(Received for publication, June 26, 1996, and in revised form, December 9, 1996)
From the Division of Hematology, Departments of Internal Medicine and Biochemistry & Molecular Biophysics, Washington University, St. Louis, Missouri 63110
Heparin cofactor II (HCII) inhibits thrombin by
forming a stable 1:1 complex. Heparin and dermatan sulfate increase the
rate of complex formation 1000-fold. Mutation of leucine 444 to
arginine at the P1 position of recombinant HCII (rHCII) increases the
rate of inhibition of thrombin ~100-fold in the absence of a
glycosaminoglycan (Derechin, V. M., Blinder, M. A., and Tollefsen, D. M. (1990) J. Biol. Chem. 265, 5623-5628). We now
report that heparin facilitates dissociation of the
thrombin-rHCII(L444R) complex. In the presence of heparin, thrombin is
inhibited rapidly and completely by a 35-fold molar excess of
rHCII(L444R), but subsequently ~50% of the thrombin activity
reappears with a t1/2 of ~20 min. At higher ratios of rHCII(L444R) to thrombin, the reappearance of thrombin activity is delayed and the final plateau of activity is decreased. Electrophoretic analysis indicates that proteolysis of excess rHCII(L444R) precedes the reappearance of thrombin activity. Addition of heparin at longer intervals after formation of the
thrombin-rHCII(L444R) complex causes a progressive decrease in the
thrombin plateau, suggesting that in the absence of heparin the complex
is slowly converted to a non-dissociable form. By contrast to heparin,
dermatan sulfate does not facilitate dissociation of the
thrombin-rHCII(L444R) complex. Our findings indicate that the P1
residue of HCII affects not only the rate of inhibition of thrombin but
also the stability of the resulting complex.
Heparin cofactor II (HCII)1 belongs to
the family of homologous proteins called serpins, most of which are
inhibitors of serine proteases (1). Inhibitory serpins function as
suicide substrates for their target proteases. During the inhibition
reaction, the protease attacks a specific peptide bond (P1-P1) in the
reactive site of the serpin and becomes trapped in a stable, 1:1
complex with the inhibitor. HCII is unusual because it inhibits both
thrombin and chymotrypsin but not a variety of other proteases (2, 3). The observation that HCII inhibits chymotrypsin 3-4 times faster than
thrombin (4) is consistent with the presence of leucine at the P1
position of the reactive site (5). When HCII is bound to certain
glycosaminoglycans, including dermatan sulfate and heparin, the rate of
inhibition of thrombin increases
1000-fold (6), while inhibition of
chymotrypsin is unaffected (3). Current evidence suggests that binding
of the glycosaminoglycan induces a conformational change in HCII that
allows its N-terminal acidic domain to interact with anion-binding
exosite I of thrombin (reviewed in Ref. 7). This interaction apparently
facilitates proteolytic attack by thrombin at the reactive site of
HCII.
We previously found that mutation of leucine 444 to arginine (L444R) at the P1 position of the reactive site of recombinant HCII (rHCII) increases the rate of inhibition of thrombin ~100-fold in the absence of a glycosaminoglycan (4). Furthermore, the L444R mutation abolishes the ability of rHCII to inhibit chymotrypsin. In the current study, we demonstrate differences in the stability of the thrombin-rHCII and thrombin-rHCII(L444R) complexes. The thrombin-rHCII(L444R) complex is much less stable in the presence of heparin than in the presence of dermatan sulfate and dissociates to yield active thrombin and a proteolytically cleaved form of rHCII(L444R). By contrast, the complex of thrombin with native rHCII is relatively stable in the presence of either heparin or dermatan sulfate. Our data indicate that the P1 residue not only determines the protease specificity of rHCII, as reflected by the relative rates of inhibition of thrombin and chymotrypsin, but also affects the stability of the resulting complex.
Human -thrombin was prepared as described
previously (8) or purchased from Hematologic Technologies (Essex
Junction, VT). Thrombin was labeled with Na125I (ICN
Biomedicals, Costa Mesa, CA) by the chloramine-T method (8) to attain a
specific radioactivity of ~5.0 × 104 cpm/pmol.
Active site-titrated human
-thrombin provided by Dr. Steven T. Olson
(University of Illinois, Chicago, IL) was used to determine the
stoichiometry of inhibition by rHCII. Bovine lung heparin was purchased
from The Upjohn Co. Porcine skin dermatan sulfate was purchased from
Sigma and treated with nitrous acid to degrade
contaminating heparin or heparan sulfate (6).
Other materials were obtained from the following sources:
isopropyl-1-thio--D-galactopyranoside,
deoxyribonuclease-1, 2-amino-2-methyl-1,3-propanediol (ammediol), and
hirudin C-terminal peptide (residues 54-65, O-sulfated at
Tyr-63, catalog no. H-6894) from Sigma;
hexadimethrine bromide (Polybrene) from Aldrich; polyethylene
glycol 8000 (PEG) from Union Carbide (Danbury, CT);
tosyl-Gly-Pro-Arg-p-nitroanilide (Chromozym TH) from
Boehringer Mannheim;
H-D-Phe-pipecolyl-Arg-p-nitroanilide (S-2238)
from Chromogenix AB (Mölndal, Sweden); heparin-Sepharose CL-6B,
Mono Q, and Mono S columns from Pharmacia Biotech Inc.; restriction
enzymes from New England Biolabs; and medium for high density bacterial
culture from BIO 101 (Vista, CA).
The full-length cDNA for native HCII was previously ligated into the pMON-5840 vector, and cassette mutagenesis was employed to construct the L444R mutation (4). For the current study, the cDNAs were removed from pMON-5840 and inserted between the NcoI and BamHI sites of the pET-3d expression vector (Novagen, Madison, WI). The mutations and ligation sites were verified by dideoxynucleotide sequencing (9).
Plasmid vectors were electroporated into Escherichia coli
BL21(DE3)pLysS cells for expression. The cells were grown to an optical
density (600 nm) of ~5-6 in a BioFlo III high density fermentor (New
Brunswick Scientific, Edison, NJ) at 37 °C and then induced with 0.3 mM isopropyl-1-thio--D-galactopyranoside for
3 h. The cells were harvested and lysed at 4 °C in 50 mM Tris-HCl buffer, pH 7.4, containing 0.1% (v/v) Triton
X-100 and 2 mM EDTA. The lysate was treated with 10 µg/ml
deoxyribonuclease-1 and 10 mM MnCl2, and
cellular debris was removed by centrifugation. rHCII was purified to
homogeneity from the lysate by chromatography on heparin-Sepharose
CL6B, Mono Q, and Mono S columns. HCII activity in the column fractions
was determined in a thrombin inhibition assay in the presence of
dermatan sulfate (4). The concentration of purified rHCII was
determined by absorbance at 280 nm using the extinction coefficient
determined for plasma HCII (1.17 ml·mg
1·cm
1) (10).
Purified rHCII (native or
L444R) was incubated with thrombin with or without a glycosaminoglycan
at room temperature in 10 mM Tris-HCl, 150 mM
NaCl, 1 mg/ml PEG, pH 7.5 (TS/PEG buffer). At specified times ranging
from 0.1 to 4500 min, 100 µl of the reaction mixture was placed in a
disposable polystyrene cuvette, 500 µl of 100 µM
Chromozym TH in TS/PEG was added, and the absorbance at 405 nm was
recorded continuously for 100 s. The rate of substrate hydrolysis
(A405/min) was proportional to the residual
thrombin activity. For short time points, 100-µl incubations were
carried out directly in cuvettes.
The second-order rate constant for thrombin inhibition by HCII in the absence of a glycosaminoglycan was determined by the discontinuous method under pseudo first-order conditions. Purified rHCII (100 nM) was incubated with thrombin (10 nM) in TS/PEG buffer. At various times, 100 µl of the reaction mixture was assayed for residual thrombin activity as described above. The pseudo first-order rate constant (kobs) was obtained from the slope of a plot of ln[E] versus time (t) according to Equation 1.
![]() |
(Eq. 1) |
![]() |
(Eq. 2) |
In the presence of a glycosaminoglycan, the second-order rate constant was determined by the continuous method according to Olson et al. (11). All reagents and incubations were held at a constant temperature of 37 °C. First, 100 µl of 0.5 mM S-2238 was mixed with 20 µl of rHCII (0.1-4.2 µM) in a cuvette. Then 480 µl of a solution containing thrombin (1.8-4.2 nM) and heparin or dermatan sulfate (62 µg/ml) was added to initiate the reaction, and the absorbance at 405 nm (A) was monitored continuously for 900 s. To obtain the pseudo first-order rate constant (kobs), we used UltraFit (Biosoft, Ferguson, MO) to fit the data to Equation 3.
![]() |
(Eq. 3) |
![]() |
(Eq. 4) |
The rate constant for dissociation of the
thrombin-rHCII complex (k1) was determined in
a continuous assay system as described by Jesty (13). Thrombin-rHCII
complexes were prepared by incubating thrombin (0.1 µM)
with rHCII (5.8 µM) in the presence or absence of
glycosaminoglycan (100 µg/ml) for 6-90 min in TS/PEG buffer at
37 °C. The reaction mixture was then diluted 25-fold into 0.4 mM S-2238, and generation of p-nitroaniline
(absorbance at 405 nm) was monitored continuously for 900 s. The
data were fit to Equation 5.
![]() |
(Eq. 5) |
Reaction mixtures containing
rHCII and 125I-thrombin were analyzed by SDS-PAGE on
5-15% linear gradient gels in an ammediol/glycine/HCl buffer system
(14). Prior to electrophoresis, the samples were heated at 100 °C
for 4 min after the addition of one-third volume of 4 × ammediol
sample buffer containing 2% -mercaptoethanol. Autoradiography was
performed as described previously (6). The radioactivity in each lane
was quantified with a GS300 scanning densitometer (Hoefer Scientific
Instruments, San Francisco, CA). Other protein samples were analyzed by
SDS-PAGE on 7.5% gels under reducing conditions according to the
method of Laemmli (15) and stained with Coomassie Blue R-250.
Quantification of the intact and cleaved rHCII(L444R) bands in the gel
was done with a Personal Densitometer using ImageQuant software
(Molecular Dynamics, Sunnyvale, CA).
Previously, we followed the reaction of thrombin with
rHCII(L444R) for short periods of time (generally 5 min) to determine the initial rate of inhibition (4). In the experiment in Fig. 1, we monitored the time course of inhibition of
thrombin by rHCII(L444R) or native rHCII for 4500 min. Thrombin
incubated for 4500 min in buffer alone remains fully active (open
squares). As previously reported (4), thrombin is inhibited much
faster by rHCII(L444R) than by native rHCII in the absence of a
glycosaminoglycan (compare closed squares, panels
A and B). In the presence of heparin (closed circles) or dermatan sulfate (open circles), however,
thrombin is inhibited rapidly by both forms of rHCII. Rate constants
for inhibition of thrombin by rHCII(L444R), native rHCII, and plasma HCII are compared in Table I.
|
After being completely inhibited by either native rHCII or
rHCII(L444R), thrombin activity remains undetectable for 4500 min in
the absence of a glycosaminoglycan or in the presence of dermatan
sulfate. In the presence of heparin, however, a significant fraction of
the thrombin activity inhibited by rHCII(L444R) reappears after a brief
lag phase (Fig. 1, panel B, closed circles).
Under the conditions of this experiment, thrombin activity reaches a plateau equal to ~50% of its initial value at ~60 min. By
contrast, thrombin remains completely inhibited by native rHCII for
4500 min in the presence of heparin (Fig. 1, panel A,
closed circles). Fig. 2 indicates the plateau
level of thrombin released from rHCII(L444R) after 720 min in the
presence of various concentrations of heparin or dermatan sulfate.
Maximal dissociation of the thrombin-rHCII(L444R) complex occurs in the
presence of low concentrations of heparin (
0.2 µg/ml), whereas no
release of thrombin is observed at dermatan sulfate concentrations as
high as 10 mg/ml. Dissociation of thrombin from rHCII(L444R), but not
from native rHCII, also occurs at very high ionic strength (NaCl
concentrations
1.25 M) (data not shown), a
phenomenon similar to that reported by Cooperman et al. (16) for the chymotrypsin-
1-antichymotrypsin complex.
The experiments in Figs. 1 and 2 were performed with a 35-40-fold
molar excess of rHCII(L444R) with respect to thrombin. When higher
concentrations of rHCII(L444R) are incubated with thrombin in the
presence of heparin, the lag phase increases and the plateau of
thrombin activity decreases (Fig. 3). Conversely, when
lower concentrations of rHCII(L444R) are used, the initial inhibition of thrombin is incomplete, the lag phase disappears, and the plateau increases.
Stoichiometry of the Thrombin-rHCII(L444R) Reaction
It is apparent from the data in Fig. 3 that the stoichiometry of inhibition of thrombin by rHCII(L444R) in the presence of heparin is >18:1 (inhibitor:protease). To determine the stoichiometry more precisely, we incubated various amounts of native rHCII or rHCII(L444R) with thrombin for 1 min in the presence of heparin or dermatan sulfate and then measured the residual thrombin activity. Under each set of conditions, the stoichiometry was determined from a plot of thrombin activity versus rHCII concentration by extrapolation to zero thrombin activity (Table II). For both native rHCII and rHCII(L444R), the stoichiometry observed in the presence of heparin is 2-3 times higher than that observed in the presence of dermatan sulfate.2 Although the apparent stoichiometry with rHCII(L444R) in the presence of heparin is very high (i.e. ~24:1), this value may be overestimated somewhat because dissociation of a portion of the thrombin-rHCII(L444R) complexes may occur during the 1-min incubation (Fig. 3).
|
When rHCII(L444R) is incubated with thrombin in the presence of heparin for 960 min (during which time ~50% of the thrombin dissociates from the complex) and then fresh rHCII(L444R) is added, all of the thrombin activity is again inhibited rapidly (data not shown). This result eliminates the possibility that dissociation yields an altered form of thrombin that cannot be inhibited by rHCII(L444R) and, furthermore, suggests that no active inhibitor is present in the incubation after the thrombin activity has reached its plateau level.
Fig. 4 (panel A) shows an SDS-PAGE analysis
of rHCII(L444R) incubated with thrombin for 0.2-180 min in the
presence of heparin. Progressive conversion of excess rHCII(L444R) to a
lower molecular weight form occurs during the 20-min lag phase, when
thrombin activity is undetectable. As the thrombin activity approaches its plateau level between 60 and 120 min, intact rHCII(L444R) is no
longer present. By contrast, only about 40% of the rHCII(L444R) is
cleaved at 120 min in the presence of dermatan sulfate, and the amount
of cleavage does not increase significantly thereafter (Fig. 4,
panel B). The change in the molecular weight of rHCII(L444R) from ~58,000 to ~54,000 is consistent with proteolytic cleavage at
the reactive site, which releases a peptide of 36 amino acids (17).
Immunoblots probed with an antibody raised against the C-terminal
decapeptide of HCII (residues 471-480) (18) indicate that this epitope
is absent from the 54,000 molecular weight product, as expected for
cleavage at the reactive site (data not shown). These results indicate
that the thrombin-rHCII(L444R) complex dissociates in the presence of
heparin to yield active thrombin and an inactive, proteolytically
cleaved form of the inhibitor. Since thrombin released from the complex
can react rapidly with intact inhibitor molecules as long as they are
present in the incubation, thrombin activity remains undetectable
during the lag phase of the reaction.
Conversion of Thrombin-rHCII(L444R) to a Non-dissociable Form
When thrombin is incubated with rHCII(L444R) for 17 min in
the presence of heparin as in Fig. 1 (panel B) and then
excess Polybrene (100 µg/ml) is added to neutralize the heparin, no
release of thrombin activity occurs (data not shown). Therefore,
thrombin-rHCII(L444R) complexes formed in the presence of heparin are
stable if the heparin is neutralized soon after formation of the
complex. Conversely, when heparin is added 17 min after initiation of
the thrombin-rHCII(L444R) reaction, ~50% of the thrombin eventually
dissociates from the inhibitor (Fig. 5, open
squares). If heparin is added at progressively later times,
however, the plateau decreases. After a 630-min preincubation in the
absence of heparin, little or no thrombin activity is released from
rHCII(L444R) by the subsequent addition of heparin. These results
indicate that the thrombin-rHCII(L444R) complex slowly converts from a
form that is dissociable by heparin to one that is not.
Conversion of the thrombin-rHCII(L444R) complex to the non-dissociable
form can be prevented by a peptide that corresponds to the C-terminal
portion of hirudin (residues 54-65), which binds to anion-binding
exosite I of thrombin (19). As shown in Fig. 6
(closed circles), the thrombin-rHCII(L444R) complex
dissociates completely in the presence of heparin when the hirudin
peptide is also present throughout the incubation. The peptide does not cause dissociation of the complex in the absence of heparin (data not
shown).
Kinetics of Dissociation of Thrombin-rHCII Complexes
We
determined rate constants (k1) for
dissociation of thrombin-rHCII(L444R) and thrombin-rHCII(native)
complexes at various times of incubation in the absence or presence of
a glycosaminoglycan. For these determinations, reaction mixtures
containing the thrombin-rHCII complex were diluted into an
~105-fold molar excess of the chromogenic substrate
S-2238. Dissociation of thrombin from the complex, indicated by a
progressive increase in the rate of substrate hydrolysis, followed the
first-order kinetic model described under "Experimental
Procedures." In the absence of a glycosaminoglycan, the rate of
dissociation of thrombin-rHCII(L444R) decreases with time (Fig.
7), corresponding with formation of the non-dissociable
form of the complex. Rate constants (k
1) calculated from the data are given in Table III. Rate
constants for dissociation of thrombin-rHCII(native) in the absence of
a glycosaminoglycan also decrease with time and are similar to those of
thrombin-rHCII(L444R). Both thrombin-rHCII complexes dissociate faster
in the presence of heparin and somewhat slower in the presence of
dermatan sulfate (Table III). However, heparin increases the rate
constant for dissociation of thrombin-rHCII(L444R) to a much greater
degree than that of thrombin-rHCII(native).
|
Serpin-protease complexes usually remain associated after
denaturation in SDS (1). As shown in Fig. 8 (panel
A), native rHCII rapidly forms an SDS-stable complex with
125I-thrombin in the presence of dermatan sulfate or
heparin. In the absence of a glycosaminoglycan, the SDS-stable complex
forms more slowly (the percentage of uncomplexed
125I-thrombin, determined by densitometry of the
autoradiograph, is indicated by the open column below each
lane). A small amount of radioactive material is also present on the
gel between the free 125I-thrombin and complex bands and
may represent proteolytically degraded complexes. Fig. 8 (panel
B) shows the results of parallel incubations performed with
rHCII(L444R). SDS-stable complexes form more rapidly in comparison with
native rHCII in the absence of a glycosaminoglycan but less rapidly in
the presence of dermatan sulfate or heparin.
In these experiments, there is an obvious discrepancy between the amount of residual thrombin activity, determined by hydrolysis of Chromozym TH (closed columns), and the amount of uncomplexed 125I-thrombin (open columns) detected in the gel. This discrepancy is most pronounced at the early time points with rHCII(L444R) (panel B, lanes 2, 5, and 8). A similar discrepancy is apparent in experiments with native rHCII in the absence of a glycosaminoglycan (panel A, lanes 2-4). These results indicate that inhibition of thrombin by either native rHCII or rHCII(L444R) may precede formation of an SDS-stable complex.
A general mechanism that describes the interactions of a protease
with a serpin is shown in Fig. 9. The protease
(E) and serpin (I) initially form a reversible Michaelis
complex (E·I), which rapidly converts to the intermediate
E-I. E-I probably represents a tetrahedral or
acyl adduct involving the catalytic serine hydroxyl group of the
protease and the carbonyl group of the P1 amino acid residue of the
serpin (20, 21). Formation of E-I may be followed by a
conformational change in the serpin, in which the reactive site loop
containing the P1 residue becomes partially inserted into -sheet A
to form a kinetically stable complex (E-I*) (reviewed in
Ref. 1). Alternatively, E-I may dissociate to yield the free
protease (E) and a modified inhibitor (IM) that
has been cleaved proteolytically at the P1-P1
peptide bond. Rapid
partitioning of the intermediate E-I along pathways leading
to E-I* (inhibition pathway) or E + IM (substrate pathway) determines the stoichiometry of
inhibition (22). In general, E-I* is stable for many hours and can be considered the final product of the inhibition pathway. In
some cases, however, E-I* may dissociate slowly to yield
E + IM (13, 23) or undergo further modification
to yield a non-dissociable or "locked" form of the complex
(E-I**) (16).
Mutation of leucine 444 to arginine at the P1 position of rHCII appears to affect several steps in this mechanism.
1) The L444R mutation alters the protease specificity of rHCII as
reflected in the rates of inhibition of thrombin and chymotrypsin in
the absence of a glycosaminoglycan. Native rHCII inhibits chymotrypsin more rapidly than thrombin (4). The L444R mutation increases the rate
of inhibition of thrombin ~100-fold (Table I) while virtually
abolishing inhibition of chymotrypsin. These results are consistent
with the observation that the reactive site peptide bond (P1-P1) of a
serpin generally mimics the substrate specificity of the target
protease. Since thrombin preferentially attacks Arg-X
peptide bonds, the presence of arginine at the P1 position of HCII
probably increases the rate of formation of the Michaelis complex
(E·I) (1).
2) The L444R mutation affects the rapid partitioning of E-I between the inhibition and substrate pathways (reactions 3 and 4, respectively, in Fig. 9). The stoichiometry of inhibition of thrombin determined after a 1-min incubation is ~2-3 times greater for rHCII(L444R) than for native rHCII in the presence of dermatan sulfate or heparin (Table II). Thus, the presence of arginine at the P1 position of rHCII increases partitioning of E-I into the substrate pathway to generate E + IM.
3) Thrombin-rHCII(L444R) complexes readily dissociate in the presence
of heparin to yield active thrombin (E) and a modified form
of the inhibitor cleaved at the reactive site (IM). This unusual phenomenon is best explained by an increase in the rate of
reaction 5 in Fig. 9 (E-I* E + IM). It is important to note that partitioning of
E-I between the inhibition and substrate pathways (reactions
3 and 4 in Fig. 9) occurs rapidly (<1 min). Thus, the relatively slow
release of thrombin cannot be explained by heparin favoring reaction 4 (E-I
E + IM), since this would simply increase the stoichiometry of inhibition observed at short time
points. When the ratio of rHCII(L444R) to thrombin exceeds the
stoichiometry of inhibition (~24:1 in the presence of heparin), there
is a lag phase during which thrombin activity is undetectable and the
excess inhibitor is being cleaved to IM (Fig. 4). Thrombin released from E-I* appears to be inhibited rapidly as long
as intact rHCII(L444R) molecules remain present in the incubation. The
net effect is that accelerated dissociation of the
thrombin-rHCII(L444R) complex in the presence of heparin leads to
progressive degradation of the excess inhibitor via reactions 4 and 5 (Fig. 9).
The experiment in Fig. 4 also provides evidence for turnover of the thrombin-rHCII(L444R) complex in the presence of dermatan sulfate. Approximately 15% of the rHCII(L444R) is cleaved after a 5-min incubation with thrombin and dermatan sulfate, in reasonable agreement with the value predicted from the stoichiometry of inhibition (7.2 × 12/378 = 23%). Further cleavage of rHCII(L444R) occurs over 180 min, consistent with the slow rate of dissociation documented in Table III. The amount of cleaved rHCII(L444R) appears to level off at ~40% as thrombin becomes trapped in the non-dissociable form of the complex. Therefore, under the conditions of this experiment, no thrombin activity is released even after much longer times of incubation.
When rates of dissociation are determined in the presence of excess
S-2238, which prevents de novo formation of thrombin-rHCII complexes, we find that thrombin-rHCII(L444R) and
thrombin-rHCII(native) dissociate with similar half-lives in the range
of 1300-2000 min (t1/2 = 0.69/k1) in the absence of a glycosaminoglycan
(Table III). These rates are somewhat faster than that of the
thrombin-antithrombin complex (t1/2 = 8000 min)
(13). Heparin increases the rate of dissociation of
thrombin-rHCII(L444R) approximately 60-fold (t1/2 = 21 min) but has much less of an effect on the rate of dissociation of
thrombin-rHCII(native) (t1/2 = 300 min). Therefore,
the L444R mutation does not affect the inherent stability of
thrombin-rHCII(L444R) but somehow enables heparin to destabilize the
complex.
4) The thrombin-rHCII(L444R) complex converts to a non-dissociable form (designated E-I** in Fig. 9) in a time-dependent manner. Thus, the plateau of thrombin activity decreases from ~50% when heparin is present throughout the incubation to <5% when heparin is added 630 min after formation of the complex (Fig. 5). Because conversion of E-I* to E-I** (reaction 6 in Fig. 9) competes with heparin-induced dissociation of E-I* (reaction 5 in Fig. 9), <100% of the thrombin is ultimately released.
The hirudin C-terminal peptide prevents conversion of
thrombin-rHCII(L444R) to the non-dissociable form (Fig. 6). This
peptide binds to anion-binding exosite I of thrombin and inhibits
proteolysis of certain macromolecular substrates (e.g.
fibrinogen) (19). Although the hirudin peptide markedly decreases the
rate of inhibition of thrombin by native rHCII in the presence of a
glycosaminoglycan (18), it has little effect on the initial rate of
inhibition of thrombin by rHCII(L444R) (Fig. 6). The effect of the
hirudin peptide would be consistent with a proteolytic mechanism in
which E-I* is converted to E-I** by a trace
amount of the free enzyme, as suggested by Cooperman et al.
(16) for the chymotrypsin-1-antichymotrypsin complex.
Alternatively, the hirudin peptide could bind directly to
thrombin-rHCII(L444R) and disrupt interactions between the N-terminal
acidic domain of rHCII and anion-binding exosite I of thrombin that may
serve to stabilize the complex.
Conversion to the non-dissociable form parallels the progressive
decrease in the rate constant for dissociation
(k1) of thrombin-rHCII(L444R) observed in the
absence of a glycosaminoglycan (Table III). Similarly, the
k
1 of thrombin-rHCII(native) decreases
with time, suggesting that this complex also converts to a
non-dissociable form. The data in Table III suggest that reaction 6 (Fig. 9) is relatively slow with a t1/2 in the range
of 30-60 min. If the reaction is not first-order as implied in Fig. 9, but instead depends on the concentration of free protease as suggested by Cooperman et al. (16), then reaction 6 could occur much
faster during the initial phase of the reaction before the protease has been inhibited completely. This could explain why <100% of the thrombin is released from thrombin-rHCII(L444R) even if heparin is present throughout the incubation (e.g. Figs. 1 and
3).
5) The inhibitory pathway of a serpin-protease reaction is generally accompanied by formation of an SDS-stable complex indicative of a covalent linkage between the two proteins. By contrast, Fig. 8 (panel B) shows that formation of an SDS-stable complex between 125I-thrombin and rHCII(L444R) does not parallel inhibition of thrombin activity. Both in the absence and in the presence of a glycosaminoglycan, incorporation of 125I-thrombin into the covalent complex (open columns) lags behind inhibition of thrombin activity (closed columns). Similarly, a delay in covalent complex formation is apparent with native rHCII in the absence of a glycosaminoglycan (Fig. 8, panel A, lanes 2-4). In the presence of a glycosaminoglycan (especially heparin), there is a longer delay in covalent complex formation with rHCII(L444R) than with native rHCII (cf. lanes 5 and 8 in panels A and B). Although appearance of SDS-stable complexes roughly parallels conversion to the non-dissociable form, interpretation of this experiment is not straightforward because it is unknown to what extent a covalent acyl ester linkage may form as an artifact of denaturation in SDS. Nevertheless, the results suggest subtle differences in the structures of the initial thrombin-rHCII(native) and thrombin-rHCII(L444R) complexes.
The mechanism by which heparin facilitates dissociation of the
thrombin-rHCII(L444R) complex remains to be determined. Apparently, heparin increases the rate at which proteolytic cleavage of the P1-P1
bond in rHCII(L444R) by thrombin proceeds to completion rather than
being arrested at the stage of a tetrahedral or acyl intermediate. The
effect of heparin does not appear to be caused by nonspecific ionic
interactions, since dissociation does not occur at >10,000-fold higher
concentrations of dermatan sulfate (Fig. 2) or in the presence of 0.5 M NaCl (data not shown). More likely, dissociation of the
complex results from changes in the conformation of the active site of
thrombin and/or the reactive site of rHCII(L444R) induced by binding of
heparin to specific amino acid residues on one or both of these
proteins. Investigation of thrombin-rHCII(L444R) has revealed aspects
of serpin biochemistry, such as induced dissociation of the complex and
conversion to a "locked" form, that are not widely recognized.
These reactions may exemplify important mechanisms by which the
activities of serpins can be regulated.
We thank Steve Olson for advice during the course of this investigation and for the gift of active site-titrated thrombin.