 |
INTRODUCTION |
Thrombin is one of the most potent physiological agonists of
platelets, inducing activation responses such as cytokinesis, aggregation, secretion, and associated metabolic changes (1, 2). The
first receptor for thrombin identified on the platelet surface was the
glycoprotein (GP)1 Ib-IX
complex (3-6), although the biological significance of this
interaction remains unknown 20 years later. In the interim, other
thrombin receptors have also been identified on the platelet surface
including three members of the seven transmembrane domain receptor
superfamily known as proteolytically activated receptor-1 (PAR-1),
PAR-3, and PAR-4 (7-10). PAR-1 was shown to mediate platelet activation (7) and took much attention away from GPIb-IX as a thrombin
receptor until it was found that PAR-1 knockout mice had no
demonstrable bleeding disorder and that their platelets retained normal
responses to
-thrombin (11). Furthermore, synthetic peptides of
PAR-3 and PAR-4 that mimic the sequences of the potentially activating
tethered ligands, either failed to show (PAR-3) or showed very low
(PAR-4) activation effects on platelets (9, 10). Therefore, attention
has again turned to the GPIb-IX complex to understand, for example, why
the platelets from patients with Bernard-Soulier syndrome, which lack
or have dysfunctional GPIb-IX complexes on their surfaces fail to
respond to low doses of thrombin (12, 13).
Glycoprotein Ib-IX consists of three polypeptides (GPIb
, GPIb
,
and GPIX) that are each transmembrane proteins that span the platelet
membrane once. They are also each members of the leucine-rich repeat
superfamily (14). Glycoprotein Ib
and GPIb
are linked together
via a disulfide bond that is situated in the extracellular space very
close to the platelet membrane, and GPIX is noncovalently associated
with these two. On the platelet surface, a fourth leucine-rich repeat
protein, GPV, appears to link two GPIb-IX trimers together and may be
responsible for even more complex aggregation states (14-16). Of this
multimeric complex it is the extracellular portion of GPIb
, known as
glycocalicin, that forms the site of ligand interaction. Glycocalicin
can be released from the surface of platelets by proteolysis near the platelet membrane and consists of two subregions known as the macroglycopeptide and the amino-terminal domain (14). It is the
amino-terminal domain, which consists of about 300 amino acids, that
provides the sites within glycocalicin for ligand interaction (3), and
within this the thrombin-binding site has been localized between
residues 269 and 287 (17). The site on thrombin, however, where GPIb
binds is more controversial. Within the structure of thrombin four
prominent regions have been identified: its active site, a sodium
ion-binding site (18, 19), and two surface electropositive patches
known as anion binding exosites I and II (20) that have both been
implicated in thrombin binding. Although some studies have indicated
that GPIb
binds to anion-binding exosite I (also known as the
fibrinogen recognition site) of thrombin (21-24), the studies of De
Cristofaro and colleagues (25-27) have implicated anion-binding
exosite II (also known as the heparin binding site) of thrombin as the
site of GPIb
interaction.
Thrombin is an allosteric serine protease (28) with changes in the
conformation of its active site being induced by the binding of ligands
at the other sites of the enzyme. For example, the binding of ligands
at exosite I changes the rate of turnover of small synthetic peptidyl
and natural substrates (29, 30), whereas the binding of heparin at
exosite II alters the kinetics of the inhibition of thrombin by hirudin
(31) consistent with allosteric linkage between exosites I and II of
thrombin (32). We wondered whether the binding of GPIb
to thrombin
also produced an allosteric response in the enzyme. Furthermore, we
sought to clarify which of the exosites of thrombin was the
GPIb
-binding site in the expectation that such knowledge would lead
to the formulation of new hypotheses about the biological role of
GPIb
-thrombin interaction, because the exosite involved should be
directly related to the consequence of this binding.
In this study, we have used four approaches to investigate the binding
interaction of GPIb
with thrombin: 1) an HPLC method measuring the
effect of ligands on thrombin-induced fibrinogen turnover that has been
well characterized in the study of thrombin-fibrinogen and
thrombin-thrombomodulin interactions (33, 34), 2) mutant thrombins with
single amino acid substitutions in either exosite I or II (35-37), 3)
a resin-based assay for studying the direct binding of thrombin to
GPIb
, and 4) competition binding assays using small ligands of known
exosite interaction as competitors of the GPIb
-thrombin interaction.
Our results clearly indicated that the binding site for GPIb
is
located in thrombin anion-binding exosite II and that the binding of
GPIb
to thrombin induces conformational changes at the active site
of thrombin by an allosteric mechanism that alters the activity of
thrombin toward both physiological and small substrates.
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EXPERIMENTAL PROCEDURES |
Preparation of Glycocalicin and the Amino-terminal Fragment
of GPIb
from Human Platelets
Glycocalicin was isolated from outdated human platelets using a
modification of methods previously reported (38, 39). Briefly, after
glycocalicin was cleaved from the surface of platelets by calpain
released as a result of their sonication, it was isolated by wheat germ
agglutinin Sepharose 4B and Q-Sepharose anion exchange chromatography.
Purified glycocalicin was dialyzed into 20 mM Tris-HCl, pH
8.0, and concentrated to 1 mg/ml, and aliquots were snap frozen in
liquid nitrogen and stored at
80 °C until use. The amino-terminal
fragment of GPIb
was generated from purified glycocalicin by
cleavage with porcine pancreatic elastase at an enzyme to substrate
ratio of 1:250 (w/w). Porcine pancreatic elastase cleaves glycocalicin
after residue Val289 yielding the amino-terminal
fragment and a macroglycopeptide, which are well separated by
Q-Sepharose. Both fragments were concentrated, dialyzed, and stored as
described for glycocalicin above.
Recombinant Expression of the Amino-terminal Fragment of
GPIb
The amino-terminal region of GPIb
was expressed from
baculovirus-infected insect cells (40) and purified as described
previously (41). Briefly, the cDNA coding for the signal peptide
and amino-terminal domain of GPIb
(residues
16 to 289) followed by
the calmodulin (CaM) gene was inserted into the baculovirus expression
vector pAcSG2 (Pharmingen, San Diego, CA). The resultant plasmid,
pWIb
wt, was then recombined with BacVector-3000 triple cut virus DNA
(Novagen, Madison, WI) to produce infective virus. A high titer
baculovirus stock was obtained from a single plaque by repeated
infection of Sf9 insect cells. Recombinant virus was then used
to infect High Five insect cells for expression of the GPIb
-CaM
fusion protein. The calmodulin moiety of the fusion protein was then exploited in the first stage of recombinant protein isolation since it
binds to N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7)-agarose (prepared as described previously (42)). Calmodulin could
be released from the GPIb
() fragment by digestion with porcine pancretaic elastase (1:100 w/w E:S ratio), and the GPIb
() could be retrieved in pure form after passage over Q-Sepharose anion exchange resin.
Recombinant Prothrombin Expression
Recombinant mutant prothrombins R68E, R70E (exosite I mutants),
R89E, and K248E (exosite II mutants) were expressed and characterized as described previously (36, 37). Amino acid residues are numbered from
the first residue of the human thrombin B chain. Purified prothrombins
were activated with Echis carinatus snake venom, and the
thrombin products were further purified to homogeneity by ion exchange
chromatography on Amberlite CG-50. The purity of each thrombin was
confirmed by SDS-polyacrylamide gel electrophoresis and silver
staining. The activities of recombinant mutant and wild-type thrombins
were tested as described (43, 44).
Characterization of the Thrombin-GPIb
Interaction
Effect of Glycocalicin on the Thrombin-Fibrinogen
Interaction--
The binding of glycocalicin to thrombin was
determined by modifying an HPLC-based assay that has been well
characterized for the study of fibrinogen binding to thrombin (33).
First, thrombin was incubated with fibrinogen under the desired
solution conditions (5 mM Tris-HCl, 0.1% PEG-8000, pH 8.0 at 25 °C) and the progress curves for fibrinopeptide A (FpA) release
were measured to determine the values of the specificity constant
kcat/Km. The fibrinogen concentration was 0.2 µM, thrombin concentrations varied
according to their specific activities in the range from 0.08-2
nM, and the ionic strength was kept constant with NaCl. The
reaction was initiated by addition of thrombin to the fibrinogen
solution (or to the fibrinogen and glycocalicin solution) and quenched
at different time intervals with 3 M perchloric acid. The
sample was then centrifuged, and the amount of FpA in the supernatant
was determined by HPLC, as described previously (33). Next,
glycocalicin at varying concentrations was added to the assay system,
and the equilibrium dissociation constants (Kd) for
glycocalicin binding to thrombin were determined by analysis of the
inhibition of FpA release as a function of glycocalicin concentration
according to the following equation.
|
(Eq. 1)
|
where
|
(1a)
|
and x is the GPIb
concentration,
eT is the active thrombin concentration,
(kcat/Km)0 and
(kcat/Km)1 are the specificity constants for FpA release in the absence of and at
saturating concentration of GPIb
, respectively, and
tc = Km/eTkcat corresponds to the point in the progress curve that gives the greatest
change in the amount of FpA released as a function of the inhibitor
concentration. The exosite mutant thrombins were substituted for
wild-type thrombin in this assay to investigate the effect that GPIb
had on their cleavage reactions of fibrinogen. Before each assay the
concentration of thrombin was adjusted to ensure the same thrombin
activity was present in each reaction.
The same assay was used for investigating the salt dependence of the
thrombin-glycocalicin interaction in the range of 100-150 mM NaCl. The data plotted as log Kd
versus log [salt] were fitted to a straight line according
to the following expression (34).
|
(Eq. 2)
|
Kd is a dissociation constant. The slope of
this line yields
salt, which represents a thermodynamic
measure of the effect of salt concentration on binding equilibria (28, 34).
Direct Thrombin Binding Assay--
The direct binding of
thrombin to GPIb
was investigated using the recombinant GPIb
-CaM
fusion protein expressed in insect cells. 50 µl of W-7-agarose
solution containing 25 µl of packed resin was first added to a
500-µl Eppendorf tube. The resin beads were then washed with washing
buffer (100 mM NaCl, 50 mM Tris-HCl, pH 7.4)
three times. 1 nmol of recombinant GPIb
-CaM fusion protein in 100 µl of binding solution (100 mM NaCl, 50 mM
Tris-HCl, pH 7.4, 20 mM CaCl2) was added to the
25 µl of washed W-7-agarose beads and incubated for 2 h at
25 °C. The beads were then washed once with binding solution and
blocked with 3% bovine serum albumin, 0.1% PEG-8000 in binding
solution for another 2 h. After blocking, duplicates of increasing
quantities (2.5, 5.0, 7.5, 10, or 12.5 pmol) of thrombin (wild type or
mutants) were added to separate tubes in binding solution in a total
volume of 70 µl and incubated for 2 h at 25 °C. The total
volume of each reaction, including W-7-agarose, was therefore 95 µl
containing final concentrations of thrombin of 26, 52, 79, 105, and 131 nM, respectively. As a negative control, W-7 beads were
incubated for the indicated time in the absence of GPIb
-CaM. The
beads were kept suspended during each incubation step by inverting the
tubes every 10 min. At the end of the incubation, the beads were
pelleted by centrifugation at 600 × g for 1 min. 50 µl of each supernatant was transferred to a 96-well microtiter plate
and incubated with 50 µl of 2.5 mM thrombin substrate
D-Phe-Pro-Arg-pNA. The release of
p-nitroaniline was monitored by spectrophotometry at 405 nm
using a THERMOmax microplate reader (Molecular Devices Sunnyvale, CA)
at 5-min intervals from 5 to 60 min.
Competition of GPIb
-Thrombin Binding Using Known Exosite
Ligands--
These assays were performed essentially as for the direct
thrombin binding assay described above but with the addition of either
hirugen (residues 54-65 of hirudin with Tyr63 sulfated;
Sigma) or low molecular weight (LMW) heparin
(Rhône-Poulenc Rorer Pharmaceuticals Inc., Collegeville, PA)
added as inhibitors of exosites I and II, respectively. 50-µl
aliquots of W-7-agarose solution containing 25 µl of packed beads
were added to tubes and washed as above. 1 nmol of recombinant
GPIb
-CaM fusion protein in binding solution was added to the beads
and incubated for 2 h at 25 °C in constant suspension. After
blocking for 2 h as above, 12.5 pmol of thrombin and various
quantities (0, 50, 100, 250, 500, 750, 1000, 1500, and 2000 pmol) of
hirugen or LMW heparin were added to duplicate tubes in a volume of 70 µl making final concentrations of 0, 0.53, 1.05, 2.63, 5.26, 7.89, 10.53, 15.79, and 21.05 µM, respectively, each in a total
volume of 95 µl. After 2 h incubation at 25 °C, 50 µl of
each supernatant was transferred to a 96-well microtiter plate and
incubated with 50 µl of 2.5 mM thrombin substrate
D-Phe-Pro-Arg-pNA. The release of
p-nitroaniline was monitored spectrophotometrically at 405 nm using a THERMOmax microplate reader, as above.
Allosteric Effect of Thrombin Exosite Ligands on the
Amidolytic Activity of Thrombin
12.5 pmol of human
-thrombin was preincubated for 10 min in a
total volume of 70 µl as above with various concentrations (between 0 and 14.29 µM) of glycocalicin, hirugen (Sigma), or LMW
heparin (Rhône-Poulenc Rorer Pharmaceuticals Inc., Collegeville, PA). After incubation, 50 µl of each solution was transferred to a
96-well microtiter plate and incubated with 50 µl of 2.5 mM thrombin substrate
D-Phe-Pro-Arg-pNA. The rate of hydrolysis of the
chromogenic substrate was determined spectrophotometrically at 405 nm
by a THERMOmax microplate reader as above.
 |
RESULTS |
Noncompetitive Inhibition of Fibrinopeptide A Release--
We
began our studies by a detailed examination of the influence that
glycocalicin had on the fibrinogen-thrombin interaction. Fibrinogen
interacts with thrombin exosite I and is thereafter cleaved in its A
chain at Arg16 and in its B
chain at Arg14,
releasing FpA and FpB, respectively. We utilized a well characterized HPLC-based assay that quantitatively measures the release of FpA after
thrombin-catalyzed hydrolysis of fibrinogen (33). Various concentrations of glycocalicin were added to solutions containing both
thrombin and fibrinogen to determine whether the glycocalicin could
inhibit the thrombin-mediated cleavage of fibrinogen. As shown in Fig.
1, the addition of glycocalicin decreased
the amount of FpA released from the fibrinogen. The FpA release could
not be totally inhibited, even by a large glycocalicin excess, however, indicating that glycocalicin was not acting in a competitive manner and
was not binding to thrombin exosite I. The curve in Fig. 1 was best fit
by Equation 1, which describes a noncompetitive mode of inhibition, and
because thrombin is an allosteric enzyme (28), we concluded that the
inhibitory effect of glycocalicin occurred by an allosteric mechanism.
The equilibrium dissociation constant (Kd) for the
thrombin-glycocalicin interaction derived from this equation was
1.04 ± 0.008 µM at 150 mM NaCl.
Identical results were obtained when the amino-terminal domain of
GPIb
(residues 1-289), whether derived from human platelets or
recombinantly expressed, was substituted for glycocalicin (data not
shown).

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Fig. 1.
The amount of FpA released at time
tc = Km/eTkcat
by thrombin-catalyzed proteolysis of fibrinogen as a function of
GPIb concentration. Experimental
conditions are: 5 mM Tris-HCl, 0.15 M NaCl,
0.1% PEG-8000 at 25 °C. The data were fit by the continuous line
according to Equation 1 with the best fit parameters:
Kd = 1.04 ± 0.08 µM,
kcat/Km = 24.4 µM 1 s 1. The concentration of
thrombin was 0.1 nM, and the fibrinogen concentration was
0.2 µM.
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|
The Effect of Salt on the Glycocalicin-Thrombin
Interaction--
The binding affinities of two other ligands that
interact with thrombin exosite II, heparin (45) and the chondroitin
sulfate moiety of thrombomodulin (34) have been shown to be very
sensitive to salt concentration, whereas the affinities of ligands
binding to thrombin exosite I are not (33, 34, 46). Fig.
2 shows that the influence of
glycocalicin on the thrombin-induced FpA release from fibrinogen was
salt-dependent, with the derived equilibrium dissociation
constants for the glycocalicin-thrombin interaction varying nearly an
order of magnitude between the NaCl concentrations of 100 and 150 mm
(K100 mM NaCl = 0.187 ± 0.009 µM;
Kd125 mM NaCl = 0.41 ± 0.03 µM;
Kd150 mM NaCl = 1.04 ± 0.008 µM). The thrombin-fibrinogen
interaction is minimally affected within this range of salt
concentration (33). At higher salt concentrations than reported here,
the affinity of glycocalicin for thrombin dropped considerably, whereas
at lower concentrations the Kd could
not be determined accurately because of changes in the kinetic
mechanism for the release of fibrinopeptides.

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Fig. 2.
The effect of NaCl on the
Kd of the
GPIb -thrombin interaction. The
experimental conditions were the same as for Fig. 1 except that each
curve was generated at a different concentration of NaCl as indicated
in the inset. Each point represents the amount of FpA
released by thrombin-catalyzed cleavage of fibrinogen at time
tc =
Km/eTkcat, which is the
time along the progress curve most sensitive to inhibition by
glycocalicin. The data at each NaCl concentration were fit by Equation 1 with the best fit parameters for 0.15 M NaCl ( ) the
same as given in the legend to Fig. 1; for 0.125 M NaCl
( ), Kd = 0.41 ± 0.03 µM,
kcat/Km = 32.5 µM 1 s 1; for 0.1 M
NaCl ( ), Kd = 0.187 ± 0.009 µM, kcat/Km = 49.2 µM 1 s 1. The concentration of
thrombin eT was 0.1 nM, and the
fibrinogen concentration was 0.2 µM.
|
|
The effect of salt on the interaction of glycocalicin with thrombin can
be further quantified by plotting the data as log Kd
verses log [salt] on a straight line according to Equation 2 (33,
34). When the log of the equilibrium dissociation constants derived for
the glycocalicin-thrombin interaction at different concentrations of
NaCl was plotted against the log of their respective
[Na+], the straight line in Fig.
3 was obtained. The value of
salt for this interaction was calculated from the slope
of the line as
4.2. For comparison, the equivalent plots reported by
other investigators for the thrombin-heparin (45) and thrombin-hirugen (34) interactions are also shown in Fig. 3 as representatives of
thrombin exosite II and exosite I interactions, respectively. The value of
salt for the glycocalicin-thrombin
interaction is compared in Table I with
the
salt values obtained for thrombin and other exosite
I and II ligands obtained from the literature. As can be seen, thrombin
ligands that bind at its exosite I and show little salt dependence
(fibrinogen and hirudin) have values of
salt around 1.0, whereas known exosite II-binding ligands (heparin and the chondroitin
sulfate moiety of thrombomodulin) are characterized by
salt values around 4-5. In this respect, glycocalicin
is behaving as a thrombin exosite II-binding ligand.

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Fig. 3.
Comparison of the NaCl concentration
dependence of the GPIb -thrombin interaction
with those of the heparin-thrombin and hirugen-thrombin
interactions. The dissociation constants for the
glycocalicin-thrombin interaction ( ) derived from the curves in Fig.
2 were plotted as a function of the respective Na+ ion
concentration on a log-log scale and fitted by linear regression
according to Equation 2. For comparison, the equivalent data previously
reported for the thrombin exosite II-heparin (45) ( ) and thrombin
exosite I-hirugen (34) ( ) interactions are shown. The
slope of the line for the glycocalicin-thrombin interaction,
salt, is listed in Table I along side the
salt values previously reported by other investigators
for other thrombin exosite-ligand interactions.
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Table I
Comparison of salt for the GPIb -thrombin interaction
with those from the literature for the interaction of thrombin with
other exosite ligands
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Interaction of Thrombin Exosite Mutants with Glycocalicin--
To
address more directly the exosite on thrombin that mediates GPIb
interaction, four thrombin exosite mutants were employed in binding
studies. Previous investigators showed that mutations in the
anion-binding exosite II of thrombin (R89E, R245E, K248E, and K252E)
greatly reduced its affinity for heparin (35, 36) and that
mutations in exosite I (R68E and R70E) reduced its affinity for
fibrinogen (37). We first determined the equilibrium dissociation constants for the interaction between each of these four mutants and
glycocalicin in the fibrinogen assay utilized above. The
Kd values derived from the curves in Fig.
4 for the interaction of glycocalicin
with the exosite I mutants were comparable with that of wild-type
thrombin (KdR68E = 0.1 µM;
KdR70E = 0.034 µM at 100 mM NaCl, although because thrombin exosite I mutants
interfere with the thrombin-fibrinogen interaction, the concentration
of exosite I mutants used in the assay was 4-40-fold more than that of
wild-type thrombin). The exosite II mutants, however, interacted very
poorly with glycocalicin having 10- and 25-fold higher
Kd values than wild-type
thrombin (KdR89E = 1.4 µM;
KdK248E = 4.9 µM at 100 mM NaCl) and showed almost no inhibitory effect on FpA
release.

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Fig. 4.
The effect of exosite mutations in thrombin
on the GPIb -mediated modulation of FpA release
from fibrinogen. Experiments were performed in the same manner as
for Fig. 1, except the NaCl concentration was 0.1 M, and
thrombin mutants containing single amino acid substitutions of alanine
for basic residues within either exosite I or II, as indicated in the
inset, were used. The concentration of fibrinogen was 0.2 µM, and the concentration of each thrombin preparation
was adjusted to ensure the same thrombin activity was present in each
reaction. The continuous lines were drawn to fit the data according to
Equation 1 for wild type and exosite I mutants or by linear regression
for exosite II mutants with the best fit parameters: for wild-type
thrombin ( ), Kd = 0.187 ± 0.009 µM, kcat/Km = 49.2 µM 1 s 1; for R68E ( ),
Kd = 0.1 ± 0.001 µM,
kcat/Km = 1.0 µM 1 s 1; for R70E ( ),
Kd = 0.0335 ± 0.001 µM,
kcat/Km = 34 µM 1 s 1; for K248E ( ),
Kd = 4.9 ± 0.001 µM,
kcat/Km = 27 µM 1 s 1; and for R89E ( ),
Kd = 1.4 ± 0.7 µM,
kcat/Km = 13.67 µM 1 s 1.
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Direct Binding of Thrombin Exosite Mutants to Recombinant
GPIb
--
This assay utilized the properties of CaM to attach the
recombinant GPIb
-CaM fusion protein to W-7-agarose beads. The
thrombin-binding properties of this GPIb
-CaM fusion protein were
indistinguishable from either glycocalicin or the amino-terminal domain
of GPIb
(data not shown). The thrombin that bound to the immobilized
GPIb
pelleted with the W-7-agarose beads and was quantified from the difference between the total amount of thrombin activity added to the
tube and that remaining in the supernatant. In this way it was found
that the thrombin exosite I mutants interacted with the GPIb
() attached to the resin in an identical way as did wild-type
thrombin (Fig. 5). In contrast, the
thrombin exosite II mutants showed minimal binding activity toward the
resin-associated GPIb
() and had similar binding curves to the
negative control in which no recombinant GPIb
() was attached
to the resin (Fig. 5). Taken together, these studies also support the
concept that the GPIb
binding-site on thrombin is within
anion-binding exosite II.

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Fig. 5.
The effect of thrombin exosite mutations on
the direct binding of thrombin to GPIb .
Recombinant GPIb () was attached to agarose beads via its
fusion partner, calmodulin, and wild-type ( ) or mutant thrombin
variants with single amino acid substitutions in either exosite I, R68E
( ) and R70E ( ), or exosite II, R89E ( ) and K248E ( ), were
added. Thrombin that bound to the immobilized GPIb () was
determined from the difference between the total amidolytic activity of
thrombin added to each tube and that remaining in the supernatant after
pelleting the beads, as determined by its hydrolysis of substrate
D-Phe-Pro-Arg-pNA. The negative control ( )
had no recombinant GPIb -CaM fusion protein attached to the agarose
beads to provide a measure of the total amount of thrombin that was
available to bind to the GPIb .
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Thrombin Exosite I and II Ligands as Inhibitors of Glycocalicin
Binding--
We next investigated the abilities of hirugen (a
thrombin exosite I ligand) and LMW heparin (a thrombin exosite II
ligand) to inhibit thrombin from binding to the GPIb
attached to the W-7-agarose beads. Again we derived the difference between the total
thrombin activity added to controls with no GPIb
-CaM fusion protein
attached to the W-7 beads, and the residual thrombin activity left in
the supernatant after pelleting the thrombin that had bound to the
GPIb
() attached to the resin particles. During these
experiments we observed that the activity of the unbound thrombin
toward the peptidyl substrate D-Phe-Pro-Arg-pNA
was itself influenced by the binding of hirugen and LMW heparin. This
is shown in Fig. 6 together with the
allosteric effect of glycocalicin on the amidolytic activity of
thrombin toward this small synthetic tripeptidyl substrate. Whereas the
binding of hirugen resulted in allosteric enhancement of the amidolytic
activity of thrombin, the binding of LMW heparin and glycocalicin
resulted in allosteric inhibition of the amidolytic activity of
thrombin.

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Fig. 6.
The allosteric effects of
GPIb and other exosite ligands on the
amidolytic activity of thrombin toward the small peptidyl substrate
D-Phe-Pro-Arg-pNA. Wild-type
-thrombin (12.5 pmol) was incubated for 10 min with the indicated
concentrations of glycocalicin ( ), hirugen ( ), and LMW heparin
( ), and the rate of hydrolysis of the chromogenic tripeptidyl
substrate, D-Phe-Pro-Arg-pNA (2.5 mM), was then determined spectrophotometrically in a
microplate reader as described under "Experimental
Procedures."
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|
It can be seen from Fig. 7A
that at a concentration of 21 µM, LMW heparin completely
inhibited thrombin binding to the GPIb
() on the resin,
because all the available thrombin activity remained unbound in the
supernatant. In contrast, hirugen had no effect on the thrombin-GPIb
() interaction, as determined by the absence of convergence of
the two curves in Fig. 7B. In the presence of all
concentrations of hirugen, most of the added thrombin binds to the
GPIb
() attached to the resin and is removed from solution resulting in a marked and constant reduction in the unbound thrombin activity in the supernatants in each tube. These results indicate that
LMW heparin, which binds to thrombin exosite II, can fully inhibit
GPIb
binding to thrombin, whereas the exosite I ligand, hirugen, is
not an inhibitor of this interaction.

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Fig. 7.
The effect of LMW heparin (an exosite II
ligand) and hirugen (an exosite I ligand) on the
GPIb -thrombin interaction. Recombinant
GPIb attached to W-7-agarose via its calmodulin fusion moiety was
incubated with thrombin (12.5 pmol) in 50 mM Tris-HCl, 0.1 M NaCl, pH 7.4, for 2 h in the presence of the
indicated concentrations of either LMW heparin (A) or
hirugen (B). The dotted lines in A and
B show the allosteric effects on the amidolytic activity of
thrombin toward the peptidyl substrate
D-Phe-Pro-Arg-pNA caused by adding just LMW
heparin or hirugen, respectively, to thrombin in a solution of
W-7-agarose with no GPIb attached to the beads. The solid
lines in each panel indicate the amount of thrombin that had bound
to the GPIb immobilized on the W-7-agarose in the presence of
increasing concentrations of either LMW heparin (A) or
hirugen (B) as competitors. Bound thrombin that had been
removed from the solution by pelleting the W-7-agarose beads was
calculated from the difference between the total thrombin activity
added to the reaction tubes minus that remaining in the supernatant
after pelleting the beads (unbound thrombin activity). The convergence
of the solid curve with the dotted curve in
A indicates that LMW heparin inhibited the binding of
thrombin to the immobilized GPIb so that all of the thrombin added
to the reaction remained unbound by the addition of 20 µM
LMW heparin. Conversely, the addition of hirugen did not inhibit
thrombin from binding to the immobilized GPIb .
|
|
 |
DISCUSSION |
Evidence has been accumulating for some time that thrombin
undergoes changes in the conformation of its active site as an allosteric response to ligands binding at other sites of the enzyme. For example, the binding of proteins or peptides corresponding to
segments of natural inhibitors and substrates that bind to exosite I
(30) and the binding of the chondroitin sulfate moiety of
thrombomodulin to exosite II (34) have been shown to alter the
amidolytic activity of thrombin toward small synthetic substrates. Thrombomodulin binding to exosite I of thrombin enhances the cleavage of protein C at least 500-fold. Most of this effect has been attributed to the influence of thrombomodulin on the conformation of protein C in
the thrombin-thrombomodulin-protein C ternary complex. However, an
approximately 15-fold rate enhancement appears to be due to the
influence of thrombomodulin on the active site architecture of thrombin
as measured by turnover of small substrates that mimic the sequence cut
by thrombin in protein C (28). Another notable example of allostery
involves the sodium-binding site the occupancy of which triggers the
transition of thrombin between the slow and the fast forms (28) that
are primarily associated with the anticoagulant and procoagulant
functions of thrombin, respectively (47).
Our present studies provide an additional example of the allosteric
nature of thrombin. This became evident when we investigated the
detailed effect of GPIb
on the thrombin-fibrinogen interaction. The
results showed that glycocalicin inhibited the cleavage of fibrinogen
by thrombin, confirming the findings of previous investigators (23, 24,
48). The inhibition was not, however, of a competitive nature because
it was not possible to completely inhibit FpA release by increasing the
concentration of glycocalicin. Instead, the equation that best fit the
inhibition curve of Fig. 1 described a noncompetitive mode of
inhibition that would be consistent with the operation of an allosteric
mechanism. Therefore, unlike the previous investigators (23, 24, 48),
we concluded that glycocalicin did not bind to thrombin exosite I. Although our conclusions differed, the experimental findings reported
here are similar to those of Jandrot-Perrus et al. (23, 24)
and De Marco et al. (48). The key to understanding the
different interpretations of the findings resides in consideration of
the magnitude of the inhibition caused by GPIb
on the
thrombin-fibrinogen interaction. In no study did GPIb
completely
inhibit thrombin-fibrinogen interaction. Our present data now indicate
that this is because GPIb
does not compete for the same binding site
on thrombin as fibrinogen but rather induces a conformational change at
the active site through an allosterical mechanism that slows the rate
of fibrinogen cleavage. As shown in Fig. 6, the alteration in the
architecture of the active site of thrombin induced allosterically by
the binding of GPIb
to its exosite II also decreases the amidolytic
activity of thrombin toward the small synthetic tripeptidyl substrate
D-Phe-Pro-Arg-pNA.
The binding of glycocalicin to thrombin was highly
salt-dependent. This finding was reminiscent of ligands
that bind to thrombin exosite II such as heparin and the chondroitin
sulfate moiety attached to thrombomodulin and implicated GPIb
as a
thrombin exosite II ligand. This would make GPIb
the first protein
to directly bind to thrombin exosite II through protein-protein
interactions. In terms of negative charge density, however, the
thrombin-binding site on GPIb
(residues 269-287 (17)) could be said
to resemble that of heparin. Of the 19 residues in this region of
GPIb
, 13 are negatively charged, including three sulfated tyrosines
(residues 276, 278, and 279 (49, 50)), which themselves give this
region of GPIb
even more resemblance to the polysulfated
glycosaminoglycan, heparin. The value of
salt is a
quantitative measure of the salt dependence of a binding interaction
and for the GPIb
-thrombin interaction was determined to be
4.2
(51), in close agreement with that recently reported by others (52)
(Table I). This value of
salt far exceeds the values
reported for fibrinogen and hirudin binding (Table I) and signals a
much larger electrostatic contribution to the binding of GPIb
to
thrombin. Consistent with this is the similarity of
salt
for GPIb
-thrombin with that of the heparin-thrombin interaction that
has been characterized as a predominantly nonspecific electrostatic
interaction (45). If it is assumed that the salt-dependent
interactions between thrombin and its exosite II ligands are solely due
to electrostatic association, then the values of
salt
will also indicate the minimum number of ionic bonds involved in the
binding (45). Thus, it might be expected that a minimum of four ionic
bonds contribute to the association of GPIb
with thrombin.
More direct evidence that GPIb
binds to exosite II of thrombin was
derived from our studies using mutant thrombins with single amino acid
substitutions in either exosite I or II. The thrombin mutants with
either R89E or K248E substitutions in exosite II both displayed
dramatically decreased interactions with GPIb
(Figs. 4 and 5). In
contrast, the exosite I mutations of R68E and R70E had little effect on
GPIb
binding (Figs. 4 and 5). The mutant thrombins were employed
here in two different assays. The first was in the same HPLC assay used
above to investigate the effect that glycocalicin had on fibrinogen
turnover by thrombin. Because, however, the thrombins with exosite I
mutations themselves had reduced interactions with fibrinogen,
potentially complicating the interpretation of the results of this
assay, we also utilized an assay we had developed to study the direct
binding of thrombin to a recombinant portion of GPIb
containing the
thrombin-binding site (51). The results from both assays indicated that
exosite II mutations, but not exosite I mutations, drastically reduced the binding of thrombin to GPIb
.
The final way in which we investigated the GPIb
interaction site on
thrombin was to employ hirugen and LMW heparin as inhibitors of binding
to exosites I and II, respectively. As seen in Fig. 6 and by the
dashed curves in Fig. 7, both of these small molecules caused allosteric conformational changes in the active site of thrombin
as determined by changes in the amidolytic activity of thrombin toward
the peptidyl substrate D-Phe-Pro-Arg-pNA. The solid lines in panels A and B of Fig.
7 represent how much thrombin was inhibited from binding to GPIb
immobilized on W-7-agarose beads by LMW heparin and hirugen,
respectively. The convergence of the two curves in Fig. 7A
indicate that at a concentration of 21 µM, LMW heparin
had inhibited all of the thrombin from binding to the immobilized
GPIb
because all of the available thrombin amidolytic activity
remained in the supernatant. Conversely, hirugen caused no detectable
increase in the activity of unbound thrombin in the supernatant
indicating that the binding of thrombin to the immobilized GPIb
was
not being inhibited by hirugen. This again shows that GPIb
is
interacting with thrombin exosite II.
In the present work we have determined the affinity for thrombin
binding to glycocalicin as an inhibitor of the thrombin-fibrinogen interaction in an assay system that has been well characterized in the
study of other inhibitors of this reaction (33, 34). Under these
conditions, glycocalicin behaved as a classical noncompetitive inhibitor of the thrombin-fibrinogen interaction with a
Kd value of 1 µM for binding to
thrombin in the presence of 150 mM NaCl. Thus, thrombin
binds more avidly to glycocalicin than to the extracellular
amino-terminal fragment of the classical thrombin receptor, PAR-1, for
which the Km is 15-30 µM, also at 150 mM NaCl (53). If thrombin binding to the platelet surface involved the formation of a GPIb
-thrombin-PAR-1 ternary complex, then thermodynamic considerations dictate that the 1 µM
thrombin-glycocalicin interaction added to that of the 15-30
µM thrombin-PAR-1 interaction could reduce the
Kd of thrombin binding to as low as 15-30 × 10
12 M, which would be compatible with the
Kd values of 10
8-10
10
M reported for thrombin binding to platelets (48, 54).
The finding that glycocalicin binds to thrombin exosite II, whereas PAR-1 binds at thrombin exosite I, would allow such a ternary complex.
Alternatively, GPIb
might act as a "ligand-passing" receptor,
initially trapping thrombin at the platelet surface to make it
available to the PAR receptors, in a manner analogous to that proposed
for the passing of tumor necrosis factor from tumor necrosis factor
receptor-2 to tumor necrosis factor receptor-1 (55). In this role it
would be advantageous for the association and dissociation of thrombin
to and from GPIb
to be rapid, and a micromolar dissociation constant
for their interaction would be consistent with this.
A further hypothesis, again involving a ternary complex mechanism,
would describe a functional role for GPIb
binding to thrombin to
retain and localize the enzyme at sites where fibrin generation is
needed for the maturation and stabilization of blood clots (56). The
platelet surface provides a major site for thrombin generation (57)
through the clotting sequence. Like other factors involved in this
pathway, the precursor of thrombin, prothrombin, is anchored to the
platelet phosopholipid membrane by
-carboxylated glutamate residues
(58-61), which do not form part of the active
-thrombin enzyme when
it is released from prothrombin by proteolysis. Perhaps at this time
thrombin binds via its anion binding exosite II to GPIb
to be
retained in the locality where fibrin generation is required.
Fibrinogen would subsequently bind to thrombin exosite I and be cleaved
to the products utilized for cross-linking into the insoluble fibrin
matrix found in mature thrombi. Binding to GPIb
through exosite II
would also prevent the inhibition of thrombin by antithrombin, as
recently shown (52), because the inhibitory mechanism requires heparin
binding (62) to thrombin exosite II. Obviously, it would be desirable
to only temporarily prevent thrombin inhibition by antithrombin until
the fibrin clot became large enough to stop further blood loss but not
so large as to totally occlude blood flow through the vessel. The
relatively weak affinity between thrombin and GPIb
described by a
micromolar dissociation rate would be consistent with this,
facilitating the ready release of thrombin from GPIb
for its
subsequent inhibition by antithrombin. Furthermore, the 50% allosteric
reduction in the rate of fibrinogen turnover, caused by the binding of
GPIb
to thrombin at physiological ionic strength (Fig. 1), may be an additional mechanism to regulate blood coagulation in vivo,
similar to the allosteric "switch" mechanism described recently for
exosite inhibitors of factor VIIa (63). The localization of
thrombin-GPIb complexes on the platelet surface might also recruit and
activate additional platelets during the thrombus formation.
Although the precise role for thrombin binding to GPIb
is not known,
several observations suggest that this interaction is physiologically
important. For example, selective inhibition of thrombin binding to
GPIb
shifts the dose-response curve of platelets induced by low
doses of thrombin (21), and the thrombin-GPIb
interaction is
necessary for thrombin-induced platelet procoagulant activity (64).
Therefore, the thrombin-GPIb interaction may contribute to the
initiation and maintenance of platelet responses during hemostasis.