From McMaster University and the Henderson Research Centre, Hamilton, Ontario L8V 1C3, Canada
Received for publication, November 22, 2000, and in revised form, March 9, 2001
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ABSTRACT |
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Although fibrin-bound thrombin is resistant
to inactivation by heparin·antithrombin and heparin·heparin
cofactor II complexes, indirect studies in plasma systems
suggest that the dermatan sulfate·heparin cofactor II complex can
inhibit fibrin-bound thrombin. Herein we demonstrate that fibrin
monomer produces a 240-fold decrease in the heparin-catalyzed rate of
thrombin inhibition by heparin cofactor II but reduces the dermatan
sulfate-catalyzed rate only 3-fold. The protection of fibrin-bound
thrombin from inhibition by heparin·heparin cofactor II reflects
heparin-mediated bridging of thrombin to fibrin that results in the
formation of a ternary heparin·thrombin·fibrin complex. This
complex, formed as a result of three binary interactions
(thrombin·fibrin, thrombin·heparin, and heparin·fibrin), limits
accessibility of heparin-catalyzed inhibitors to thrombin and induces
conformational changes at the active site of the enzyme. In contrast,
dermatan sulfate binds to thrombin but does not bind to fibrin.
Although a ternary dermatan sulfate· thrombin·fibrin complex
forms, without dermatan sulfate-mediated bridging of thrombin to
fibrin, only two binary interactions exist (thrombin·fibrin and
thrombin· dermatan sulfate). Consequently, thrombin remains
susceptible to inactivation by heparin cofactor II. This study explains
why fibrin-bound thrombin is susceptible to inactivation by heparin
cofactor II in the presence of dermatan sulfate but not heparin.
Heparin, a sulfated polysaccharide, acts as an anticoagulant by
accelerating the inhibition of thrombin and factor Xa by antithrombin (1). Although heparin is widely used for the treatment of acute coronary ischemic syndromes, it has limitations in patients undergoing percutaneous coronary interventions (2) or when used as an adjunct to
thrombolytic therapy (3-5). These limitations have been attributed to
the inability of the antithrombin·heparin complex to inactivate
clotting enzymes bound to components of the thrombus, particularly
thrombin bound to fibrin (6, 7).
Resistance of fibrin-bound thrombin to inactivation by the
antithrombin·heparin complex reflects the incorporation of thrombin into a ternary heparin·thrombin·fibrin complex (8-10). To form this complex, heparin interacts with both exosite II on thrombin (11-13) and the D domain of fibrin (14), thereby bridging thrombin to
fibrin via exosite II (8). This heightens exosite I-mediated binding of
thrombin to fibrin and likely increases the overall affinity of
thrombin for fibrin. The formation of the ternary complex, therefore,
is a consequence of the presence of two exosites on thrombin, which
independently bind fibrin and heparin. Recently, we demonstrated that
protection requires ligation of both of thrombin's exosites within the
ternary heparin·thrombin·fibrin complex, a process that impairs
access of inhibitor-bound heparin to exosite II on thrombin (15).
Thrombin within the ternary complex is protected from inactivation by
the heparin·heparin cofactor II (HCII)1 complex to a greater
extent than the heparin·antithrombin complex (15), because access of
the amino terminus of HCII to exosite I on thrombin, an obligatory part
of the HCII inhibitory mechanism (16, 17), is reduced. Allosteric
changes in the active site of thrombin induced upon formation of the
ternary heparin·thrombin·fibrin complex may also contribute to the
protection of fibrin-bound thrombin from inactivation by
heparin·antithrombin and heparin·HCII complexes by limiting
inhibitor reactivity with fibrin-bound thrombin (18).
Dermatan sulfate (DS), a sulfated glycosaminoglycan that has
antithrombotic activity in laboratory animals (19, 20) and in humans
(21-23), acts as an anticoagulant by catalyzing only HCII. Because
thrombin is the exclusive plasma target of HCII, DS is considered a
selective inhibitor of thrombin (16). Although fibrin-bound thrombin is
protected from inactivation by the heparin·HCII complex (15),
indirect studies done in plasma systems suggest that fibrin-bound
thrombin is susceptible to inactivation by the DS·HCII complex (24,
25). The purpose of this study was to confirm these findings using
purified reagents and to elucidate the mechanism by which the DS·HCII
complex, but not the heparin·HCII complex, inactivates fibrin-bound thrombin.
Materials
Human HCII, isolated from plasma by affinity chromatography, was
from Affinity Biologicals Inc. (Hamilton, Ontario, Canada). Human Preparation of Soluble Fibrin--
Human fibrinogen, treated
with gelatin-agarose to remove fibronectin, was used to prepare soluble
fibrin (SF), as described previously (15). Briefly, fibrin clots were
centrifuged and dialyzed versus water. The fibrin was then
dissolved by dialysis versus 20 mM acetic acid,
aliquoted, and stored at a concentration of about 100 µM
at Preparation of Fibrin Monomer-Sepharose--
Fibrinogen was
coupled to cyanogen bromide-activated Sepharose 4B, treated with
thrombin to convert it to fibrin monomer (FM), and washed with 20 mM Tris-HCl, pH 7.4, 150 nM NaCl (TBS) as
described previously (15).
Methods
Rates of Thrombin Inhibition by HCII in the Absence or Presence
of SF, Glycosaminoglycans, or Both--
The influence of varying
concentrations of heparin or DS on the second-order rate constants
(k2) for inhibition of thrombin by HCII were
determined under pseudo-first-order conditions in the absence or
presence of SF. Thrombin (10 nM) was incubated for 5 min at
room temperature in TBS containing 0.6% PEG 8000 in the presence of
various concentrations of heparin or DS (0-11 µM), SF
(0-4 µM), and 10 mM GPRP-NH2.
Reaction mixtures (10 µl) were aliquoted to 96-well round bottom
microtiter plates and an equal volume of HCII (in a concentration at
least 10-fold higher than that of thrombin) was added to each well at
time intervals ranging from 2 s to 5 min. All reactions were
terminated concomitantly by the addition of 200 µM
chromogenic substrate (tGPR-pNA) in 200 µl of TBS containing 10 mg/ml
Polybrene. Residual thrombin activity was calculated by measuring
absorbance at 405 nm for 5 min using a Spectra Max 340 Microplate
Reader (Molecular Devices, Menlo Park, CA). The pseudo-first-order rate
constants (k1) for thrombin inhibition were
determined by fitting the data to the equation
k1·t = ln([P]o/[P]t), where [P]o is
initial thrombin activity and [P]t is thrombin activity at
time t. The second-order rate constant,
k2, was then determined by dividing
k1 by the HCII concentration (27).
Effect of Heparin and DS on the Binding of
125I-FPR-thrombin to FM-Sepharose--
Thrombin blocked at
its active site with FPRCK (FPR-thrombin) was radiolabeled with
Na125I as described previously (28). The binding of
125I-FPR-thrombin to FM-Sepharose in the absence or
presence of either heparin or DS (from 0 to 10 µM) was
studied in TBS containing 0.6% PEG 8000 and 0.01% Tween 20. FM-Sepharose (9 µM) in 0.5 ml of buffer was incubated
with 50 nM 125I-FPR-thrombin by inverted mixing
for 2 min. The tube was centrifuged in a microcentrifuge for 2 min at
15,000 × g, and a 100-µl aliquot of supernatant was
removed for gamma counting. The aliquot was returned to the tube. This
procedure was repeated following each addition of heparin or DS to the
tube. The fraction of 125I-FPR-thrombin bound to
FM-Sepharose as a function of the total amount of
125I-FPR-thrombin present was calculated.
Effect of Thrombin or Effect of Ternary Thrombin·Fibrin·Heparin or
Thrombin·Fibrin·DS Complex Formation on the Active Site Environment
of Thrombin--
Thrombin labeled at the active site with
anilinonaphthalene-6-sulfonic acid (ANS-FPR-thrombin) was prepared
using ATA-FPR (N-(acetylthio)acetyl-D-Phe-Pro-Arg-CH2Cl)
as described by the supplier (Molecular Innovations Inc., Royal Oak,
MI). Briefly, 10.6 µM thrombin was incubated for 30 min
at 23 °C with a 2.5-fold molar excess of ATA-FPR in 20 mM HEPES-NaOH, pH 7.0, 100 mM NaCl, 1 mM EDTA (HBSE buffer). The reaction mixture was dialyzed
against HBSE buffer, incubated with a 10-fold molar excess of
2-(4-(iodoacetamide)anilino)naphthalene-6-sulfonic acid (Molecular
Probes Inc.) in the presence of hydroxylamine for 60 min at 23 °C in
the dark and then redialyzed. 800 µl of 140 nM
ANS-FPR-thrombin in 50 mM HEPES, pH 7.5, 0.125 M NaCl, 1 mg/ml PEG 8000, 5 mM
GPRP-NH2 was added to a semi-micro quartz cuvette. Using a
PerkinElmer Life Sciences LS50B luminescence spectrometer with
excitation wavelength set to 334 nm and excitation and emission slit
widths set to 5 nm, the fluorescence emission spectrum from 380-580 nm
of ANS-FPR-thrombin was monitored before and after the addition of 310 nM SF and/or 100 nM heparin or 3 µM DS. Soluble fibrin was neutralized by the addition of
40% v/v 1 M Tris-HCl, pH 7.5, just prior to use. Addition
of 1 M Tris-HCl, pH 7.5, had no effect on the fluorescence
spectrum of ANS-FPR-thrombin (data not shown).
Determination of the Affinities of f-heparin, f-DS, and
f-FPR-thrombin for Fibrin Clots--
Fluorescent derivatives of
heparin and DS were prepared as follows (29). 10 mg of heparin or DS
were incubated with 15 mg of FITC in 2.5 ml of 1.0 M
Na2CO3, pH 9.0 for 5 h at 23 °C. After centrifugation at 13,000 × g for 5 min, 1 ml of the
supernatant was applied to duplicate PD-10 gel filtration columns
(Millipore Corp., Bedford, MA), equilibrated with H2O, and
eluted with H2O under gravity. 0.5-ml fractions were
collected, frozen, and lyophilized. Recovered material was pooled,
weighed, and dissolved in TBS to a stock concentration of 10 mg/ml.
FITC-labeled active site-blocked thrombin (f-FPR-thrombin) was prepared
as previously described (28). Briefly, thrombin was incubated with
fluorescein-D-Phe-Pro-Arg chloromethyl ketone (f-FPRCK)
until no residual thrombin chromogenic activity was detected. After
dialysis to remove unincorporated f-FPRCK, the concentration of
fluorescently labeled thrombin was determined by measuring absorbance
using
The affinity of FITC-labeled ligands for fibrin was determined by
measuring unbound ligand in supernatants of clots prepared by clotting
varying concentrations of fibrinogen with 1 nM thrombin. Briefly, 200 µl of 2 mM CaCl2, 100 nM FITC-labeled ligand, various concentrations of
fibrinogen (30-3000 nM), and 1 nM thrombin
were mixed in a series of microcentrifuge tubes. After 1-h incubation at 23 °C, fibrin was pelleted by centrifugation for 5 min at
15,000 × g and 100 µl of supernatant was removed and
added to 300 µl of TBS. The fluorescence intensity of the samples was
monitored with excitation and emission wavelengths set to 492 and 522 nm, respectively, and excitation and emission slit widths both set to
15 nm. Kd values were calculated by plotting
I/Io versus fibrinogen
concentration, where Io and I
represent the fluorescence intensities in the absence and presence of
the varying concentrations of fibrinogen, respectively. The parameters
Kd and Determination of the Affinities of Thrombin for Heparin and
DS--
The affinities of heparin or DS for thrombin were determined
by monitoring changes in intrinsic thrombin fluorescence when the
enzyme was titrated with heparin or DS. The initial intensity reading
of 100 nM thrombin in a 2-ml quartz cuvette
(Io) was determined with excitation and emission
wavelengths set to 280 and 340 nm, respectively, and excitation and
emission slit widths set to 6 nm. Aliquots of either heparin or DS were
then added to the cuvette, and, after mixing, changes in fluorescence
were monitored (I). Kd values were
calculated by plotting I/Io
versus glycosaminoglycan concentration and the data were fit
by nonlinear regression to the equation given above.
Determination of the Affinities of HCII for Heparin, DS, and
SF--
The association between HCII and either heparin, DS, or fibrin
was monitored by the ligand-dependent fluorescence
intensity change of anilinonaphthalene-6-sulfonic acid labeled HCII
(ANS-HCII). ANS-HCII, prepared as previously described (30), was
added to a concentration of 100 nM to a quartz cuvette. The
initial fluorescence intensity (Io) of ANS-HCII
was determined at excitation and emission wavelengths set to 280 and
437 nm, respectively, and excitation and emission slit widths set to 10 nm, and an emission filter of 290 nm. Known quantities of either
heparin, DS, or SF were then added to the cuvette and, after mixing,
changes in fluorescence was monitored (I).
Kd values were calculated by plotting I/Io versus ligand
concentration, and the data were fit by nonlinear regression to the
equation given above.
Displacement of Thrombin from FM-Sepharose by HCII--
500-µl
suspensions of FM-Sepharose (9 µM FM) containing 50 nM 125I-FPR-thrombin without glycosaminoglycan
or with 2.5 µM DS in TBS/0.6% PEG/0.01% Tween 20 were
mixed for 2 min. The amount of unbound 125I-FPR-thrombin in
the suspension after each addition of an aliquot of 80 µM
HCII was determined as described above.
Comparison of the Effect of SF on Heparin- and DS-catalyzed Rates
of Thrombin Inhibition by HCII--
To verify the protective effect of
SF on thrombin inhibition by HCII, we determined the rates of thrombin
inhibition in the absence or presence of SF and heparin. As shown in
Fig. 1A, SF caused a
dose-dependent decrease in the heparin-catalyzed rates of
thrombin inhibition by HCII, which by two-way analysis of variance performed using Minitab (software version 11, State College, PA), was
highly significant (p < 0.001). At 1 µM
heparin and 4 µM SF, a maximal 240-fold decrease in the
rate was observed, a value consistent with that reported previously
(15). In contrast, at a concentration of 4 µM SF, a
significant (p < 0.001) but modest 3-fold decrease in
the DS-catalyzed rates of thrombin inhibition by HCII was observed
(Fig. 1B).
Quantification of Binary Interactions That Comprise Ternary
Thrombin·Fibrin·Glycosaminoglycan Complexes--
The assembly of
the ternary heparin·thrombin·fibrin complex is postulated to occur
through a series of binary interactions between thrombin·fibrin,
thrombin·heparin, and fibrin·heparin (8, 10, 18). In this study, we
determined the dissociation constants for these interactions, as well
as those involving DS. The affinities of DS and heparin for thrombin
were determined by monitoring changes in intrinsic protein fluorescence
of thrombin when titrated with DS or heparin (not shown). DS and
heparin bind saturably to thrombin with Kd values of
2600 and 116 nM, respectively (Table
I). The Kd value for
the thrombin·heparin interaction is in agreement with that determined
by titration of ANS-labeled thrombin with increasing concentrations of
heparin (Kd = 59 nM) (10).
Current evidence suggests that both heparin and DS bind to exosite II
on thrombin (11-13, 31). This was confirmed in two ways. First,
competitive binding studies were performed where thrombin-bound
f-heparin was displaced by DS (Fig. 2).
In this experiment, addition of thrombin to f-heparin resulted in an
approximate 3.5% decrease in fluorescence intensity. Subsequent
titration of the sample with DS resulted in a
dose-dependent increase of fluorescence intensity to the
value observed for f-heparin prior to binding to thrombin
(I/Io of 1). Likewise, in the
reciprocal experiment, the fluorescence decrease that occurred upon
addition of thrombin to f-DS was negated in a
dose-dependent fashion by titration with heparin (not
shown). In a second approach, we used thrombin variants with impaired
exosites to identify the DS binding site (not shown). DS binds to
The affinities of f-FPR-thrombin, f-heparin, and f-DS for fibrin were
monitored by clotting varying concentrations of fibrinogen with a
catalytic amount of thrombin in the presence of a fluorescently labeled
ligand, and quantifying unbound ligand in the clot supernatant (not
shown). As listed in Table I, f-FPR-thrombin and f-heparin bind to
fibrin with Kd values of 1500 and 187 nM, respectively, values consistent with previous reports
(8, 10). In contrast, f-DS does not bind to fibrin.
Both heparin and DS bind to ANS-HCII, but the affinity of heparin for
HCII is 5-fold higher (13 and 71 µM, respectively). Thus,
DS binds to both HCII and thrombin with lower affinity than heparin. No
binding of ANS-HCII to fibrin was detected (Table I).
Effect of Heparin and DS on the Binding of
125I-FPR-thrombin to Fibrin--
It has been shown
previously that heparin enhances the binding of thrombin to fibrin, an
effect that occurs regardless of whether heparin has high or low
affinity for antithrombin, but occurring only with heparin chains of
11,200 Da or more (8). In this study, we compared the ability of DS to
promote thrombin binding to FM-Sepharose with that of heparin and
low-molecular-weight heparin. As shown in Fig.
3, DS has little effect on
125I-FPR-thrombin binding to FM-Sepharose, even at
concentrations up to 10 µM. In contrast, at
concentrations up to 250 nM, heparin enhances
125I-FPR-thrombin binding to FM-Sepharose in a
dose-dependent manner. At heparin concentrations above 250 nM, 125I-FPR-thrombin binding to fibrin monomer
decreases, likely reflecting the accumulation of distinct
heparin·fibrin and heparin·thrombin populations (8). When thrombin
and FM-Sepharose were titrated with low-molecular-weight heparin
(enoxaparin), there was only a small increase in the amount of thrombin
bound, comparable to that observed with DS. Similar results were
obtained with fibrin clots in place of FM-Sepharose (not shown).
Incorporation of Thrombin into Ternary
Glycosaminoglycan·Thrombin·Fibrin Complexes--
Our binding
studies indicate that thrombin can interact with both fibrin and DS,
via exosites I and II, respectively. These findings suggest that, like
heparin, DS can form a three-component complex with thrombin and
fibrin, even though DS does not bind fibrin. To explore this
possibility, a mixture of 500 nM 125I-labeled
DS and 5 µM FM-Sepharose was titrated with increasing concentrations of thrombin or Influence of Ternary Thrombin·Fibrin·Glycosaminoglycan Complex
Formation on the Conformation of the Active Site of
Thrombin--
Incorporation of thrombin into a ternary
heparin·thrombin·fibrin complex alters the rate of
thrombin-mediated hydrolysis of chromogenic substrates (9, 10, 15),
suggesting that ternary complex formation induces conformational
changes in the active site of thrombin that may limit its reactivity
with macromolecular inhibitors (18). To determine whether thrombin in a
DS·thrombin·fibrin complex also experiences conformational changes
in its active site, the fluorescence of ANS-FPR-thrombin was monitored
upon addition of DS and/or SF, as was described previously for heparin or SF (10). As illustrated in Fig.
5A, addition of 310 nM SF or 100 nM heparin to 140 nM
ANS-FPR-thrombin produces a 19 and 48% increase in fluorescence,
respectively, compared with the fluorescence of ANS-thrombin alone.
When 100 nM heparin is added to the ANS-FPR-thrombin/SF
mixture, however, there is an 86% increase in fluorescence. The
observation that the addition of both SF and heparin produces a greater
change in the emission spectrum of ANS-FPR-thrombin than the two
components alone is in agreement with a previous report by Hogg
et al. (10) and suggests that formation of the ternary
heparin· thrombin·fibrin complex induces conformational
changes in the active site of thrombin beyond those produced by
formation of binary thrombin·fibrin or thrombin·heparin complexes.
In contrast to the results obtained with heparin, the addition of 3 µM DS, a concentration above the Kd
value for the DS·thrombin interaction (Table I), to either
ANS-FPR-thrombin alone or to the ANS-FPR-thrombin/SF mixture has
minimal effect on the emission spectra (Fig. 5B). These
findings suggest that, unlike heparin, DS does not induce
conformational changes in the active site environment of thrombin in
the absence or presence of fibrin.
Effect of Heparin and Low-molecular-weight Heparin on the Rate of
Inhibition of Fibrin-bound Thrombin by the DS·HCII
Complex--
Although fibrin-bound thrombin is susceptible to
inactivation by the DS·HCII complex (Fig. 1), we predicted that
heparin, by virtue of its higher affinity for both fibrin and thrombin than DS, would impair DS activity by bridging thrombin to fibrin, thereby enhancing the fibrin·thrombin interaction. As a control, we
used low-molecular-weight heparin, because only heparin chains of
>11.2 kDa are of sufficient length to bridge thrombin to fibrin (8).
In the presence of 4 µM SF, heparin concentrations of 0.1 µM or higher cause a decrease in the 3.3 µM
DS-catalyzed rate of thrombin inhibition by HCII (Fig.
6). In contrast, a low-molecular-weight heparin, enoxaparin, has no effect on the rate of inhibition of fibrin-bound thrombin by the DS·HCII complex. Neither heparin nor
low-molecular-weight heparin influenced the DS-catalyzed rate of
thrombin inhibition in the absence of SF (data not shown).
Displacement of IIa from FM-Sepharose by HCII--
Because
catalysis of thrombin inhibition by HCII is not impaired by fibrin in
the presence of DS, exosite I on thrombin must be accessible to the
DS·HCII complex even though thrombin binds to fibrin via this site.
This observation predicts that the DS·HCII complex is capable of
displacing thrombin from fibrin by binding the amino-terminal tail of
HCII to exosite I of thrombin. This was tested by monitoring the amount
of 125I-FPR-thrombin displaced from FM-Sepharose by
increasing concentrations of HCII in the absence or presence of DS
(Fig. 7). HCII alone, at concentrations
up to three times physiological concentration, had limited
capacity to displace thrombin from FM-Sepharose. However, in the
presence of 2.5 µM DS, dose-dependent
displacement of thrombin by HCII was observed. Maximal displacement was
achieved with physiological concentrations of HCII, with half-maximal
effect at about 250 nM HCII. Thus, the amino terminus of
HCII is able to compete effectively with fibrin for binding to thrombin
exosite I.
In this study, we demonstrate that fibrin-bound thrombin is
readily inhibited by the DS·HCII complex, but not by the
heparin·HCII complex, even though heparin and DS demonstrate
comparable catalysis of HCII in the absence of fibrin (Fig. 1). The
resistance of fibrin-bound thrombin to inhibition by the heparin·HCII
complex is a consequence of formation of a ternary
heparin·thrombin·fibrin complex (15). This ternary complex forms as
a result of mutual binary interactions between each of the three
components, resulting in increased affinity of each of the individual
interactions (10, 18). Formation of this ternary complex protects
thrombin from inhibition by heparin·HCII by reducing access of the
amino terminus of HCII to exosite I on thrombin, impairing the ability
of HCII-bound heparin to bridge HCII to exosite II on thrombin, and
inducing allosteric changes in the active site of thrombin that may
limit its reactivity with HCII (10, 15).
Like heparin, DS binds to exosite II on thrombin, even when thrombin is
fibrin-bound (Fig. 2). However, DS and heparin are distinguished by the
fact that only heparin binds fibrin (Table I). With high affinity for
thrombin and fibrin, heparin augments exosite I-mediated binding of
thrombin to fibrin, impairing the ability of HCII to bind and inhibit
thrombin (Fig. 3). Because DS does not heighten the thrombin·fibrin
interaction, the amino terminus of DS-activated HCII retains access to
exosite I on thrombin. The failure of DS to induce a conformational
change in the active site of thrombin (Fig. 5B) may also
permit reactivity of HCII with fibrin-bound thrombin. These results
confirm the hypothesis that all three binary interactions
(thrombin·fibrin, thrombin·glycosaminoglycan, glycosaminoglycan·fibrin) within the ternary complex are
integral to convey protection of fibrin-bound thrombin from inhibition by heparin-catalyzed inhibitors (15).
Heparin compromises the DS-catalyzed rate of thrombin inhibition by
HCII in the presence of fibrin (Fig. 6). This reduction in rate is
consistent with the formation of a ternary thrombin·fibrin·heparin complex. Heparin can bridge thrombin to fibrin in the presence of DS,
because the affinity of heparin for thrombin is higher than that of DS
(Table I). In contrast, low-molecular-weight heparin (enoxaparin) does
not reduce the DS-catalyzed rate of thrombin inhibition by HCII,
because only heparin chains greater than 11.2 kDa are of sufficient
length to bind simultaneously to thrombin and fibrin (8).
The DS·HCII complex displaces thrombin from FM-Sepharose with an
IC50 value of 0.17 µM (Fig. 7). This value
reflects the ability of the amino terminus of DS-activated HCII to bind
to thrombin exosite I. A previous study determined that a 22-residue
peptide, corresponding to amino acids 54-75 of HCII, inhibited the
clotting activity of thrombin with an IC50 of 28 µM (33). The lower IC50 value with intact
HCII suggests that there are additional interactions with thrombin
provided by the remainder of the amino terminus or the body of the HCII
molecule. A homologous peptide derived from hirudin residues 54-66
inhibited thrombin clotting activity with an IC50 of 0.8 µM (33). This compares with the Ki value of 0.6 µM derived for the initial encounter of
intact hirudin with thrombin (34). Thus, in addition to its ability to
inhibit fibrin-bound thrombin, the DS·HCII complex displaces thrombin from fibrin allowing both HCII and antithrombin to inhibit
thrombin. A similar displacement role for the
hirudin53-64 peptide has been proposed (35).
Our observation that fibrin-bound thrombin is susceptible to
inactivation by the DS·HCII complex suggests that DS should be effective for the prevention and treatment of thrombosis. In a randomized trial comparing subcutaneous DS with low dose subcutaneous heparin for thromboprophylaxis in patients undergoing general surgical
procedures, both agents had comparable efficacy (22). More recently, DS
was shown to be more effective than heparin at preventing postoperative
venous thrombosis in patients undergoing general surgical procedures
(36). Studies in rabbits suggest that DS suppresses thrombus accretion
to a greater extent than heparin when the two agents are given in doses
that have the same inhibitory activity against thrombin in
vitro (25).
DS produces only minimal inhibition of platelet and fibrin
deposition in a high shear, arterial-type thrombosis model in baboons (37). The limited efficacy of DS in this setting may reflect the fact
that DS only inhibits thrombin and has no effect on factor Xa-mediated
thrombin generation, an important contributor to thrombus growth (38).
Consequently, if selective thrombin inhibitors are used as monotherapy
for treatment of arterial thrombosis, high concentrations may be
necessary to prevent thrombus propagation. This concept is supported by
our recent studies demonstrating that both DS and hirudin, agents that
inhibit fibrin-bound thrombin, as well as free thrombin, are limited in
their ability to block clotting in a thrombogenic extracorporeal
circuit (30). In contrast, Vasoflux, a drug that not only inhibits
fibrin-bound thrombin but also blocks factor Xa generation, prevents
clotting in the circuit (30). These findings raise the possibility that
adding heparin to DS may improve its efficacy, because heparin will
block factor Xa-mediated thrombin generation. Our observation that
unfractionated heparin compromises the rate of thrombin inhibition by
the DS·HCII complex, whereas low-molecular-weight heparin does not
(Fig. 6), suggests that low-molecular-weight heparin may be a better
adjunct to DS than unfractionated heparin. The observation that DS and low-molecular-weight heparin display additive anticoagulant activity in vitro provides further support for this approach
(39).
In summary, our results indicate that fibrin-bound thrombin is readily
inhibited by the DS·HCII complex because, unlike heparin, DS does not
bridge thrombin to fibrin. Although DS and heparin can form
thrombin·fibrin·glycosaminoglycan complexes, the additional heparin·fibrin interaction strengthens the ternary complex, thereby limiting accessibility of thrombin exosites and inducing conformational changes in the active site of thrombin. These findings reveal that
resistance of thrombin to inhibition by serpins results from a
glycosaminoglycan·fibrin interaction, as well as interactions between
thrombin·fibrin and thrombin·glycosaminoglycan. These mechanistic
insights also demonstrate the advantages of glycosaminoglycans whose
activities are not compromised by fibrin.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-
and
-thrombin and fibrinogen were from Enzyme Research Laboratories
(South Bend, IN). Dermatan sulfate (DS) was from Mediolanum
Farmaceutici (Milan, Italy). Heparin,
N-p-tosyl-Gly-Pro-Arg-p-nitroanilide acetate (tGPR-pNA), gelatin-agarose, and Gly-Pro-Arg-Pro-amide (GPRP-NH2) were from Sigma Chemical Co. (St. Louis, MO).
Enoxaparin, a commercial low-molecular-weight heparin, was from
Rhône-Poulenc Rorer Canada (Montreal). Based on high performance
liquid chromatography gel filtration analysis, the mean molecular
masses of DS, heparin, and enoxaparin are 20 kDa (range 7-34 kDa), 15 kDa (range 5-30 kDa), and 4.5 kDa (range 3.5-5.5 kDa), respectively.
Fluorescein-5'-isothiocyanate (FITC) was from Molecular Probes Inc.
(Eugene, OR). D-Phe-Pro-Arg-chloromethyl ketone (FPRCK) was
from Calbiochem Novabiochem Corp. (San Diego, CA). FITC-FPRCK was from
Hematologic Technologies, Inc. (Essex Junction, VT). Hexadimethrine
bromide (Polybrene) was obtained from Aldrich Chemical Co.
(Milwaukee, WI). Cyanogen bromide-activated Sepharose 4B was from
Amersham Pharmacia Biotech (Dorval, Quebec).
70 °C. Polymerization of SF was blocked by addition of 5 mM GPRP-NH2 (26), and the material was
neutralized with 1 M Tris-HCl, pH 7.5, in a volume
corresponding to 40% of the volume of SF, just prior to use. A
molecular weight of 340,000 and
-Thrombin on the Binding of
125I-Labeled DS to FM-Sepharose--
DS (3 mg) was labeled
with 1.0 mCi of Na125I (PerkinElmer Life Sciences, Markham,
Ontario, Canada) in a volume of 300 µl using the IODO-BEAD iodination
reagent as described by the manufacturer (Pierce, Rockford, IL).
Labeled DS catalyzed the inhibition of thrombin by HCII to the same
extent as its unlabeled counterpart, indicating that the labeling
procedure did not influence DS activity. A mixture of 500 nM 125I-labeled DS and 5 µM
FM-Sepharose was titrated with increasing concentrations of thrombin or
-thrombin (from 0 to 3 µM). After centrifugation,
residual 125I-labeled DS in the supernatant was quantified
by gamma counting and used to determine the fraction of
125I-labeled DS bound to FM-Sepharose.
1.7 × A320.
I were calculated by nonlinear
regression ("Tablecurve," Jandel Scientific, San Rafael, CA) using
the equation,
where
(Eq. 1)
I is the maximum fluorescence change,
P is the initial concentration of f-heparin, f-DS, or
f-FPR-thrombin, L is the total fibrinogen concentration, and
a stoichiometry of 1 is assumed (28).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (17K):
[in a new window]
Fig. 1.
Effects of SF on the second-order rate
constants for the inhibition of thrombin by heparin- or DS-catalyzed
HCII. A, the second-order rate constants for
inhibition of 10 nM thrombin by at least 10-fold molar
excess of HCII in the presence of heparin and in the absence ( ) or
presence of 0.25 µM (
) or 4 µM (
) SF
were determined under pseudo-first-order conditions. B, the
second-order rate constants for inhibition of 10 nM
thrombin by 100 nM HCII in the presence of DS and in the
absence (
) or presence of 2 µM (
) or 4 µM (
) SF were determined under pseudo-first-order
conditions. Each point represents the mean of two
determinations, and the bars represent the standard error of
the mean.
Dissociation constants for the binary interactions constituting ternary
thrombin · fibrin · heparin and thrombin · fibrin · DS complexes
-thrombin, a proteolytic derivative of
-thrombin lacking exosite
I (11), with a 2.6-fold lower affinity than
-thrombin
(Kd values of 6.8 µM and 2.6 µM, respectively). In contrast, DS binds RA-thrombin, a
thrombin variant with three point mutations in exosite II that lower
its affinity for heparin 20-fold (32), with a 7-fold lower affinity (Kd = 17 µM). These studies confirm
that, like heparin, DS also binds to exosite II on thrombin, albeit
with lower affinity (Table I).
View larger version (9K):
[in a new window]
Fig. 2.
Displacement of f-heparin from thrombin by
DS. The fluorescence (Io) of 50 nM f-heparin was monitored at ex = 492 nm
and
em = 535 nm with an emission filter at 515 nm.
Addition of 200 nM thrombin caused an approximate 3.5%
decrease in intensity. The sample was then titrated with DS, and the
intensity was determined after each addition (I). Values for
I/Io are plotted versus DS
concentration, and the line represents nonlinear regression
analysis of the fit of the data to the binding isotherm equation.
View larger version (17K):
[in a new window]
Fig. 3.
Effect of heparin or DS on the binding of
125I-FPR-thrombin to FM-Sepharose. Suspensions
containing 50 nM 125I-FPR-thrombin and 9 µM FM-Sepharose in 0.5 ml were centrifuged, and 0.1-ml
aliquots were counted for radioactivity to determine the percentage of
125I-FPR-thrombin bound. Aliquots of heparin ( ),
low-molecular-weight heparin (
), or DS (
) were added to the
tubes, and the samples were mixed for 2 min. After each addition of
glycosaminoglycan, the samples were centrifuged and the supernatants
recounted. Each point represents the mean of two
determinations, and the bars represent the standard error of
the mean.
-thrombin (Fig.
4). After centrifugation, residual
125I-labeled DS in the supernatant was quantified and used
to determine the fraction of 125I-labeled DS bound to
FM-Sepharose. In the absence of thrombin, minimal amounts of
125I-labeled DS bind to FM-Sepharose. With thrombin
addition, 125I-labeled DS binds to FM-Sepharose in a
concentration-dependent fashion. In contrast, when
-thrombin, a proteolytic derivative of thrombin that lacks exosite
1, is substituted for thrombin, there is no increase in the amount of
125I-labeled DS that binds to FM-Sepharose. These data
indicate that a DS·thrombin·fibrin complex can form despite the
lack of direct DS·fibrin interactions.
View larger version (19K):
[in a new window]
Fig. 4.
Effect of thrombin or
-thrombin on the binding of
125I-labeled DS to FM-Sepharose. Binding of 500 nM 125I-labeled DS to 5 µM
FM-Sepharose was determined in the presence of thrombin (
) or
-thrombin (
) at the indicated concentrations. After each thrombin
addition, FM-Sepharose was sedimented by centrifugation, aliquots of
supernatant were counted for radioactivity, and the fraction of
125I-labeled DS bound to FM-Sepharose was calculated. Each
point represents the mean of at least two determinations,
and the bars reflect the standard error of the mean.
View larger version (11K):
[in a new window]
Fig. 5.
Influence of SF and/or heparin or DS on the
emission spectrum of ANS-thrombin. A, the emission
spectrum of ANS-thrombin, at excitation wavelength of 334 nm, was
monitored from 380 to 580 nm in the absence ( ) or presence of SF
(
), heparin (
), or the combination of SF and heparin (
).
B, the emission spectrum of ANS-thrombin was monitored in
the absence (
) or presence of SF (
), DS (
), or the combination
of SF and DS (
). For clarity, only every 15th point of each spectrum
is plotted.
View larger version (13K):
[in a new window]
Fig. 6.
Effect of heparin or low-molecular-weight
heparin on DS-catalyzed rates of thrombin inhibition by HCII in the
presence of SF. The second-order rate constants for the inhibition
of 5 nM thrombin by HCII with 3.3 µM DS in
the presence of 4 µM SF were determined under pseudo
first-order conditions. Experiments were repeated in the presence of
increasing concentrations of heparin ( ) or low-molecular-weight
heparin (
). Each point represents the mean of at least
two determinations, and the bars represent the standard
error.
View larger version (15K):
[in a new window]
Fig. 7.
Displacement of thrombin from FM-Sepharose by
HCII in the absence or presence of DS or heparin. Suspensions of
10 µM FM-Sepharose containing 50 nM
125I-FPR-thrombin in the absence ( ) or presence of 2.5 µM DS (
) were prepared. The amount of
125I-FPR-thrombin not bound to FM-Sepharose, determined by
measuring radioactivity of aliquots of the supernatant after
centrifugation of the suspension, was used to calculate the fraction of
125I-FPR-thrombin bound. Aliquots of HCII were added to the
suspensions and the amount 125I-FPR-thrombin bound was
determined after each addition.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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FOOTNOTES |
---|
* This work was supported in part by grants-in-aid from the Medical Research Council of Canada (MT-3992), the Heart and Stroke Foundation of Ontario (T-2268 and MT-3664), and the Ontario Research and Development Challenge Fund.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.
Recipient of a Research Fellowship from the Heart and Stroke
Foundation of Canada.
§ Recipient of a Career Investigator Award from the Heart and Stroke Foundation of Canada and holder of the Heart and Stroke Foundation of Ontario/J. Fraser Mustard Chair in Cardiovascular Research and the Canada Research Chair in Thrombosis at McMaster University. To whom correspondence should be addressed: Henderson Research Center, 711 Concession St., Hamilton, Ontario L8V 1C3, Canada. Tel.: 905-574-8550; Fax: 905-575-2646; E-mail: jweitz@ thrombosis.hhscr.org.
Published, JBC Papers in Press, April 9, 2001, DOI 10.1074/jbc.M010584200
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ABBREVIATIONS |
---|
The abbreviations used are: HCII, heparin cofactor II; ANS, anilinonaphthalene-6-sulfonic acid; DS, dermatan sulfate; f, fluorescein-labeled; SF, soluble fibrin; FM, fibrin monomer; FITC, fluorescein-5'-isothiocyanate; FPR, D-Phe-Pro-Arg; FPRCK, D-Phe-Pro-Arg-chloromethyl ketone; GPRP-NH2, Gly-Pro-Arg-Pro-amide; tGPR-pNA, N-p-tosyl-Gly-Pro-Arg-p-nitroanilide acetate; ATA, N-(acetylthio)acetyl; TBS, Tris-buffered saline; PEG, polyethylene glycol.
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