From the
Recombinant
Thrombin plays a pivotal role in both the platelet activation
and fibrin generation inherent to thrombosis. In the blood, thrombin is
produced through the action of the prothrombinase complex (Factors Xa,
Va, Ca
Heparin is the current mainstay of
antithrombotic therapy. The antithrombotic effect of heparin is
primarily due to its acceleration of the inhibition of thrombin (and
factor Xa) by antithrombin
(3) . One of the principal drawbacks
of the therapeutic use of heparin is the requirement for intensive
laboratory monitoring of its anticoagulant effect because of pronounced
individual variation in its anticoagulant response. Moreover, heparin
can be inactivated by platelet factor 4 and heparinase
(4) , both
of which are released by activated platelets, and fibrin monomers have
been found to protect thrombin from inactivation by
heparin-antithrombin
(5) . Heparin will also be ineffective in
patients with antithrombin deficiency. Many of the limitations of
heparin are not observed with the direct thrombin inhibitors currently
in clinical trials. These inhibitors (most notably hirudin and hirulog)
do not require cofactors for their activity and are not subject to
inactivation by platelet proteins. Moreover, the anticoagulant
responses of these compounds are reproducible and, therefore, intensive
laboratory monitoring of anticoagulant effects is not
required
(6, 7) . However, one potential disadvantage
that hirudin and hirulog share with heparin is their short plasma
half-life (<1 h
(8, 9) . Intravenous infusion or
repeated subcutaneous injections would be required for a persistent
anticoagulant effect. The necessity for such procedures could be
obviated by increasing the plasma half-life of a direct thrombin
inhibitor. Engineered plasma serine protease inhibitors (serpins)
appear to be good candidates for such a thrombin inhibitor as their
plasma half-life can be as long as several
days
(10, 11) . The engineered serpin would have to be a
rapid inhibitor of thrombin and not require cofactors such as heparin.
In this respect, the Pittsburgh variant of
The primary determinant of the specificity of a
particular serpin is the sequence of the reactive site loop which binds
to the active site cleft of the protease. The most important residue
within this sequence is the P
On-line formulae not verified for accuracy
On-line formulae not verified for accuracy
On-line formulae not verified for accuracy
On-line formulae not verified for accuracy
For the reaction of LS-AT with APC, the k
The results in indicate that
the mutations did not improve the selectivity of PRS-AT. The
replacement of the P
The effects of the replacement of
the P
The effects of the P
The effects
of the double mutations demonstrated cooperative interactions between
the P
Despite the improved selectivity of LS-AT, the
k
The cooperativity between serpin
reactive site loop residues observed here is in notable contrast to the
Kazal inhibitor ovomucoid third domain. Over 100 variants of the
ovomucoid third domain, many of which can be arranged into double
mutant cycles, have been examined; the effect of the mutations was
shown to be additive, the contribution of a particular residue being
independent of the other residues in the
inhibitor
(34, 35, 36, 37) .
In
plasma, the activity of APC is controlled by the serpin protein C
inhibitor (PCI). Antithrombotics should not augment the rate of
inhibition of this protease. The k
Mutants were made and their
association constants against thrombin and APC were determined in the
absence of heparin as described under ``Experimental
Procedures.'' All estimates of k
-antitrypsin with a P
arginine residue (Arg-
-antitrypsin) is a rapid
inhibitor of both thrombin and activated protein C (APC). A series of
mutants were made in an attempt to increase the specificity of this
serpin for thrombin over APC. Initially, P
and P`
residues of Arg-
-antitrypsin were replaced in
single and double mutations by the corresponding residues in
antithrombin and C1 inhibitor which are very poor inhibitors of APC. No
improvement in selectivity was achieved by these mutations. In fact,
all P
/P`
substitutions led to a decrease in
selectivity for thrombin over APC. For example, replacement of the
P
proline of Arg-
-antitrypsin by glycine
decreased the association rate constant (k
) with
thrombin by 37-fold while the k
value with APC
was reduced by only 16-fold. Co-operative effects were observed with
the double P
and P`
substitutions; the
mutational effects were not additive. The decrease in the
k
for thrombin caused by the mutation of the
P
proline to alanine or glycine was 3-fold greater when
threonine was present in the P`
position instead of the
normal serine. In contrast to the disappointing results with the
P
/P`
mutations, replacement of the P
to P`
residues of
-antitrypsin by
those of antithrombin led to a dramatic increase in selectivity.
Although this substitution only affected the k
value with thrombin by 10-fold, a 12,500-fold decrease in this
value with APC was observed. Substitution of proline for the P
glycine of this chimeric serpin increased the k
values with thrombin and APC by 7- and 90-fold, respectively. The
effect of the P
substitution was again found to depend on
the sequence surrounding the residue; the change in the
k
for APC caused by the P
Pro
Gly replacement was 6-fold larger in the chimeric serpin. Evaluation of
the k
values of the chimeric serpin with a
P
proline in light of the likely rates of inhibition of
thrombin and APC during antithrombotic therapy with heparin suggested
that this serpin may have kinetic parameters suitable for an
antithrombotic agent.
, and phospholipid). Thrombin further
stimulates its own production by activating factors V, VIII, and
platelets
(1) . These procoagulant activities of thrombin are
blocked by the binding of thrombin to the endothelial cell surface
protein thrombomodulin. In complex with thrombomodulin, thrombin
becomes a more efficient activator of protein C, a protease with
anticoagulant activity. In addition, the thrombomodulin-thrombin
complex is no longer able to cleave fibrinogen and factor V and cannot
activate platelets. Furthermore, activated protein C (APC)
(
)
turns off the coagulation cascade by inactivating factors
Va and VIIIa which are essential cofactors for the formation of
thrombin
(2) . Thus, hemeostasis depends on a balance between the
activity of thrombin, which promotes clot formation, and of APC, which
prevents excessive clotting
-antitrypsin is a good starting point for the
development of a recombinant serpin with antithrombotic properties. In
this variant, the P
methionine of
-antitrypsin has been replaced with an arginine, and
the resulting molecule (PRS-AT) is a moderately rapid inhibitor of
thrombin. This inhibitor has been tested in a baboon model of septic
shock
(12) . Activation of coagulation is the main cause of death
associated with septic shock. Arg-
-antitrypsin was,
however, unable to prevent the activation of coagulation. In addition
to inactivating thrombin, Arg-
-antitrypsin was an
effective inhibitor of APC
(13) with the result that APC
anticoagulant pathway was inhibited, and persistent activation of the
coagulation cascade occurred. Thus, although rapid activation of
thrombin is a requirement for an antithrombotic serpin, specificity is
also important; in particular, inhibition of APC should be avoided.
This is vital in the treatment of septic shock where APC appears to
have a protective role
(14) . Consequently, the aim of the
current investigation was to engineer PRS-AT to increase its
specificity for thrombin over APC while maintaining its fast inhibition
rate with thrombin.
residue which interacts with
the S
pocket of the protease. The P
residue is,
however, not the sole residue in the reactive site loop which
contributes to specificity; many serpins have a P
arginine
residue yet have quite different inhibitory profiles. It has been shown
that mutating the P`
residue of antithrombin can
dramatically affect its inhibition of thrombin, while having a
relatively minor effect on the inhibition of factor Xa
(15) .
Mutagenesis of the P
residue of a number of different
serpins has shown that it also can contribute to serpin
specificity
(16, 17, 18, 19) .
Antithrombin and C1 inhibitor are very poor inhibitors of APC and
differ from PRS-AT in the P
and/or P`
positions. The P
-P
-P`
sequences of antithrombin and C1 inhibitor are Gly-Arg-Ser and
Ala-Arg-Thr, respectively. The significance of the P
alanine/glycine and the P`
threonine in regulating
inhibitory specificity toward APC was tested by making a series of
single and double mutations in these positions. The results indicated
that the inhibitory specificity of antithrombin and C1 inhibitor was
not due solely to the P
-P
-P`
sequence. The poor inhibition of APC by antithrombin and C1
inhibitor could have been due to other sequences within the reactive
site loop or regions in other parts of the molecule. For antithrombin,
it was found that the poor interaction with APC was due mainly to
sequences within the reactive site loop. Substitution of the reactive
site loop of antithrombin (P
-P`
) into
-antitrypsin resulted in a chimeric serpin whose
association rate constant with APC was reduced 10
-fold
compared with PRS-AT.
Materials
The chromogenic substrate S-2266
(D-Val-Leu-Arg-p-nitroanilide) was purchased from
Kabi-Pharmacia (Mölndal, Sweden). Oligonucleotides were
synthesized by the Department of Biochemistry, University of Cambridge.
Protease-free bovine serum albumin and p-nitrophenyl
p`-guanidinobenzoate were purchased from Sigma (Poole, United
Kingdom). Thrombin was prepared as described previously
(20) .
APC was a gift of Dr. J. Stenflo and Dr. A. hlin (Malmö, Sweden).
All other chemicals were of the highest grade commercially available.
Production of Mutants of
-Antitrypsin
-Antitrypsin
mutants were produced as recombinant protein in Escherichia coli using the vector p
N15. This vector was derived from the
vector pTermat
(21) by deletion of the 15 5` codons of the
-antitrypsin sequence; as described previously
(22) expression levels of
-antitrypsin were
significantly higher with the 15 5` codons deleted than with the intact
N terminus. Reactive loop mutants were made by PCR exploiting the
AvaI restriction site that lies at a position corresponding to
the codons for residues P`
and P`
. Mutagenic
oligonucleotides contained the desired mutations as well as the
AvaI site, and these were used in a PCR reaction with an
oligonucleotide complementary to an upstream region. The PCR product
could be cut with BstXI and AvaI and cloned into the
expression vector cut with the same two restriction enzymes. The entire
cloned region was sequenced to ensure the presence of the desired
mutation and the absence of PCR-induced error. Proteins were expressed
and purified as described previously
(21) .
Determination of Association Rate Constants
All
serpins were found to be slow tight-binding inhibitors of thrombin and
APC, indicating that there was no significant problem associated with
modification of the single cysteine, which is reported to convert
-antitrypsin to a reversible competitive inhibitor
(23). All assays were performed at 37 °C in 30 mM sodium
phosphate buffer, pH 7.4, containing 0.16 M NaCl, 0.1%
polyethyleneglycol (M
4000), 0.2 mg/ml
protease-free bovine serum albumin, and 200-400 µM
S-2266. Slow-binding kinetics were performed using a Hewlett-Packard
diode-array spectrophotometer. The production of
p-nitroanilide was measured between 400 and 410 nm. For the
analysis of APC inhibition, the plastic cuvettes were coated with
bovine serum albumin and polyethyleneglycol by incubating overnight
with a solution of 10 mg/ml bovine serum albumin and 0.1%
polyethyleneglycol (M
8000). After washing in
distilled water, the cuvettes were dried at 37 °C before use. Each
slow-binding inhibition experiment consisted of six to seven assays
with one in the absence of serpin and five to six others with
increasing serpin concentrations. After initiation of the reactions by
the addition of either thrombin or APC, the release of
p-nitroaniline was monitored. Data points where substrate
utilization was in excess of 10% of total substrate concentration were
excluded from the analyses. The mechanism of inhibition was well
described by where E, S, P, and I represent enzyme,
substrate, product and serpin respectively; K
and k
are the Michaelis and catalytic rate
constants for the enzyme-substrate reaction, and k
and k
are the association and
dissociation rate constants for the enzyme-inhibitor complex. Values of
K
for S-2266 with thrombin and APC were
262 ± 14 and 654 ± 62 µM,
respectively
(13) . Progress curve data were analyzed as in
Hermans and Stone
(13) to yield estimates for
k
. For each enzyme-inhibitor combination, at
least two slow-binding experiments were performed, and the
k
values given represent the weighted mean of
the determinations. The estimates obtained for
K
were 1 nM or less in all
cases.
value was too low to be estimated by slow-binding kinetics; the
k
values for this mutant were therefore
determined as described by Beatty et al.(24) .
Determination of the Active Concentrations of Proteases
and Serpins
Thrombin and APC were titrated with
p-nitrophenyl p`-guanidinobenzoate
(25) .
Serpins were titrated against thrombin in microtiter plates by
incubating increasing amounts of serpin in 50 µl of buffer
containing 50 nM thrombin for 3-4 h at 37 °C. The
microtiter plate was then allowed to reach room temperature prior to
adding 150 µl of reaction buffer containing 300 µM
S-2266, and the hydrolysis of the substrate was monitored at 405 nm
using a Thermomax microtiter plate reader. Regression analysis of the
substrate conversion rate against volume of inhibitor added
(13) was used to calculate the concentration of inhibitor. All
recombinant serpins displayed roughly the same specificity activity of
over 70%.
RESULTS AND DISCUSSION
Mutation of P
In order to function as an antithrombotic agent, a
serpin should inhibit thrombin rapidly. Rapid inhibition of thrombin
will not only hinder the formation of fibrin and thrombin-dependent
platelet aggregation, but it will also prevent the feedback activation
of the coagulation cascade by thrombin (1). At the same time, an
antithrombotic agent should not rapidly inhibit APC, since the activity
of APC is required to shut down the coagulation cascade
(2) . We
have used PRS-AT as a starting point for engineering a serpin with a
suitable kinetic profile for an antithrombotic agent. PRS-AT rapidly
inactivates thrombin (50-100 times more rapidly than
antithrombin), but it also reacts rapidly with APC ().
Thus, if the rapid inactivation of APC could be dissociated from that
of thrombin, PRS-AT would a exhibit a suitable kinetic profile for use
as an antithrombotic. In a previous study, it was found that
antithrombin and C1 inhibitor inactivated APC very slowly
(13) .
An earlier study with peptidyl chloromethane inhibitors had shown that
compounds with a P and P`
Residues
in PRS-AT
glycine failed to inhibit APC
(26) and, thus, it was suggested that the P
glycine
of antithrombin may be responsible for its slow reaction with
APC
(13) . Examination of the reactive site loop sequence of C1
inhibitor did not reveal any obvious reasons for its poor inhibition of
APC apart from the fact that the usual P`
serine was
replaced by a threonine in this serpin
(13) . Another serpin
which displayed a reasonable selectivity for thrombin over APC was
protease nexin 1. The ratio of the association rate constants
(k
) for this serpin with thrombin and APC was
290 compared with 7 for PRS-AT (). Like C1 inhibitor,
protease nexin 1 contains an alanine in the P
position.
Thus, on the basis of these previous studies, five mutations were made
in PRS-AT to examine the possibility of increasing the selectivity of
this serpin: the P
proline was mutated to glycine or
alanine, and the P`
serine was replaced by threonine. In
addition, double mutations of the P
and P`
residues were made. These mutations reproduced the
P
-P
-P`
sequences of antithrombin
(Gly-Arg-Ser), protease nexin 1 (Ala-Arg-Ser), and C1 inhibitor
(Ala-Arg-Thr) in
-antitrypsin. The effect of these
mutations on the k
values with thrombin and APC
are given in .
proline of PRS-AT by alanine resulted
in decreases in the k
values with thrombin and
APC of 7- and 4-fold, respectively. The k
values
determined for ARS-AT with thrombin and APC agree with those previously
reported (16, 17, 27). The effects of the substitution of the P
residue by glycine were larger; 37- and 16-fold decreases in the
k
values were observed with thrombin and APC,
respectively. Thus, for both substitutions of the P
proline, the decrease in the k
value with
thrombin was about twice as large as that observed with APC with the
result that the selectivity of the mutants was decreased
(). The replacement of the P`
serine of PRS-AT
by threonine did not markedly affect the k
values with either protease (). These results
indicated that the poor inhibition of APC by antithrombin and C1
inhibitor cannot be attributed solely to the P
and P`
residues in these serpins.
proline on the inhibition of thrombin can be
rationalized on the basis of the structure of the covalent complex
between thrombin and
D-Phe-Pro-ArgCH
(28, 29) . In this
structure, the P
proline of the inhibitor is bound in a
hydrophobic pocket formed by the side chains of Tyr
Ala,
Trp
Asp, His
, Trp
, and
Leu
. All the contacts made by the P
proline
would be lost upon replacement of the residue by glycine or proline,
and the loss of these contacts would be more than sufficient to explain
the observed decreases in k
due to the P
Pro
Ala/Gly mutations. Since the tertiary structure of APC
has not been determined, the results with this protease cannot be
interpreted reliably in terms of tertiary structure. However,
comparison of the sequences of APC and thrombin indicates that several
of the residues in thrombin that interact with the P
proline are not present in APC. Tyr
Ala and
Trp
Asp are found in an insertion that is not present in
APC, and Leu
in thrombin is replaced by a threonine in
APC. These changes in APC would result in an S
pocket less
suited for a proline and, thus, a smaller decrease in
k
with the P
Pro
Ala/Gly
substitutions may be expected.
Pro
Ala/Gly mutations on the k
values with
thrombin and APC illustrate the difficulties in predicting the effects
of mutations in the reactive site loop of serpins from results obtained
with small peptide substrates and inhibitors. Studies with peptide
substrates and inhibitors have demonstrated that both thrombin and APC
displayed a marked preference for proline at P
over
glycine
(26, 30, 31) . The effects of the
mutations on the k
values of PRS-AT with
thrombin and APC are, however, smaller than those that would be
expected from these studies. The average
k
/K
value for
thrombin with a series of seven p-nitroanilide substrates with
a P
proline was 130-fold higher than the corresponding
average value with six substrates containing a P
glycine
(30). A similar difference in
k
/K
values was
also observed for p-nitroanilide substrates with APC; the
average k
/K
for
substrates containing a P
proline was 300-fold higher than
those containing a glycine in this position
(26) . Thus, although
the results from studies with synthetic peptide substrates and
inhibitor predicted the observed decrease in the k
value upon the replacement of the P
proline, they
could not accurately predict the magnitude of the change. The magnitude
of the effect of a P
substitution is clearly modulated by
the context of the individual serpin reactive site loop. Serpins
interact with proteases over a more extended area than peptide
substrates and inhibitors. In particular, p-nitroanilide
substrates and chloromethane inhibitors only bind to the
S
/S
-S
sites and do not interact
with the S` sites. As discussed below, interactions with the P`
residue can modulate those made by the P
amino acid
and Madison et al.(32) have demonstrated that contacts
with P`
residues of plasminogen activator inhibitor I are
important in determining the specificity of this serpin.
and P`
residues in the inhibition of
thrombin; the effects of the mutations were not additive.
Non-additivity in mutational effects is conveniently interpreted in
terms of double mutant cycles
(33) as shown in Fig. 1A for the effect of the P
Pro
Ala and P`
Ser
Thr mutations on the k
for
thrombin. With threonine in the P`
position, the effect of
replacing the P
proline by alanine (or glycine) was about
3-fold larger. The P
Pro
Ala mutation caused a
22-fold decrease in k
with threonine in P`
compared with the 7-fold decrease with serine in this position
(Fig. 1A). If the mutational effects were additive, the
P
Pro
Ala mutation would cause the same decrease in
k
irrespective of the P`
residue. It
follows from the symmetry of the cycle shown in Fig. 1A that the P`
Ser
Thr mutation was approximately
3-fold more detrimental with alanine as the P
residue. The
P
Pro
Ala and P`
Ser
Thr
mutations are coupled; the magnitude of the mutational effect in one
postion depends on the nature of the amino acid in the second position.
A similar degree of coupling was observed for the P
Pro
Gly and P`
Ser
Thr mutations; the decreases
in k
with thrombin caused by the P
Pro
Gly substitution were 96- and 37-fold when threonine
and serine, respectively, were the P`
residues. Similar,
but smaller, cooperativity effects between P
and P`
substitutions were also observed with APC; the effect of the
P
Pro
Ala/Gly substitutions were about twice as
large with threonine in the P`
positions. These results
indicate that the interactions made by the P
residue within
the reactive site loop of serpins are influenced by the nature of the
P`
residue. The presence of a threonine in the P`
position of PRS-AT has made it more difficult for thrombin to
accommodate the P
Pro
Ala/Gly substitutions.
Figure 1:
Non-additivity of
mutational effects in the reactive site loops of serpins. Additivity of
mutational effects can be analyzed by constructing double mutant cycles
as described by Horovitz (33, 45). The effects of the P Pro
Ala and P`
Ser
Thr substitutions on the
k
with thrombin are shown in A; changes
in k
for APC caused by the
-antitrypsin
antithrombin loop swap (PRS
LS) and the P
Pro
Gly mutation are shown in
B. If the effects of the mutations are independent of each
other, the magnitude of change in k
should be
equal on parallel sides; i.e. the effect of a mutation should
be independent of the residues in the other positions. Cycle A shows
that the effects of P
Pro
Ala and P`
Ser
Thr substitutions are not independent of each other; the P
Pro
Ala replacement causes a 3-fold greater decrease in
k
when threonine is the P`
residue
(similarly, the effect of the P`
Ser
Thr
substitution is three times larger with glycine in the P
position). Cycle B shows the cooperative effect between the
P
glycine and the rest of the antithrombin reactive loop
sequence; the decrease in the k
with APC caused
by the P
Pro
Ala mutation is about 6-fold greater in
the antithrombin loop.
Replacement of the Reactive Site Loop of
PRS-AT
The results of the above experiments indicate that the
slow inhibition of APC by antithrombin and C1 inhibitor cannot be
attributed solely to the P-P
-P`
sequence within the reactive site loop. Additional sequences in
these serpins must be responsible for their poor inhibition of APC.
These sequences could reside within the reactive site loop;
alternatively, the structural motifs responsible for the poor
inhibition could be found elsewhere in the molecules. In order to test
these two alternative explanations for antithrombin, a chimeric serpin
was constructed in which the reactive site loop of PRS-AT was replaced
by that of antithrombin. The results obtained with this serpin (LS-AT)
indicated that most of the structural information that causes the poor
inhibition of APC is found within the reactive site loop. The
k
value for LS-AT with thrombin was 10-fold
lower than that observed with PRS-AT and slightly higher than the value
observed for antithrombin (). The effect of the
substitution on the inhibition of APC was dramatic. The
k
value for APC was reduced by 12,500-fold. This
value is about 40-fold higher than antithrombin in the absence of
heparin and only 3-fold higher than antithrombin in the presence of
heparin. The improvement in selectivity of LS-AT over PRS-AT was
striking. The ratio of the k
values for thrombin
and APC increased from 7 for PRS-AT to 8200 for LS-AT; the value of
this ratio was only 10-fold lower than that observed with antithrombin
().
value of this serpin with thrombin is still
somewhat lower than would be required for an antithrombotic agent (see
below). It was only slightly higher than that observed for antithrombin
in the absence of heparin. In an attempt to improve the
k
value of LS-AT with thrombin, the P
glycine was replaced by a proline (LS-Pro-AT). From the results
obtained with PRS-AT, the P
Gly
Pro would be
expected to yield increases in the k
values for
thrombin and APC of about 40- and 15-fold, respectively. In the context
of the antithrombin reactive site loop, however, the relative increases
in the k
were opposite to those expected from
the PRS-AT substitutions. The increase for thrombin was only 7-fold
while a 90-fold increase was seen with APC (). However,
LS-Pro-AT still remained reasonably selective; the k
value with thrombin was similar to that observed with PRS-AT (3.1
10
M
s
) while the corresponding value with APC (4.9
10
M
s
) was 140-fold lower than that of PRS-AT. The
increase in selectivity of LS-Pro-AT over PRS-AT was 90-fold. It was
observed above that the effect of the P
Pro
Ala/Gly
substitution was influenced by the nature of the P`
residue. The results observed with the P
Gly
Pro substitution in LS-AT indicated that residues within the reactive
site loop other than the P`
residue were able to affect the
magnitude of the change observed upon the mutation of the P
residue. Fig. 1B shows the double mutant cycle for
the inhibition of APC by PRS-AT, GRS-AT, LS-Pro-AT, and LS-AT. The
decrease in k
caused by the P
Pro
Gly substitution was six times larger (90- versus 16-fold) in the antithrombin reactive site loop compared with the
-antitrypsin loop. Evidently, the interactions made by
a particular residue in the reactive site loop are influenced by the
nature of other residues in the loop, and cooperativity effects in the
binding of residues to the active site of proteases play a role in
determining serpin specificity.
Suitability of PRS-AT Variants for Use as Antithrombotic
Agents
For use as an antithrombotic agent, a serpin should
inhibit thrombin rapidly and APC slowly. Some indication of required
rates of inhibition can be obtained by examining the rates of
inhibition of these two proteases by their natural inhibitors. The rate
constant (k) for the inactivation of a protease
will be given by k
=
k
[I] where [I] is the
concentration of the serpin. The half-life (t
)
for the inactivation will be t
=
ln(2)/k
=
ln(2)/(k
[I]). The major inhibitor of
thrombin in plasma is antithrombin which is present at a concentration
of 2.3 µM and has a k
value of
about 10
M
s
in the absence of heparin
(38) . Thus, the rate constant
(k
) for the inactivation of thrombin by
antithrombin in the absence of heparin is about 0.03
s
. However, since the rate of inhibition of thrombin
during thrombus formation is too slow, an antithrombotic agent should
exhibit a higher k
value. Heparin is the current
mainstay of antithrombotic therapy, and the rate of inhibition thrombin
by the heparin-antithrombin complex represents a target rate for other
antithrombotics. The plasma level of heparin during effective
antithrombotic therapy is usually 0.2-0.4 units/ml which
corresponds to a concentration of about 60-120 nM. Using
values for the kinetic constants of the inhibition of thrombin by
heparin-antithrombin
(38) , it can be calculated that at this
concentration of heparin, the k
value for the
inactivation of thrombin will be about 1-2 s
.
From this value of k
, it is possible to calculate
concentrations of the recombinant serpins that would be necessary to
achieve an equivalent antithrombotic effect to that achieved usually
during heparin therapy. Assuming that a k
value
of 1 s
is sufficient, the plasma concentrations of
serpins required will be 1/k
. Values calculated
on this basis ranged from 2 to 3 µM for PRS-AT, PRT-AT,
and LS-Pro-AT to 185 µM for GRT-AT. For the best
inhibitors of thrombin (PRS-AT, PRT-AT, LS-Pro-AT), the concentrations
required (2-3 µM) are about equal to the plasma
concentration of antithrombin and represent about 0.1 g/liter.
value for PCI
with APC is 8
10
M
s
(13) , and the plasma concentration of
the inhibitor is 0.1 µM (39) which yields
k
value for the formation of the inhibited
complex of 8
10
s
. In the
presence of heparin, the k
for PCI with APC is
moderately increased, and in the presence of therapeutic levels of
heparin (0.2-0.4 units/ml) the k
value will
be about 2
10
s
(13) . An inhibitor will not
significantly increase the intrinsic rate of inhibition of APC provided
it reacts more slowly than PCI and the k
value of
PCI in the presence of therapeutic levels of heparin gives some
indication of the magnitude of k
that would be
appropriate. Using the minimum k
value for
thrombin and the maximum k
value, it is possible
to calculate a required selectivity ratio
k
(thrombin)/k
(APC)
(=
k
(thrombin)/k
(APC)) of
5000. LS-AT was the only recombinant serpin for which this selectivity
ratio was achieved (). However, the concentration of LS-AT
that would be required for an antithrombotic effect was 22
µM (1/(4.6
10
M) which is
equivalent to 1 g/liter and is probably too high to make its use as an
antithrombotic agent feasible. The calculated values for LS-Pro-AT are
more encouraging. Its selectivity ratio is only 8-fold higher than the
suggested target value and as noted above, it was calculated that a
concentration of about 0.1 g/liter of LS-Pro-AT should be
antithrombotic. Thus, the kinetic parameters of LS-Pro-AT with thrombin
and APC are sufficiently promising to warrant further investigation of
its antithrombotic potential in animal models of thrombosis. The plasma
half-life of
-antitrypsin is severely affected by its
glycosylation state
(40, 41) , thus material produced in
E. coli could have a short plasma half-life and may be
antigenic. This situation may be alleviated by producing
-antitrypsin variants in the mammary glands of sheep
as described previously
(42) which produces fully glycosylated
-antitrypsin.
Table:
Association rate constants of natural and mutant
serpins with thrombin and APC
had
standard errors of less than 5%. Each of the values were determined at
least twice and the weighted means of the estimates are given. Protease
nexin 1 is abbreviated to PN1.
-antitrypsin are identified by the sequence of their
P
-P
-P`
residues, e.g. PRS-AT and ART-AT represent recombinant
-antitrypsin with the
P
-P
-P`
sequences Pro-Arg-Ser and
Ala-Arg-Thr, respectively; LS-AT; recombinant
-antitrypsin with the P
to P`
residues of the reactive site loop replaced with those from the
antithrombin loop; LS-Pro-AT, LS-AT with the P
glycine
mutated to proline; PCR, polymerase chain reaction; PCI, protein C
inhibitor.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.