 |
INTRODUCTION |
Heparin acts as an anticoagulant by activating antithrombin
(AT),1 a serine protease
inhibitor (serpin) that inhibits thrombin and factor Xa (1-5).
Catalysis of AT is dependent on a unique pentasaccharide sequence found
on about one-third of naturally occurring heparin chains.
Pentasaccharide-containing heparin chains bind AT with high affinity
and induce a conformational change in the reactive center loop of AT
that facilitates its interaction with target proteinases. Whereas this
conformational change is sufficient to accelerate the reactivity of AT
with factor Xa, inhibition of thrombin is only effected by heparin
chains that are of sufficient length to bridge the inhibitor and the
proteinase (6).
In addition to catalyzing AT, heparin also activates heparin cofactor
II (HCII), a naturally occurring serpin whose only target in the
coagulation system is thrombin. Heparin-induced catalysis of HCII has
been described as a "double bridging" effect because, in addition
to the heparin bridge between thrombin and HCII, a second bridge is
formed when heparin displaces the acidic amino terminus of HCII which
then binds thrombin (7). In contrast to AT, HCII does not bind with
high affinity to a unique sequence of heparin (8).
Interactions of thrombin with inhibitors and substrates are mediated by
positively charged exosites (9-11). Exosite 1 binds fibrinogen, the
fifth and sixth growth factor domains of thrombomodulin, hirudin, and
the amino terminus of HCII. As a recognition site, exosite 1 is
commonly employed as an initial docking site that orients the substrate
or inhibitor with the active site of thrombin (12). A second positively
charged region, exosite 2, is the heparin-binding site. To bridge
thrombin to AT or HCII, heparin binds simultaneously to exosite 2 on
thrombin and to the serpin (13). In addition to serving as docking
sites, exosites 1 and 2 may also serve allosteric regulatory roles by
influencing reactivity of the active site (14).
Although heparin is an effective anticoagulant, it has limitations,
particularly in patients with acute coronary ischemic syndromes
(15-18). These limitations have been attributed, at least in part, to
the inability of the heparin-AT complex to inactivate thrombin bound to
fibrin. Thrombin bound to fibrin remains enzymatically active and is
protected from rapid inhibition by plasma inhibitors, in the presence
of heparin (19, 20). The basis of this resistance is thought to reflect
formation of a ternary thrombin-heparin-fibrin complex wherein thrombin
is protected from inactivation by AT (21, 22). The ternary complex is
formed because heparin augments thrombin binding to fibrin, presumably
by bridging the protease to fibrin (22). Since thrombin binds fibrin
via exosite 1, and heparin binds to thrombin exosite 2, we predicted
that both exosites were essential not only for formation of the ternary
complex but also for rendering thrombin within the complex resistant to
inhibition by AT and other heparin-catalyzed inhibitors. To explore
this possibility, we measured the binding of thrombin to immobilized fibrin monomer in the absence or presence of heparin, and we compared thrombin binding with the binding of thrombin variants with impaired exosites 1 or 2. In addition, protection of thrombin from inhibition by
AT in the presence of fibrin was examined using the same thrombin variants. We also studied the extent to which thrombin is protected from inactivation by HCII and by
1-antitrypsin
Met358
Arg, inhibitors whose mode of interaction with
thrombin differs from that of AT. It was observed that only when both
exosites of thrombin are ligated is fibrin-bound thrombin protected
from inactivation by either the heparin-AT or heparin-HCII complex. In
contrast, fibrin-bound thrombin is susceptible to inhibition by
1-antitrypsin Met358
Arg even in the
presence of heparin suggesting that formation of the ternary
thrombin-heparin-fibrin complex does not impair access of
macromolecular inhibitors to the active site of the enzyme. The use of
a fourth inhibitor, consisting of a covalent complex of heparin and AT
(23), revealed that accessibility of AT-bound heparin to thrombin
within the ternary complex is impaired. Thus, these studies not only
elucidate the mechanism of ternary thrombin-fibrin-heparin complex
assembly but also explain why thrombin within this complex is protected
from inhibition by AT and HCII.
 |
EXPERIMENTAL PROCEDURES |
Proteins and Reagents--
Human
-thrombin,
-thrombin,
plasminogen-free fibrinogen, and factor Xa were obtained from Enzyme
Research Laboratories (South Bend, IN). The naturally occurring
thrombin variant Quick 1, with a Cys to Arg mutation at position 67 in
exosite 1 (24), was kindly provided by Ruth Ann Henriksen, University
of North Carolina School of Medicine. RA-thrombin (exosite 2 mutations
Arg93
Ala, Arg97
Ala, and
Arg101
Ala) (25) was a generous gift from Charles T. Esmon, Howard Hughes Medical Institute, Oklahoma City, OK. Recombinant
1-antitrypsin Met358
Arg (mutation
Met358
Arg (26) and truncation of five amino-terminal
amino acids) was a gift from Dr. R. Bischoff, Transgene S. A. (Strasbourg, France). Human plasma AT and HCII were from Affinity
Biologicals (Hamilton, Ontario, Canada). The covalent
antithrombin-heparin (ATH) complex (23) was generously provided by L. Berry, McMaster University. Thrombin blocked at its active site with
D-Phe-Pro-Arg chloromethyl ketone (FPRCK; Calbiochem) was
prepared as described previously (14). The chromogenic thrombin
substrate N-
-tosyl-Gly-Pro-Arg-
-nitroanilide (tGPR-pNA), hexadimethrine bromide (Polybrene),
gelatin-agarose, and Gly-Pro-Arg-Pro-amide (GPRP-NH2) were
from Sigma-Aldrich Canada Ltd. (Oakville, Ontario, Canada).
Heparin--
Unless otherwise specified, all experiments were
performed with heparin with high affinity for AT. High affinity heparin
was prepared by affinity chromatography of unfractionated heparin from
porcine intestinal mucosa (grade II; Sigma) using an AT-Sepharose column (27). Human AT and cyanogen bromide-activated Sepharose (Amersham Pharmacia Biotech) were used to prepare the column according to the manufacturer's instructions. Approximately 2-5 mg of AT were
immobilized per ml of Sepharose. The column (50 ml bed volume) was
equilibrated with 0.05 M Tris-HCl, 0.15 M NaCl,
pH 7.4 (TBS). Unfractionated heparin (10 mg) was dissolved in 1 ml of
TBS and pumped onto the column at an approximate flow rate of 15 ml/h at 4 °C, followed by another 90 ml of starting buffer. Bound
material was eluted with 90 ml of 2.0 M NaCl, collecting
3-ml fractions. The heparin-containing fractions, identified by Alcian
blue staining (23), were pooled, precipitated with 3 volumes of
ethanol, dissolved in starting buffer, and reapplied to the
re-equilibrated column. After repeating the procedure three times, the
final product was dissolved in 0.15 M NaCl, and its
concentration was determined using a protamine sulfate turbidometric
assay (28). The high affinity heparin prepared in this way had a
specific activity of 280 anti-Xa units/mg when compared with the 1st
International Unfractionated Heparin Standard and had an average
molecular mass of 15,000 Da, as determined by high pressure liquid
chromatography (29).
Preparation of Soluble Fibrin--
Plasminogen-free human
fibrinogen was subjected to gelatin-agarose adsorption to remove
fibronectin. Approximately 15 ml of fibrinogen (~130
µM) was mixed in a tube for 30 min with 5 ml of
gelatin-agarose. After centrifugation for 10 min, the supernatant was
removed and exposed a second time to a new aliquot of gelatin-agarose. The fibrinogen concentration in the supernatant was determined using a
molecular weight of 340,000 and by measuring the absorbance using
2801% of 15.1 (21) after correction
for light scatter at 320 nm using the relationship
A280corr = A280
1.7 × A320
(30). Soluble fibrin (SF) was prepared by clotting fibrinogen (60-100
µM) at 37 °C for 4-6 h with 2 nM thrombin
(21). The resultant fibrin was sedimented by centrifugation at
2000 × g for 5 min and transferred to dialysis tubing
(12,000-14,000 molecular weight cut-off, 2.5 cm wide). After dialysis
against distilled water (>200 volume) at 4 °C overnight to remove
fibrinopeptides A and B, the material was dialyzed against 20 mM acetic acid for approximately 8 h until dissolved.
A molecular weight of 340,000 and
2801% value of 14.0 was used to
calculate the SF concentration (31), which was typically about 100 µM. Aliquots were stored at
70 °C. Prior to use, SF
was neutralized with 40% v/v 1 M Tris-HCl, pH 7.5, and
polymerization was blocked by the addition of 5 mM GPRP-NH2 (32).
Preparation of Fibrin Monomer-Sepharose--
Approximately 35 mg
of fibrinogen was coupled to 3 ml of cyanogen bromide-activated
Sepharose, as described above for AT. To block unreacted amino groups,
the resin was packed in a small column (0.7 × 9 cm) and washed
over 2 h with 15 ml of coupling buffer and then with 10 ml of TBS.
Finally, the washed resin was removed from the column and diluted 1:2
with TBS, and the immobilized fibrinogen was converted to fibrin
monomer (FM) by addition of thrombin at a final concentration of 2 nM. After 3 h incubation with gentle end-over-end
mixing in a 15-ml conical tube at room temperature (33), the
FM-Sepharose was transferred to a column (0.7 × 9 cm) and washed
with 20 mM Tris-HCl, pH 7.4, 1 M NaCl (10 volumes) followed by TBS (10 volumes). The amount of FM conjugated to
the Sepharose was determined by performing a BCA Protein Assay (Pierce)
on an aliquot of the FM-Sepharose using fibrinogen as the standard and
unconjugated Sepharose as a blank.
Proteinase Inhibition Assays--
The rates of inactivation of
(a) thrombin,
-thrombin, Quick 1 dysthrombin, or
RA-thrombin by AT, HCII, or
1-antitrypsin Met358
Arg and (b) factor Xa by AT were
measured discontinuously under pseudo first-order rate conditions (13)
in the absence or presence of heparin, or SF, or both. Either thrombin
(20 nM), factor Xa (60 nM), or a thrombin
variant (20-60 nM) was incubated for 5 min at room
temperature in 20 mM Tris-HCl, 150 mM NaCl,
0.6% PEG 8000, pH 7.4 (TSP), containing 10 mM
GPRP-NH2 and various concentrations of heparin (0-100
µM) and SF (0-12 µM). Reaction mixtures
were aliquoted to 96-well round bottom microtiter plates (Fisher,
Nepean, Ontario, Canada), and an equal volume of inhibitor (in a
concentration at least 10-fold higher than that of the proteinase) was
added to each well at time intervals ranging from 2 s to 10 min.
Reactions were terminated simultaneously by the addition of 200 µl of
a solution containing 222 µM tGPR-pNA and 10 mg/ml Polybrene, and residual proteinase activity was determined by the
initial rate of substrate hydrolysis measured at 405 nm using a Spectra
Max 340 Microplate Reader (Molecular Devices, Menlo Park, CA). The pseudo first-order rate constant of inhibition was calculated by
fitting the data to the first-order rate Equation 1.
|
(Eq. 1)
|
where Vo represents proteinase
activity at time = 0; Vt represents proteinase
activity at time = t, and k1
represents the pseudo first-order rate constant. The apparent second-order rate constant (k2) of inhibition
was then obtained by dividing k1 by the
inhibitor concentration (13). When high concentrations of heparin were
used, the rates of proteinase inactivation were too fast to be measured
under these conditions. To circumvent this problem, reactions were done
in the presence of tGPR-pNA, which serves as a competitive
inhibitor (34). To correct for the presence of chromogenic substrate,
the pseudo first-order rate constant of inhibition was given by
Equation 2.
|
(Eq. 2)
|
where kapp is the apparent pseudo
first-order rate constant, [S] is the concentration of
competitor, and Km is the Michaelis-Menten constant
of the proteinase for tGPR-pNA (13). The
Km values of thrombin and factor Xa for
tGPR-pNA were determined to be 14 and 34 µM,
respectively. In experiments where SF reduced the rate of proteinase
inactivation, fold inhibition was calculated by dividing the
k2 value in the absence of SF by that measured
in its presence.
Inhibition of Thrombin by Covalent ATH Complex--
Inhibition
of thrombin in the presence of ATH was determined by continuous assay
of thrombin activity, monitored by cleavage of a fluorescent substrate.
A 2-ml solution containing 18 nM ATH and 50 µM
methoxysuccinyl-Ile-Glu-Gly-Arg-7-amino-4-trifluoromethyl coumarin
(AFC-67, Enzyme System Products, Dublin, CA) in TBS was incubated at
room temperature with stirring in a 3-ml quartz cuvette. Fluorescence
(
ex 400 nm and
em 505 nm; slit widths 10 nm) was monitored at 0.5-s intervals in a Perkin-Elmer LS 50B
spectrofluorimeter. SF and heparin were added, alone or in combination,
to the cuvette at final concentrations of 4 µM and 500 nM, respectively. Finally, thrombin was added to a final
concentration of 2 nM. Plots of I505
versus time were analyzed by nonlinear least squares
analysis of Equation 3 (25).
|
(Eq. 3)
|
where k1 and Imax,
the maximal rate of fluorescence change, were calculated by nonlinear
regression analysis using TableCurve (Jandel, San Rafael, CA).
Effects of Soluble Fibrin and GPRP-NH2 on Thrombin
Chromogenic Activity--
The chromogenic activity of 5 nM
thrombin in TSP was determined with 185 µM
tGPR-pNA in the presence of various concentrations of
GPRP-NH2 (0.5-25 mM) or SF (1-6
µM) in 96-well round bottom microtiter plates. The
initial rates of substrate hydrolysis, measured as the change in
absorbance at 405 nm per min, were determined over 10 min using a
microplate reader and were compared with the activity of thrombin in
the absence of GPRP-NH2 and SF.
Effects of Thrombin-Heparin-Fibrin Ternary Complex Formation on
the Rate of tGPR-pNA Hydrolysis by Thrombin,
-Thrombin, or
RA-Thrombin--
Chromogenic substrate reactions were performed in
96-well round bottom microtiter plates in TSP at room temperature.
Solutions of 5 nM thrombin,
-thrombin, or RA-thrombin,
SF (0 or 4 µM), 5 mM GPRP-NH2,
and heparin (0 or 500 nM) were mixed and then added to a
range of tGPR-pNA concentrations (0-250 µM).
The initial rates of substrate hydrolysis were determined by monitoring
the reaction at 405 nm using a microplate reader and were plotted versus tGPR-pNA concentration. The data were then
analyzed by nonlinear regression of the Michaelis-Menten Equation 4
|
(Eq. 4)
|
to solve for Km, the Michaelis-Menten
constant, and Vmax, the maximum rate of
substrate hydrolysis, given V, the rate of substrate
hydrolysis, and S, the substrate concentration.
Effect of Heparin on Thrombin,
-Thrombin, or RA-Thrombin
Binding to Fibrin Monomer-Sepharose--
Binding studies were carried
out in 20 mM Tris-HCl, 150 mM NaCl, 0.6% PEG
8000, 0.01% Tween 20, pH 7.4, at room temperature in 1.5-ml
microcentrifuge tubes (Rose Scientific, Edmonton, Alberta, Canada) in
total volumes of 500 µl. FM-Sepharose (50 µl; 1.4 µM final FM concentration) was mixed with thrombin,
-thrombin, or RA-thrombin (100 nM final concentration) in the absence or
presence of various concentrations of unfractionated heparin (25 nM to 15 µM final concentration). After
mixing for 2 min, FM-agarose was sedimented by centrifugation for 1 min
in a microcentrifuge at 15,000 × g. An aliquot of each
supernatant was transferred to a 96-well round bottom microtiter plate,
and a solution of 222 µM tGPR-pNA and 10 mg/ml
Polybrene was added simultaneously to each well. Hydrolysis of the
chromogenic substrate was monitored by measuring the increase in
absorbance at 405 nm over time using a plate reader. The quantity of
thrombin bound to FM-Sepharose was calculated by subtracting the
thrombin activity in the supernatant from a control measurement of
thrombin activity in the absence of FM-Sepharose. Thrombin activity was
converted to thrombin concentration using the specific activity of
thrombin, determined under these conditions.
Statistical Analysis--
All values reported represent the
mean ± S.D. of at least two separate experiments, each done in duplicate.
 |
RESULTS |
Thrombin Inactivation by AT--
To quantify the consequences of
ternary thrombin-heparin-fibrin complex formation, the influence of SF
on thrombin inactivation by AT was determined in the absence or
presence of 100 nM heparin (Fig.
1A). SF produced a
concentration-dependent and saturable decrease in the
heparin-catalyzed rate of inactivation of thrombin. In the presence of
100 nM heparin, the addition of 6 µM SF
decreased k2 by a factor of 50, from a value of
2.97 ± 1.01 × 108 to 6.04 ± 1.20 × 106 M
1 min
1.
Half-maximal reduction in k2 occurred with
approximately 25 nM SF. With 10 nM heparin, 6 µM SF had slightly less of an inhibitory effect, causing
a 41-fold reduction in k2 at 6 µM
SF (not shown). Hogg and Jackson (21) observed a 310-fold reduction at
10 nM heparin. These differences could reflect the fact
that the high affinity heparin used in the present studies had lower
specific activity. In the absence of heparin, less than a 2-fold
reduction in the rate of thrombin inhibition occurred when SF was
present at concentrations up to 6 µM.

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Fig. 1.
Reduction in the heparin-catalyzed rate of
thrombin inhibition by AT in the presence of soluble fibrin.
Second-order rate constants of thrombin inhibition by AT were
determined under pseudo first-order conditions. A, effect of
varying concentrations of soluble fibrin on the rate of thrombin
inactivation by AT in the absence ( ) or presence of 100 nM heparin ( ). B, effect of varying heparin
concentrations on the rates of thrombin inactivation by AT in the
absence ( ) or presence 4 µM soluble fibrin ( ). Each
point represents the mean of at least two experiments done in
duplicate, and the bars represent the standard
deviation.
|
|
In parallel experiments, the heparin dose-response for thrombin
inhibition was determined in the presence of 4 µM SF
(Fig. 1B). With all concentrations of heparin tested, SF
reduced the heparin-catalyzed rate of thrombin inactivation by AT. A
maximal reduction in the rate of thrombin inhibition of 58-fold
occurred with 100 nM heparin. In addition to a persistent
reduction in k2, 4 µM SF also
altered the shape of the heparin titration curve. The heparin
dose-response curve is biphasic in the absence of SF, characteristic of
a surface approximation phenomenon (13, 34). In the presence of SF,
there is a saturable increase in k2 with a
significantly lower maximal rate of inhibition. This result suggests
that SF alters the mechanism of thrombin inactivation by the AT-heparin complex.
As a control experiment, the rates of thrombin inactivation in the
absence and presence of both 10 nM heparin and 4 µM SF were determined over a range of AT concentrations
(not shown). As predicted, varying AT concentration from 50 to 500 nM (the range of AT concentrations used in our studies) had
no effect on k2 because calculations take the
inhibitor concentration into account (13). As further controls, the
effects of SF or GPRP-NH2 on the chromogenic activity of
thrombin for tGPR-pNA were examined. The presence of SF up
to 6 µM or GPRP-NH2 up to 25 mM
had no effect on the rate of tGPR-pNA hydrolysis by thrombin
(data not shown). Furthermore, the effect of GPRP-NH2 on
the heparin-catalyzed and uncatalyzed rates of thrombin inactivation by
AT were determined to ensure that the decreases in rates were not
caused by the peptide. GPRP-NH2, at concentrations up to 5 mM, caused a less than 2-fold reduction in
k2 (data not shown). To account for this small
effect, 5 mM GPRP-NH2 was present in all
reactions, even when the sample did not contain SF.
Factor Xa Inactivation by AT--
To examine directly the effect
of SF on the catalytic activity of heparin, factor Xa was substituted
for thrombin. Since factor Xa binds neither heparin nor fibrin, only
the heparin-fibrin interaction would remain. As with thrombin, a range
of SF concentrations had no effect on the uncatalyzed rate of factor Xa
inhibition by AT. In contrast to thrombin, SF produced only a modest
dose-dependent reduction in the rate of factor Xa
inhibition in the presence of 10 or 100 nM heparin (not
shown). At 10 nM heparin, 4 µM SF reduced
k2 by a factor of 6 (from a value of 4.70 ± 0.90 × 106 to 7.63 ± 1.0 × 105 M
1 min
1) with
half-maximal inhibition occurring at approximately 0.4 µM
SF. With 100 nM heparin, SF reduced the rate constant only 2-fold. The relatively small magnitude of these inhibitory effects suggests that the heparin-SF interaction does not compromise the ability of heparin to catalyze AT to any appreciable extent.
Thrombin Inactivation by Heparin Cofactor II--
The influence of
SF on the heparin-catalyzed and uncatalyzed rates of thrombin
inactivation by HCII was examined because, unlike AT which interacts
solely with the active site of thrombin, the amino-terminal region of
HCII requires an additional interaction with thrombin exosite 1 (35-37), the domain also responsible for the interaction of thrombin
with fibrin (38). Experiments were performed by varying the
concentrations of each of the following three components: SF, heparin,
and HCII. Similar to AT, SF caused a dose-dependent and
saturable decrease in the heparin-catalyzed rates of thrombin
inactivation by HCII, although the magnitude of the effect was much
greater (Fig. 2A). In contrast
to the 50-fold reduction in the rate of thrombin inactivation by AT in
the presence of 100 nM heparin, 6 µM SF
reduced the heparin-catalyzed rate of HCII by 222-fold (from 2.14 ± 0.05 × 108 to 9.64 ± 4.49 × 105 M
1 min
1).
Half-maximal inhibition occurred at approximately 5 nM SF. When the heparin concentration was reduced from 100 to 10 nM, the inhibitory effect of SF was slightly less, reaching
a maximum reduction in rate of 185-fold with 6 µM SF (not
shown). Similar to AT, SF did not influence the uncatalyzed rate of
thrombin inhibition by HCII.

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Fig. 2.
Reduction in the heparin-catalyzed rate of
thrombin inhibition by HCII in the presence of soluble fibrin.
Second-order rate constants of thrombin inhibition by HCII were
determined under pseudo first-order conditions. A, effect of
varying concentrations of soluble fibrin on the rates of thrombin
inactivation by HCII in the absence ( ) or presence of 1 µM heparin ( ). B, effect of varying heparin
concentrations on the rates of thrombin inactivation by HCII in the
absence ( ) or presence of 4 µM soluble fibrin ( ).
Each point represents the mean of at least two experiments done in
duplicate, and the bars represent the standard
deviation.
|
|
The effect of varying concentrations of heparin on the rate of thrombin
inhibition by HCII in the presence of 4 µM SF is shown in
Fig. 2B. Whereas 250 nM SF caused a modest
reduction in k2 values at the various heparin
concentrations (not shown), 4 µM SF had a more dramatic
effect. Maximal 247-fold reduction occurred at 1 µM
heparin. As with AT, the addition of SF changed the shape of the
heparin titration curve from a biphasic curve to a curve showing a
heparin dose-dependent and saturable increase. This finding
suggests that SF alters the mechanism of thrombin inactivation by the
heparin-HCII complex.
As controls, the k2 values for thrombin
inactivation in the absence and presence of 10 nM heparin
and 4 µM SF were determined at various HCII
concentrations (not shown). Over the range of HCII concentrations used
in these experiments there was no systematic change in
k2.
Inactivation of Thrombin Exosite Variants--
The exosites of
thrombin are implicated in the protective effect of the ternary complex
due to their known interactions with fibrin and heparin (38). To
investigate directly the involvement of thrombin exosite 1 and exosite
2 in the mechanism of resistance of fibrin-bound thrombin to
inactivation by heparin-serpin complexes, variants of thrombin with
impaired exosites were compared with native thrombin in inhibition
assays in the absence or presence of 4 µM SF. The exosite
1 variants utilized were
-thrombin, a proteolytic derivative lacking
residues in exosite 1 (39), and Quick 1 thrombin, a dysthrombin
characterized by mutation of Arg67 in exosite 1 to Cys
(24). The exosite 2 variant, RA-thrombin, has three mutations in
exosite 2 that impair heparin binding (25). Table
I compares the rates of inactivation of
thrombin versus the thrombin variants by AT and 100 nM heparin or HCII and 1 µM heparin in the
absence or presence of 4 µM SF. In the presence of 4 µM SF, the rates of thrombin inactivation by heparin-AT
and heparin-HCII were inhibited 58-fold and 247-fold, respectively, as
illustrated in Figs. 1 and 2. In contrast, when the exosite 1 variants
-thrombin or Quick 1-thrombin were substituted for thrombin, SF only
decreased the heparin-catalyzed rates of inactivation by either AT or
HCII by 4- or 10-fold, respectively. Similarly, the protective effect
of SF was essentially eliminated when the exosite 2 mutant,
RA-thrombin, was used in place of thrombin because its rate of
inactivation by heparin-AT or heparin-HCII was reduced only 2- or
7-fold, respectively. As with thrombin, SF had no effect on the
uncatalyzed rates of inactivation of the thrombin variants by either AT
or HCII (data not shown). These results reveal a strict requirement for
the integrity of both exosites in formation of productive
thrombin-heparin-fibrin ternary complexes that attenuate thrombin
inhibition by either AT or HCII.
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Table I
Influence of soluble fibrin on the heparin-catalyzed rate of inhibition
of thrombin or thrombin variants by antithrombin and heparin cofactor
II
Second-order rate constants (k2) for
heparin-catalyzed inhibition of thrombin (IIa) or thrombin variants by
antithrombin or heparin cofactor II were determined under pseudo
first-order conditions as described under "Experimental
Procedures." Experiments were performed in the absence and presence
of 4 µM soluble fibrin. The values are the mean of at
least four determinations ± S.D.
|
|
Binding of Thrombin and Exosite Variants to Fibrin
Monomer-Sepharose--
To explore the role of the exosites of thrombin
in formation of the ternary complex, the influence of heparin on the
binding of thrombin exosite 1 and 2 variants to FM-Sepharose was
examined. In the absence of heparin, approximately 22% thrombin or
RA-thrombin bound to FM-Sepharose (Fig.
3). In contrast, less than 4%
-thrombin bound, confirming that thrombin binds fibrin via exosite
1. The addition of 250 nM unfractionated heparin increased
thrombin binding from 22 to 69%. Although a defective exosite 1 prevents
-thrombin from binding in the absence of heparin, binding
increased from 4 to 32% in the presence of heparin reflecting
heparin-facilitated binding via its intact exosite 2. In contrast to
both thrombin and
-thrombin, 250 nM heparin only
marginally increased the binding of RA-thrombin to fibrin (from 22 to
29%), presumably because the mutations in exosite 2 decrease the
enzyme's affinity for heparin, thereby preventing heparin from
bridging RA-thrombin to fibrin. These results are consistent with the
proposal that thrombin forms a ternary complex with fibrin and heparin
and that both thrombin exosites are involved (38); exosite 1 binds
fibrin directly, whereas exosite 2 binds fibrin indirectly via
fibrin-bound heparin.

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Fig. 3.
Effect of 250 nM unfractionated
heparin on thrombin (IIa), -thrombin
( -IIa), or RA-thrombin (RA-IIa) binding to
fibrin monomer-Sepharose. Binding of 100 nM thrombin
or thrombin variant to 1.4 µM fibrin monomer-Sepharose
was accomplished by determination of the concentration of free thrombin
in the supernatant after pelleting the Sepharose by centrifugation. The
open bars represent the percent thrombin bound in the
absence of heparin; the solid bars show the percent thrombin
bound in the presence of 250 nM unfractionated heparin, and
the lines represent the standard deviation.
|
|
Thrombin Active Site--
To determine whether thrombin bound
within the ternary thrombin-heparin-fibrin complex undergoes allosteric
changes at its active site, the reactivity of thrombin and thrombin
variants with a chromogenic substrate and with a heparin-independent
macromolecular inhibitor was assessed. Neither SF nor heparin alone
altered the Km for thrombin hydrolysis of
tGPR-pNA. However, SF and heparin in combination increased
Km of thrombin for tGPR-pNA 3-fold, from
a value of 25 ± 1.5 to 84 ± 0.7 µM (not
shown). To explore the contribution of exosite 1 and exosite 2 ligation
by fibrin and heparin, respectively, to this increase in
Km,
-thrombin or RA-thrombin was substituted for
thrombin. In contrast to thrombin, the ability of
-thrombin or
RA-thrombin to hydrolyze the chromogenic substrate was unaffected by
heparin, SF, or the combination. In the presence of both heparin and
SF, the Km of
-thrombin for tGPR-pNA
increased only slightly (from 58 ± 2.7 to 63 ± 4.1 µM), whereas the Km of RA-thrombin for the substrate decreased slightly (from 29 ± 1.5 to 24 ± 2.3 µM). These results confirm those of previous studies
indicating that a conformational change occurs at the catalytic site of
thrombin only when the ternary complex is formed (21, 40). Furthermore, our data indicate that ligation of both exosite 1 by fibrin and exosite
2 by heparin is essential to elicit this change.
The P1 mutation Met358
Arg in
1-antitrypsin yields a potent heparin-independent
inhibitor of thrombin (26) that does not require interaction with
either exosite on thrombin. To confirm this, we found that
1-antitrypsin Met358
Arg inactivated
both
-thrombin and RA-thrombin at the same rate as thrombin
(4.60 ± 0.15 × 106, 5.18 ± 0.97 × 106, and 4.22 ± 0.93 × 106
M
1 min
1, respectively). The
effects of 500 nM heparin and 4 µM SF alone, and in combination, on the rate of thrombin inactivation by
1-antitrypsin Met358
Arg are shown in
Fig. 4. Heparin or SF alone inhibited the rate of thrombin inactivation by 1.4- and 1.7-fold, respectively. When
both were added to form thrombin-heparin-fibrin ternary complexes, the
rate of thrombin inactivation was reduced only 4-fold (from 4.22 ± 0.93 × 106 to 1.05 ± 0.28 × 106 M
1 min
1). Thus,
the protective effect of ternary complex formation is less with
1-antitrypsin Met358
Arg than with AT or
HCII, whose rates were inhibited 58- and 247-fold, respectively. These
results indicate that allosteric changes at the active site induced by
ternary complex formation have minimal effects on the interaction of
thrombin with a macromolecular heparin-independent inhibitor.

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Fig. 4.
Influence of soluble fibrin and heparin on
the rate of thrombin inhibition by
1-antitrypsin Met358
Arg or a covalent antithrombin-heparin complex
(ATH). The second-order rate constants for thrombin inhibition by
1-antitrypsin Met358 Arg or ATH were
determined. The experiments were repeated in the presence of 4 µM SF, 500 nM heparin, or both SF and
heparin. The open bars represent
1-antitrypsin Met358 Arg, and the
solid bars represent ATH. Each bar represents the
mean of at least two experiments done in duplicate, and the
lines represent the standard deviation.
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Mechanism of Protection--
The preceding experiments demonstrate
that the two thrombin exosites participate in ternary complex
formation. They also confirm the proposal that heparin bridges thrombin
to fibrin, effectively increasing the affinity of thrombin for fibrin
(38). To begin to elucidate the mechanism by which fibrin-bound
thrombin is protected from inactivation by the heparin-AT complex, we
examined whether the heparin component of the ternary complex is able
to activate AT. Although heparin is able to catalyze factor Xa
inhibition by AT in the presence of fibrin (not shown) (21), heparin
and fibrin alone do not constitute a ternary complex. Therefore,
complete ternary complexes were assembled by the addition of
FPRCK-thrombin to heparin and SF, and their effect on factor Xa
inhibition by AT was determined. FPRCK-thrombin was added at
concentrations equivalent to, or 10-fold higher than, that of heparin
(10 nM), conditions under which the rate of inhibition of
active thrombin by AT is reduced 42-fold. The addition of
FPRCK-thrombin caused no further reduction in the rate of factor Xa
inhibition than the 2-3-fold observed with SF alone (not shown). These
results suggest that heparin within the ternary thrombin-heparin-fibrin complex retains its ability to bind and activate AT. Thus, formation of
a ternary complex does not appear to compromise the function of either
heparin or AT. Instead, the protective effects of ternary complex
formation likely reside with thrombin.
Results with the heparin-independent inhibitor,
1-antitrypsin Met358
Arg, demonstrated
that access of macromolecular inhibitors to the active site of thrombin
is not impaired upon formation of the ternary complex, thereby making
steric mechanisms of protection unlikely (Fig. 4). Comparison of the
results with
1-antitrypsin Met358
Arg
with those with AT-heparin or HCII-heparin also reveals that the
protective effect is heparin-dependent. Therefore, the next
most likely scenario involves impairment of the ability of heparin to
act as a catalyst in thrombin inhibition in the presence of ternary
complexes. Functional studies to address this possibility are
complicated by the fact that heparin serves two roles, bridging thrombin to fibrin and bridging thrombin to AT. To circumvent this
problem, we used a covalent AT-heparin complex (ATH) to ensure that
these two roles were performed by separate heparin molecules. The ATH
complex is prepared by incubating heparin and AT so that a Schiff base
is formed that subsequently rearranges into a covalent complex (23).
ATH is a more potent inhibitor of thrombin and factor Xa than
heparin-catalyzed AT, presumably because of the covalent binding of
heparin to AT in the ATH complex (23, 41). In support of this, the
second-order rate constant for thrombin inhibition is 9.3 × 108 M
1 min
1 for ATH
and 4 × 108 M
1
min
1 for AT with 100 nM heparin, the
concentration of heparin that provides maximal catalysis (Fig. 1). In
the presence of 500 nM heparin, the rate of inhibition by
ATH decreased only 2-fold, whereas 4 µM SF caused only a
5.4-fold reduction in the rate of inhibition by ATH (Fig. 4). These
findings demonstrate that fibrin has minimal effects on the heparin
component of ATH. When exogenous heparin was added to bridge thrombin
to fibrin thereby forming a ternary complex, there was a 66-fold
reduction in the rate of thrombin inhibition by ATH, a decrease
comparable to that produced by SF for AT and heparin (Fig. 1). Since
the heparin moiety of ATH must still bind thrombin to catalyze
inhibition (41), the reduction in the rate of thrombin inhibition
reflects impaired access of the heparin moiety of ATH to thrombin
within the thrombin-heparin-fibrin complex. Therefore, the major source
of protection from inhibition resides in the inability of AT-associated
heparin to access exosite 2 of thrombin and perform its obligatory
bridging role.
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DISCUSSION |
Exosites 1 and 2 of thrombin have been implicated in the formation
of ternary thrombin-heparin-fibrin complexes that protect thrombin from
inhibition by AT (38). To investigate the role of the thrombin exosites
directly, the protective effect of the ternary complex was investigated
from the perspective of both the inhibitor and the enzyme. AT,
HCII, and
1-antitrypsin Met358
Arg have
distinct structural requirements for inhibition of thrombin. AT and
HCII, when catalyzed by heparin, require the participation of thrombin
exosite 2, the heparin-binding site, whereas
1-antitrypsin Met358
Arg is
heparin-independent (26, 42). HCII has an additional requirement
for exosite 1 for the initial docking of its amino terminus (8). The
effect of ternary complex formation on thrombin inhibition was
exaggerated with HCII compared with AT and abrogated with
1-antitrypsin Met358
Arg, with 250-, 50-, and 4-fold reductions in the rate of thrombin inhibition,
respectively. These data reveal that protection is observed only with
heparin-catalyzed inhibitors, confirming the importance of the exosites
in the protective mechanism.
In order to discern more directly the role of thrombin exosites on the
protective effect of ternary complex formation, variants of thrombin
with impaired exosites 1 or 2 were employed. Involvement of the
exosites can be inferred from a lack of protective effect of SF on the
heparin-catalyzed rate of factor Xa inhibition, because factor Xa does
not bind fibrin and, in the absence of calcium (43), does not interact
with heparin. Thrombin Quick 1, a dysthrombin (24), and
-thrombin, a
proteolytic derivative of native thrombin (39), both have defects in
exosite 1, whereas RA-thrombin, a recombinant variant, has mutations in
exosite 2 (25). SF caused only minor reductions in the
heparin-catalyzed rate of inhibition by AT for both exosite 1 and 2 thrombin variants. The lack of protection cannot be attributed to low
rates of inhibition of the thrombin variants because the
heparin-catalyzed rates of inhibition of
-thrombin and thrombin
Quick 1 by AT are comparable to that of native thrombin. This
cannot be said of the exosite 2 thrombin variant because the magnitude
of heparin catalysis is modest. These data demonstrate that the
protective effect of ternary complex formation is lost when the
function of either exosite is impaired.
Both exosites on thrombin also are critical for assembly of the ternary
thrombin-heparin-fibrin complex. Heparin promoted the binding of both
native thrombin and
-thrombin to immobilized fibrin monomer,
although little
-thrombin bound in the absence of heparin. In
contrast, the binding of RA-thrombin to fibrin was only marginally
enhanced in the presence of heparin, presumably because its reduced
affinity for heparin prevents the heparin from bridging RA-thrombin to
fibrin. Although this bridging is independent of the exosite 1-fibrin
interaction, native thrombin demonstrates a greater increase in binding
than
-thrombin, possibly reflecting a cooperative effect of binding
via both exosites in the ternary complex.
Although these experiments demonstrate the interactions required to
support ternary complex formation, they do not reveal the mechanism
responsible for protection of thrombin within the ternary complex from
inhibition by AT or HCII. A number of models can be envisioned that
involve impairment of accessibility or function of thrombin, heparin,
or AT (38, 44). Briefly, these models can be classified as allosteric
or steric. The former model proposes that the active site of thrombin
is altered in such a way as to limit its reactivity with macromolecular
inhibitors. Steric models suggest physical impairment of the ability of
AT or heparin to bind thrombin, heparin to bind AT, or the AT-heparin complex to bind the thrombin-heparin-fibrin complex. Models proposing functional changes at the active site of thrombin or the formation of a
nonproductive AT-heparin-fibrin complex also have been suggested (38).
The possibility that formation of the ternary complex caused steric
inhibition at the active site of thrombin which altered its
accessibility to macromolecular inhibitors was addressed with the use
of
1-antitrypsin Met358
Arg, a serpin
that reacts exclusively with the active site of thrombin. The
observation that ternary complex formation does not protect thrombin
from inhibition by this inhibitor suggests that the active site of
thrombin remains accessible to macromolecules. This concept is
corroborated by previous work indicating that thrombin within the
ternary complex retains its ability to convert prothrombin to
prethrombin 1 (40). Although our study and those of others (40, 45)
reveal structural and functional changes in the active site upon
ternary complex formation, these changes do not appear to influence the
reactivity of the enzyme with macromolecules. Since the protective
effect is observed with heparin-dependent inhibitors, and
not heparin-independent inhibitors, the results, to this point, suggest
that the protective effect of the ternary complex results from
limitations of heparin catalysis and not changes at the active site of thrombin.
Since heparin binds both thrombin and AT, hampered catalysis could
result from impaired interaction with either protein. This was
addressed with the use of factor Xa since its inhibition by AT is
catalyzed by heparin solely through heparin-AT interactions. Factor Xa
inhibition by heparin-AT was only slightly affected by fibrin monomer
(22). These findings suggest that heparin associated with fibrin is
able to catalyze AT. It could be argued, however, that heparin in a
binary heparin-fibrin complex is not identical to that in a ternary
thrombin-heparin-fibrin complex. To address this possibility, factor Xa
inhibition also was monitored in the presence of heparin, fibrin, and
FPRCK-thrombin. Since heparin bridges FPRCK-thrombin to fibrin (Fig.
3), a productive ternary complex is formed. That there was only a
modest effect on factor Xa inhibition reveals that the catalytic
properties of heparin are retained, even when heparin is associated
with both thrombin and fibrin.
There are two mechanisms of protection of thrombin that could involve
an impaired ability of heparin to catalyze thrombin inhibition by AT.
The first is one in which thrombin and AT are bound with high affinity
to the same heparin chain but are unable to associate with each other
because of physical separation of the binding sites. In this scenario,
AT is bound to the high affinity pentasaccharide sequence on heparin at
a site remote from the portion of the heparin chain involved in the
formation of the thrombin-heparin-fibrin complex. This mechanism is
unlikely because thrombin within this ternary complex is protected from
inactivation by HCII to a greater extent than by AT, even though HCII
does not utilize the pentasaccharide or any other high affinity binding site on heparin (8). The second mechanism of protection involving heparin could result from impaired access of serpin-bound heparin to an
already occupied heparin-binding site on thrombin. This possibility was
assessed using a covalent ATH complex to ensure that the catalytic
heparin species was distinct from the heparin associated with thrombin
in the ternary complex. Inhibition of thrombin by the covalent ATH
complex was reduced 66-fold in the presence of the ternary complex
suggesting that the heparin moiety of ATH was unable to adequately bind
to exosite 2 on thrombin. This is consistent with the observation that
the protective effect of the ternary complex is obtained with
heparin-catalyzed inhibitors, such as AT and HCII, but not with the
heparin-independent
1-antitrypsin Met358
Arg. These data suggest that formation of the ternary complex increases
the affinity of heparin for exosite 2 on thrombin such that heparin
associated with AT or HCII is unable to bind to fibrin-bound thrombin
(Fig. 5). With HCII, the effect is
exacerbated because heparin increases the affinity of fibrin for
exosite 1 on thrombin, a requisite binding site for HCII. Thus,
although all the binary interactions between heparin, thrombin, and
fibrin are strengthened in the ternary complex, the heightened
heparin-thrombin interaction appears to be responsible for the
impairment of AT function.

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Fig. 5.
Proposed mechanism for protection of
fibrin-bound thrombin from inactivation by AT and HCII in the presence
of heparin. Thrombin (IIa) is held in a ternary complex through
interaction with fibrin via exosite 1 and heparin via exosite 2. A, heparin, in complex with antithrombin (AT), cannot access
exosite 2 because the domain is occupied by heparin from the ternary
complex. B, heparin, in complex with heparin cofactor II
(HCII) has restricted access to exosite 2 on thrombin. In addition, the
amino-terminal acidic domain of HCII cannot interact with exosite 1 on
thrombin because this site is bound to fibrin. This additional
interaction explains why fibrin-bound thrombin is more resistant to
inhibition by HCII than by AT.
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