From the
Utilizing site-directed mutagenesis, 77 charged and polar
residues that are highly exposed on the surface of human thrombin were
systematically substituted with alanine. Functional assays using
thrombin mutants identified residues that were required for the
recognition and cleavage of the procoagulant substrate fibrinogen
(Lys
Thrombin is a multifunctional serine protease that plays a
prominent role in the maintenance and regulation of hemostasis by blood
coagulation(1) . Upon generation by factor Xa activation of
prothrombin (2), thrombin recognizes and hydrolyzes multiple
macromolecular substrates that have either procoagulant or
anticoagulant functions and alters their activity (reviewed in Refs. 3
and 4). Thrombin cleaves two peptide bonds in fibrinogen, producing
fibrin monomers that polymerize to form an insoluble clot. The fibrin
polymers are subsequently stabilized through cross-linking by factor
XIIIa which also results from thrombin-catalyzed activation(5) .
Platelet activation and aggregation is mediated by the
thrombin-catalyzed hydrolysis of the platelet thrombin
receptor(6) , and the procoagulant stimulus is maintained by
thrombin-mediated feedback activation of the serine protease factor XI (7) and the cofactors V and VIII(8) . Binding of thrombin
to the cofactor thrombomodulin on the surface of endothelial cells
alters the specificity of thrombin such that it no longer recognizes
the procoagulant substrates described above but instead recognizes and
activates the anticoagulant substrate, protein C. Activated protein C
attenuates the coagulant stimulus by the cleavage and inactivation of
activated cofactors Va and VIIIa and may function to localize blood
coagulation at the site of vascular injury(9) .
The crystal
structure of human
The current thrombin inhibitors can be divided into four groups
according to the location of their putative binding sites on the
surface of thrombin: active site only (e.g. PPACK(10, 11) ), active site and exosite 1 (e.g. hirudin(14, 15) , heparin/heparin
cofactor II(17) ), active site and exosite 2 (e.g. heparin/antithrombin III(18, 19) ), exosite 1 only (e.g. hirugen(20) , exosite 1 specific
antisera(21) ). An ideal thrombin inhibitor would inhibit the
procoagulant activities of thrombin while preserving its anticoagulant
function. However, all the current inhibitors inhibit the activity of
thrombin toward both the procoagulant substrates and the anticoagulant
substrate, protein C. Thus, preliminary mutagenesis studies that
indicated that the binding sites for fibrinogen and the platelet
thrombin receptor can be dissociated from those of thrombomodulin and
protein C (22) provoked speculation that there may be an epitope
on the surface of thrombin, specific for procoagulant function, that
could be selectively targeted by an inhibitor.
In this study we
undertook an extensive mutagenesis study in order to map the secondary
binding sites on the surface of thrombin required for fibrinogen
clotting, thrombomodulin-dependent activation of protein C, and
inhibition by the thrombin aptamer, an oligonucleotide-based thrombin
inhibitor identified by a combinatorial selection strategy(23) ,
that does not interact with the active site and has been proposed to
interact with both exosites 1 and
2(24, 25, 26) . The approach used involved the
systematic substitution of all the charged and polar amino acids that
are highly exposed on the surface of thrombin with the small neutral
amino acid alanine. Functionally important residues were found only on
a single hemisphere of the thrombin surface lining and flanking the
active site cleft. Although residues were identified that are
specifically required for unique functions, these residues were
generally not spatially separated from residues with other activities.
Expression
levels varied between 0.12-2.0 µg/1
Concentrated cell culture medium containing
prothrombin mutants was analyzed by Western blotting of reducing
SDS-PAGE gels before and after processing to thrombin with E. carinatus venom (Fig. 1A). Processing was
demonstrated to be complete for all mutants by the disappearance of the
band (
The specific amidolytic activity of each thrombin mutant
toward the chromogenic peptidyl substrate S-2238 was assessed as a
measure of the structural integrity of each mutant (). The
mean specific activity of recombinant wild-type thrombin in cell
culture medium from 16 separate transfections was 837 ± 168
mOD/min/µg compared to 1012 ± 61 mOD/min/µg for purified
wild-type thrombin validating the slot-blot method for determination of
prothrombin concentration and thrombin specific activity. The
concentration of purified wild-type thrombin was determined by direct
protein assay using the BCA (bicinchoninic acid) assay kit (Pierce).
Medium from mock-transfected cells and from cells transfected with the
active site mutant S205A displayed no detectable activity. Two
additional mutants (E229A,R233A,D234A) and (R245A, K248A,Q251A) had
undetectable amidolytic activity. Amidolytic activity was recovered
when the residues that were simultaneously replaced in these triple
mutants were substituted separately. All remaining mutants retained
greater than 40% of the specific amidolytic activity of wild-type
thrombin indicating that the overall tertiary conformation of each
thrombin mutant was not perturbed.
Using a site-directed mutagenesis strategy to probe the
highly exposed residues on the surface of thrombin, we have identified
residues that are important for the recognition and cleavage of
fibrinogen and protein C, interactions with thrombomodulin, and
inhibition by the thrombin aptamer. Our approach was designed to
minimize nonspecific structural disruption, and amidolytic activity
toward the peptidyl substrate S-2238 was generally preserved among all
the mutants () indicating that conformational integrity was
unperturbed.
Nineteen residues were identified as being important
for fibrinogen clotting activity (Lys
Additional residues implicated in
fibrinogen recognition include residues Asp
The crystal structure of fibrinopeptide A bound to
thrombin predicted that residue Arg
Fourteen residues were identified that are important
for thrombomodulin-dependent protein C activation (Lys
Unlike fibrinogen, protein C does
not have an acidic domain on the COOH-terminal side of the cleavage
site analogous to the COOH terminus of hirudin. However, an analogous
domain can be found in thrombomodulin, and numerous studies have
suggested that thrombomodulin binds to exosite 1 (21, 22, 32, 33, 42,
46-48). In our mutagenesis study, the replacement of 11 residues
in exosite 1 (Lys
A thrombin-based
synthetic peptide corresponding to thrombin residues 147-158 was
reported to bind thrombomodulin and block thrombin binding to
thrombomodulin(46) . Analysis of modified peptides suggested
residues Asn
Five residues were
identified as being important for inhibition by the thrombin aptamer
(Lys
The functional epitope for a procoagulant activity of thrombin,
fibrinogen clotting is distinct from but overlaps with the residues
required for thrombomodulin-dependent protein C activation, an
anticoagulant function. Although unique residues involved in the
contrasting functions of thrombin can be identified, they are not
spatially separated and are located on a single face of thrombin that
surrounds the active site cleft and includes exosite 1 (Fig. 6A). Thus, thrombin utilizes the same general
surface for substrate recognition regardless of substrate function
although the specific contact residues may vary. The lack of spatial
separation of these two epitopes is not conducive to the generation of
thrombin inhibitors that can distinguish the procoagulant and
anticoagulant functions of thrombin.
We are grateful to Regan Shea, Terry Terhorst, Kim
Sweetnam, Cathy Sueoka, Lisa Crow, and Melissa Klute for the synthesis
of the oligonucleotides used in this study and Lisa Paborsky for
critical comments on this manuscript.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
, Trp
, Lys
,
Asn
+Thr
, Lys
,
His
, Arg
, Tyr
, Arg
,
Lys
, Lys
+Lys
,
Asp
+Lys
, Glu
,
Glu
, Arg
, Asp
) and the
anticoagulant substrate protein C (Lys
, Trp
,
Lys
, His
, Arg
, Tyr
,
Arg
, Lys
,
Lys
+Lys
, Glu
,
Arg
), interactions with the cofactor thrombomodulin
(Gln
, Arg
) and inhibition by the thrombin
aptamer, an oligonucleotide-based thrombin inhibitor (Lys
,
His
, Arg
, Tyr
,
Arg
). Although there is considerable overlap between the
functional epitopes, distinct and specific residues with unique
functions were identified. When the functional residues were mapped on
the surface of thrombin, they were located on a single hemisphere of
thrombin that included both the active site cleft and the highly basic
exosite 1. No functional residues were located on the opposite face of
thrombin. Residues with procoagulant or anticoagulant functions were
not spatially separated but interdigitated with residues of opposite or
shared function. Thus thrombin utilizes the same general surface for
substrate recognition regardless of substrate function although the
critical contact residues may vary.
-thrombin bound to the peptidyl inhibitor D-Phe-Pro-Arg chloromethylketone
(PPACK)
(
)(10, 11) revealed that
prominent structural features of the thrombin molecule are the location
of the catalytic triad (His
, Asp
,
Ser
) within a deep canyon-like active site cleft and the
presence of two extensive surfaces (referred to as exosite 1
(Arg
, Lys
, Arg
,
Arg
, Arg
, Lys
,
Lys
, Lys
, Lys
) and exosite 2
(Arg
, Arg
, Arg
,
Arg
, Arg
, Lys
,
Arg
, Arg
, Lys
,
Lys
, Lys
)) that are mainly comprised of
positively charged residues. Considerable insight into the interaction
of the thrombin active site with substrate residues immediately
proximal to the cleavage site has been provided by the structure of the
PPACK
thrombin complex and the complex of human
-thrombin
with fibrinopeptide A(12) . However, the exquisite specificity
of thrombin for the macromolecular substrates described above is
thought to involve interactions with secondary sites on the surface of
thrombin (13). The crystal structure of thrombin bound to hirudin, an
inhibitory protein from the medicinal leech, revealed that while the
amino-terminal domain of hirudin occupies the active site of thrombin,
the carboxyl terminus makes extensive contacts with exosite
1(14, 15) . Although some insight into the interactions
of fibrinogen, thrombomodulin, and the platelet thrombin receptor with
thrombin has been derived by analogy with the COOH terminus of hirudin
(reviewed in Ref. 16), the interactions of these macromolecular
substrates with secondary sites on thrombin are not well defined.
Construction of the Vector for Expression of Human
Prothrombin
The human prothrombin coding sequence from cDNA
clone BS(KS)-hFII provided by Ross MacGillivray, University of British
Columbia (27) was inserted into eukaryotic expression vector
pRc/CMV (Invitrogen Corp.) to generate the construct, pRc/CMV-hPT which
can be used to express prothrombin in mammalian cells and to generate a
single-stranded template for mutagenesis and sequencing in Escherichia coli in the presence of a helper phage, M13K07
(28).
Oligonucleotide-directed Mutagenesis
The method
utilized has been described previously in detail(29) .
Oligonucleotide-directed mutagenesis was performed on a
uracil-containing single-stranded template of pRc/CMV-hPT produced in dutung
E. coli strain CJ236 (30) to allow selection against the parental
strand in dut
ung
E.
coli strain, XL1-Blue. Single-stranded DNA from individual
transformants was sequenced using dideoxy chain termination and
Sequenase 2.0 (United States Biochemical) to confirm the identity of
each mutation. 500 ml cultures of each pRc/CMV-hPT mutant in XL1-Blue
were used to isolate plasmid DNA using the QIAGEN Maxi plasmid
preparation kit for transfection of cultured COS-7 cells.
Expression and Activation of Recombinant
Prothrombins
Plasmid DNA encoding pRc/CMV-hPT mutants (10
µg) was introduced into 1 10
COS-7 cells, grown
in a 35-mm well, by the DEAE-dextran method of
transfection(31) . Two days post-transfection, the cell
monolayer was washed twice with PBS and incubated with 1 ml of
serum-free Dulbecco's modified Eagle's medium at 37 °C
for 24 h. The conditioned medium was concentrated 20-fold with a
Centricon-30 ultrafiltration apparatus (Amicon). Prothrombin in 50
µl of this concentrated medium was activated with 1.5 µg of Echis carinatus venom (Sigma) at 37 °C for 45 min.
Twenty-five µl of concentrated conditioned medium before and after
venom activation were analyzed by Western blotting of reducing SDS-PAGE
gels to ensure that processing was complete.
Quantitation of Recombinant Prothrombins in Conditioned
Cell Culture Medium
Thrombin protein concentration was
determined by quantitative Western slot-blotting using a Schleicher and
Schuell Minifold II vacuum slot-blot apparatus. Prothrombin in 20-fold
concentrated conditioned medium and purified prothrombin standards
(American Diagnostica) were activated as described above, complete
processing was demonstrated by Western blotting of reducing SDS-PAGE
gels (Fig. 1A). Samples and standards were diluted with
PBS and adjusted to the same concentration of conditioned medium from
mock transfected cells. Duplicate 100-µl aliquots containing
approximately 50 ng of the activated prothrombin unknown or duplicate
aliquots of purified, activated prothrombin standards (1-200 ng)
were aspirated through a 0.45-µm nitrocellulose filter in the
slot-blot apparatus. Each slot was washed twice with 200-µl
aliquots of PBS. The blot was washed twice with PBS and blocked with 5%
non-fat skim milk (Carnation) in PBS. The blot was incubated with 11
µg/ml rabbit polyclonal immunoglobulins against human prothrombin
that cross-react with thrombin (Dako) in 5% non-fat skim milk in PBS.
The blot was washed with PBS containing 0.05% Tween-20 and incubated
with 1 µCi/ml S-labeled donkey F(ab`)
directed against rabbit immunoglobulins (Amersham Corp.). The
blot was washed with PBS containing 0.05% Tween-20, and the
radioactivity at each position was determined using an Ambis 4000
radioanalytic imaging detector. The thrombin concentration in each
sample was determined from the standard curve which was linear over the
range of 1-200 ng of prothrombin (Fig. 1B).
Figure 1:
Processing and quantitation of
prothrombin mutants. A, alignment of two Western blots
(positions 1-3 and 4-8) of SDS-PAGE gels probed with the
polyclonal antibody directed against prothrombin (Dako) used for
quantitation of prothrombin mutants in the quantitative Western
slot-blot. Lane 1, purified prothrombin (1 µg), fragments
corresponding to prothrombin (72 kDa) and partially processed
intermediates are visible. Lane 2, purified prothrombin (1
µg) processed with E. carinatus venom, fragments
corresponding to the thrombin B chain (
34 kDa), and the pro region
fragments F1 (
21 kDa) and F2 (
12 kDa) are visible. Lane
3, purified thrombin (1 µg), a fragment corresponding to the
thrombin B chain (
34 kDa) is visible along with a possible
degradation product. Lanes 4-8, (
0.5 µg)
recombinant prothrombin mutants (wild-type, mutants 3a, 8b, 10a, 36b)
in concentrated cell culture medium following processing with E. carinatus venom, fragments corresponding to the
thrombin B chain (
34 kDa), and the pro-region fragment F1 (
21
kDa) are visible. B, standard curve for quantitation of
prothrombin mutants by Western slot-blot. Binding of the rabbit
polyclonal antibody directed against prothrombin (Dako) to duplicate
samples of purified prothrombin was quantitated following binding of
S-labeled donkey antibodies (Amersham) directed against
rabbit immunoglobulins. Linear correlation coefficient, r
= 0.998.
Amidolytic Assay
The hydrolysis by thrombin of the
chromogenic substrate S-2238 (H-D-Phe-Pip-Arg-pNA) was
performed as described previously(22) . Purified wild-type
thrombin (1 µg) gave a rate of hydrolysis of 1012 mOD/min in 300
µl of 100 µM S-2238.
Fibrinogen Clotting
Fibrinogen clotting activity
was determined using a standard amount of thrombin as measured by
amidolytic activity. An aliquot of venom-activated conditioned medium
containing 335 mOD/min of S-2238 amidolytic activity was added to each
reaction. The reaction mixture contained 20 µl of conditioned
medium and 180 µl of selection buffer (20 mM Tris acetate,
pH 7.5, 140 mM NaCl, 5 mM KCl, 1 mM
MgCl, 1 mM CaCl
). The reaction was
initiated by addition of 50 µl of human fibrinogen at 2 mg/ml,
freshly diluted in selection buffer from a stock of 10 mg/ml made in
calcium-free PBS. The time in seconds from addition of fibrinogen to
clot formation was measured with a fibrometer. A plasma thrombin
standard clotting curve was used to convert the clotting times into
microgram/milliliter equivalent of plasma thrombin.
Inhibition of Clotting by the Thrombin Aptamer
The
thrombin aptamer, an oligonucleotide thrombin inhibitor
(GGTTGGTGTGGTTGG), was synthesized on an Applied Biosystems solid-phase
synthesizer. For screening the susceptibility of thrombin mutants to
inhibition by the thrombin aptamer, the fibrinogen clotting assay
described above was used except that the thrombin aptamer was added in
180 µl of selection buffer to give a final concentration of 250
nM in 250 µl. For the determination of IC for
selected mutants, a standard amount of venom-activated conditioned
medium (S-2238 amidolytic activity that gives a clotting time of 30 s
for wild-type thrombin) was assayed in the presence of increasing
concentrations of thrombin aptamer (0-20 µM).
Protein C Activation
Cell lysates were prepared
from TMnc cells expressing recombinant human thrombomodulin at the
level of 504 ± 34 fmol/1 10
cells (32) as described previously (33). About 8
10
cells were lysed in 800 µl, giving a thrombomodulin
concentration of
5 nM in the lysate. The commercially
available human plasma protein C used contains detectable levels of
contaminating prothrombin (
0.005-0.02 pmol for each pmol of
protein C). To circumvent this problem, 444 pmol of protein C were
first treated with 10 µg of E. carinatus venom for 30 min
at 37 °C to convert the contaminating prothrombin into thrombin,
which was then inactivated by titration with PPACK. This
venom-processed and PPACK-titrated protein C preparation was then used
in a protein C activation assay(33) . The assay mixture
contained venom-activated conditioned medium corresponding to a
standard amount of S-2238 amidolytic activity (8.5 mOD/min), 20 µl
of TMnc cell lysate and 887 nM protein C in a total volume of
50 µl. This mixture was incubated at 37 °C for 1 h and the
reaction terminated by the addition of antithrombin III and heparin.
For determination of thrombomodulin-independent protein C activation,
the TMnc lysate was omitted and 2 mM CaCl
was
replaced with 5 mM Na
EDTA. The activated protein C
generated was assayed by hydrolysis of chromogenic substrate S-2366
(PyrGlu-Pro-Arg-pNA).
Mutagenesis Strategy to Identify Functional Residues on
the Surface of Thrombin
A mutagenesis strategy was designed to
systematically scan the surface of human -thrombin to identify
residues important for fibrinogen clotting, thrombomodulin-dependent
protein C activation and inhibition of clotting by the thrombin
aptamer. The strategy was designed to maximize the chances of
identifying functional residues while minimizing the possibility of
nonspecific disruption of protein conformation. Only the charged (Arg,
Lys, Asp, Glu, His) and polar (Ser, Thr, Gln, Asn, Tyr, Trp) amino
acids were considered for mutation, as these residues are capable of
participating in hydrogen bonds and electrostatic interactions that are
likely to be important for the binding of charged ligands. Secondly,
only the charged and polar residues on the surface of
-thrombin
that are highly exposed to solvent were selected for mutation. These
residues are available for interactions with ligands and are more
tolerant of sequence variation (34). The fractional accessibility (35, 36) to a solvent probe of radius 1.4 Å was
determined for each residue in the 1.9-Å crystal structure of
human
-thrombin complexed with PPACK(10) . The 70 charged
and polar residues with a fractional accessibility of >35% were
selected for mutation. Only a single residue, Arg
, was
excluded from this list. Arg
is located at the junction
between the A and B chains of
-thrombin and is required for the
processing of prethrombin-2 to mature, two-chain
-thrombin.
However, several residues were added to the list (Arg
,
His
, Lys
, Arg
, Lys
,
Arg
, Trp
) because functional studies (24, 25) and crystallography studies (26) indicated that residues in these locations may be involved
in interactions with the thrombin aptamer. In addition, the catalytic
serine residue (Ser
) that participates in the formation
of the acyl-enyzme intermediate was mutated to be used as a negative
control in activity assays. A total of 77 residues were replaced with
alanine by oligonucleotide-directed mutagenesis. Alanine was used for
all substitutions because alanine is compatible with both
and
secondary structures(37) , tolerated in both buried and
exposed locations in proteins(35, 37) , and the
nonpolarity and small size of its side chain ensures that substitution
with alanine is less likely to disrupt protein conformation. Multiple
substitutions were made simultaneously when 2 or 3 targeted residues
were clustered together. If such multiple mutants displayed a
functional phenotype, then each residue was substituted individually.
The complete list of alanine replacement mutants is included in .
Expression, Quantitation, and Amidolytic Activity of
Prothrombin Mutants
Human prothrombin and prothrombin mutants
were transiently expressed in COS-7 cells. Recombinant wild-type human
thrombin produced in COS-7 cells was demonstrated to be functionally
comparable to wild-type thrombin purified from human plasma
(Haematologic Technologies Inc.) in the assays used in this study. In
the fibrinogen clotting assay, recombinant thrombin displayed 88% of
the clotting activity displayed by plasma-derived thrombin. The
specific activity for generation of activated protein C by recombinant
wild-type thrombin was 91.2% of plasma-derived thrombin.
10
cells. Mutants that expressed poorly at 37 °C showed improved
expression at 27 °C. Only two mutants (K23aA,R26aA,E27aA) and W249A
could not be expressed even at 27 °C (). When the 3
residues Lys
, Arg
, and Glu
were replaced separately, all three mutants could be expressed at
levels sufficient for analysis. The inability to express mutant W249A
suggests that Trp
plays a critical role in maintaining
the structural integrity of prothrombin. All mutants except (N53A,T55A)
displayed the same molecular weight as wild-type prothrombin as
assessed by Western blots of SDS-PAGE gels, indicating that expression
was stable with no evidence of proteolytic degradation and that there
was no heterogeneity in glycosylation and proteolytic processing. The
mutant (N53A,T55A) involves the 2 residues that define the single N-linked glycosylation site in mature
-thrombin. Thus,
this mutant is not glycosylated and exhibits a corresponding loss of
molecular weight.
72 kDa) corresponding to prothrombin and the appearance of a
band (
34 kDa) corresponding to the B chain of mature
-thrombin. Following processing there were no bands of higher
molecular weight than the thrombin B chain, and there was no
heterogeneity in the products except for the occasional appearance of
trace amounts (<5%) of material corresponding to the thrombin
autodegradation products
-thrombin and
-thrombin. The
concentration of processed prothrombin was determined by quantitative
Western slot-blot using a polyclonal antibody raised against
prothrombin that recognizes prothrombin, thrombin A chain, thrombin B
chain, and fragments (F1 and F2) derived from the pro region of
prothrombin (Fig. 1B). A constant ratio of band
intensities in Western blots of SDS-PAGE (Fig. 1A)
indicated that there was no differential recognition of the B chain and
pro-region fragments for different mutants, suggesting that mutations
in the thrombin B chain did not affect recognition by the polyclonal
antibody.
Fibrinogen Clotting Activity of Thrombin
Mutants
The procoagulant activity of each thrombin mutant was
tested by determining their ability to clot fibrinogen (Fig. 2).
Eighteen mutants had less than 50% of wild-type activity.
Clotting-deficient mutants with multiple substitutions were reanalyzed
with the residues replaced individually. When double mutant (W50A,D51A)
was split, the mutation W50A was found to be solely responsible for
impairing fibrinogen clotting. In double mutant (N74A,K77A) both
mutations N74A and K77A decreased the clotting activity, with K77A
having the greater effect. Both single mutants from mutant
(K106A,K107A) were minimally affected in clotting activity (>50% of
wild-type activity) but their effects were additive in the double
mutant. Six mutants involving single amino acid substitutions retained
less than 5% of the fibrinogen clotting activity of wild-type thrombin.
Because this assay was normalized with respect to S-2238 amidolytic
activity the effects of these mutations on clotting are not due to
generalized disruption of the active site. Therefore the residues
substituted in these mutants (Trp, Lys
,
His
, Tyr
, Glu
,
Arg
) are likely to be critical for the recognition of
fibrinogen.
Figure 2:
Fibrinogen-clotting by thrombin mutants.
The clotting times were converted into equivalent concentrations of
plasma thrombin using a plasma thrombin standard clotting curve.
Clotting activity of the mutant thrombins was then expressed as percent
of wild-type activity. Wild-type thrombin gave a clotting time of 38
± 6 s. Error bars represent the standard deviations of
at least two independent experiments. The filled circle indicates that a mutant thrombin retained <50% of wild-type
clotting activity, and the open circle indicates that a mutant
thrombin retained <50% of wild-type protein C activation
activity.
Protein C Activation by Thrombin Mutants
The
anticoagulant properties of the mutant thrombins were initially
screened for their ability to activate protein C in the presence of
thrombomodulin (Fig. 3). Sixteen mutants had less than 50% of
wild-type activity (). When the triple mutant
(S22A,Q24A,E25A) was split into single mutations, mutation Q24A was
found to be mainly responsible for the decrease in protein C
activation. Similarly, mutation W50A in mutant (W50A,D51A) and mutation
K77A in mutant (N74A,K77A) had the major effect on protein C
activation. The result of splitting the double mutant (K106A,K107A)
showed that neither mutation alone had much effect on protein C
activation. Eight mutants involving single amino acid residues
(Lys, Gln
, Lys
,
His
, Arg
, Tyr
, Lys
,
Glu
) displayed less than 15% of the
thrombomodulin-dependent protein C activating activity of wild-type
thrombin. These residues are candidates for those that are important
for the recognition of thrombomodulin or protein C. Again the effects
of these mutations are not due to generalized disruption of catalytic
activity because the assay was normalized with respect to S-2238
amidolytic activity.
Figure 3:
Protein C activation by thrombin mutants.
Protein C activation activity of the mutant thrombins is expressed as
percent of wild-type activity. Wild-type thrombin activated protein C
at a rate of 800 ± 122 pmol aPC/µg/h. The error bars represent the standard deviations of at least two independent
experiments (see also Table II). The open circle indicates
that a mutant thrombin retained <50% of wild-type protein C
activation activity, and the filled circle indicates that a
mutant thrombin retained <50% of wild-type clotting
activity.
The mutants with less than 50% of wild-type
activity in the thrombomodulin-dependent protein C assay were
subsequently assayed for protein C activation in the absence of
thrombomodulin and calcium ions (). Eleven of these also
showed decreased activity in the thrombomodulin-independent protein C
assay suggesting that the thrombin-protein C interaction was affected
in these mutants. In contrast, mutants (S22A,Q24A,E25A), Q24A, and to a
lesser extent R70A showed relatively unaltered or even enhanced
activity, suggesting that the residues involved (Gln and
Arg
) participate in the thrombin-thrombomodulin
interaction.
Inhibition of Clotting Activity of Thrombin Mutants by
the Thrombin Aptamer
The ability of the thrombin aptamer to
inhibit the mutant thrombins at 250 nM was assessed in a
fibrinogen clotting assay (Fig. 4). Mutant E229A had virtually no
clotting activity and was not tested in this assay. Five mutants were
inhibited by less than 30% in this assay and were further assayed for
their dose response to inhibition of clotting by the thrombin aptamer (Fig. 5). Mutant R70A had a 2800-fold increase in IC and was most refractory to inhibition. It was followed by K65A,
R73A, H66A, and Y71A which increased the IC
over wild-type
by 220-, 94-, 28- and 7-fold, respectively. These residues are likely
to be critical for interactions with the thrombin aptamer.
Figure 4:
Inhibition of thrombin mutants by the
thrombin aptamer. The inhibitory activity of 250 nM thrombin
aptamer toward each thrombin mutant was assayed in a clotting assay.
The inhibitory activity was expressed as percent inhibition of clotting
activity relative to uninhibited control. The error bars represent the standard deviations of at least two independent
experiments.
Figure 5:
Dose dependence of inhibition by the
thrombin aptamer for thrombin mutants. Inhibitory activity was
determined in a clotting assay where the concentration of the thrombin
aptamer was varied from 0 to 20 µM. The error bars represent the standard deviations of at least two independent
experiments. The activities were normalized to that of uninhibited
control. Filled circle, wild-type; open circle, Y71A; filled diamond, H66A; open diamond, R73A; filled
triangle, K65A; open triangle, R70A; filled
square, R70A with the following scrambled sequence of the thrombin
aptamer: GGTGGTGGTTGTGGT. Values for IC were determined by
curve fitting to the data using the SigmaPlot curve fitter (Jandel
Scientific). Wild-type 0.058 ± 0.002 µM; K65A 21.9
± 3.4 µM; H66A 1.5 ± 0.2 µM;
R70A 161 ± 88 µM; Y71A 0.428 ± 0.038
µM; R73A 5.4 ± 0.5
µM).
, Trp
,
Lys
, Asn
+Thr
,
Lys
, His
, Arg
, Tyr
,
Arg
, Lys
,
Lys
+Lys
,
Asp
+Lys
, Glu
,
Glu
, Arg
, Asp
) (Fig. 2). Only 8 of these residues are absolutely conserved among
11 vertebrate thrombins (Lys
, Trp
,
Lys
, Asn
+Thr
,
Arg
, Glu
, Glu
)(38) .
When the residues above were mapped onto the surface of thrombin (Fig. 6A) they all mapped to a single face of thrombin
surrounding the active site cleft. Highly conserved residues,
Trp
, Lys
, Glu
, and Glu
project into the active site cleft where they are well placed to
interact with substrates. Trp
occludes entry to the active
site cleft from above and forms part of the apolar binding pocket and
is in contact with the valine residue at P2 in the crystal structure of
fibrinopeptide A bound to thrombin(12, 39) . Deletion of
Trp
along with Pro
and Pro
was
previously shown to decrease k
for
fibrinopeptide A release(40) . In the complex with
fibrinopeptide A, Glu
is in contact with P5 glycine.
Glu
occludes entry to the active site cleft from below
and could potentially contact residues at P3 or P3`(12) .
Previous substitution of Glu
with glutamine caused no
change in fibrinopeptide A release but enhanced fibrinopeptide B
release(41) . Modeling the interaction of fibrinogen residues on
the carboxyl-terminal side of the cleavage site suggested that
Lys
could form part of the S2` subsite(12) .
Previously Lys
was nonconservatively substituted with
glutamate and demonstrated to be important for fibrinogen
clotting(22) .
Figure 6:
Localization of functional epitopes on the
surface of thrombin. Models generated are based on the coordinates of
the PPACK-thrombin complex (10, 11) with PPACK removed, using MidasPlus
(University of California, San Francisco). A, space filling
model of human thrombin looking directly into the active site cleft.
Exosite 1 is on the right side of the cleft. Catalytic residues
(His and Ser
) are colored red.
Residues implicated only in recognition of fibrinogen are colored blue, residues implicated only in thrombomodulin-dependent
protein C activation are colored yellow, and residues
implicated in both functions are colored green. B,
identical model as in A but rotated 180° about the
vertical axis, illustrating that no functional residues were located on
the opposite face of the molecule. C, same view as in A but highlighting residues implicated in binding the thrombin
aptamer (Lys
= blue, His
= green, Arg
= yellow, Tyr
= aqua, Arg
= magenta), clustered in the lower right corner
of exosite 1.
Numerous studies suggested that a cluster of
acidic amino acids located 20-24 residues COOH-terminal to the
cleavage site of fibrinogen interacts with the highly basic exosite 1
on thrombin(21, 33, 42, 43) , including
mutagenesis studies which demonstrated that the thrombin mutant R68E
had no clotting activity(22) . The crystal structure of thrombin
with the acidic COOH-terminal fragment of hirudin (hirugen) bound to
exosite 1 has been used as a model for the interaction of fibrinogen
with exosite 1(12, 20, 39) . In our mutagenesis
study the loss of clotting activity for mutants involving residues
Lys, His
, Arg
, Tyr
,
Arg
, Lys
, Lys
, and Lys
(Fig. 2) suggests that exosite 1, located to the right of
the active site cleft (Fig. 6A), is extensively involved
in the recognition of fibrinogen.
,
Lys
, Arg
, and Asp
. These
residues are located below and to the left of the active site cleft (Fig. 6A). Residues Asn
and Thr
define the only site of N-linked glycosylation in
-thrombin. Although the enzymatic removal of the sugar moiety was
previously reported to have no effect on clotting activity(44) ,
the mutation of these residues caused a mild defect in fibrinogen
clotting (43% of wild-type) that may be due to the loss of the
oligosaccharide.
could form an ion
pair with the glutamate residue at P6 in fibrinopeptide A (12) This interaction is conserved in the thrombin-hirudin
complex. In our study Arg
was substituted along with 2
other residues in mutant (R178A,R180A,D183A), and this mutant had
fibrinogen clotting activity comparable to wild-type thrombin.
Arg
is non-conserved among vertebrate
thrombins(38) , and the glutamate at P6 in fibrinopeptide A can
be varied without effect(45) . Collectively, the data suggest
that Arg
is not as important for fibrinogen recognition
by thrombin as the crystal structure of the fibrinopeptide A complex
would suggest.
,
Gln
, Trp
, Lys
, His
,
Arg
, Arg
, Tyr
, Arg
,
Lys
, Lys
+Lys
,
Glu
, Arg
) () Of these, only 5
(Lys
, Gln
, Trp
,
Arg
, Glu
) are absolutely conserved among 11
vertebrate thrombins(38) . Many of these residues are the same
as those required for fibrinogen clotting, and they all map to the same
hemisphere on the thrombin structure surrounding the active site cleft (Fig. 6A). No residues affecting protein C activation or
fibrinogen clotting were found on the opposite face of thrombin (Fig. 6B). Residues Trp
, Glu
,
and Arg
line the active site cleft on the proximal side
of the cleavage site. Deletion of Trp
along with
Pro
and Pro
was previously shown to decrease
protein C activation(40) .
, Gln
, Lys
,
His
, Arg
, Arg
, Tyr
,
Arg
, Lys
, Lys
,
Lys
) (Fig. 6A) resulted in a decrease in
thrombomodulin-dependent activation of protein C suggesting that
protein C or thrombomodulin are involved in extensive interactions with
exosite 1. Of these residues, substitution of Gln
and to a
lesser degree Arg
did not affect
thrombomodulin-independent activation of protein C ()
suggesting that these 2 residues may be exclusively involved in
interactions with thrombomodulin. Previously, Arg
and
Arg
were nonconservatively replaced with glutamate (22) with the same results as reported here.
, Lys
, and Gln
were essential for thrombomodulin binding(47) . In our
study, residues Thr
, Trp
,
Thr
, Asn
, Lys
, and
Ser
were all substituted with alanine in mutants
27-30 without any effect on thrombomodulin-dependent activation
of protein C, suggesting that this region is not required for
thrombomodulin binding. Human thrombomodulin can be isolated from
transfected 293 cells in two forms with or without a covalently
attached chrondroitin sulfate moiety that has been reported to enhance
the affinity of thrombomodulin for thrombin 10-fold by interacting with
exosite 2(48, 49) . In our study, mutation of residues
in exosite 2 had no effect on thrombomodulin-dependent activation of
protein C consistent with the observation that less than 10% of the
thrombomodulin derived from the CV-1 cell line used in our study
contains chrondroitin sulfate(32) .
, His
, Arg
,
Tyr
, Arg
) (Fig. 5). All of these
residues are tightly clustered in the lower right corner of exosite I (Fig. 6C). Chemical modification studies demonstrated
that Lys
and Lys
were protected by the
thrombin aptamer (25) and nonconservative substitution of
Arg
with glutamate decreased affinity for the thrombin
aptamer(24) . Crystallography studies of the thrombin aptamer
complex with thrombin revealed that the thrombin aptamer was sandwiched
between two symmetry related thrombin molecules, contacting exosite I
on one thrombin molecule and exosite 2 on the other(26) . All
the residues in exosite 1 predicted to make electrostatic interactions
with the thrombin aptamer (His
, Arg
,
Arg
) were identified in our analysis. The residues in
exosite 2 predicted to form hydrogen bonds or ion pairs with the
thrombin aptamer (Arg
, Arg
,
Arg
, Lys
, Trp
,
Lys
) were substituted without affecting inhibition (Fig. 4) suggesting that either the thrombin aptamer does not
bind to exosite 2 or that exosite 2 binding is noninhibitory. Binding
of the thrombin aptamer to the exosite 1 mutant R70A was not detectable
in solid-phase binding assays (data not shown) indicating that
interactions with exosite 2 may be unique to the crystal complex.
Table: Thrombin mutants: residues substituted and
specific amidolytic activity
Table: Thrombin mutants
with thrombomodulin-dependent protein C activation activity <50% of
wild-type
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.