 35-mm well, and tested for
prothrombin secretion by incubation of the washed monolayer with 1 ml
of serum-free CHO media for 24 h at 37 °C or 30 °C. Mutant
prothrombins (100 µl of conditioned media) were processed to thrombin
with 5 µg of Echis venom at 37 °C for 1 h and
assayed for thrombin activity with S-2238. Two or three of the clones with the highest expression were kept and expanded into roller bottles.
For each mutant, 6-10 roller bottles were grown to confluence, washed
extensively with phosphate-buffered saline, and then incubated with
serum-free medium. Conditioned medium was harvested every 5-7 days.
Purification of Thrombin Mutants--
The purification
procedures for prothrombin and thrombin were modified from previously
described protocols (41). For each mutant, ~2 liters of conditioned
medium containing 7-50 mg of crude prothrombin, was batch-extracted
with 50 ml of swollen QAE-Sephadex (equivalent to 2.5 g of dry
resin) for 1 h at room temperature. The resin was collected on a
glass-sintered funnel, and prothrombin was eluted immediately by the
addition of 50 ml of 20 mM HEPES, pH 8.0, 2 M
NaCl for 15-30 min. After centrifugation, the supernatant was
collected and dialyzed against 0.1 M potassium phosphate, pH 7.5. The dialysate was loaded onto a Q-Sepharose fast flow column
(10 ml) and washed, and prothrombin was eluted with a 0.1-0.7 M potassium phosphate, pH 7.5, gradient. Fractions were
activated with Echis venom and tested for amidolytic
activity. Peak fractions were pooled and dialyzed against 20 mM HEPES, pH 8.0, and 0.1 M NaCl. 25 ml of
mutant prothrombin was processed to thrombin with 1 mg of
Echis venom that had been preincubated with Amberlite CG50.
After incubation for 45 min at 37 °C, the thrombin mutant was loaded
directly onto an Amberlite CG50 column (10 ml) equilibrated in 20 mM HEPES, pH 8.0, and washed, and thrombin was eluted with a 0.1-1.0 M NaCl gradient. Fractions were assayed for
amidolytic activity, pooled, and dialyzed against 10 mM
potassium phosphate, pH 7.4, 150 mM NaCl, and 0.1%
polyethylene glycol-8000. After dialysis, the thrombin was concentrated
with a Centriprep-10 (Amicon), and concentration was determined by
A280 using an extinction coefficient (0.1%) of
1.83.
Steady-state Kinetics of S-2238 Hydrolysis--
The initial
rates of S-2238 hydrolysis, monitored by an absorbance change at 405 nm
(SpectraMAX, Molecular Devices), were measured for each of the mutants
using 2 nM purified mutant with 2, 4, 6, 8, 10, 15, 20, 25, 50, 100, and 150 µM substrate in 20 mM Tris,
pH 8.3, 150 mM NaCl. Km and
kcat were determined by fitting the data to the
Michaelis-Menton equation.
Fibrinogen Clotting--
Fibrinogen clotting activity was
determined for each of the mutants by using a purified fibrinogen
solution and measuring the time to clot formation with a fibrometer.
150 µg of fibrinogen in 290 µl of Dulbecco's phosphate-buffered
saline with calcium and magnesium (Irvine Scientific, Santa Ana, CA)
was incubated at 37 °C for 5 min. Clotting was initiated by the
addition of 10 µl of thrombin mutant. A standard curve with wild-type
(WT) thrombin ranging from 8.3 nM to 50 nM was
used to convert clotting times of the mutants to nM
equivalents of WT thrombin. Mutants were tested initially at 16.7 and
33.3 nM in duplicate. For those mutants with diminished
clotting activity, increasing amounts were used to give at least 2 points within the standard curve (range: 25-75 s).
Protein C Activation--
Activation of PC was carried out in 20 mM HEPES, pH 7.4, 150 mM NaCl, 5 mM
CaCl2, 0.2% CHAPS in the presence and absence of 50 nM soluble TM. In the presence of TM, 3 nM
mutant thrombin was incubated with TM at room temperature for 5 min. PC
was added at a final concentration of 200 nM and incubated
for 60 min at 37 °C. The reaction was terminated by the addition of
10 µM PPACK, and PC activity was determined by hydrolysis
of the substrate S-2366. In separate experiments, PPACK at this
concentration had no effect on the cleavage of S-2366 by activated PC
yet fully inhibited thrombin activity. A standard curve with WT
thrombin ranging from 0.25-16 nM was constructed for each
experiment to convert mutant activity to wild-type equivalent. For
mutants with <15% WT activity, increasing amounts were used to give
at least two points within the standard curve. In the absence of TM, PC activation was carried out in the same buffer, but 100 nM
mutant thrombin was incubated with 1 µM PC at 37 °C
for 2 h. The WT standard curve ranged from 25-400 nM thrombin.
TAFI Activation--
Activation of TAFI was carried out in the
same buffer as for PC. 100 pM mutant thrombin was incubated
with 50 nM TM for 5 min at room temperature. Then
recombinant TAFI was added at a final concentration of 85 nM and incubated for 20 min at room temperature. The
reaction was terminated by the addition of 5 µM PPACK,
and TAFI activity was determined from the initial rate of hydrolysis of
the substrate 3-(2-furyl)acryloyl-Ala-Arg-OH (final concentration 600 µM) at 37 °C by monitoring the absorbance change at
336 nM (44). A WT standard curve ranging from 15.6-1000 pM thrombin was constructed for each experiment to convert
mutant activity to WT equivalent. For mutants with <15% WT activity, increasing amounts were used to obtain at least two points within the
standard curve.
Expression, Amidolytic Activity, and Clotting Activity of Thrombin
Mutants--
In the previous studies employing this large collection
of thrombin alanine mutants, a strategy of transient transfection in
COS-7 cells followed by concentration, activation, and quantitation was
undertaken to study a number of the enzymatic activities of thrombin
(38, 39). In the present study, we have successfully converted the
collection of mutants to purified preparations (Table I). Permanent CHO cell lines were
established for each of the 56 mutant prothrombins, and crude
prothrombin was obtained for all of them except one mutant, N53A/T55A,
which is the site of N-linked glycosylation in the protein.
It is possible that mutation of this site leads to inefficient
secretion by the CHO cells. All of the remaining mutants were
successfully processed to
The catalytic efficiency of S-2238 hydrolysis was determined for each
of the mutants (Table I) and was found to be comparable to WT except
for 2 mutants, W50A and E229A, which were reduced 5-10-fold compared
with WT. In the fibrinogen clotting assay, 16 of the mutants had less
than 50% WT activity, with 7 of these <15% (Table I). These results
are similar to those obtained previously with transient transfection in
COS-7 cells (38, 39).
Protein C and TAFI Activation by Thrombin Mutants--
All 56 mutants were screened for activation of PC and TAFI in the presence of
soluble TM (Fig. 2). Activation of these
substrates was carried out under identical buffer conditions and
similar substrate concentrations for direct comparison. The majority of the mutants displayed wild-type activity, with no effect on either PC
or TAFI activation. 12 mutants showed less than 50% WT activity in
activation of both substrates: K21A, Q24A, W50A, R62A, K65A, H66A,
R68A, T69A, Y71A, R73A, K77A, and K106A. All of these residues except
Trp50 are located in exosite I. These effects are not due
to generalized disruption of the molecule, because all of the mutants
(except W50A) have WT catalytic efficiency for S-2238 cleavage; in
addition, the Q24A mutant has WT clotting activity. Interestingly,
mutants were identified with selective effects on the two substrates. Three mutants, E25A, D51A, and the triple mutant R89A/R93A/E94A, showed
diminished TAFI activation but maintained WT PC activation, whereas
three others, R178A/R180A/D183A, E229A, and R233A, showed WT TAFI
activation but diminished PC activation. These residues are candidates
for those that are important in the activation of the individual
substrates.
Protein C Activation in the Absence of TM--
Activation of the
substrates PC and TAFI by thrombin-TM requires formation of a ternary
complex. Thrombin mutations that affect activation of both substrates
may do so simply by disrupting the thrombin-TM interaction, thereby
preventing complex formation. To determine which residues may be
involved in the thrombin-TM interaction, the activation of PC in the
presence and absence of TM was determined for the 56 thrombin mutants
(Fig. 3). Those mutants that display
diminished activation of PC in the presence of TM but normal activation
in the absence of TM are likely to mediate binding of TM to thrombin.
Activation of PC was carried out under identical buffer conditions for
direct comparison. Again, the majority of mutants displayed WT
activity, having no effect on PC activation in the absence or presence
of TM. Three mutants (R20A, S22A/Q24A/E25A, E25A) were identified with
enhanced activation of PC in the absence of TM, showing a 2.6-3-fold
enhancement in activation. Of the 15 mutants with diminished PC
activation in the presence of TM, 11 displayed normal or elevated basal
activation in the absence of TM (K21A, Q24A, R62A, K65A, H66A, R68A,
T69A, Y71A, R73A, K77A, K106A) (Fig. 4).
These mutations are all located in exosite I, supporting the hypothesis
that these residues are involved in interactions with TM. Four mutants
(W50A, R178A/R180A/D183A, E229A, R233A) showed decreased PC activation
in both the presence and absence of TM, suggesting these mutations
affect direct interactions with PC (Fig. 4), and only one of these,
W50A, demonstrated defective TAFI activation, suggesting that this is
the only site of overlap on thrombin for these two substrates.
Localization of the PC-, TAFI-, and TM-interacting Residues on
Thrombin--
Mapping of the functional residues involved in PC and
TAFI activation demonstrates that discrete domains in the molecule are involved (Fig. 5). Residues involved in
the thrombin-TM interaction localize exclusively to the exosite I
domain, are primarily positively charged, and overlap significantly
with those residues important in fibrinogen clotting. Residues
mediating TAFI interaction are located along the "rim" above the
active site and are primarily negatively charged. Residues important in
PC activation are located along the rim below the active site and are
intimately involved in the sodium binding pocket. Trp50 is
the only identified residue that is involved with both substrates but
not involved directly in the thrombin-TM interaction.
This study describes the use of a large collection of thrombin
mutants to identify key residues involved in the interaction of
thrombin with thrombomodulin, protein C, and TAFI. This stategy of
alanine scanning has been described previously (38, 39), in which 79 charged or polar surface residues on thrombin have been systematically
mutated to alanine to minimize nonspecific structural pertubations in
the molecule while focusing on those residues most likely to mediate
the interactions of thrombin with macromolecular substrates. These
residues cover 62% of the solvent-accessible surface of the molecule,
allowing for a comprehensive structural-functional analysis of the
molecule. To obtain a readily usable set of reagents in sufficient
quantity for detailed studies, the library of mutants was converted
into purified thrombin preparations. The thrombin mutants used here
were purified after expression in CHO cells and have similar enzymatic
and clotting activities to those of transiently expressed thrombins in
crude cell supernatants used in earlier studies. Despite the
heterogeneity in glycosylation of the proteins, there was no evidence
for any significant effects on the enzymatic properties of thrombin
when produced in CHO cells rather than COS-7 cells.
By comparing the activation of two thrombin-TM-dependent
substrates, we were able to identify specific domains on thrombin mediating its interaction with each of these molecules. Mutations in
thrombin were found that either had selective effects on PC activation,
TAFI activation, or both. Those mutations affecting both substrates
were found to do so primarily by disruption of the thrombin-TM
interaction, and all of these residues are located in exosite I. In the
previous study (38), only two residues (Arg70,
Gln24) were identified that had normalized PC activation in
the absence of TM compared with 11 residues in the current study. This
difference is most likely due to differences in the assay conditions.
In the original study, PC activation without TM was carried out in the
absence of calcium and compared with activation with TM in the presence
of calcium. Calcium has been shown to have direct effects on PC
activation (45), so that a direct comparison of thrombin mutant PC
activation under different buffer conditions could be misleading. In
the current study, PC activation was determined under identical buffer
conditions. In addition, we found only a minor effect for the R70A
mutant on PC activation (~50% WT activity), which differs from the
previous study. There may be differences between the soluble TM used in
this study and the full-length membrane-bound form used previously
(38), although a recent study suggests that there is little difference
in TAFI activation between these two forms of TM (46). The current PC
activation results have been repeated using a second preparation of
R70A, and the presence of the R70A mutation in the purified protein was
confirmed by using Lys-C digestion followed by mass spectroscopy of the
thrombin fragments (data not shown). This residue was also studied by
Wu et al. (26), who found more profound effects, but these
may be attributed in part to the mutant used in their studies, R70E,
which involves a charge reversal that could have more dramatic effects
on thrombin-TM binding.
Enhanced activation of PC by thrombin in the presence of TM is
postulated to occur by formation of a ternary complex, which could lead
to either conformational changes in thrombin, which change its
substrate specificity (7), or changes in PC, which make it a better
substrate for thrombin (47). Mutations in thrombin could interfere with
formation of the thrombin-TM complex, could affect its ability to
undergo the allosteric changes needed for proper substrate activation,
or could directly affect interactions with PC. In this communication,
we have used the panel of purified thrombin mutants as a general screen
to identify interesting residues involved in TAFI and PC activation for
future studies.
Enhanced PC activation in the absence of TM has been described
previously for two thrombin mutants, E25K and E202Q (28, 29), which has
led to the hypothesis that there are repulsive interactions between
thrombin and the P'3 and P3 aspartate residues in PC, making PC a poor
substrate for free thrombin. We confirmed this finding for the E25A
mutant and observed the same effect for R20A. This is not completely
unexpected, as Arg20 is hydrogen-bonded with
Glu25, and mutation of either residue would disrupt this
interaction (29). Because of autodegradation, E202A could not be
produced and tested in this system. Whether or not a similar mechanism for TAFI activation exists remains to be tested. The P3 and P'3 residues of TAFI are not acidic
(NDTVSPRASAYY), and in the absence
of TM, thrombin will cleave at additional sites leading to TAFI
inactivation. Furthermore, mutation of acidic residues in thrombin
(Glu25, Asp51, and possibly Glu94)
diminishes TAFI activation, suggesting there may be important basic
residues in TAFI mediating the thrombin interaction. Further studies
will be necessary to elucidate the mechanism for enhanced TAFI
activation with TM.
Although mapping of the residues important in fibrinogen clotting and
PC activation (with TM) showed considerable overlap in the molecule,
those involved in PC and TAFI activation show that distinct domains are
involved. The residues important in TAFI activation lie above the
active site, involving and in close proximity to the Trp50
insertion loop. Trp50 itself appears to be a critical
residue for all of the activities of thrombin, as mutation or deletion
of this residue results in diminished small molecule substrate
hydrolysis, fibrinogen cleavage, PC activation, and antithrombin III
inhibition. This is unlikely to be due to nonspecific stuctural
pertubations within the loop, as mutations of neighboring residues have
very selective effects; D51A has normal clotting activity and PC
activation but diminished TAFI activation, whereas K52A has diminished
clotting activity but normal PC and TAFI activation. The residues
important in PC activation are below the active site and clustered
around the sodium binding pocket. Mutation of these residues may lead
to allosteric changes in thrombin affecting PC activation, or
alternatively, represent residues in direct contact with the substrate,
which then affects sodium binding. In either case, these residues are not involved in TAFI activation.
Previous studies with EGF deletion mutants (48, 49) have demonstrated
the functional TM requirements for enhanced PC and TAFI activation.
EGF5-6 of TM binds with high affinity to thrombin but does not enhance
PC activation, whereas EGF i4-6 supports enhanced PC but not TAFI
activation. EGF3-6 supports both enhanced PC and TAFI activation, but
deletion of EGF4 alone (to give EGF35-6) does not support activation
of either TAFI or PC. These data, taken together with the current
thrombin mapping data, suggests one possible model for formation of the
ternary complexes. Namely, EGF5-6 would mediate the binding of TM to
thrombin exosite I and orient EGF4 and EGF3 such that they are adjacent
to the lower and upper lips of the active-site cleft, respectively.
EGF4 would then align PC along the lower cleft, where it could interact
with the residues around the sodium binding pocket, which is located on
the opposite side of the molecule from exosite I. This assembly process
is further augmented by the endothelial cell PC receptor (50), and the
current mapping data suggest that thrombin and PC would be bridged on
opposite sides by TM and the endothelial PC receptor in a quaternary
complex. On the other hand, EGF3 would align TAFI along the upper lip
to contact the acidic residues in and around the Trp50
loop. Although perhaps overly simplistic, this model could be tested
directly using TM alanine mutants in conjunction with the thrombin
alanine mutants or TAFI mutants to map potential sites of interaction
between all of these molecules. In addition, the thrombin mutant data
along with the TM deletion data strongly suggest that the mechanisms of
activation of TAFI and PC by the thrombin-TM complex are quite different.
In summary, a large collection of purified thrombin alanine mutants was
used to map the interaction of thrombin with TM, PC, and TAFI. These
studies demonstrated that specific mutations can dissociate PC from
TAFI activation in the presence of TM. This library represents a
powerful set of reagents for studying the numerous macromolecular
interactions of thrombin. By examining the functional requirements for
a particular thrombin activity, one may identify candidate residues
that effectively dissociate one thrombin activity from another. Since
knock-out of the prothrombin gene in mice results in either embryonic
lethality or death soon after birth from hemorrhage (22, 51),
introduction of (pro)thrombin mutants with selected deficient
activities might allow one to salvage these mice and examine the
interplay of the numerous and complex macromolecular interactions of
thrombin in vivo.
We are grateful to Dr. Craig Gibbs for
assistance in preparation of the thrombin models and critical reading
of the manuscript.
*
This work was supported in part by a postdoctoral fellowship
award from the American Heart Association (to S. W. H.), by National Institutes of Health Grant R01 HL57530-01, and by 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 should be addressed: Div. of Hematology, Rm.
S-161, Stanford University School of Medicine, Stanford, CA 94305-5112. Tel.: 650-723-5007; Fax: 650-723-1269;
E-mail:shall@leland.stanford.edu.
2
Numbering is by the thrombin B chain. Table I
lists both B chain and bovine chymotrypsinogen numbering of the
residues for comparison.
The abbreviations used are:
TM, thrombomodulin;
PC, protein C;
TAFI, thrombin-activable fibrinolysis inhibitor;
EGF, epidermal growth factor;
CHAPS, 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonic acid;
PPACK, Phe-Pro-Arg-chloromethyl ketone;
WT, wild-type;
aPC, activated
protein C. CMV, cytomegalovirus;
CHO, Chinese hamster ovary.
Thrombin Interacts with Thrombomodulin, Protein C, and
Thrombin-activatable Fibrinolysis Inhibitor via Specific and
Distinct Domains*
§,
Division of Hematology, Stanford University
School of Medicine, Stanford, California 94305-5112 and ¶ Berlex
Biosciences, Richmond, California 94804
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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
-thrombin with Echis venom and
purified to 95% homogeneity (Fig.
1A), with the exception of one
mutant, E202A. Although initially processed correctly, after
cation-exchange purification and dialysis, this preparation
consistently and repeatedly degraded to a lower molecular weight form.
The glutamate 202 resides in the S3 specificity pocket of the active
site, and mutation to alanine may convert thrombin to a
"trypsin-like" enzyme, which slowly autodegrades (data not shown).
Approximately half of the thrombin mutants demonstrated a doublet at
Mr 36,500 by SDS-polyacrylamide gel
electrophoresis, with the upper band comigrating with plasma-purified
thrombin. Digestion of these proteins with N-glycanase
resulted in a single sharp band of identical mobility to digested
plasma-purified thrombin, suggesting that there is heterogeneity in the
glycosylation of the recombinantly expressed proteins (Fig.
1B).
Clotting and S-2238 amidolytic activities of purified thrombin mutants
View larger version (42K):
[in a new window]
Fig. 1.
Processing and purification of thrombin
mutants. A, SDS-polyacrylamide gel electrophoresis gel
demonstrating the purification steps for one of the mutants (R233A).
Lane 1, QAE-batch-extracted material; lane 2,
prothrombin peak from Q-Sepharose column; lane 3,
Echis-processed prothrombin; lane 4, Amberlite
CG-50-purified thrombin. B, N-glycanase treatment
of purified plasma thrombin and thrombin mutant E229A. 5 µg of
thrombin was incubated with 1 unit of N-glycanase for 1 h at 37 °C in phosphate-buffered saline. Lane 1,
untreated purified plasma thrombin; lane 2,
N-glycanase-treated purified plasma thrombin; lane
3, untreated thrombin mutant E229A; lane 4, N-glycanase-treated E229A.
View larger version (49K):
[in a new window]
Fig. 2.
Protein C and TAFI activation by thrombin
mutants in the presence of TM. Activation of PC (black
bars) and TAFI (gray bars) by the mutants is expressed
as a percentage of recombinant WT thrombin activity by comparison to a
standard curve. For PC activation, mutants were screened at a
concentration of 3 nM. At this concentration, WT thrombin
generated aPC at a rate of 3.8 pmol of aPC/pmol of thrombin/h. For TAFI
activation, mutants were screened at 100 pM. At this
concentration, WT thrombin generated aTAFI at a rate of 2.0 fmol of
aTAFI/fmol of thrombin/min. Error bars are S.D. of at least
two independent experiments.
View larger version (43K):
[in a new window]
Fig. 3.
Protein C activation in the presence and
absence of TM. Activation of PC by the mutants in the presence
(black bars) and absence (gray bars) of TM is
expressed as a percentage of WT thrombin by comparison to a standard
curve. In the presence of TM, WT thrombin generated aPC at a rate of
3.8 pmol of aPC/pmol of thrombin/h, whereas in the absence of TM, this
rate was 21 fmol of aPC/pmol of thrombin/h. Error bars are
S.D. of at least two independent experiments.
View larger version (32K):
[in a new window]
Fig. 4.
Protein C activation in the presence and
absence of TM Identification of thrombin mutants with
TM-dependent PC activation <50% WT, as taken from Fig.
3. Mutants are grouped by whether there is increased activity in
the absence of TM or no change in activity. Mutants that showed
significantly reduced PC activation only in the presence of TM were
designated as TM-interacting residues, whereas those that showed
reduced activity either in the presence or absence of TM were
designated as PC-interacting residues.
View larger version (45K):
[in a new window]
Fig. 5.
Localization of residues in thrombin
mediating its interactions with TM, PC, and TAFI. A, space-filling
model of thrombin looking directly into the active-site cleft (standard
orientation) with catalytic serine (Ser205) colored
red. Exosite I is on the right. Residues implicated in
interacting with TM are colored green, residues important in
TAFI activation are blue, and residues involved in PC
activation are yellow. The light blue residue is
Trp50, which is involved in both PC and TAFI activation.
B, identical model as in A but rotated 90° to
the left about the vertical axis, demonstrating exosite I and the
residues implicated in the TM interaction. C, identical
model as in A, but rotated 90° to the right about the
vertical axis, demonstrating the residues implicated in PC and TAFI
interaction.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
ACKNOWLEDGEMENT
FOOTNOTES
ABBREVIATIONS
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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