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INTRODUCTION |
Platelet activation by the coagulation protease
-thrombin plays
a crucial role in physiologic hemostatic processes and in thrombotic
diseases. The activation of human platelets by thrombin is mediated by
at least two receptors belonging to the family of protease-activated
receptors (PARs),1
i.e. PAR-1 and PAR-4 (1-2). These receptors are activated
upon cleavage by thrombin and mediate transmembrane signaling by
coupling to G-proteins (1-2).
-Thrombin also binds with high
affinity to the platelet glycoprotein Ib (GpIb) that belongs to the
leucine-rich repeat family of proteins (3). Thrombin binding to GpIb
contributes to platelet activation by the enzyme, as demonstrated by
the finding that Bernard-Soulier platelets, which lack the GpIb-IX-V
complex, have a delayed response to thrombin stimulation (4). In
addition, several in vitro studies have demonstrated that
the inhibition of thrombin binding to GpIb by different strategies
causes a reduction in thrombin-induced platelet activation (5-9).
The mechanism by which the binding of
-thrombin to GpIb contributes
to platelet activation is not clear. Numerous studies have shown that a
proteolytic-active enzyme is required to activate platelets.
PPACK-thrombin, which retains its ability to bind to GpIb, does not
induce platelet aggregation (10). Moreover, GpIb does not undergo
cleavage by thrombin. On the other hand, the cleavage of
G-protein-coupled PARs seems to be essential in platelet activation and
transmembrane signaling. The finding that thrombin binding to GpIb
involves a distinct thrombin domain, the HBS, which is far from the
thrombin catalytic site and the fibrinogen recognition site (7-9),
would suggest that a ternary complex thrombin·GpIb·PAR-1 may form
on the platelet membrane that could be responsible for optimal
hydrolysis and signal transduction.
In the present study, the hypothesis that thrombin binding to GpIb may
affect the hydrolysis of PAR-1 by the enzyme on intact platelets was
investigated. The hydrolysis of PAR-1 was evaluated as a paradigm to
construct a model where GpIb acts as a cofactor for PAR(s) cleavage.
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EXPERIMENTAL PROCEDURES |
Materials--
Human
-thrombin was purified and characterized
as previously reported (11). Mutant thrombin R98A, which contains an
alanine substitution at Arg-98 (thrombin B-chain
numbering),2 and
recombinant wild-type (WT) human thrombin were obtained and characterized as described previously (12). The mutant form retains its
catalytic activity, although its ability to interact with heparin was
severely impaired, as demonstrated by in vitro studies on
heparin-catalyzed thrombin inhibition by anti-thrombin III.
PPACK-thrombin was obtained and characterized as previously reported
(13).
Mocarhagin was purified from cobra venom, as described previously (14),
at the Baker Medical Research Institute, Praharan, Australia. HD22, a
single-stranded DNA oligonucleotide with the sequence
5'-AGTCCGTGGTAGGGCAGGTTGGGGTGACT-3', which binds with high affinity to
the heparin binding site of thrombin (15), was a kind gift of Prof.
J. I. Weitz, (McMaster University, Hamilton, Ontario).
The PAR-1-(38-60) peptide, LDPRSFLLRNPNDKYEPFWEDEE, was synthesized
and characterized by mass spectrometry at Primm s. r. l. (Milan,
Italy). The monoclonal antibody LJ1b10, which is able to selectively
inhibit thrombin-GpIb interaction, was a generous gift from Dr. Zaverio
Ruggeri (The Scripps Research Institute, La Jolla, CA).
Preparation of Platelets--
Platelets from normal donors were
obtained by gel filtration of platelet rich plasma onto Sepharose 2B
columns (25 × 1 cm) equilibrated with a buffer containing 20 mM Hepes, 135 mM NaCl, 5 mM KCl, 5 mM glucose, 0.2% albumin, pH 7.4. EDTA (1-mM
final concentration) was added to PRP prior to gel filtration to avoid activation during washing procedures. The platelet count was adjusted to 1 × 105/µl with washing buffer in all the experiments.
GpIb-depleted platelets were prepared by using the cobra venom
metalloproteinase, mocarhagin, which selectively cleaves GpIb between
Glu-282 and Asp-283 residues, according to a previously reported method
(9). Briefly, gel-filtered platelets were incubated with 10 µg/ml
mocarhagin and 1 mM Ca2+ for 20 min at
37 °C. The mocarhagin activity was stopped by 2 mM EDTA.
An aliquot of intact platelets (i.e. not exposed to
mocarhagin) from the same donor was subjected to the same incubation
with 1 mM Ca2+ for 20 min at 37°, addition of
2 mM EDTA and was used as a control. Evaluation of GpIb
cleavage was performed by flow cytometry using the fluorescein
isothiocyanate (FITC)-conjugated anti-GpIb monoclonal antibody CD42b
(SZ2 clone) (Immunotech, Marseille, France), whose epitope on GpIb
is lost following mocarhagin treatment (5).
Measurement of PAR-1 Hydrolysis by Thrombin on Intact
Platelets--
Gel-filtered platelets, intact or GpIb-depleted, were
exposed to
-thrombin, whose final concentration ranged from 1 nM to 10 nM in different experiments. At
different times (from 15 to 600 s), aliquots of 50 µl of
stimulated platelets were drawn into tubes containing 100 nM hirudin, which abolished the thrombin activity. The
tubes were placed in ice to stop internalization of cleaved PAR-1
molecules (16).
In different experiments, intact platelets were stimulated by
-thrombin in the presence of either 130 µg/ml anti-GpIb SZ2 mAb,
400 nM HD22 aptamer, or 140 nM
PPACK-thrombin.
In the experiments using the R98A thrombin mutant and the recombinant
WT human thrombin, gel-filtered platelets were stimulated by the
enzymes at a concentration of 1 nM.
The presence of intact PAR-1 molecules on the platelet membrane at
different times after thrombin stimulation was measured by using a
phycoerythrin (PE)-conjugated anti-PAR-1 mAb, SPAN12 clone
(Immunotech). SPAN12 recognizes the NH2-terminal peptide of
PAR-1 residues 35-46 (NATLDPR
SFLLR), where "
" indicates the cleavage site by thrombin. This mAb reacts only with uncleaved PAR-1
molecules (16). In preliminary experiments, PE-SPAN12 was added to
1 × 106 platelets at increasing amounts (0.5, 1, 1.5, 2, 2.5, and 3 µg) for 30 min at 4 °C. Platelet suspensions were
subsequently washed in phosphate-buffered saline/EDTA at 1500 rpm for 5 min. An optimal resolution of control and test histograms was obtained
with 2 µg of anti-PAR-1 mAb/1 × 106 platelets.
Thus, this saturating dose was chosen for subsequent experiments.
Background fluorescence was determined using a PE-conjugated isotype-matched irrelevant mAb.
At the end of stimulation by thrombin, 2 µg of SPAN-12 was added to
each tube containing platelets and hirudin. After incubation at 4 °C
for 30 min, platelets were washed in phosphate-buffered saline/EDTA,
and samples were run through a FACScan flow cytometer (Becton
Dickinson, Mountain View, CA) equipped with an argon laser emitting at
488 nm. PE signals were collected and recorded at 575 nm; gain setting
stability was verified daily with Calibrite BeadsTM®
(Becton Dickenson). A minimum of 20,000 events were acquired in list
mode using CellQuestTM software (Becton Dickenson); forward
(FSC) and side (SSC) scatter were collected with logarithmic
amplification, and fluorescent emissions were collected on a 4-decade
logarithmic scale. Levels of silver expression were measured in terms
of the geometric mean of specific fluorescence.
Measurement of PAR-1-(38-60) Peptide Hydrolysis by WT Thrombin
and R98A Thrombin--
Hydrolysis of the PAR-1-(38-60) peptide by
thrombin was followed by measuring the release of the peptide LDPR,
resulting from the cleavage of the NH2 terminus of PAR-1,
according to a previously described method (7). Briefly, 0.5 µM PAR-1-(38-60) peptide was incubated with 0.1 nM WT thrombin or 0.1 nM R98A thrombin in HEPES
(10 mM), NaCl (0.15 M), polyethylene glycol
Mr 6000 (0.1%) pH 7.5 at 25°. At time
intervals (1, 2, 3, 4, 8, 12, and 15 min) the reaction was stopped with
0.3 M HClO4, and the cleaved peptide was
measured by reversed-phase HPLC, using a 250 × 4.6 mm RP-304 column (Bio-Rad).
Experimental concentrations of PAR-1-(38-60) peptide cleaved at time
t (Pt), were fitted to the following
equation,
|
(Eq. 1)
|
where P
is the peptide concentration
at t =
and kobs is the
pseudo first-order rate of PAR-1 hydrolysis, equal to e0 × kcat/Km (e0 is
the thrombin concentration).
Aggregometry Studies--
Gel-filtered platelets were suspended
in HEPES buffer, as detailed above, containing 2 mM
CaCl2 and used at a final count of 200,000/µl. Platelet
aggregation by thrombin was studied using a 4-channel PACKS-4
aggregometer (Helena Laboratories, Sunderland, UK), according to the
Born method. In some experiments, platelets were stimulated by
recombinant human WT thrombin and R98A thrombin at concentrations
ranging from 0.39 nM to 50 nM. In other
experiments, platelets were stimulated by WT thrombin at concentrations
ranging from 0.625 to 20 nM, or by R98A thrombin at
concentrations ranging from 3.125 to 100 nM, in the absence
and in the presence of monoclonal antibody LJIb10 (0.15 mg/ml, final
concentration). The velocity of absorbance change was measured and
expressed as percent/min.
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RESULTS |
Measurement of Thrombin Hydrolysis of PAR-1 on Intact
Platelets--
Measurement of PAR-1 hydrolysis by
-thrombin was
accomplished by a cytofluorimetric method, using a fluorescent
monoclonal antibody, SPAN-12, that recognizes only the intact
NH2-terminal portion of the receptor. After cleavage, the
mAb does not interact with the receptor, so that disappearance of the
mAb signal reflects the hydrolytic reaction. Internalization of the
receptor, which could also lead to loss of signal, has been shown to
occur only for cleaved PAR-1 molecules (16-18). Under the conditions
of the study, using intact gel-filtered platelets, the concentration of
PAR-1 present on the platelet membrane (100-2000 copies of PAR-1/platelet, Refs. 19-20) is much lower (nanomolar) than the Km value of its hydrolysis by thrombin (micromolar
range), so that the kinetics of PAR-1 cleavage can be fitted to an
exponential equation, whose rate constant,
kobs, is proportional to the
kcat/Km value of
thrombin-PAR-1 hydrolysis, according to the following equation
(21).
|
(Eq. 2)
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In the experimental setup, the fluorescent signal given by the
SPAN-12 mAb was monitored as a function of time at fixed thrombin concentrations. Although the antibody staining procedure is relatively long compared with PAR-1 cleavage kinetics at the concentrations of
thrombin used, this limitation was overcome by performing the staining
on ice and using high concentrations of hirudin to completetly inhibit
thrombin activity. Fluorescence signals measured as a function of time
were fitted to the following equation,
|
(Eq. 3)
|
where Flt is the fluorescence at time t,
Fl0 is the initial, Fl
is the final
fluorescence value, and kobs is the observed rate constant for the single exponential decay. Knowing the enzyme concentration and using Eq. 2, the
kcat/Km value could be
calculated. Additional experiments were carried out to confirm the
validity of this experimental approach. From Eq. 2 it follows that if
the kobs value actually reflects the
kcat/Km value of the
hydrolytic reaction, then it must depend on the enzyme concentration.
Thus, the observed rate constant was measured at different thrombin
concentrations, ranging from 0.5 to 8 nM. The above
analysis assumes that the concentration of the uncleaved receptor does not change during the time course of the experiments. To test this
hypothesis, control experiments were carried out with a different mAb,
WEDE-15, directed against the COOH-terminal domain of the PAR-1
cleavage site. Using thrombin in the nanomolar range, only a decrease
in WEDE-15 binding to gel-filtered platelets was observed, consistent
with an internalization process of the hydrolyzed PAR-1 receptor, which
has been previously reported (16-18). This implies that over the time
course of the experiments, newly exposed receptors (present in the
surface connecting system) did not significantly alter the total PAR-1
concentration and that loss of WEDE-15 binding to platelets is likely
because of an internalization process involving the cleaved receptor
molecules. These findings therefore allow calculation of the apparent
kcat/Km value pertaining to
thrombin-PAR-1 interaction as outlined above.
As shown in Fig. 1, the pseudo
first-order rate constant of PAR-1 cleavage increased as a function of
-thrombin concentration ranging from 0.5 to 8 nM,
consistent with the canonical Michaelis scheme for serine protease
activity that predicts a linear relation between the catalytic rate and
the enzyme concentration (Eq. 2). The inset in Fig. 1
demonstrates this relationship with the slope of the straight line
expressing the apparent
kcat/Km value, which is equal
to 1.5 ± 0.1 × 107
M
1
sec
1. This value is similar to that for
thrombin hydrolysis of the PAR-1-(38-60) peptide in solution (see
below), supporting the validity of the cytofluorimetric strategy used
in this investigation.

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Fig. 1.
Kinetics of PAR-1 cleavage by different
thrombin concentrations, measured as a loss of SPAN12 mAb fluorescence
over time. The experimental conditions reported under
"Experimental Procedures" included 60,000 gel-filtered
platelets/µl and 0.12 µg/ml of the mAb. Solid lines are
drawn according to Eq. 3 with the bestfit kobs
values: ( ), 6.6 ± 0.6 × 10 3
sec 1; ( ), 1.2 ± 0.3 × 10 2
sec 1; ( ), 2.9 ± 0.6 × 10 2
sec 1; ( ), 3.7 ± 0.4 × 10 2
sec 1; ( ), 1.2 ± 0.3 × 10 1
sec 1. Data are presented as mean ± S.E. from two
different determinations. In the inset, the experimental
values of kobs pertaining to PAR-1 cleavage on
intact platelets are plotted as a function of thrombin concentration,
according to Eq. 2. The straight line was drawn according to
the bestfit kcat/Km value of
1.5 ± 0.1 × 107 M 1
sec 1.
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Effect of Inhibiting Thrombin-GpIb Interaction on Hydrolysis of
PAR-1 in Intact Platelets--
The effect of inhibiting thrombin-GpIb
interaction on the enzyme hydrolysis rate of PAR-1 was evaluated by
five different experimental strategies: 1) elimination of the thrombin
binding site on GpIb
by mocarhagin treatment of gel-filtered
platelets, 2) use of a monoclonal antibody SZ2, which binds to GpIb
and inhibits thrombin interaction, 3) use of the DNA aptamer HD22, which specifically interacts with the heparin binding site (HBS) of
thrombin and inhibits enzyme binding to GpIb
, 4) use of a thrombin
mutant, R98A, which bears a perturbation of the HBS structure, and 5)
use of PPACK-thrombin in competition experiments with
-thrombin.
The removal of the NH2-terminal 1-282 region of GpIb
by
mocarhagin severely impaired the PAR-1 hydrolysis by thrombin. As shown
in Fig. 2, the
kobs value for PAR-1 hydrolysis by 1 nM
-thrombin was reduced by about 6-fold in
GpIb-depleted platelets compared with untreated platelets. The
inset in Fig. 2 shows that the
kcat/Km value of PAR-1
hydrolysis in mocarhagin-treated platelets, which was 2.8 ± 0.3 × 106 M
1
sec
1, decreased 5.4-fold in comparison with untreated
platelets. Similar results were obtained by using two different
competitive inhibitors of thrombin binding to GpIb, the mAb SZ2, which
interacts with the thrombin binding site on GpIb
(5, 22), and the
DNA aptamer HD22, which interacts with the thrombin HBS. Fig.
3 clearly shows that in both cases a
roughly 5-6-fold reduction in kobs was obtained using a large excess of the inhibitors. Although the targets of the
inhibitors were different, the effect was the same, as in both cases
the thrombin-GpIb interaction was inhibited. Altogether these results
show that inhibition of thrombin binding to GpIb
causes a marked
reduction of the apparent catalytic specificity constant of the
thrombin-PAR-1 interaction.

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Fig. 2.
Measurement of the
kobs value pertaining to
PAR-1 hydrolysis on intact and mocarhagin-treated platelets.
Experimental conditions were similar to those reported in the legend to
Fig. 1. Intact ( ) and mocarhagin-treated ( ) (10 µg for 10 min
at 37 °C) platelets were exposed to 1 nM -thrombin,
and the cleaved PAR-1 was estimated cytofluorimetrically by the SPAN 12 mAb, as described under "Experimental Procedures." The solid
lines were drawn by nonlinear regression according to Eq. 3, with
the bestfit kobs values: ( ), 1.8 ± 0.3 × 10 2 sec 1; ( ), 0.3 ± 0.07 × 10 2 sec 1. Data are presented
as mean ± S.E. from two different determinations. In the
inset, the experimental values of
kobs pertaining to PAR-1 cleavage on
mocarhagin-treated ( ) and, for comparison, intact platelets ( )
are plotted as a function of thrombin concentration, according to Eq. 2. The straight lines were drawn according to the bestfit
kcat/Km value: ( ),
1.5 ± 0.1 × 107 M 1
sec 1; ( ), 2.8 ± 0.3 × 106
M 1 sec 1.
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Fig. 3.
Measurement of the
kobs value pertaining to
PAR-1 hydrolysis in the presence of inhibitors of thrombin-GpIb
interaction. Quantification of PAR-1 hydrolysis was accomplished
as reported in the legend to Fig. 1, using 2 nM
-thrombin, but in the presence of 400 nM HD22 aptamer
( )or 130 µg/ml anti-GpIb SZ2 mAb ( ), both inhibiting the
thrombin-GpIb interaction. The kinetics of PAR-1 hydrolysis in the
absence of any inhibitor, but in the presence of 130 µg/ml human IgG
used as control, is reported as well ( ). Data are presented as
mean ± S.E. from two different determinations. The
lines were drawn by nonlinear regression according to Eq. 3,
with the bestfit kobs values: ( ), 3.2 ± 0.6 × 10 2 sec 1 (solid
line); ( ), 0.48 ± 0.06 × 10 2
sec 1 (dashed line); ( ), 0.6 ± 0.09 × 10 2 sec 1 (dotted
line).
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The findings reported above raise the question of whether thrombin
interaction with GpIb
might induce an intracellular signal that
could alter the interaction of thrombin with PAR-1, for instance through liberation of a second messenger molecule acting on the expression/conformation of PAR-1 molecules. PPACK-thrombin, which is
catalytically inactive, yet still able to interact with GpIb, did not
cause aggregation of gel-filtered platelets, even at 200 nM
and did not induce any intracytoplasmic Ca2+ flux (data not
shown), in accordance with numerous previous reports (6, 10). On the
other hand, competition experiments showed that a high concentration of
PPACK-thrombin (140 nM), by displacing
-thrombin from
GpIb
, reduced by about 3-fold the apparent
kcat/Km value of PAR-1
hydrolysis on intact platelets, as shown in Fig. 4. Again, this experiment is in accord
with the other functional experiments described above and corroborates
the hypothesis that thrombin-GpIb interaction is able to enhance the
specificity of thrombin cleavage of PAR-1 on intact platelets.

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Fig. 4.
Effect of displacement of
-thrombin binding to GpIb by high PPACK-thrombin
concentration on PAR-1 hydrolysis. Kinetics of PAR-1 hydrolysis,
as described in the legend to Fig. 1, using 1 nM
-thrombin, in the absence ( ) or presence ( ) of 140 nM PPACK-thrombin. Data are presented as mean ± S.E.
from two different determinations. Solid lines were drawn by
nonlinear fitting according to Eq. 3, with the bestfit
kobs values: ( ), 1.6 ± 0.4 × 10 2 sec 1. ( ), 0.6 ± 0.08 × 10 2 sec 1.
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This hypothesis was further supported by the finding that a thrombin
mutant, R98A, bearing an alanine substitution in the HBS of thrombin at
Arg-98, showed a similar reduction of the kobs value of PAR-1 hydrolysis compared with recombinant WT human thrombin, as shown in Fig. 5. In control
experiments, this diminished activity of PAR-1 hydrolysis on platelets
could not be attributed to an intrinsic reduction of the catalytic
efficiency of the thrombin mutant. Solution experiments employing the
PAR-1-(38-60) peptide, bearing both the cleavage and the recognition
site for thrombin (23), demonstrated that thrombin R98A cleaved the
solution peptide with a
kcat/Km that was slightly
higher than that of WT thrombin, as shown in Fig.
6. This finding is consistent with the
hypothesis that the reduction of the
kcat/Km value of PAR-1
hydrolysis on intact platelets is likely because of a defective
interaction of the thrombin mutant with GpIb
. Moreover, the
biological effect of this mutation on platelet activation can be seen
in Fig. 7, which shows that the
EC50 value for platelet aggregation by R98A is roughly
10-fold higher compared with WT thrombin.

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Fig. 5.
Measurement of the
kobs value pertaining to
PAR-1 hydrolysis by wild-type and the R98A thrombin mutant.
Kinetics of PAR-1 cleavage by wild-type ( ) and the R98A thrombin
( ) at 1 nM concentration. Gel-filtered platelets were
used at a concentration of 60,000/µl. Data are presented as mean ± S.E. from two different determinations. The solid lines
were drawn by nonlinear regression according to Eq. 3, with the bestfit
kobs values: ( ), 1.9 ± 0.2 × 10 2 sec 1, ( ), 0.34 ± 0.05 × 10 2 sec 1.
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Fig. 6.
Measurement of the
kcat/Km value
pertaining to the PAR-1-(38-60) peptide hydrolysis by wild-type and
the R98A thrombin mutant. Hydrolysis of the PAR-1-(38-60) peptide
was measured by the HPLC method, using 0.1 nM wild-type
( ) and R98A thrombin mutant ( ) and 0.5 µM peptide.
The experimental points are the mean of two determinations. Solid
lines were drawn by nonlinear regression according to Eq. 1, with
the bestfit kobs values: ( ), 4.3 ± 0.3 × 107 M 1
sec 1, ( ), 7 ± 0.8 × 107
M 1 sec 1.
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Fig. 7.
Platelet aggregation capacity of the
wild-type and the R98A thrombin mutant. Gel-filtered platelets
used at 200,000/µl were exposed to different concentrations of
wild-type ( ) or R98A thrombin mutant ( ). Data are presented as
mean ± S.E. from two different determinations. The solid
lines were drawn by nonlinear regression with the bestfit
EC50 values equal to 1.1 ± 0.2 and 10 ± 2 nM for the wild-type and the R98A thrombin mutant,
respectively.
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Effect of Anti-GpIb mAb LJIb10 on Platelet Aggregation by WT
Thrombin and R98A Mutant Thrombin--
These experiments were carried
out to evaluate whether the thrombin HBS could be involved in the
interaction with other thrombin receptors. PAR-1 was already
demonstrated not to be involved in HBS ligation (6-9), but PAR-4 could
potentially contain a binding site for the thrombin HBS, although a
close inspection of the PAR-4 primary sequence (24) does not show a
negatively charged domain to support this hypothesis. The mAb LJ1b10
specifically inhibits the interaction of thrombin with GpIb
, without
affecting the binding of von Willebrand factor to GpIb
(22, 25). The use of this mAb allows one to rule out a potential inhibitory effect on
the aggregation of gel-filtered platelets that could be attributed to
an impaired interaction of GpIb with platelet von Willebrand factor,
which is released upon thrombin stimulation.
As shown by Fig. 8A, platelets
stimulated by WT thrombin had a rightward shift in the aggregometric
dose-response curve in the presence of 0.15 mg/ml LJIb10 (with
EC50 values of 4.43 ± 0.4 nM and
10.15 ± 0.77 nM, respectively). This finding is in agreement with the hypothesis that specific inhibition of thrombin binding to GpIb causes a reduced platelet response to the agonist. In
contrast, the dose-response curve obtained by stimulating platelets with thrombin mutated at the HBS (Fig. 8B) was not affected
at all by the presence of the mAb (with EC50 values of
23.5 ± 2.8 nM and 22.1 ± 5 nM,
respectively). This result is in agreement with the concept that the
thrombin-GpIb interaction involves the enzyme HBS, as the LJ-Ib10 mAb
could not induce any inhibition of the R98A thrombin-induced platelet
activation. This result would also suggest that the thrombin HBS is
involved only in GpIb binding and not in the interaction with other
platelet receptors, such as PAR-4.

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Fig. 8.
Effect of the anti-GpIb mAb LJIb10 on
platelet aggregation by WT and R98A mutant thrombin. Aggregation
of gel-filtered platelets induced by WT thrombin (A) and the
R98A thrombin mutant (B) was measured in the absence ( )
and presence ( ) of the LJ-Ib10 mAb, as described under
"Experimental Procedures." Data are presented as mean ± S.E.
from two different determinations. Solid lines were drawn
using the bestfit EC50 values: 4.43 ± 0.4 nM and 10.15 ± 0.77 nM for WT-thrombin
(in the absence and in the presence of 0.15 mg/ml LJ-Ib10,
respectively); 23.5 ± 2.8 nM and 22.1 ± 5 nM for the R98A mutant (in the absence and in the presence
of 0.15 mg/ml LJ-Ib10, respectively).
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 |
DISCUSSION |
In this study, the role of GpIb
in the hydrolysis of PAR-1 by
thrombin and in thrombin-induced platelet activation was examined. GpIb
binds thrombin with high affinity and contributes to platelet activation by the enzyme; however, there is no evidence that thrombin ligation to GpIb per se is able to trigger platelet
activation. Results from the present study show that GpIb
can
function as a cofactor for PAR-1 cleavage and activation in human
platelets. In fact, by directly measuring the hydrolysis of PAR-1 on
intact platelets, it was possible to evaluate the effect of the
inhibition of thrombin binding to GpIb
on PAR-1 cleavage. The
cytofluorimetric strategy used to detect PAR-1 hydrolysis on platelets
allowed measurement of the apparent specificity constant,
kcat/Km, pertaining to
thrombin hydrolysis of the receptor. It is noteworthy that the value
measured in intact cells is similar to that for hydrolysis of the
PAR-1-(38-60) peptide in solution, as confirmation of the validity of
the experimental method. Interestingly, the inhibition of thrombin
interaction with GpIb on platelets, obtained by different experimental
strategies, consistently showed a 5- to 6-fold reduction of the
specificity rate constant of PAR-1 hydrolysis by thrombin. This finding
might suggest that in the absence of GpIb on the platelet membrane, the
thrombin-PAR-1 interaction is partially hampered or less productive,
given that the kcat/Km value
is much lower than that measured with the PAR-1-(38-60) peptide in solution.
Thrombin bound to GpIb
via its HBS, whose blockade caused a
reduction in both PAR-1 hydrolysis and platelet aggregation. That the
HBS is not directly involved in the interaction with PAR-1 was
demonstrated by the experiment showing that the HBS mutant thrombin,
R98A, cleaves the PAR-1-(38-60) peptide in solution with an apparent
kcat/Km similar to or even
higher than that pertaining to WT thrombin. Platelet aggregation by
R98A thrombin was not sensitive to the presence of the anti-GpIb mAb
LJ-Ib10, whereas the aggregation by WT thrombin showed a
rightward-shift of the dose-response curve (Fig. 8). In the former
case, in fact, thrombin binding to GpIb was already impaired by HBS
perturbation, whereas in the latter case use of the mAb blocked the
GpIb contribution to platelet aggregation. The effect of the anti-GpIb
mAb on the aggregation by WT thrombin is in agreement with the
inhibitory effect of the mAb on PAR-1 activation by thrombin via the
inhibition of enzyme binding to GpIb.
Because inhibition of the thrombin-GpIb interaction, obtained through
different methods, caused in all cases a reduction of the PAR-1
hydrolysis rate, this finding was interpreted as a consequence to a
positive linkage between thrombin binding to GpIb and the catalytic
interaction of this enzyme complex with PAR-1. It was previously shown
that GpIb
in solution does not alter the kinetic constants of
hydrolysis of the PAR-1-(38-60) peptide (7, 26). This implies that
membrane phenomena are responsible for the effect observed using intact platelets.
One possible mechanism of action of GpIb could be that this
transmembrane glycoprotein mediates a signal transduction event that
could modify the membrane and/or PAR-1-folding, so that PAR-1 would be
more activatable by thrombin. However, experiments with PPACK-thrombin
did not confirm this hypothesis. In fact, intraplatelet Ca2+ flux was not observed using PPACK-thrombin that binds
to GpIb but does not cleave PAR-1 (data not shown). Moreover, when
PPACK-thrombin was used along with
-thrombin, it caused, as shown in
Fig. 4, a 3-fold reduction of the apparent
kcat/Km value pertaining to
PAR-1 cleavage, in agreement with the effect of the other inhibitors of
thrombin-GpIb binding. PPACK-thrombin would have caused an enhancement
of PAR-1 cleavage, if a positive intracellular signal were generated.
It is postulated that von Willebrand factor binding to GpIb induces
signal transduction (27), whereas this phenomenon has not yet been
demonstrated for thrombin binding to GpIb. Accordingly, very recent
findings have indicated that thrombin-GpIb interaction is a necessary
but not sufficient condition to induce a procoagulant capacity in the
platelet membrane (28). In addition to thrombin binding to GpIb, the
procoagulant platelet activity has been demonstrated to arise from both
the involvement of the GpIIb-IIIa complex and the platelet-platelet
interaction as well (28).
It may be hypothesized that GpIb would enhance the productive
collisions between the enzyme and the substrate, PAR-1, on the membrane
surface, essentially acting as a catalyst for the reaction. This
hypothesis hinges on the structural properties of the components involved in the interaction. The formation of a ternary
thrombin·GpIb·PAR-1 complex can be hypothesized, as GpIb binds to
the thrombin HBS, whereas PAR-1 interacts with both the exosite
referred to as fibrinogen recognition site and the catalytic pocket (6,
9, 23). The positive effect of GpIb interaction with thrombin might be considered as a template function, which could enhance the number of
productive collisions between the enzyme and the PAR-1 substrate. The
mechanisms through which this effect takes place is not in the realm of
this study. Further studies are needed to demonstrate experimentally
the formation of the ternary thrombin·GpIb·PAR-1 complex, as well
as to unravel some structural issues pertaining to the components of
this complex. GpIb is in fact an elongated molecule with a longitudinal
axis of ~550 Å (29). Because the thrombin binding site on GpIb
and the NH2-terminal PAR-1 domain are expected to be
located at roughly 300 and 65-70 Å from the membrane surface,
respectively, it has to be demonstrated how a ternary
thrombin·GpIb·PAR-1 adduct could form on the platelet membrane.
From a physical standpoint, one can reasonably assume that the kinetics
of thrombin binding to GpIb is faster than kinetics of formation of the
thrombin·PAR-1 Michaelis adduct. Thus, although the association rate
constants of thrombin·GpIb and thrombin·PAR-1 may be similar, the
concentration of GpIb is at least 1-2 orders of magnitude higher than
that of PAR-1 on the platelet membrane (19-20), and thus the
bimolecular interaction of thrombin with GpIb is faster than the
formation of the thrombin·PAR-1 adduct. Hence GpIb may function as a
cofactor for PAR-1 activation by thrombin in situ. It is not
clear from this investigation whether this cofactor activity is
exclusively mediated by GpIb alone, or through more complex
interactions involving the entire glycoprotein adduct, GpIb-IX-V. GpV
in fact is known to be cleaved by thrombin, but its role in platelet
activation remains unknown (30).
The cofactorial function of GpIb would resemble the one recently
identified for thrombin interaction with PAR-3 and PAR-4 on mouse
platelets (31). Similarly to GpIb for human platelets, PAR-3 in mouse
platelets does not mediate transmembrane signaling, but its loss
inhibits the mouse platelet activation by low thrombin concentrations.
The model for PAR-3 function predicts that this receptor binds thrombin
that remains available on platelet membrane to cleave and activate
nearby PAR-4 molecules. Although PAR-3 and GpIb are completely
different molecules, they might share a cofactorial function for PARs
activation. This model may be of interest for human platelets, because
it introduces the concept that cofactors for PARs activation might
regulate the specificity of response to proteases of target cells.
Regulation of cofactor function, rather than that of the receptor
itself, might thus become crucial in modulating the effects of
proteases on cells. This concept seems particularly to fit for proteins
and enzymes that do not bear phospholipid binding sites. Membrane
surface is one of the most relevant cofactors usually involved in
strongly accelerating coagulation reactions (32). Many coagulation
factors, such as Factor V, and Factor VIII, operating on a membrane
surface, bear specific phospholipid binding sites. The interaction of
thrombin with its PAR-1 substrate, inserted in the platelet membrane,
could be hampered by the lack in the thrombin molecule of a domain
capable of binding to membrane phospholipids. The high affinity
interaction of thrombin with GpIb could overcome this limitation. The
thrombin-GpIb interaction could pay the energetic cost to favor an
otherwise hampered interaction between the enzyme and its
macromolecular substrate. Likewise, an interesting model for this kind
of cofactorial function of protease receptors is that recently
described for the endothelial Protein C receptor involved in activating
Protein C along with the thrombin·thrombomodulin complex on the
surface of endothelial cells (33).
At this point one might question whether the 5-6-fold increase of the
kcat/Km value of PAR-1
hydrolysis caused by thrombin interaction with GpIb could be of
physiological relevance. The present data cannot indicate whether the
increase of the kcat/Km value
arises from either an increase of kcat, or a
decrease of Km value, or else a combination of both
phenomena. The present study shows that, under physiological
conditions, thrombin cleaves PAR-1 according to a pseudo first-order
kinetics. Under this condition, whatever the mechanism, an increase of
5-6-fold of the kcat/Km
value of PAR-1 hydrolysis leads to a 5-6-fold increase of the net
velocity of its cleavage, according to Eq. 2. Although the GpIb effect
on PAR-1 activation is not dramatic, if compared with other similar
phenomena of the blood coagulation system (33), it could likely be
physiologically relevant. In fact, the PAR-1 shut-off is a very rapid
process after cell activation by thrombin (34), and thus a very
efficient enzyme interaction with PAR-1 molecules is needed to generate
an optimal amount of activated receptors required for a full cell activation.
The results from the present and previous studies show that
-thrombin binding to GpIb
has a net prohemostatic effect, as not
only is this interaction able to enhance the hydrolysis of PAR-1 on the
platelet membrane, but also protects the enzyme from the
heparin-catalyzed inhibition by anti-thrombin III (9). The
demonstration that the specific inhibition of thrombin-GpIb interaction
leads to reduced thrombin cleavage of PAR-1 on intact platelets, might
open the way to new strategies for specific modulation of platelet
responses to thrombin stimulation in different clinical settings.