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J Biol Chem, Vol. 273, Issue 47, 31203-31208, November 20, 1998
From the Site-directed mutagenesis was used to investigate
the role of basic residues in the thrombin anion-binding exosite-I
during formation of thrombin-antithrombin III (ATIII),
thrombin-protease nexin 1 (PN1), and thrombin-heparin cofactor II
(HCII) inhibitor complexes, in the absence and presence of
glycosaminoglycans. In the absence of glycosaminoglycan, association
rate constant (kon) values for the inhibition
of the mutant thrombins (R35Q, K36Q, R67Q, R73Q, R75Q,
R77aQ, K81Q, K109Q, K110Q, and
K149eQ) by ATIII and PN1 were similar to
wild-type recombinant thrombin (rIIa), whereas
kon values were decreased 2-3-fold for HCII
against the majority of the exosite-I mutants. The exosite-I mutants
did not have a significant effect on heparin-accelerated inhibition by
ATIII with maximal kon values similar to rIIa.
A small effect was seen for PN1/heparin inhibition of the exosite-I
mutants R35Q, R67Q, R73Q, R75Q, and R77aQ,
where kon values were decreased 2-4-fold,
compared with rIIa. For HCII/heparin, kon
values for inhibition of the exosite-I mutants (except R67Q, R73Q, and
K149eQ) were 2-3-fold lower than rIIa. Larger
decreases in kon values for HCII/heparin were
found for R67Q and R73Q thrombins with 441- and 14-fold decreases,
respectively, whereas K149eQ was unchanged. For
HCII/dermatan sulfate, R67Q and R73Q had kon
values reduced 720- and 48-fold, respectively, whereas the remaining
mutants were decreased 3-7-fold relative to rIIa. The results suggest
that ATIII has no major interaction with exosite-I of thrombin with or
without heparin. PN1 bound to heparin uses exosite-I to some extent,
possibly by utilizing the positive electrostatic field of exosite-I to
enhance orientation and thrombin complex formation. The larger effects
of the thrombin exosite-I mutants for HCII inhibition with heparin and
dermatan sulfate indicate its need for exosite-I, presumably through
contact of the "hirudin-like" domain of HCII with exosite-I of thrombin.
Hemostasis is dependent on the intricate balance between
thrombin's procoagulant activity, which promotes clot formation, and
its anticoagulant activity in complex with thrombomodulin that
activates protein C and restricts excessive clotting (1, 2). Serine
protease inhibitors (serpins) further regulate the procoagulant
activity of thrombin. The most important inhibitors of thrombin are
antithrombin III (3), protease nexin 1 (4), and heparin cofactor II
(5). These serpins inhibit thrombin by forming tight 1:1 complexes
between the serpin P1-P1' reactive center and
the active site of thrombin1
(5-7). Complex formation for
ATIII2 and PN1 occurs by a
two-step mechanism where there is an initial formation of a loose
complex followed by a conformational change that yields an essentially
irreversible complex (8). The final complex exists either as a
tetrahedral intermediate or as an acyl enzyme in which the
P1 residue is covalently bound to Ser195 of
thrombin3 after hydrolysis of
the P1-P1' scissile bond (9).
The inhibitory activities of the three serpins are accelerated by the
presence of glycosaminoglycans such as heparin (5-7, 10), whereas the
inhibitory activity of HCII is also accelerated by dermatan sulfate
(11, 12). Site-directed mutagenesis studies of thrombin (13, 14), ATIII
(15), and PN1 (16) have shown that heparin-accelerated inhibition by
ATIII and PN1 occurs by a template mechanism. As heparin binds to ATIII
and PN1 with a higher affinity than for thrombin (17), the template
mechanism involves the binding of heparin to a conserved site on the
serpin (18, 19). The serpin-heparin complex then quickly associates with the heparin binding site on thrombin (anion-binding exosite-II) through electrostatic interactions (20). However, the mechanism for
glycosaminoglycan-accelerated inhibition of thrombin by HCII seems to
be different to that of ATIII and PN1. Inhibition studies of This paper investigates the interaction of three heparin-binding
serpins with the thrombin anion-binding exosite-I. Past work has not
fully examined the contribution of all the basic residues of
thrombin's exosite-I with ATIII and HCII (20, 25) or PN1 (26) in the
absence and presence of glycosaminoglycans. An extensive study will
define those residues involved in interactions between thrombin's
exosite-I and various domains on ATIII, HCII, and PN1. These studies
further emphasize mechanistic differences in how different
glycosaminoglycan-dependent serpins recognize thrombin, especially through exosite-I-dependent mechanisms. This
work clearly establishes a very prominent, a less prominent, and a
nonessential role for thrombin's exosite-I in HCII-, PN1-, and
ATIII-thrombin inhibition reactions, respectively.
Materials--
Human plasma thrombin was prepared as described
previously (27) and active site titrated with p-nitrophenol
p'-guanidinobenzene to determine the concentration of active
thrombin molecules (28). The expression, purification, and
characterization of recombinant and mutant
thrombins4 (R35Q, K36Q, R67Q,
R73Q, R75Q, R77aQ, K81Q, K109Q, K110Q, and
K149eQ) have been described
elsewhere.5 ATIII was
purified from human plasma as described by McKay (29). HCII was
purified to homogeneity from human plasma as described previously (30).
PN1 was purified as described previously (26). The chromogenic
substrates S2238 (H-D-Pro-Pip-Arg-pNA) and S2266 (H-D-Val-Leu-Arg-pNA) were purchased from Chromogenix
(Quadratech, Surrey, UK). Heterogeneous porcine heparin was purchased
from Grampian Enzymes (Arthrath, Scotland), and dermatan sulfate
(Chondroiten) was purchased from Sigma (Poole, Dorset, UK).
Thrombin Inhibition by Serpins in the Absence of
Glycosaminoglycan--
The value for the association rate constant
(kon) for thrombin(s) and the serpins HCII,
ATIII, and PN1 in the absence of glycosaminoglycan were determined
using pseudo-first order kinetics. For HCII, the value of
kon was determined by preincubating a 400-µl
volume containing 1 µM HCII and 0.1 µg/ml polybrene in
1 × assay buffer at 37 °C. Adding thrombin to a final
concentration of 50 nM started the reaction. At various
time points, a 20-µl aliquot was removed and added to 230 µl of 200 µM S2266 in assay and incubated at 37 °C for 4 min in
a Thermomax plate reader (Molecular Devices) to measure the initial
velocity of residual thrombin. A control reaction was also performed
with the same components except that HCII was omitted. The amount of
uninhibited thrombin present is proportional to its initial velocity
and obeys the equation At =
A0 e-k't, where
t is the time the sample was taken, At
and A0 are the velocities at times
t and zero, respectively, and k' is the apparent first order rate constant. The value for the second order rate constant
kon was determined by dividing k' by
the inhibitor concentration.
For the serpins ATIII and PN1, the value for kon
was determined using progress curve kinetics. Cuvettes containing
100-200 µM S2238 and serpin (15 µM ATIII
or 150 nM PN1) were preheated at 37 °C for 10 min and
then transferred to a Hewlett-Packard spectrophotometer. Reactions were
started by the addition of thrombin to 100 pM, and the
release of p-nitroaniline from the hydrolysis of S2238 was
followed by measuring the absorbance at 400-410 nm. Progress curves
for the formation of product are described by the following equation
(31-33):
Glycosaminoglycan-accelerated Inhibition of Thrombin by
Serpins--
Progress curve kinetics were used to estimate the value
of kon for the interaction of native and variant
thrombins with the serpins HCII, ATIII, and PN1 in the presence of the
glycosaminoglycans heparin (heterogeneous) and dermatan sulfate under
pseudo-first order conditions. The dependence of the
kon value for the inhibition of recombinant
native and plasma thrombin on the concentration of glycosaminoglycans
was determined by incubating ATIII (200 nM) or PN1 (2 nM) with heparin concentrations ranging from 1 to 1000 nM, in the presence of S-2238 (50-400 µM)
for 10 min at 37 °C. Reactions were followed by the release of
p-nitroaniline on a Hewlett Packard spectrophotometer after
the addition of thrombin to a final concentration of 200 pM. The effects of both heparin and dermatan sulfate on the
kon value were examined for HCII. HCII (25 nM) was incubated with varying concentrations of heparin (25 nM-25 µM) and dermatan sulfate (2-50
µM), and the reactions were started as described above.
Values for kon were calculated using Equations 1
and 2. The optimum concentrations of glycosaminoglycan and S2238 were
used to determine kon for all the recombinant
thrombin mutants.
Molecular Modeling--
The heparin cofactor II-thrombin complex
was constructed essentially as described previously (34) using the
Homology module of the Insight II molecular modeling package (Version
2.3.0, BIOSYM Technologies, Inc., San Diego). The x-ray crystal
structure of Inhibition of Thrombin Exosite-I Mutants by ATIII and PN1 in
the Absence of Heparin--
Values for the association rate constant
(kon) for the inhibition of native plasma
thrombin (pIIa), recombinant native thrombin (rIIa), and mutant
thrombins by ATIII and PN1 were determined under pseudo-first order
conditions using progress curve kinetics. Plasma thrombin and rIIa were
inhibited by ATIII (Table I) and PN1
(Table II) with
kon values similar to previously reported values
(16, 36).
The inhibition of the exosite-I mutants by ATIII showed no major
differences for kon values (Table I). The
largest deviations found for ATIII inhibition were 1.5-fold for the
mutants R73Q (0.82 × 104 M Inhibition of Thrombin Exosite-I Mutants by ATIII and PN1 in the
Presence of Heparin--
The optimum heparin concentration required to
achieve the maximum kon value for the inhibition
of pIIa and rIIa by the serpins ATIII and PN1 was determined by
comparing kon values over a range of heparin
concentrations (1-1000 nM). Both plasma and recombinant thrombins showed typical bell-shaped inhibition rate curves with dependence of the kon values for PN1 and ATIII
on heparin concentration (Fig. 1). The
inhibition of pIIa and rIIa by ATIII gave maximal kon values (0.75 × 108
M
There were no major differences in the kon
values for the exosite-I mutants when compared with rIIa for ATIII,
with kon varying no more than ~1.4-fold (Table
I). The increase in kon for each thrombin
exosite-I mutant in the presence of heparin and ATIII was similar to
rIIa (9,675-fold), varying from 8,626-fold
(K149eQ) to 15,172-fold (R67Q). Likewise, many
of the thrombin exosite-I mutants did not have any significant effects
on inhibition by PN1 in the presence of 20 nM heparin
(Table II). However, the exosite-I mutants R35Q and
R77aQ showed decreases in
kon (3.7- and 3.2-fold, respectively) when compared with rIIa, whereas there were 2-fold decreases in
kon for the inhibition of R67Q, R73Q, and R75Q
by PN1 with heparin. Rate enhancement by heparin for the inhibition of
R35Q and R77aQ by PN1 was only 553- and 743-fold
compared with rIIa (2,049-fold), whereas the rest of the mutants ranged
from 1,018-fold (R67Q) to 2,194-fold (K81Q).
Inhibition of Thrombin Exosite-I Mutants by HCII in the Absence of
Glycosaminoglycans--
Pseudo-first order kinetics were used to
examine the effects of mutations on the inhibition of thrombin by HCII.
Recombinant and plasma native thrombins were inhibited with similar
values for kon (both 1.1 × 103
M Inhibition of Thrombin Exosite-I Mutants by HCII in the Presence of
Glycosaminoglycans--
Maximal kon values for
the inhibition of rIIa and pIIa by HCII were determined by comparing
kon values with various concentrations of these
glycosaminoglycans (Fig. 1). Heparin titration of HCII gave optimum
kon values of 7.6 × 107
M
The degree to which heparin-accelerated HCII inhibition of the
exosite-I mutants R35Q, K36Q, R75Q, R77aQ, K81Q,
K109Q, K110Q, and K149eQ varied from 45,714-fold
(K81Q) to 67,692-fold (R75Q) compared with rIIa (67,568-fold) (Table
III). Interestingly, heparin accelerated the inhibition of the two
mutants, R67Q and R73Q, by HCII by only 347- and 15,882-fold,
respectively (Table III). The kon values for all
other exosite-I mutants were approximately 2-3-fold lower than that
for rIIa. These decreases in kon values in the
presence of heparin paralleled the differences observed in the absence of heparin. In contrast, HCII inhibition of R73Q displayed a larger decrease (14-fold) in the kon value in the
presence of heparin, whereas a very large decrease (441-fold) was
observed with R67Q.
Dermatan sulfate-catalyzed HCII inhibition of the exosite-I mutant
K149eQ was similar to recombinant wild-type
thrombin (Table III). The effect of the other exosite-I mutations was
generally greater on the degree of dermatan sulfate acceleration
compared with heparin (Table III). In particular, R67Q and R73Q were
only accelerated 61- and 1323-fold, respectively, in the presence of
dermatan sulfate. Additionally, the reduction of the
kon values of HCII inhibition of the other
exosite-I mutations was about 2-fold greater in the presence of
dermatan sulfate than in the presence of heparin (Table III).
The kon values for the inhibition of
recombinant native thrombin by the serpins were not very different from
those observed with plasma thrombin. The recombinant thrombins were
expressed in a baculovirus system and were devoid of
Asn60g-linked carbohydrate (37, 38). Thus, the
absence of carbohydrate on recombinant thrombin does not influence the
inhibitory activity of the serpins in the absence of glycosaminoglycan.
The kon values for inhibition of the exosite-I
mutants by ATIII and PN1 in the absence of heparin did not differ
substantially from rIIa, suggesting that there are no significant
interactions between these serpins and exosite-I. However, the
inhibitory activity of HCII was affected by most of the exosite-I
mutants, where kon values were decreased
~2-3-fold. This is comparable with decreases in inhibition of
The inhibitory activities of all three serpins are greatly accelerated
by the presence of glycosaminoglycans. Interestingly, kon values for the interaction of recombinant
native thrombin with ATIII and HCII in the presence of heparin were
consistently higher (1.8-fold) than those with plasma thrombin. This
suggests that the Asn60g carbohydrate may
slightly retard the association of HCII and ATIII with thrombin in the
presence of heparin. This could be due to either charge repulsion or
steric hindrance.
ATIII-Thrombin Exosite-I Interactions in the Presence of
Heparin--
The inhibition of thrombin by ATIII occurs by a two-step
mechanism that is accelerated by heparin (7, 8). This acceleration proceeds primarily by the formation of ATIII-heparin complexes, which
then bind to exosite-II, using complementary electrostatic fields in a
manner similar to the interaction between thrombin and the C-terminal
tail of hirudin (13, 14). Extensive site-directed mutagenesis of the
thrombin exosite-I (R35Q, K36Q, R67Q, R73Q, R75Q,
R77aQ, K81Q, K109Q,K110Q, and
K149eQ; Fig. 2,
top panel) has shown that inhibition by ATIII in the absence
and presence of heparin does not require any of the basic residues that
form exosite-I. Our results complement past studies that suggested the
interaction between ATIII/heparin and thrombin uses exosite-II for
electrostatic interactions and that exosite-I has no valuable
contribution to complex formation (Fig. 2, middle panel),
either in "electrostatic steering" or in direct interactions by
salt-bridging (13, 14).
PNI-Thrombin Exosite-I Interactions in the Presence of
Heparin--
PN1 also inhibits thrombin in a two-step mechanism (8),
and inhibition is heparin accelerated by a template mechanism similar to ATIII (26). Thrombin inhibition by PN1 in the presence of heparin
shows a small effect for the exosite-I mutants R35Q, R67Q, R73Q, R75Q,
and R77aQ (Fig. 2), where there were decreases
in the kon values of 3.7-, 2.2-, 2.0-, 1.8-, and
3.2-fold, respectively. The association rate constant for thrombin-PN1
interaction in the presence of heparin was 2.5 × 109
M HCII-Thrombin Exosite-I Interactions in the Presence of
Glycosaminoglycan--
Thrombin inhibition by HCII is unique compared
with the serpins ATIII and PN1 for three reasons: (a) the
P1 residue is a leucine as opposed to the P1
arginine of ATIII and PN1; (b) the glycosaminoglycans heparin and dermatan sulfate increase the rate of inhibition; and
(c) the unique N-terminal acidic domain (21, 22, 24). Like
other thrombin macromolecular substrates, the N-terminal domain
(homologous to the C terminus of hirudin) is rich in acidic and polar residues that are postulated to bind exosite-I (Fig. 3). There are two current models that
explain the mechanism for glycosaminoglycan-accelerated inhibition of
thrombin by HCII: the double-bridge mechanism (21, 24) and the
allosteric only model (20). In both models, it is assumed that the
N-terminal acidic domain is displaced from the glycosaminoglycan
binding site of HCII, thereby allowing the N-terminal domain to bind
exosite-I (20, 21, 24, 42).
The role of thrombin's exosite-I during inhibition by HCII has been
partially characterized by studies using dysthrombin Quick I (39, 40)
and exosite-I mutants R73E and R75E (20, 25). In the current study, the
effects of the majority of exosite-I mutants (R35Q, K36Q, R75Q,
R77aQ, K81Q, K109Q,K110Q, and
K149eQ) were minimal (Fig. 2, top and
middle panels), which suggests that most of the basic
residues in exosite-I make no major contacts with the acidic domain of
HCII with or without glycosaminoglycans. In contrast, Arg67
and Arg73 play a major role in complex formation with HCII
in the presence of glycosaminoglycans (Fig. 2, bottom
panel). It is interesting to note that the hirudin peptide
(similar to the acidic domain of HCII) strongly interacts with both
Arg67 and Arg73 of thrombin (Fig. 2,
bottom panel). A large decrease in
kon was found with R67Q thrombin in the presence
of both glycosaminoglycans and HCII (441-fold for heparin, 721-fold for
dermatan sulfate), which confirms that the structural integrity of the
thrombin 70-80 loop (43) is important for interactions between the
HCII acidic domain and exosite-I. More moderate decreases of
kon were seen for the mutant R73Q when inhibited
in the presence of heparin (14-fold) and dermatan sulfate (48-fold),
also suggesting that Arg73 may interact with the N-terminal
domain of HCII (Fig. 2, bottom panel). The reasons for an
overall greater effect by the thrombin exosite-I mutations in the
presence of dermatan sulfate could indicate how different
glycosaminoglycans might influence the binding mode of the N-terminal
domain to exosite-I. Finally, dermatan sulfate is more dependent on the
allosteric mechanism for HCII-thrombin inhibition than is heparin and
might be more affected by alterations in exosite-I of thrombin (see below).
The Mechanism for HCII-Thrombin Inhibition in the Presence of
Glycosaminoglycans--
The mechanism for
glycosaminoglycan-accelerated inhibition of thrombin by HCII is
dependent on the displacement of the N-terminal domain from the HCII
glycosaminoglycan binding site so that it is free to interact with
exosite-I of thrombin. The difference between the proposed
double-bridge model (21, 24) and the allosteric model (13) is the role
of glycosaminoglycan acting as a secondary bridge between HCII and
thrombin's exosite-II. When our thrombin exosite-I site-directed
mutagenesis data are compared with those obtained from Sheehan et
al. (20, 25), some interesting observations can be made. First,
the effect of the K149eQ mutation in this study
is similar to that for the K149eA mutant (25),
which suggests that this residue plays no significant part in the
interaction of HCII with thrombin. However, the effect of the two
exosite-I mutants R73E and R75E have more pronounced effects when
compared with residues substituted with glutamine replacements used in
this study (20, 25). In particular, the association rate constant for
R73E in the presence of heparin was decreased 376-fold compared with
14-fold for R73Q. Likewise for the R73E substitution in the presence of
dermatan sulfate, the decrease is 620-fold compared with a 48-fold
decrease for R73Q. Thus, charge reversal has a major effect on the
association rate constant. It seems likely that the charge reversal has
affected a number of contacts in the exosite-I important for the
formation of a stable complex.
On the basis of a large effect of exosite-I mutations and the small
effects by exosite-II mutations, Sheehan et al. (20) concluded that the role of exosite-II for glycosaminoglycan-stimulated inhibition of thrombin by HCII was unimportant. They proposed that the
major determinant for inhibition by HCII was allosteric, i.e. glycosaminoglycans bind to HCII and displace the N
terminus that can then interact with exosite-I. However, many of the
decreases in kon for the exosite-II mutants in
the presence of heparin (14, 20) are of similar magnitude to the
decreases in kon seen for the exosite-I mutants
in this study. Taking into account the charge reversal of the
exosite-II mutants, it seems that both exosites play an important part
in heparin-mediated inhibition of thrombin by HCII. In contrast,
exosite-II mutants had no effect on dermatan sulfate-accelerated
inhibition of thrombin by HCII (20). This suggests that the two
glycosaminoglycans employ different mechanisms for the inhibition of
thrombin by HCII. Heparin employs the double-bridge model, whereby it
binds to HCII displacing the acidic N-terminal region from the
glycosaminoglycan binding site, allowing it to interact with the
thrombin exosite-I through complementary electrostatic fields and also
by acting as a template, bridging the HCII to exosite-II. Dermatan
sulfate does not use exosite-II at all; therefore, the mode of thrombin
inhibition is strictly allosteric.
*
This work was supported in part by the British Heart
Foundation of Great Britain (to S. R. S.) and by National Institutes of Health Research Grant HL-32656 (to F. C. C).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
¶
To whom reprint requests may be addressed. Fax: 41-61-6973976;
E-mail: myles{at}fmi.ch.
**
To whom reprint requests may be addressed. Fax: 919-966-7639;
E-mail: fchurch{at}email.unc.edu.
The abbreviations used are:
ATIII, antithrombin
III; PN1, protease nexin 1; HCII, heparin cofactor II; pIIa, plasma
thrombin; rIIa, recombinant native thrombin.
1
The nomenclature of Schechter and Berger (44) is
used to describe the interaction between the protease reactive site
cleft subsites and the corresponding side chain of one residue of the target ligand.
3
The numbering of thrombin residues is according
to Bode et al. (45), which is based on chymotrypsinogen
numbering. Insertion residues are shown as superscripted lowercase
letters in alphabetical order (e.g.
Lys149e is the 5th residue inserted at position 149).
4
Standard nomenclature is used to describe mutant
thrombins. The first letter shows the amino acid in the wild type to be
replaced, and the second letter shows the amino acid used for the
substitution (e.g. the mutant thrombin R67Q represents the
substitution of Arg67 with Gln.
5
T. Myles and S. R. Stone, manuscript in preparation.
Role of Thrombin Anion-binding Exosite-I in the Formation of
Thrombin-Serpin Complexes*
§¶,
**,
,
Department of Haematology, University of
Cambridge, MRC Centre, Hills Road, Cambridge, CB2-2QH, United
Kingdom, the Department of Pathology and Laboratory Medicine,
Department of Medicine, and Center for Thrombosis and Hemostasis, The
University of North Carolina School of Medicine, Chapel Hill, North
Carolina 27599-7035, and § Friedrich Miescher Institut,
Postfach 2543, Basel CH-4058, Switzerland
ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
-,
T- (cleaved at Arg77a), and
T-thrombin (cleaved at Arg67,
Arg77a, and Lys149e) by
HCII and ATIII in the absence and presence of glycosaminoglycan (21)
have shown that anion-binding exosite-I of thrombin is important for
rapid inhibition by HCII. HCII contains an N-terminal acidic region
(residues 56-75) similar to the C-terminal tail of hirudin (22). The
HCII acidic domain is required for inhibition of thrombin in the
presence of glycosaminoglycan by interacting with thrombin's
exosite-I, and it is also postulated to bind to the glycosaminoglycan
binding site in the absence of glycosaminoglycans (22-24). Thus, HCII
inhibition of thrombin with glycosaminoglycan is consistent with a
"double-bridge" mechanism where (i) glycosaminoglycan binds both to
the glycosaminoglycan binding site of HCII and exosite-II of
-thrombin, forming a ternary complex, and (ii) the displaced acidic
domain of HCII interacts with thrombin's exosite-I, thereby facilitating rapid complex formation (21, 24). Sheehan et al. (20) examined the HCII inhibition of thrombin exosite-I and
exosite-II mutants in the presence of glycosaminoglycans. These data
suggest that dermatan sulfate acceleration of HCII inhibition is
primarily due to only an "allosteric" mechanism in which dermatan
sulfate binding to the serpin promotes an interaction with exosite-I
(essentially the same as in (ii) above). It was also suggested that the
ternary complex (described in (i) above) played a minor role during the
heparin-accelerated HCII inhibition of thrombin (20).
EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
where P is the amount of p-nitroanilide at
time t, k' is the apparent first order rate constant, and
v0 and vs are the initial and steady-state velocities, respectively. Progress curves fitted to
the equation gave estimates for k',
v0, and vs. The
association rate constant kon was calculated
using the following equation (33):
(Eq. 1)
where [S] is the concentration of S2238 and
Km is the Michaelis constant. At least two progress
curves were performed for each enzyme-inhibitor combination, with the
calculated values for kon reported as a weighted mean.
![k<SUB><UP>on</UP></SUB>=k′<FENCE>1−v<SUB>s</SUB>/v<SUB>0</SUB></FENCE><FENCE>1+[<UP>S</UP>]/K<SUB>m</SUB></FENCE>/[<UP>I</UP>]](/content/vol273/issue47/fulltext/31203/img002.gif)
(Eq. 2)
-thrombin complexed with residues 55-65 of hirudin,
and D-Phe-Pro-Arg-chloromethyl ketone was obtained from the
Brookhaven Protein Data Bank (Ref. 35; Brookhaven entry 1DWE). The
coordinates of the backbone atoms in the two thrombin molecules were
aligned in Insight II with a root mean square deviation of 0.595. Connolly solvent-accessible surfaces for heparin cofactor II, thrombin,
D-Phe-Pro-Arg-chloromethyl ketone, and hirudin were created
in Insight II using a probe radius of 1.4 Å.
RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References
Effect of thrombin anion-binding exosite-I mutants during antithrombin
III inhibition in the absence and presence of heparin
Effect of thrombin anion-binding exosite-I mutants during protease
nexin-1 inhibition in the absence and presence of heparin
1
s1) and K149eQ (1.82 × 104 M1 s1) compared
with rIIa (1.23 × 104 M1
s1). A similar trend was seen for PN1, where the greatest
differences were only 1.5-fold for R73Q (0.89 × 106
M1 s1) and
K149eQ (1.84 × 106
M1 s1) when compared with rIIa
(1.23 × 106 M1
s1) (Table II).
1 s1 and 1.19 × 108 M1 s1,
respectively) at 50 nM heparin (Fig. 1, Table I). Maximal
kon rates for PN1 were seen with pIIa (2.56 × 109 M1 s1) and
rIIa (2.52 × 109 M1
s1) at 20 nM heparin (Fig. 1, Table II). PN1
and ATIII were then used at their optimal concentration of heparin for
inhibition studies on the thrombin exosite-I mutants.
View larger version (30K):
[in a new window]
Fig. 1.
Serpin-glycosaminoglycan inhibition of
wild-type recombinant and plasma thrombins. Inhibition of
wild-type recombinant thrombin (squares) and plasma thrombin
(circles) was determined with antithrombin III
(A), protease nexin-1 (B), and heparin cofactor
II (C and D) with various concentrations of
glycosaminoglycans as described under "Experimental
Procedures."
1 s1) (Table
III), which were approximately 2-fold
higher to previously published values (21). This variance is most
likely due to differences in reaction temperature (37 °C
versus ambient temperature) and buffer systems. With the
exception of K149eQ, the exosite-I mutants show
between 2-3-fold reductions in kon values
(Table III).
Effect of thrombin anion-binding exosite-I mutants during heparin
cofactor II inhibition in the absence and presence of
glycosaminoglycans
1 s1 and 4.5 × 107 M1 s1 at 500 nM heparin for rIIa and pIIa, respectively (Fig. 1, Table III). Dermatan sulfate at 30 µM gave optimal
kon values for rIIa and pIIa of 2.2 × 107 M1 s1 and
1.8 × 107 M1
s1, respectively (Fig. 1, Table III). The
kon values were slightly different than
previously reported (20, 21), likely due both to differences in
glycosaminoglycans and in buffer/temperature conditions. HCII was then
used at its optimal concentration of heparin and dermatan sulfate for
inhibition studies on the thrombin exosite-I mutants.
DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
T-thrombin and thrombin Quick I
(Arg67
Cys) by HCII (21, 39, 40). Although the slightly
reduced kon values for the exosite-I mutants
with HCII without glycosaminoglycan suggest that the acidic N-terminal
domain is unlikely to make direct contact with exosite-I, these
decreases indicate the possibility that a significant electrostatic
field generated by the bound acidic domain of HCII can interact with
the positive electrostatic potential of exosite-I. Therefore, exosite-I
mutations that decrease this electrostatic field strength could
slightly reduce the association rate constant between HCII and
thrombin, which is consistent with our data.
View larger version (66K):
[in a new window]
Fig. 2.
Molecular model of thrombin, thrombin-HCII
bimolecular complex, and thrombin-HCII-hirudin peptide trimolecular
complex. Above, the three-dimensional structure of
thrombin with D-Phe-Pro-Arg-chloromethyl ketone (depicted
in white) in the active site. Anion-binding exosite-I
residues of thrombin that were mutated in this study are highlighted in
orange (clockwise from the southern rim of the structure
Lys149e, Arg75,
Arg77a, Arg35, Lys36,
Lys109, Lys110, Lys81) and
red (the upper residue shown is Arg67 and the
lower is Arg73). The rest of the residues composing
exosite-I are shown in yellow. Center, thrombin
complexed with HCII. The first 99 residues (including both hirudin-like
acidic domains from the N-terminal region) are not included in the
model. The first amino acid residue (Gly100) of the HCII
model is shown in red. As the acidic domain of HCII is
missing from this model, ATIII and PN1 should have a similar
interaction surface area with thrombin. Below, thrombin-HCII
complex as shown in the center panel, except the C-terminal
hirudin peptide (residues 55-65) shown in purple has been
added to the molecular model. The position of the hirudin peptide was
taken from the PDB file 1DWE and represents the probable interaction of
HCII's first acidic domain with anion-binding exosite-I of
thrombin.
1 s1, which is more rapid than
that usually observed for neutral molecules. The magnitude of the
kon value suggested that electrostatic forces accelerated the interaction. The heparin-PN1 complex could use the
negative electrostatic field generated by the heparin molecule to
interact with the positive electrostatic field of exosite-II to
accelerate the interaction and to promote a correct orientation for
productive complex formation (41). Complex formation could be further
enhanced if heparin-bound PN1 could also utilize the electrostatic
field generated by thrombin's exosite-I through electrostatic
steering. Our results indicate that several of the exosite-I residues
may participate to some extent to optimally align the PN1-heparin
complex during thrombin inhibition.
View larger version (46K):
[in a new window]
Fig. 3.
Alignment of acidic domains from various
macromolecules involved in binding to thrombin's anion-binding
exosite-I. Alignment of hirudin, hirullin, thrombin receptor, and
thrombomodulin is based on the comparison of crystallographic
structures of each ligand complexed to -thrombin. Common to all of
the structures was a Phe or Tyr (arrowhead) that binds in a
pocket formed by Phe34, Arg73, and
Thr74. Also, salt-bridge interactions with
Arg73, Arg75, and Arg77a
were considered for these alignments. The remaining alignments were
arbitrary and, where possible, based on importance of Phe and Tyr that
bind in the pocket formed by Phe34, Arg73, and
Thr74.
FOOTNOTES
Supported by National Institutes of Health Research Grant
5-T32- HL-07149.
Deceased December 16, 1996. This paper is dedicated to his memory.
REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.
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