(Received for publication, April 30, 1997)
From the Department of Pathology, Vanderbilt
University School of Medicine, Nashville, Tennessee 37232, the
¶ Center for Molecular Biology of Oral Diseases, University of
Illinois-Chicago, Chicago, Illinois 60612, and the
Department of
Veterinary Medical Chemistry, Swedish University of Agricultural
Sciences, S-75123 Uppsala, Sweden
Exosite I of the blood clotting proteinase, thrombin,
mediates interactions of the enzyme with certain inhibitors,
physiological substrates and regulatory proteins. Specific binding of a
fluorescein-labeled derivative of the COOH-terminal dodecapeptide of
hirudin ([5F] Hir54-65) to exosite I was used to probe
changes in the function of the regulatory site accompanying
inactivation of thrombin by its physiological serpin inhibitor,
antithrombin. Fluorescence-monitored equilibrium binding studies
showed that [5F]Hir54-65 and
Hir54-65 bound to human -thrombin with
dissociation constants of 26 ± 2 nM and 38 ± 5 nM, respectively, while the affinity of the peptides for the stable thrombin-antithrombin complex was undetectable (
200-fold weaker). Kinetic studies showed that the loss of binding sites for [5F]Hir54-65 occurred with the same
time-course as the loss of thrombin catalytic activity. Binding of
[5F] Hir54-65 and Hir54-65 to thrombin
was correlated quantitatively with partial inhibition of the rate of
the thrombin-antithrombin reaction, maximally decreasing the
bimolecular rate constants 1.7- and 2.1-fold, respectively. These
results support a mechanism in which thrombin and the
thrombin-Hir54-65 complex can associate with antithrombin
and undergo formation of the covalent thrombin-antithrombin complex at
modestly different rates, with inactivation of exosite I leading to
dissociation of the peptide occurring subsequent to the rate-limiting
inactivation of thrombin. This mechanism may function physiologically
in localizing the activity of thrombin by allowing inactivation of
thrombin that is bound in exosite I-mediated complexes with regulatory proteins, such as thrombomodulin and fibrin, without prior dissociation of these complexes. Concomitant with inactivation of thrombin, the
thrombin-antithrombin complex may be irreversibly released due to
exosite I inactivation.
The activity of the blood clotting enzyme, thrombin, is determined by a balance between proteolytic action on physiological substrates, and irreversible inactivation by the serpin, antithrombin. The substrate specificity of thrombin is regulated by macromolecular effectors which interact with the enzyme at either of two electropositive sites, exosites I and II (1, 2). Exosite I has been defined in crystallographic studies as the site occupied by the extended COOH-terminal sequence of the specific thrombin inhibitor, hirudin (3-5). Exosite II is a distinct site which binds heparin and other ligands (1). Specific binding of COOH-terminal hirudin peptides, particularly N-acetyl-hirudin53-64 (Hirugen1; Ref. 6), to thrombin has been used to establish an important functional role for exosite I in mediating thrombin interactions. The peptide inhibits thrombin hydrolysis of the substrates, fibrinogen (7), factors V and VIII (8), and activation of the thrombin receptor (9, 10); it dissociates thrombin from regulatory interactions with fibrin I and II (7, 11) and thrombomodulin (12, 13); and it inhibits thrombin inactivation by the serpin, heparin cofactor II (14, 15). By contrast to these interactions, the rate of thrombin inactivation by antithrombin is decreased only 1.9-fold by Hirugen (7), contributing to the conclusion that recognition of antithrombin by thrombin does not involve exosite I significantly (7, 15-18). Binding of regulatory macromolecules to exosite I results in similarly modest effects on the rate of thrombin inactivation by antithrombin. Thrombin binding to fibrin I increases the rate 2.8-fold (19, 20), while fibrin II reduces the rate 1.6-fold (21). The rate of inactivation of thrombin bound to thrombomodulin is unaffected or decreased ~2-fold for thrombomodulin lacking covalently attached chondroitin sulfate (22-25), and increased 2-8-fold for thrombomodulin containing the attached glycosaminoglycan (23-27).
The information available indicates that the catalytic site of thrombin is accessible to antithrombin when the proteinase is bound in exosite I complexes with hirudin peptide, fibrin I and II, or thrombomodulin, and the reaction rate is not greatly affected (7, 19, 20, 24, 26, 28). On the basis of the mechanism of the thrombin-antithrombin reaction (see Refs. 29 and 30 for reviews), this implies that thrombin can associate with antithrombin and undergo irreversible formation of the covalent thrombin-antithrombin complex with exosite I occupied. This suggests that exosite I may remain accessible in the proteinase-inhibitor complex, which could inhibit further exosite I-dependent reactions by accumulation of this bound product. Results of other studies, however, suggest that this is not the case. The thrombin-antithrombin complex formed in the presence of thrombomodulin does not inhibit the rate of subsequent thrombomodulin-enhanced inactivation of thrombin by antithrombin or activation of protein C, and it has been proposed that loss of affinity of the thrombin-antithrombin complex for thrombomodulin allows the cofactor protein to recycle following thrombin inactivation (22, 27).
Collectively, the observations available present a paradox concerning the mechanism that can accommodate the relatively small, nonessential effects of exosite I interactions on the rate of thrombin inactivation by antithrombin, and also result in loss of affinity of the thrombin-antithrombin complex for exosite I ligands. The present studies were undertaken to investigate this question by determining the fate of exosite I in the thrombin-antithrombin reaction. A fluorescent analog of hirudin54-65 (Hir54-65), similar to the derivative of hirudin53-64 described previously by Liu et al. (31), was prepared and used as a specific probe of the function of the exosite. The affinities of fluorescein-labeled Hir54-65 and unlabeled Hir54-65 for exosite I were decreased >200-fold in the thrombin-antithrombin complex. Results of kinetic studies indicate that thrombin with Hir54-65 bound to exosite I can associate with antithrombin, and that effectively irreversible inactivation of exosite I and dissociation of the peptide occur subsequent to the rate-limiting formation of the covalent thrombin-antithrombin complex. These observations support a mechanism in which thrombin bound to regulatory macromolecules through exosite I may be inactivated by antithrombin without the requirement for dissociation of these complexes. Formation of the thrombin-antithrombin stable complex may drive the release of the inactive complex, thereby providing a mechanism for regulatory macromolecules to act as catalytic effectors of thrombin inactivation. This mechanism may function physiologically in clearing thrombin from regulatory macromolecules and participate in localizing thrombin activity.
Human
-thrombin was purified as described previously (32) or obtained from
Dr. John Fenton (New York State Department of Health, Albany, NY).
Thrombin concentration was determined by active site titration and
absorbance at 280 nm with an absorption coefficient of 1.83 (mg/ml)
1 cm
1 and a molecular weight of
36,600 (33). Preparations of thrombin used in these studies were >95%
active. Thrombin inactivated with FPR-CH2Cl (FPR-thrombin)
was prepared by incubation of 30 µM enzyme with a 2-fold
excess of FPR-CH2Cl for 30 min. at 25 °C, and the residual activity (<0.02%) was determined by chromogenic substrate assay, as described below. Thrombin was labeled at the active site with
4
-{[(iodoacetyl)amino]methyl}fluorescein and
N
-[(acetylthio)acetyl]-D-Phe-Pro-Arg-CH2Cl
([4
-AF]FPR-T) and characterized as described previously (34, 35).
The preparation used in these studies contained 0.87 mol of
fluorescein/mol of thrombin. Antithrombin was purified from human
plasma as described previously (36), and its concentration was
determined by absorbance at 280 nm with an absorption coefficient of
0.65 (mg/ml)
1 cm
1 and a molecular weight of
58,000. Preparations of antithrombin were fully active, as measured by
thrombin reaction stoichiometries of 1.08 ± 0.08 mol/mol and a
second-order rate constant for thrombin inhibition of 0.89 × 104 M
1 s
1 under the
conditions used previously (37). Thrombin-antithrombin complex (T-AT*)
was prepared by reaction of 28-39 µM thrombin with a
1.7-2.2-fold excess of antithrombin for 10-120 min at 25 °C in 50 mM Hepes, 0.125 M NaCl, 1 mM EDTA,
1 mg/ml polyethylene glycol 8000, pH 7.40. The concentration of T-AT*
was taken as the initial thrombin concentration, consistent with the
essentially complete disappearance of thrombin observed by SDS-gel
electrophoresis. The residual thrombin concentration (<0.19%) was
calculated from the residual chromogenic substrate activity.
Solutions of the
Tyr63-sulfated hirudin dodecapeptide (Sigma or Bachem) were
prepared in water and their concentrations calculated from the purity
specified by the manufacturers. The fluorescent derivative,
[5F]Hir54-65, was prepared by modifications of the
procedure described previously for a similar derivative (31).
5-Carboxyfluorescein succinimidyl ester (Molecular Probes) in
Me2SO was added to 0.4-0.5 mM peptide to give
a 5-9-fold molar excess in 50 mM sodium phosphate buffer, pH 8.0, and incubated for 2 h in the dark at 22 °C. The
reaction was stopped by addition of 1 M NH4Cl,
pH 7.4, to 0.1 M and the labeled peptide was separated from
excess dye by chromatography on Sephadex G10 (1 × 119 cm) in
water. The labeled peptide was purified by reverse-phase HPLC on a
Beckman Ultrasphere 5-µm C18-silica column (4.6 × 150 mm) equilibrated with 0.1% trifluoroacetic acid in water and
developed at 0.5 ml/min with 0.1% trifluoroacetic acid in
CH3CN. The column was washed with trifluoroacetic
acid/H2O for ~20 min after application of the sample,
increased to 20% CH3CN over 10 min and washed for ~50
min, and eluted finally with a linear gradient from 20% to 50%
CH3CN over 60 min. Fractions containing the major
fluorescent peak eluting at ~36% CH3CN were neutralized
by addition of 1 M NH4HCO3 to 50 mM and lyophilized. The purified peptide was dissolved in
water, and its concentration was determined from the fluorescein
absorbance at 490 nm in 10 mM NaOH with an absorption
coefficient of 89,125 M1 cm
1
(38). Purified [5F]Hir54-65 eluted from the above column
with a linear gradient as a single peak at 39% CH3CN.
Solutions of [5F]Hir54-65 were stored frozen in water.
The labeled peptide was stable in water for at least 2 days at room
temperature in the dark, as shown by the unchanged HPLC elution
profile.
All experiments were performed
in 50 mM Hepes, 0.125 M NaCl, 1 mM
EDTA, 1 mg/ml polyethylene glycol 8000, pH 7.4, and at 25 °C. Fluorescence measurements were made with an SLM 8000 fluorometer in the
ratio mode, using acrylic cuvettes (Sarstedt) coated with polyethylene
glycol 20,000 to minimize protein adsorption (39). Measurements of
[5F]Hir54-65 binding to thrombin were performed with 491 nm excitation (4 nm band pass) and 515 nm emission (8 nm band pass),
corresponding to the difference maxima from spectra of 0.2 µM peptide in the absence and presence of saturating (1 µM) thrombin. Fluorescence changes of
[4-AF]FPR-thrombin were measured with 500 nm excitation (4-8 nm
band pass) and 519 nm emission (8-16 nm band pass), the excitation and
emission difference maxima from spectra of 175 nM labeled
thrombin in the absence and presence of 5 µM
Hir54-65. Titrations were done by successive addition of
small volumes of titrant with <10% dilution, and, when necessary,
corrected for background by subtraction of measurements on solutions
lacking the labeled species. Fluorescence data were expressed as the
fractional change in the initial fluorescence
(
F/Fo = (Fobs - Fo)/Fo). Titrations of
several fixed concentrations of [5F]Hir54-65
([P]o) as a function of the total concentration of thrombin ([L]o) were fit simultaneously by the quadratic equilibrium binding equation (Equation 1) to obtain the maximum fluorescence change
(
Fmax/Fo), dissociation
constant (KP), and stoichiometric factor
(n).
![]() |
![]() |
(Eq. 1) |
Fluorescence studies of native thrombin (L) binding to
[5F]Hir54-65 (P) with unlabeled Hir54-65
(C) as a competitor were performed by measuring the fluorescence changes after addition of fixed concentrations of thrombin to [5F]Hir54-65, and titration with Hir54-65.
Data collected at a fixed concentration of [5F]Hir54-65
([P]o) as a function of the total Hir54-65
concentration ([C]o) and several fixed total thrombin concentrations ([L]o), along with data for titration of the
same concentration of [5F]Hir54-65 with thrombin alone,
were fit simultaneously with Equation 2 to determine the dissociation
constant (KC) and stoichiometric factor
(m) for competitive binding of Hir54-65 to
thrombin, as well as KP and
Fmax/Fo (40, 41).
![]() |
(Eq. 2) |
![]() |
(Eq. 2a) |
![]() |
(Eq. 2b) |
![]() |
(Eq. 2c) |
![]() |
(Eq. 2d) |
The time course of thrombin inactivation by
antithrombin was measured under pseudo-first-order reaction conditions
([AT]o 10[T]o), from the loss of thrombin
chromogenic substrate activity. Chromogenic substrate activity was
determined as the initial rate of hydrolysis of 100 µM
H-D-Phe-Pip-Arg-p-nitroanilide at pH 7.4 and
25 °C from the linear increase in absorbance at 405 nm with time.
Residual thrombin concentration ([T]t) was expressed as the
fraction of the initial activity measured for an identical control
reaction mixture lacking AT, to account for small effects of
transferred hirudin peptide on the assay rate. Reaction progress curves
were fit by Equation 3, for a single exponential decay, to obtain the
observed pseudo-first-order rate constant
(kobs), the reaction amplitude ([T]o),
and end point (B).
![]() |
(Eq. 3) |
![]() |
(Eq. 4) |
Kinetic studies of the dissociation of [5F]Hir54-65 from
T upon its reaction with AT were done by first measuring the decrease in fluorescence on addition of thrombin to the fluorescent peptide, and
then the increase with time subsequent to initiation of the reaction
with AT. The fluorescence changes measured with time (Ft) were transformed into the total
concentrations of thrombin binding sites for the peptide probe (P) at
the corresponding times ([T]t) with Equation 5, which is a
rearrangement of the binding equation (Equation 1), using the
parameters determined in the binding studies.
![]() |
(Eq. 5) |
Least-squares fitting, numerical integration, and simulation were performed with SCIENTIST software (MicroMath Software). All reported estimates of error represent ± 2 S.E.
A fluorescent analog
of Hir54-65 that was modified at the amino terminus with
5-carboxyfluorescein ([5F]Hir54-65) was synthesized, and
its binding to thrombin was characterized from changes in fluorescence
intensity. As shown in Fig. 1A, thrombin titrations of fixed concentrations of [5F]Hir54-65 from
10 nM to 500 nM were well described by binding
of the peptide to 0.91 ± 0.04 sites on thrombin with a
dissociation constant of 26 ± 2 nM, and a maximum
decrease in fluorescence of 26 ± 0.3% (Table I).
Binding of unlabeled Hir54-65 to thrombin was examined
from its effect on the [5F]Hir54-65 interaction.
Addition of Hir54-65 to mixtures of thrombin and the
labeled peptide resulted in return of the quenched fluorescence toward
the initial value (Fig. 1B). Simultaneous nonlinear
least-squares analysis of titrations with Hir54-65 at
fixed thrombin concentrations ranging from 20 to 460 nM
indicated a good fit with the equation for high affinity competitive
binding of the labeled and unlabeled peptides, with
Hir54-65 binding to 0.89 ± 0.07 sites on thrombin
with a dissociation constant of 38 ± 5 nM (Table I).
These results indicated that Hir54-65 and the
fluorescein-labeled analog bound competitively and with similar, high
affinity to exosite I of thrombin.
|
In contrast to the results for thrombin, the
thrombin-antithrombin stable complex (T-AT*) or antithrombin (AT)
decreased the fluorescence of [5F]Hir54-65 by <3% at
concentrations up to 3 µM T-AT* or 10 µM AT
(Fig. 2). This indicated either an undetectably low affinity
of the peptide for T-AT*, or that the peptide bound but there was no
significant change in fluorescence resulting from the interaction. To
investigate this further, the effect of the T-AT* complex on binding of
[5F]Hir54-65 to thrombin that had been active
site-blocked with D-Phe-Pro-Arg-CH2Cl (FPR-thrombin) was examined. Active site-blocked thrombin was used in
these experiments to prevent reaction with excess antithrombin present
in the mixture of thrombin and antithrombin used to form the T-AT*
complex. As shown by the titrations of 11 nM and 110 nM [5F]Hir54-65 with FPR-thrombin in Fig.
3, binding of the peptide to active site-blocked thrombin
was accompanied by a slightly larger, 30 ± 0.2% change in
fluorescence when compared with native thrombin, and the same
dissociation constant (Table I). The presence of 4.2 µM
T-AT* complex had no significant effect on titrations of the labeled
peptide with FPR-thrombin (Fig. 3). Simulation of the effect of
competitive binding of [5F]Hir54-65 to T-AT* and
FPR-thrombin indicated that an affinity of the labeled peptide for
T-AT* complex up to 200-fold lower than the affinity for thrombin would
have been detected (Fig. 3).
To determine if the low affinity of the T-AT* complex for
[5F]Hir54-65 was due to the presence of the fluorophore
on the peptide, binding of unlabeled Hir54-65 was studied
using an active site-labeled fluorescent thrombin derivative as a
binding probe. To identify a fluorescent derivative with suitable
properties, six derivatives were prepared by active site-specific
inactivation of thrombin with either of the two thioester tripeptide
chloromethyl ketones, ATA-FPR-CH2Cl and
ATA-FFR-CH2Cl (32, 34). The thiol generated from the
thioester on the incorporated peptides was labeled with each of three
structural isomers of fluorescein-iodoacetamide in which the
iodoacetamide group was linked through the 5-, 6-, or 4-positions of
the fluorescein ring system (34). Results of screening these thrombin
derivatives for those that signaled binding of Hir54-65
showed, remarkably, that five of the six derivatives exhibited small,
1 to +8% changes in fluorescence (data not shown), while only
[4
-AF]FPR-thrombin reported the interaction with a large fluorescence change. Titrations of [4
-AF]FPR-thrombin with
Hir54-65 showed an enhancement of 52 ± 0.6%
accompanying binding of the peptide with a dissociation constant of
136 ± 7 nM (Fig. 4, Table I). Analysis of
the effect of native thrombin on binding of Hir54-65 to
[4
-AF]FPR-thrombin (Fig. 4) was consistent with competitive binding
of the peptide to native thrombin with a dissociation constant of
35 ± 9 nM, in agreement with the value of 38 ± 5 nM determined from the previous experiments (Table I).
The presence of 4.0 µM T-AT* or 10.1 µM AT
had no significant effect on binding of Hir54-65 (Fig. 4).
Simulation of the effect of competitive binding indicated that, similar
to the previous results with [5F]Hir54-65, the affinity
of Hir54-65 for T-AT* was at least 200-fold lower than the
affinity for native thrombin (Fig. 4). Titrations of the active
site-labeled thrombin in the absence of Hir54-65 with up
to 2 µM native thrombin, 4 µM T-AT*, or 10 µM AT produced ±1% changes in fluorescence (data not
shown), indicating no significant nonspecific interactions.
Kinetics of Thrombin Inactivation by Antithrombin and the Loss of Affinity for the Hirudin Peptide
Addition of antithrombin to a
mixture of thrombin and the fluorescent hirudin peptide resulted in a
time-dependent return of the quenched fluorescence of
[5F]Hir54-65 to the initial value, consistent with
dissociation of the peptide from thrombin upon its reaction with
antithrombin. On the basis of the previous results, the changes in
fluorescence were taken as measures of the concentrations of thrombin
containing the peptide binding site, which were calculated using the
binding parameters that had been determined (see "Experimental
Procedures"). As shown in Fig. 5, the loss of thrombin
binding sites for [5F]Hir54-65 closely paralleled the
loss of thrombin activity following addition of antithrombin, over the
full course of the reaction. Similar comparisons were done at other
concentrations of [5F]Hir54-65 (see below). The measured
fluorescence changes of 5-17% for these reactions corresponded to
calculated changes in the concentration of thrombin able to bind the
peptide that were indistinguishable from the initial thrombin
concentrations (89 ± 30%). The large error in this calculation
reflected the diminishing reaction amplitude at higher concentrations
of the fluorescent peptide, and a deviation of the fluorescence
amplitudes of 2% from the predicted maxima. For all of the
reactions, the final fluorescence was within 1% of the initial
fluorescence of the peptide alone, indicating complete dissociation of
the peptide.
Effect of Hirudin Peptides on the Kinetics of Thrombin Inactivation by Antithrombin
Kinetic studies were undertaken to characterize the mechanism of the effect of the hirudin peptides on the reaction of antithrombin with thrombin, from the time courses of enzyme inactivation and peptide dissociation. The initial results, together with previous observations (7), suggested that the reactions under bimolecular conditions would follow the mechanism shown in Scheme I.
![]() |
![]() |
![]() |
(Eq. 6) |
![]() |
(Eq. 7) |
![]() |
(Eq. 8) |
![]() |
(Eq. 9) |
The effect of Hir54-65 on the kinetics of the thrombin-antithrombin reaction was studied similarly from the loss of thrombin catalytic activity. Results of these experiments showed similar behavior (Fig. 6B), with a decrease in the rate constant of 2.1-fold and an apparent dissociation constant of 31 ± 24 nM, in agreement with the value of 38 ± 5 nM determined for binding of the unlabeled peptide to thrombin (Table I).
The above analysis of the kinetics supported the mechanism of Scheme I but resulted in rather large uncertainties in the parameters because of the small effect of the peptides on the rate, and error inherent in the measurements. To test the mechanism further, all of the full time course fluorescence and activity data collected as a function of [5F]Hir54-65 concentration, represented by the results in Fig. 6A, were fit without approximation by numerical integration of the differential equation (Equation 6) to solve for the thrombin concentration as a function of time, with changes in the concentration of the thrombin-peptide complex with time calculated with Equation 9. This analysis gave second-order rate constants indistinguishable from those determined by the initial exponential analysis (Table I) and an apparent peptide dissociation constant of 19 ± 6 nM, in closer agreement with the dissociation constant obtained in the equilibrium binding studies. Application of the same analysis to all of the progress curve data for Hir54-65 gave a dissociation constant of 37 ± 7 nM and rate constants that were similarly in good agreement with the values obtained in the preceding analysis and binding studies (Table I). These results supported the adequacy of the mechanism in Scheme I as a quantitative description of the effect of the hirudin peptides on the kinetics of thrombin inactivation and peptide dissociation.
The effect of the peptides on the inhibition reaction stoichiometry was examined to determine if binding of the peptides to thrombin decreased the rate by enhancing an alternative substrate reaction of antithrombin with thrombin (42, 43). The stoichiometry of 1.01 ± 0.03 mol of AT/mol of T obtained in the absence of peptide was unchanged by 5.3 µM [5F]Hir54-65 (1.00 ± 0.01 mol/mol) or 4.9 µM Hir54-65 (0.98 ± 0.06 mol/mol), indicating no significant effect of the peptides on the reaction stoichiometry.
These studies were undertaken to investigate possible changes in the function of regulatory exosite I of thrombin accompanying its inactivation by antithrombin. The current understanding of the mechanism of antithrombin inactivation of thrombin provides a foundation for interpretation of the results. Previous studies support a branched-pathway, suicide substrate mechanism (42, 44), as shown in the upper part of Scheme 2.
[View Larger Version of this Image (9K GIF file)]Scheme 2.
A rapidly reversible Michaelis-type of enzyme-inhibitor complex (T·AT) is formed initially between the proteinase active site and the reactive-site sequence of the inhibitor, exposed on a flexible loop (45-48). The reactive-site bond is subsequently engaged chemically by the proteinase as a normal protein substrate, in an irreversible reaction that results in a covalent intermediate complex (T-AT) (44, 49). A conformational change in the T-AT complex results finally in irreversible trapping of thrombin in the stable product complex (T-AT*). In parallel with this reaction, the covalent intermediate can undergo completion of the substrate reaction, ultimately deacylating to yield reactive-site cleaved inhibitor (ATm) and active proteinase (42-44, 50, 51). The rate-limiting step of thrombin inactivation is the formation of the covalent T-AT intermediate, which is thought to trigger the subsequent, faster serpin conformational change (52, 53).
The thrombin-antithrombin mechanism can be expanded to include all of
the exosite I binding equilibria as shown in Scheme 2. The results of
the binding studies indicate that the affinity of thrombin in the
product, T-AT* complex, for the hirudin peptides is reduced at least
200-fold compared with free thrombin. Characterization of binding of
the fluorescein-labeled hirudin54-65 peptide to human
-thrombin gave results consistent with those reported previously for
bovine thrombin and an analogous derivative of
hirudin53-64 in which the probe was coupled through
slightly different chemistry (31). The ~4-fold higher affinity of the
peptide used here and slightly larger fluorescence change are probably
accounted for by the structural differences between the peptides and
the lower affinity of such peptides for bovine thrombin compared with
the human enzyme (6). In the present studies,
[5F]Hir54-65 and the unlabeled peptide bound
competitively and with similar, high affinity to a single site on
thrombin, while [5F]Hir54-65 had no detectable affinity
for T-AT* in direct fluorescence titrations or in competitive binding
experiments with FPR-thrombin. Evidence of a similar loss of affinity
for the unlabeled peptide was obtained in experiments in which active
site labeled thrombin was used as a macromolecular fluorescence probe
of binding. These results demonstrated that the fluorescence changes of
[5F]Hir54-65 reported the peptide binding equilibrium,
rather than perturbations due to nonspecific interactions. The absence
of detectable binding of the peptides to the T-AT* complex is concluded
to be due to loss of specific interactions with exosite I.
Although the thrombin-antithrombin reaction intermediates in Scheme 2 were not resolved in the present studies, the results of the kinetic studies support the conclusion that the loss of affinity of exosite I for the hirudin peptide occurs in the rate-limiting chemical reaction or a subsequent step. This is supported by the correspondence between the time courses of peptide dissociation and loss of thrombin activity, and between the dissociation constants for the peptides determined kinetically and by direct binding. The relatively small inhibition of the reaction, with 1.7- and 2.1-fold lower bimolecular rate constants at saturating peptide, rules out mechanisms of inhibition in which peptide binding competes with antithrombin for formation of the T·AT complex, or prevents the subsequent chemical reaction (Scheme 2). These mechanisms would show complete inhibition at saturating peptide concentration. These findings are in agreement with the previously reported effect of Hirugen (7), and the conclusion that recognition of antithrombin by thrombin does not depend significantly on exosite I (7, 15-17). The significant residual rate at saturating peptide concentrations implies that the thrombin-peptide complex reacts with antithrombin and undergoes conversion to the covalent P·T-AT complex. On this basis, it is concluded that dissociation of the peptide occurs in a step following either the irreversible reaction that produces the covalent complex, or following a subsequent step that occurs at a faster rate (Scheme 2).
Because the kinetic studies were restricted to bimolecular reaction
conditions, the 1.7- and 2.1-fold inhibition of the rate by binding of
the peptides could be due to an increase in the dissociation constant
for antithrombin binding to the thrombin-peptide complex, or to a
decrease in the rate constant for covalent complex formation. Previous
observations indicate that initial recognition of antithrombin by
thrombin involves binding of the reactive-site sequence at the primary
substrate specificity site and secondary subsites (45, 48, 53-55). In
other studies, binding of hirudin peptides to exosite I has been shown
to change thrombin specificity toward tripeptide substrates, which
interact at these sites (12, 31, 56). This linkage between interactions
at exosite I and the active site is thought to account for the
difference observed here in the affinity of the hirudin peptide for
[4-AF]FPR-thrombin and native thrombin (Table I). This linkage may
be similarly responsible for the decrease in the bimolecular rate
constant for thrombin reaction with antithrombin.
The results of the kinetic studies do not resolve whether the loss of
affinity of thrombin for hirudin peptides occurs as a result of the
formation of the covalent T-AT complex, or the subsequent serpin
conformational change leading to the final T-AT* complex (Scheme 2).
The apparently small effect of the peptide on steps prior to the
chemical reaction implies that the exosite is not greatly affected by
the initial proteinase-inhibitor association, and more likely is
disrupted or blocked as a result of the major structural changes which
accompany the conformational change. The conformational change is
thought to involve incorporation of the reactive site loop into
-sheet A of antithrombin, accompanied by a significant movement of
the proteinase relative to the inhibitor (30, 47, 48, 57), and loss of
specific binding affinity of antithrombin for heparin (52, 58). Other
studies indicate that a conformational change also occurs in the
thrombin component of the thrombin-antithrombin complex, as evidenced
by an increased susceptibility to proteolysis by free thrombin (59,
60). An initial site of cleavage was identified as Arg68
(Arg73 in the chymotrypsin numbering system) (60), which is
located in the
-loop that forms an integral part of exosite I
involved in binding of hirudin peptides (1, 4, 5). Cleavage of this
loop in
-thrombin to produce
-forms of thrombin, or mutation of
Arg68 are accompanied by loss of exosite I functions,
including cleavage of fibrinogen, and binding of hirudin and
thrombomodulin (18, 61). These observations suggest that exosite I is
accessible to thrombin cleavage in the stable thrombin-antithrombin
complex and that this occurs at an enhanced rate, supporting the idea that conformational changes in this region of the proteinase are likely
responsible for the loss of affinity. Significant levels of degradation
of the T-AT* complex requires excess thrombin and is relatively slow
(59, 60). The little degradation observed by SDS-gel electrophoresis
(data not shown), and the correspondence between peptide dissociation
and enzyme inactivation showed that the loss of affinity observed here
was not due to proteolysis.
The results obtained for the hirudin peptide are thought to mimic the
behavior of regulatory proteins that interact with exosite I. The
finding that the loss of affinity of exosite I for hirudin peptide
occurs concomitant with or after the rate-limiting irreversible inactivation step implies that a binding cycle exists for the peptide
that may also apply to regulatory proteins, as illustrated by the model
in Fig. 7. Previous binding and kinetic studies suggest that
this model applies to interactions of thrombin with thrombomodulin and
fibrin. Results of kinetic studies showed that thrombin can react with
antithrombin while remaining bound to these regulatory proteins (7, 19,
20, 24, 26, 28). Thrombin is displaced from complexes with fibrin I and
II (7, 11), and thrombomodulin (12, 13) by hirudin peptides, indicating
that these interactions overlap in specificity for exosite I and that
this binding accounts for a large portion of the affinity. In the case
of thrombomodulin, the situation is more complicated because of a form
of thrombomodulin that contains a covalently attached chondroitin
sulfate chain, which contributes to the thrombin binding affinity by
interaction with exosite II (24, 62). The results of the present
studies suggest that inactivation of exosite I accompanying formation of the T-AT* complex will result in substantially reduced affinity for
fibrin and thrombomodulin, and result in clearance of thrombin from
these complexes as the inactivated antithrombin complex. This mechanism
is thought to be responsible for the lack of inhibition by the T-AT*
complex of the effects of thrombomodulin on thrombin reactions with
antithrombin (27) and protein C (22). The finding that thrombin bound
to the rabbit heart microvascular endothelium is released as the
thrombin-antithrombin complex may similarly involve inactivation of
exosite I (63). In the mechanism proposed here, a regulatory protein
that interacts with exosite I can act catalytically as an effector of
thrombin inactivation. This mechanism may play an important role
physiologically in localizing the activity of thrombin by coupling the
inactivation of the proteinase at the sites of its exosite I-mediated
interactions to the obligatory release of the proteinase-inhibitor
complex. Such coupling would maintain the competency of regulatory
proteins to bind thrombin and prevent dissemination of the enzyme from
sites of vascular injury.
We are grateful to Renee Harrington, Ray Manley, and Udeni Dharmawardana, Ph.D., for their expert technical assistance in purification of proteins and peptides.