From the Servizio Immunotrasfusionale e Analisi Cliniche, Centro di
Riferimento Oncologico, 33081 Aviano, PN, Italy, the
§ Department of Medicine, State University of New York,
Stony Brook, New York 11794, and the ¶ Roon Research Center
for Arteriosclerosis and Thrombosis, Division of Experimental
Hemostasis and Thrombosis, Departments of Molecular and Experimental
Medicine and of Vascular Biology, Scripps Research Institute,
La Jolla, California 92037
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INTRODUCTION |
Platelet deposition at sites of vascular injury is thought to be
enhanced by
-thrombin during normal hemostasis as well as pathological arterial thrombosis (1-3), but the mechanisms responsible for this effect have yet to be fully understood. There is evidence that
-thrombin-induced platelet activation is initiated by coupling of
the agonist to specific receptors (4-6), whose nature is still a topic
of debate. In this context, it is recognized that glycoprotein (GP)1 Ib
, a component of
the GP Ib-IX-V complex (7, 8), binds
-thrombin possibly with
affinity higher than other sites on platelets (6, 9, 10). This
interaction has been variably judged as functionally relevant (10-12)
or irrelevant (13) or even as a negative regulator acting through
sequestration of the enzyme (14). It is also known that
-thrombin
cleaves GP V (15, 16) but with no apparent relation to platelet
activation (17, 18).
The agonist activity of
-thrombin depends on proteolysis, a fact
explained with the identification of a seven-transmembrane domain
receptor stimulated by a tethered ligand sequence exposed as the new
amino terminus of the molecule after cleavage of an internal Arg-Ser
bond (19). This protease-activated receptor, PAR1, exemplifies an
effector mechanism common to a family of related proteins exhibiting
distinct specificities as substrates for different proteases (20).
Because the function of PAR1 seemed to explain many of
-thrombin
effects on platelets, it was surprising that deletion of the homologous
mouse gene, albeit lethal in many homozygous embryos, failed to result
in decreased thrombogenic potential in the animals born alive (21). The
subsequent demonstration on platelets of PAR3, another member of the
family with specificity similar to PAR1 (22), provided a reasonable
solution to the puzzle and reinforced the concept that a
protease-activated receptor pathway is crucially involved in mediating
responses to
-thrombin. Yet the participation of GP Ib
in these
processes remains a possibility that must be addressed
conclusively.
There are apparent contradictions in the reported characteristics of
-thrombin binding to platelets. Only few hundred high affinity sites
have been ascribed to GP Ib
(6, 10), but the latter is present in
greater number on the membrane (23). Moreover, a specific anti-GP Ib
antibody has been shown to block the interaction of approximately 5,000
-thrombin molecules with platelets, abolishing higher affinity sites
but also decreasing markedly the moderate affinity ones (10). Finally,
the apparent kd of the highest affinity sites on
platelets, 0.25-1.3 nM (10), is substantially lower than
that reported for
-thrombin interaction with the isolated
amino-terminal domain of GP Ib
, approximately 20 nM
(24). Platelet binding parameters have been typically deduced from
experiments with relatively long incubation times, in contrast with the
rapidity of platelet responses to
-thrombin stimulation (25-27),
and may reflect events not relevant for activation. Indeed, the results
presented here indicate that GP Ib
accounts for most of the initial
-thrombin binding to platelets but apparently with the same
"moderate" affinity assigned to the corresponding isolated
functional domain (24). Different conclusions in this regard may be
explained by time- and temperature-dependent deviations
from equilibrium conditions. The fully reversible
-thrombin interaction with GP Ib
supports the association with platelets of a
proteolytically active enzyme that may contribute to activation.
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EXPERIMENTAL PROCEDURES |
Purification and Iodination of
-Thrombin--
Purified human
-thrombin with specific clotting activity between 2,180 and 2,800 NIH units/mg (28) (a gift of Dr. John W. Fenton II, Wadsworth Center
for Laboratories and Research, New York Department of Health, Albany)
was radiolabeled with 125I (Amersham Corp.) using IODO-GEN
(Pierce) (29). The radiolabeled ligand, with specific radioactivity
between 5 and 7 mCi/mg, was characterized and stored as described (10)
and was used within 2 weeks of iodination.
Preparation of Washed Platelets--
Blood was drawn from normal
volunteers, who had denied ingestion of drugs known to interfere with
platelet function for at least 1 week and given their informed consent
to these experimental studies according to the declaration of Helsinki,
and was collected into one-sixth final volume of citric
acid/citrate/dextrose, pH 4.5, containing 25 nM
prostaglandin E1 (Sigma). Platelets were washed free of
plasma constituents using the albumin density gradient method (30),
with modifications previously described (31).
Antibodies for Inhibition Studies--
The epitope of LJ-Ib10
lies between residues 238 and 290 of the amino-terminal domain of GP
Ib
. This monoclonal antibody inhibits
-thrombin binding to
platelets (10, 32) without effect on von Willebrand factor binding (33,
34). The rabbit polyclonal antibody, anti-TR1-160, binds
to one or more epitopes within the 160 amino-terminal residues of PAR1
and abolishes platelet activation induced by low doses of
-thrombin
and by the SFLL peptide ligand (35). Purified IgG and divalent
F(ab
)2 fragments, prepared as reported (36), were stored
at
80 °C in 20 mM Tris, 150 mM NaCl, pH
7.4, until used.
Binding of
-Thrombin to Platelets--
Binding was measured
according to a procedure described previously in detail (6, 9), in the
presence of a binding buffer composed of 25 mM Tris-HCl and
136 mM CH3CO2Na, pH 7.3, containing 0.6% polyethylene glycol (average molecular weight 6,000-7,000; Serva, Heidelberg, Germany) and 1% bovine serum albumin (Sigma). Washed platelets were kept at the temperature selected for any given
experiment for 15 min before use. Binding as a function of ligand
concentration was measured by adding platelets (2.8 × 108/ml, final count) to a mixture composed of a constant
concentration (0.1 nM) of 125I-labeled
-thrombin and increasing concentrations (0.1-1000 nM) of nonlabeled
-thrombin. Each experimental mixture had a total volume of 125 µl. After incubation, platelet-bound and free
-thrombin were separated by centrifugation through a 20% sucrose
layer at 12,000 × g for 4 min. Binding isotherms, each
consisting of 20 experimental points, were analyzed using the COLD
option of the computer-assisted program LIGAND, calculating
nonsaturable binding as a fitted parameter (37, 38). A concentration of
1 nM 125I-
-thrombin was employed in time
course and dissociation assays. In the latter, a 1,000-fold excess of
nonlabeled
-thrombin was added to platelets after incubation with
the labeled ligand.
Binding of
-Thrombin to Immobilized Glycocalicin--
The
extracytoplasmic domain of GP Ib
was purified from fresh platelet
concentrates as reported (39). The glycoprotein was immobilized onto
Sepharose CL 4B beads (Sigma) bearing covalently bound anti-GP Ib
monoclonal antibody, LJ-P3 (33), by incubating 800 µl of packed beads
with 2 ml of glycocalicin solution (0.5-2 mg/ml) for 1 h at
22-25 °C with constant mixing. The beads were then washed twice
with a buffer composed of 100 mM Tris, 500 mM LiCl2, 1 mM EDTA, pH 7.4, and 1 volume of
packed beads was resuspended into 6 volumes of binding buffer
containing 0.6% polyethylene glycol and 4.1% bovine serum albumin.
This suspension was used immediately. The presence of purified
glycocalicin on the beads was confirmed by SDS-polyacrylamide gel
electrophoresis of protein eluted at pH 2.9 (39) and by measuring the
binding of two different 125I-labeled anti-GP Ib
monoclonal antibodies, LJ-P19 and LJ-Ib10 (24). The binding of
125I-labeled
-thrombin was evaluated by mixing 20 µl
of bead suspension (corresponding to 3 µl of packed beads) with 65 µl of binding buffer, or other appropriate reagent, and 40 µl of
the desired ligand concentration. After incubation at the desired
temperature, the radiolabeled ligand bound to the beads was separated
from free ligand by centrifugation at 12,000 × g for 4 min through a 20% sucrose layer. Binding isotherms were analyzed with
the computer-assisted program LIGAND (37, 38).
Flow Cytometric Analysis--
Surface membrane expression of
P-selectin (40, 41) and GP Ib
was determined by using, respectively,
an anti-CD62P monoclonal antibody (Becton-Dickinson) labeled with
phycoerythrin (PE) and the anti-GP Ib
monoclonal antibody, LJ-Ib1
(33), labeled with fluorescein isothiocyanate (42). Washed platelets
were stimulated with increasing concentrations of
-thrombin
(0.01-100 nM) at a desired temperature, until recombinant
hirudin (Iketon, Milan, Italy) was added at the final concentration of
200 NIH units/ml to neutralize the proteolytic activity. Platelets were
then fixed with 1% paraformaldehyde for 30 min at 4 °C, washed
twice in Tris buffer, incubated for 15 min at 22-25 °C with the
specific antibodies or, in control experiments, with mouse IgG-labeled
with the same fluorochromes (43), and analyzed in a flow cytometer
(Becton-Dickinson). Fluorescence intensity was measured on an arbitrary
scale, and platelets were considered positive for a given marker when
their level of fluorescence was at least twice that of background or control platelets.
Measurement of Platelet ATP Secretion--
The release of ATP
from the dense granules of platelets was measured by the
luciferin-luciferase assay (44). Washed platelets were resuspended in
calcium-free Tyrode buffer (31) at a count of 2 × 108/ml, and 0.4 ml were mixed with 150 µg/ml
F(ab
)2 fragment of the anti-GP Ib
monoclonal antibody,
LJ-Ib10, or the rabbit polyclonal anti-TR1-160 antibody.
In control experiments, the same F(ab
)2 fragment
concentration of the anti-GP Ib
monoclonal antibody, LJ-Ib1, and of
preimmune rabbit IgG was used as control. The mixtures were placed in a glass cuvette and stirred at 1,200 revolutions per min (rpm) with a
Teflon-coated magnetic bar for 5 min at 37 °C in a lumiaggregometer (Chrono-log Corp.). At the end of the incubation, 50 µl of
luciferin-luciferase (Chrono-lume reagent, Chrono-log Corp.) was added,
and platelet release was induced by the addition of
-thrombin at
final concentration between 0.5 and 3 nM. Luminescence was
recorded to monitor ATP release, measured by comparing peak height with
that generated by known standard amounts of ATP.
Amidolytic Activity of
-Thrombin--
Washed platelets at a
count of 2.8 × 108/ml, treated with control buffer or
test antibodies, were mixed with 3 nM
-thrombin and
binding buffer to a volume of 0.6 ml. After incubation for 1 min at
37 °C, platelets were sedimented by centrifugation through a 20%
sucrose layer at 12,000 × g for 2 min. The supernatant
containing free
-thrombin was removed, the sucrose layer was
discarded, and the platelet pellet was resuspended with 600 µl of
binding buffer. The chromogenic substrate S-2238 (Kabi) (45) was added into the supernatant as well as the resuspended platelets at the concentration of 0.4 mM, and the incubation was continued
for 5 min at 37 °C. The hydrolysis reaction was then stopped with 4% acetic acid, and the release of p-nitroaniline was
measured at 405 nm in a spectrophotometer (Beckman DU-65) after
removing the platelets by centrifugation at 12,000 × g
for 2 min.
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RESULTS |
Time Course and Reversibility of
-Thrombin Binding to
Platelets--
The binding of 125I-
-thrombin to washed
platelets reached a maximum in 5 min at 37 °C but required 10 min
and was about 30% lower at 4 °C (Fig.
1). Concurrent addition of 1000-fold
excess of nonlabeled
-thrombin inhibited the binding of labeled
ligand by greater than 90% at either temperature (not shown). In
contrast, addition of nonlabeled ligand 20 min after
125I-
-thrombin, when binding of the latter was maximum,
resulted in 80-95% dissociation of bound ligand at 4 °C but only
about 50-60% at room temperature (22-25 °C) and 25-30% at
37 °C (Fig. 2). At the latter
temperature, dissociation was approximately 70% when nonlabeled ligand
was added 1 min after 125I-
-thrombin, 60% when it was
added after 2 min, and 45% when it was added after 10 min (Fig. 2).
The dissociation of bound ligand at 4 °C was not only greater in
extent but occurred more rapidly than at higher temperatures, being
almost maximal in 1 min as opposed to 5 min (Fig. 2).

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Fig. 1.
Time course of -thrombin binding to
platelets. Washed platelets, 2.8 × 108/ml final
count, were incubated at 37 °C (filled circles) or
4 °C (open circles) with a constant concentration (1 nM) of 125I-labeled -thrombin. At each
indicated time point, the reaction was terminated by centrifuging the
platelets through 20% sucrose in modified Tyrode buffer (31) for 4 min
at 12,000 × g, and the radioactivity in the pellet was
measured in a counter. Nonspecific binding, determined by mixing a
1000-fold excess of nonlabeled -thrombin with the labeled ligand,
was less than 0.02% of the total counts added and 10% or less of
total bound ligand both at 37 and 4 °C and was subtracted from total
binding to give the values shown in the figure. The number of
125I- -thrombin molecules bound per platelet was
calculated assuming a molecular mass of 37 kDa. Each point represents
the mean ± S.E. of three experiments.
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Fig. 2.
Dissociation of bound -thrombin from
platelets. a-c, washed platelets were mixed with 1 nM 125I-labeled -thrombin, as indicated in
the legend to Fig. 1. After 20 min at either 4 °C (a),
22-25 °C (room temperature; (b) or 37 °C
(c), 1 µM nonlabeled -thrombin
(filled circles) or the same volume of binding buffer
(open circles) was added, and the incubation was continued.
All indicated concentrations were in the final mixture. Bound
125I- -thrombin was calculated (see legend to Fig. 1)
immediately before and at selected time intervals after addition of the
nonlabeled ligand. Each point represents the mean ± S.E. of three
experiments at 4 °C or two each at 37 °C and room temperature.
d, washed platelets were incubated at 37 °C with
125I- -thrombin; nonlabeled ligand was added after 1, 2, or 10 min (arrows). Binding was measured immediately before
and at selected time points after addition of the nonlabeled ligand.
Similar results were obtained in two experiments.
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Inhibitory Effect of Antibody LJ-Ib10 on
-Thrombin Binding to
Platelets and Purified Glycocalicin as a Function of Incubation Time
and Temperature--
The anti-GP Ib
monoclonal antibody, LJ-Ib10,
inhibited the maximum binding of 125I-
-thrombin to
platelets, measured after incubation of 20 min, by approximately 75%
at 4 °C but only 60% at 22-25 °C and less than 50% at 37 °C
(Fig. 3). The same antibody inhibited the
maximum binding to glycocalicin, the isolated extracytoplasmic domain of GP Ib
, by at least 90% at all temperatures tested (Fig. 3). In
the latter case, the degree of inhibition was equivalent to that
produced by a 1000-fold excess of nonlabeled ligand added concurrently
with 125I-
-thrombin (not shown). At 37 °C, the time
of incubation between 125I-
-thrombin and platelets
influenced the inhibitory effect of LJ-Ib10 on the interaction.
Inhibition was essentially complete, i.e. equivalent to that
caused by a 1000-fold excess of unlabeled ligand, during the first 2 min of incubation, when binding reached approximately 50% of maximum
(Fig. 4). With continuing incubation, however, the inhibitory effect of the antibody progressively decreased as compared with that seen with excess unlabeled ligand (Fig. 4).
Altogether, the results shown in Figs. 1-4 are compatible with the
hypothesis that
-thrombin binding to GP Ib
on platelets becomes
progressively less reversible as a consequence of changes related to
activation, as they occur better at 37 °C than at lower temperatures. The fact that
-thrombin binding to isolated
glycocalicin was inhibited by the antibody LJ-Ib10 in identical manner
at all the temperatures tested is in agreement with such a concept.

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Fig. 3.
Effects of temperature on the inhibition of
-thrombin binding to platelets or purified glycocalicin by the
anti-GP Ib antibody LJ-Ib10. Twenty µl of washed platelets
(shaded bars) or beads with immobilized glycocalicin
(solid bars) were incubated for 10 min at room temperature
with 65 µl of F(ab )2 fragment of the anti-GP Ib
antibody, LJ-Ib10 (150 µg/ml concentration in the final mixture), or
binding buffer. The samples were placed at either 4, 22-25, or
37 °C for 10 min, as indicated, and then mixed with 40 µl of
125I-labeled -thrombin (1 nM concentration
in the final mixture). After an additional incubation of 20 min at the
indicated temperatures, bound and free ligand were separated as
indicated in the legend to Fig. 1. Residual binding in the presence of
LJ-Ib10 was expressed as percentage of that measured in control
mixtures without antibody. Results shown are the mean ± 95%
confidence limits of six experiments with platelets (shaded
bars) or four with glycocalicin (solid bars).
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Fig. 4.
Time course of -thrombin binding to
platelets at 37 °C in the presence of the anti-GP Ib antibody
LJ-Ib10. Washed platelets (2.8 × 108/ml final
count) were incubated for 10 min at room temperature, followed by 10 min at 37 °C, with either binding buffer (circles), or
150 µg/ml F(ab )2 fragment of the anti-GP Ib antibody
LJ-Ib10 (squares), or 1 µM nonlabeled
-thrombin (triangles). A constant concentration (1 nM) of 125I-labeled -thrombin was then added
to all the samples, and the incubation was continued at 37 °C. All
indicated concentrations were in the final mixture. At selected time
points (indicated on the abscissa), bound -thrombin was
measured as described in the legend to Fig. 1. Upper panel,
results obtained in the 1st min after addition of labeled ligand.
Lower panel, results obtained between 1 and 30 min after
addition of labeled ligand. Similar findings were observed in three
separate experiments.
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Markers of
-Thrombin-induced Platelet Activation--
The
following experiments were performed to evaluate the time course of
platelet stimulation by
-thrombin and correlate the membrane
expression of an activation marker, P-selectin, with changes in the
accessibility of GP Ib
to antibody probes. Greater than 50% of
platelets incubated with
-thrombin concentrations as low as 0.1 nM for 20 min at 37 °C exhibited increased P-selectin membrane expression, and greater than 80% was positive when the agonist concentration was in the range of 1-10 nM; in
contrast, at 4 °C there was no significant change relative to
nonstimulated platelets even with concentrations as high as 100 nM (Fig. 5). The number of
platelets displaying surface expression of P-selectin increased rapidly
after stimulation with
-thrombin, reaching a maximum in 20-40 s at
37 °C (Fig. 5).

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Fig. 5.
Platelet membrane expression of P-selectin.
Upper panel, washed platelets (final count 2.8 × 108/ml) were incubated for 20 min at 37 °C (filled
circles) or 4 °C (open circles) with increasing
concentrations of -thrombin (0.01-100 nM, corresponding
approximately to 0.001-10 NIH units/ml); at the end of the incubation,
hirudin was added at the final concentration of 200 units/ml. After 2 min, platelets were fixed with 1% paraformaldehyde, washed twice with
Tris buffer, resuspended in 100 µl of the same buffer to the initial
count of 2.8 × 108/ml, and then mixed with 5 µl of
PE-labeled anti-CD62P (P-selectin) monoclonal antibody (corresponding
to a final concentration of 10 µg/ml purified IgG). After 30 min at
22-25 °C, positive (fluorescent) platelets that had bound the
anti-P-selectin antibody were detected by flow cytometric analysis.
Background fluorescence was measured by adding the same concentration
of PE-labeled nonspecific mouse IgG and corresponded to that measured
with the anti-CD62P antibody in the absence of -thrombin
stimulation. At either temperature less than 10% of untreated washed
platelets reacted with the anti-P-selectin antibody. Each point
represents the mean ± S.E. of the percent of
fluorescence-positive platelets (see "Experimental Procedures") measured in two experiments at 4 °C and six at 37 °C. Lower
panel, these experiments were performed at 37 °C as described
above, except that a constant concentration of 1 nM
-thrombin was incubated with platelets for the indicated periods
before adding hirudin. The results shown represent the mean ± S.E. of three separate experiments.
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Exposure of platelets to
-thrombin at 4 °C had no significant
effect on the binding of an anti-GP Ib
antibody, whereas
progressively lower binding as a function of agonist concentration was
seen at 37 °C (Fig. 6). Identical
results were observed whether or not 2 mM Ca2+
and/or 1 mM Mg2+ was present in the incubation
mixtures (data not shown). The observed changes in anti-GP Ib
antibody binding started after a time interval approximately 10-fold
longer (Fig. 6) than required for increase in P-selectin surface
expression (Fig. 5) following
-thrombin stimulation.

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Fig. 6.
Effect of -thrombin on anti-GP Ib
antibody binding to platelets. a and b, these
experiments were performed as described in the legend to Fig. 5,
upper panel, except that 100 µg/ml of fluorescein
isothiocyanate-labeled anti-GP Ib antibody, LJ-Ib1, was used instead
of the anti-CD62P antibody. a, platelets stimulated at
37 °C; b, platelets stimulated at 4 °C. Curve
1, platelets stimulated with 1 nM -thrombin;
curve 2, platelets stimulated with 100 nM
-thrombin. The curves obtained at other thrombin concentrations have
been omitted for graphical clarity. Curve NS, fluorescence
distribution of nonstimulated platelets. The curve at the
extreme left corresponds to background fluorescence. Similar
results were obtained in three separate experiments. c, platelets were incubated with 1 nM -thrombin at 37 °C
for the indicated periods before measuring anti-GP Ib antibody
binding (see above). Each point is the mean ± S.E. of three
experiments in which the median fluorescence intensity of stimulated
platelets was expressed as percentage of that of nonstimulated
ones.
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Effects of Temperature on
-Thrombin Binding to Platelets and
Immobilized Glycocalicin--
The concentration-dependent
binding of
-thrombin to platelets was different at 4 °C as
compared with 37 °C (Fig. 7).
Scatchard-type analysis of data generated at 37 °C, performed for
comparative purposes even though binding was not at equilibrium (see
above), resulted in a plot with downward concavity in the range of
ligand concentrations between 0.1 and 3.5 nM and upward
concavity at higher concentrations. The data generated at 4 °C, on
the other hand, yielded an upward concave Scatchard plot (Fig. 7). The
results obtained at 4 °C could be represent by a two-site model,
with fitted nonsaturable binding in good agreement with experimentally determined values on the order of 2 × 10
2 (Table
I). In contrast to the results with
platelets, the binding of 125I-labeled
-thrombin to
immobilized glycocalicin was the same at 4 and 37 °C, yielding a
linear Scatchard plot indicative of a single class of binding sites
(Fig. 7) with a kd of 4 ± 1 × 10
8 M (mean ± S.E. of four experiments
at each temperature). The latter corresponded closely to the
kd of the lower affinity sites detected on platelets
at 4 °C (Table I). Binding isotherms generated at 4 °C in the
presence of the antibody LJ-Ib10 (Fig. 8)
revealed selective and complete inhibition of the lower affinity sites
but no effect on the higher affinity ones (Table I). Of note, the
function blocking anti-PAR1 rabbit polyclonal antibody, anti-TR1-160, did not interfere with
-thrombin binding
to platelets at 4 °C (Table I).

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Fig. 7.
Isotherms of -thrombin binding to
platelets or purified glycocalicin. Upper panel, washed
platelets (final count 2.8 × 108/ml) were incubated
for 20 min at 37 °C (filled circles) or 4 °C (open circles) with a constant concentration of
125I-labeled -thrombin (0.1 nM,
corresponding to 0.0125 pmol in each binding mixture) mixed with
increasing concentrations of nonlabeled -thrombin (0.1-1000
nM, corresponding to 0.0125-125 pmol in the binding
mixtures). At the end of the incubation, platelet-bound radioactivity
was determined as described in the legend to Fig. 1. The COLD option of
the computer-assisted program LIGAND was used to construct binding
isotherms representing total bound as a function of added ligand as
shown. The inset contains the Scatchard-type plot of the
data (bound/free versus bound ligand). Similar results were
obtained in four separate experiments. Lower panel, 20 µl of a suspension of beads bearing immobilized glycocalicin (see "Experimental Procedures") was mixed with 65 µl of binding buffer and 40 µl of increasing concentrations of
125I- -thrombin, as indicated. After 30 min at 37 °C
(filled circles) or 4 °C (open circles), the
amount of bound ligand was calculated on the basis on its specific
activity, and expressed as a function of added ligand. The
inset contains the Scatchard-type plot of the data. Similar
results were obtained in four experiments.
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Table I
Parameters of -thrombin binding to platelets
Control indicates normal platelets; LJ-Ib10 and anti-TR1-160
indicate normal platelets treated with saturating amounts (150 µg/ml) of F(ab )2 fragment of the anti-GP Ib or anti-PAR1 antibody, respectively. All parameters and the corresponding standard error of
estimated values were calculated with LIGAND (37, 38).
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Fig. 8.
Effect of antibody LJ-Ib10 on -thrombin
binding to platelets at 4 °C. Washed platelets (final count
2.8 × 108/ml) were incubated for 10 min at room
temperature with 150 µg/ml F(ab )2 fragment of the
anti-GP Ib antibody LJ-Ib10 (open circles) or binding
buffer (filled circles). The samples were kept for 10 min at
4 °C before adding a constant concentration of
125I-labeled -thrombin (0.1 nM) mixed with
increasing concentrations (0.1-500 nM) of nonlabeled
-thrombin. After additional incubation for 20 min at 4 °C,
platelet-bound -thrombin was separated from free ligand and measured
as described in the legend to Fig. 1. Binding isotherms were generated
as described in the legend to Fig. 7. Upper panel, bound
-thrombin as a function of added ligand. The inset shows
an enlargement of the curves representing the results obtained at the
lower ligand concentrations. Lower panel, Scatchard-type
plot of the data evidencing complete inhibition of the lower affinity
binding sites by the antibody LJ-Ib10. The inset shows the
plot obtained in the presence of LJ-Ib10 on an expanded scale.
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Inhibition of
-Thrombin-induced ATP Secretion by Anti-GP Ib
and Anti-PAR1 Antibodies--
Washed platelets stimulated with 3 nM
-thrombin at 37 °C exhibited rapid ATP release.
This was inhibited approximately 50% by LJ-Ib10 and 80% by
anti-TR1-160 but greater than 90% when the two were used
together (Fig. 9). The difference between
the effect of anti-TR1-160 alone and in combination
with LJ-Ib10 was significant (t test: p = 0.01). Since release of ATP always occurs in parallel with that
of the platelet agonist, ADP (46), these results indicate that
-thrombin binding to GP Ib
is associated with a response capable
of enhancing platelet pro-thrombotic functions.

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Fig. 9.
Inhibition of -thrombin-induced ATP
secretion by anti-GP Ib and anti-TR1-160
antibodies. Washed platelets (2.8 × 108/ml final
count) were incubated for 5 min at 22-25 °C with either a mixture
of monoclonal anti-GP Ib antibody, LJ-Ib1, and preimmune rabbit IgG
as control; or monoclonal anti-GP Ib antibody, LJ-Ib10; or rabbit
polyclonal anti-TR1-160 antibody directed against epitopes
within the 160 amino-terminal residues of the thrombin receptor PAR-1;
or a mixture of the latter two antibodies, as indicated. All antibodies
were F(ab )2 fragments at the final concentration of 150 µg/ml. At the end of the incubation, each sample was placed in a
lumiaggregometer cuvette, at 37 °C, and stirred at 1,200 rpm. After
the addition of 50 µl of luciferin-luciferase reagent and 3 nM -thrombin (equivalent to 0.3 NIH units/ml), ATP
secretion was recorded continuously. A known amount of ATP (2 nmol) was
used to calibrate the instrument and obtain a quantitative estimate of
that released from platelets. The values shown are the mean of three
experiments with 95% confidence limits.
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Amidolytic Activity of
-Thrombin Associated with GP
Ib
--
Measurable amidolytic activity could be partitioned with
platelets after incubation with
-thrombin, and this association was
selectively inhibited by LJ-Ib10 (Fig.
10). The appearance of
platelet-associated
-thrombin activity corresponded to an equivalent
decrease measured in the suspension medium after platelet separation,
and such a decrease was also blocked selectively by LJ-Ib10 (Fig. 10).
Thus,
-thrombin that had become associated with GP Ib
retained
similar amidolytic activity as the free enzyme.

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Fig. 10.
Amidolytic activity of -thrombin
associated with platelet GP Ib . Washed platelets at the final
count of 2.8 × 108/ml were incubated for 10 min at
room temperature with either binding buffer; or 150 µg/ml
F(ab )2 fragment of the monoclonal anti-GP Ib antibody
LJ-Ib10; or 150 µg/ml F(ab )2 fragment of the monoclonal
anti-GP Ib antibody LJ-Ib1 as control; or 20 units/ml hirudin, as
indicated. -Thrombin at the final concentration of 0.3 NIH units/ml
(3 nM) was then added into each mixture and incubated for 1 min at 37 °C. At this point, part of each mixture was kept intact
and part was rapidly centrifuged to separate the platelet pellet. The
chromogenic substrate S-2238 (0.4 mM) was then added either
into the total mixture (left bar), or into the platelet pellet resuspended in buffer to the original volume (middle
bar), or into the supernatant separated from the platelet pellet
(right bar). The amidolytic activity of -thrombin was
measured after 5 min at 37 °C. The activity shown here was
calculated as mean percentage ± S.E. (two experiments) of that
obtained in control mixtures without platelets.
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DISCUSSION |
The effects of
-thrombin on platelets are rapidly manifest
(27), and most of the induced responses may reach completion in a time
frame of seconds (25, 26). Here we show that within 1 min from exposure
to the agonist at 37 °C, a time sufficient for maximal activation as
judged by surface expression of P-selectin, the initial
-thrombin
interaction with platelets is rapidly reversible and completely blocked
by a specific anti-GP Ib
antibody. By 5-10 min, however,
-thrombin binding to platelets is less promptly reversible and only
partially blocked by the anti-GP Ib
antibody. These changes occur in
temporal relationship with decreased accessibility of GP Ib
to
antibody probes (43, 47-49), an event that is also known to correlate
with decreased von Willebrand factor binding (50), and both are
prevented at 4 °C. The latter temperature, therefore, appears to
preserve in time the characteristics of
-thrombin binding to GP
Ib
as during the initial interaction with platelets, thus providing
appropriate conditions to obtain equilibrium binding isotherms.
Accordingly, in the time frame relevant for agonist-induced activation,
GP Ib
may bind
-thrombin with "intermediate" or moderate
affinity (kd on the order of 10
8
M), rather than being the "highest" affinity site
(kd on the order of 10
10
M) as currently thought (6, 10, 12). Our findings also indicate that selective blockade of
-thrombin interaction with GP
Ib
dampens responses to the agonist and prevents the association with platelets of a proteolytically active, thus potentially
procoagulant, enzyme. Such conclusions are in agreement with recent
evidence showing that kininogens inhibit
-thrombin-induced platelet
aggregation because they interfere with agonist binding to GP Ib
(51). Indeed, owing to its relatively high membrane density, the
function of GP Ib
may be relevant in localizing
-thrombin at
sites of vascular injury, thus facilitating its action on specific
substrates.
Application of Scatchard-type analysis to
-thrombin-platelet binding
isotherms has usually resulted in curvilinear, upwardly concave plots
indicative of a deviation from the simplest model of reversible ligand
interaction with a homogeneous class of noninteracting receptors (52).
This lack of uniformity has been interpreted as evidence for the
presence of more than one type of receptor (6, 10, 12), leading to the
proposed existence of three
-thrombin binding sites with high,
intermediate, and low affinity (kd of approximately
0.3, 30, and 3000 nM, respectively) without nonspecific
binding (6). Even considering the more likely possibility that the low
affinity site represents nonspecific binding with respect to
physiologic significance (32), accepting the existence of the other two
sites with the reported characteristics requires prior exclusion of
alternative explanations for the observed curvilinear Scatchard plots
and more direct experimental evidence. Regardless of the method used
for analysis of experimental results, the validity of estimated binding
parameters, such as dissociation constant and receptor density, depends
on the assumption that ideal thermodynamic conditions, including
reversibility of ligand-receptor coupling (37, 38), are satisfied in
the assay. We show here that, using intact platelets and active
-thrombin, this condition is met at 4 °C but not at 22-25 or
37 °C, owing to partly irreversible ligand binding at 37 °C as
well as room temperature (usually 22-25 °C). Notably, previous
studies proposing the concept that GP Ib
is the high affinity
-thrombin binding site were performed at room temperature (6, 10,
12) and, thus, may have resulted in incorrect estimates for the
parameters of interaction.
The results obtained with platelets at 4 °C are still best fitted
with a two-site model represented by an upwardly concave Scatchard plot
but are compatible with the conclusion that the binding of
-thrombin
to GP Ib
occurs with a kd of between 4 and 9 × 10
8 M, as shown by obliteration of this
class of sites by the monoclonal antibody LJ-Ib10 without any influence
on the putative higher affinity sites. The latter observation is
relevant, since previous evaluation of the effects of this antibody at
room temperature had shown apparent inhibition of both high and
intermediate affinity receptors, as well as appearance of a new class
of binding sites, not present on control platelets, with affinity
halfway between high and intermediate (10). This finding, later
confirmed independently (12), could be taken to reflect the existence
of negative cooperativity between two distinct receptors but, in view
of the above considerations on equilibrium binding, is more likely to
indicate that the parameters estimated at 22-25 °C were erroneous.
It is also relevant to note that the sum of presumed high and
intermediate affinity sites inhibited by LJ-Ib10 at room temperature
(10, 32) is of the same order of magnitude as the number of
homogeneously intermediate affinity sites inhibited at 4 °C, in
agreement with the notion that the sites may be the same and the
estimated affinities at room temperature may be misleading.
The conclusion that
-thrombin interaction with GP Ib
on platelets
has a kd on the order of 10
8
M is substantiated both by results obtained with isolated
glycocalicin, as shown here and in agreement with independent data
reported elsewhere (24, 32), and by previous findings with the
recombinant amino-terminal domain of GP Ib
(24). In the case of
isolated receptor fragments containing the
-thrombin binding site,
interactions with the ligand are fully reversible at 4 °C as well as
37 °C and occur with kd between 1 and 5 × 10
8 M, reflecting the initial attributes of
-thrombin pairing with GP Ib
on platelets. These results cannot
support the proposed alternative possibility that high and intermediate
affinity sites are both expressed on GP Ib
(9). They also indicate
that other components of the GP Ib-IX-V complex have no direct
influence on the function of the
-thrombin binding site on GP Ib
,
suggesting that data to the contrary obtained in heterologous
expression systems (53) may depend on specific experimental conditions. The nature and physiologic significance of the
-thrombin binding sites not inhibited by LJ-Ib10, of higher affinity than GP Ib
sites
on the basis of the results obtained at 4 °C, remain undetermined at
present. Candidates for their identification may include PAR1 (13, 19),
PAR3 (22), and protease nexin 1 (54).
Despite evidence to the contrary from independently performed
experiments (10, 12), others have reached the conclusion that
antibodies against the proposed
-thrombin binding site on GP Ib
,
such as LJ-Ib10, have no inhibitory effect on platelet interaction with
the agonist nor on activation (13). As in the case of studies aimed at
determining binding characteristics, the methodology used may have
influenced the conclusions reached. Lack of inhibitory effect by
LJ-Ib10 was reported in experiments in which platelets were fixed after
incubation with
-thrombin, then processed for indirect detection of
bound ligand after repeated washing steps. It may be that, after such a
procedure, the
-thrombin remaining associated with platelets, not
defined in terms of quantity or binding characteristics, is interacting
with PAR1 rather than GP Ib
, as suggested (13); such a conclusion is
compatible with the two-site model discussed here. On the other hand,
the present studies provide direct evidence that the reversible
-thrombin binding to platelets at 37 °C can be blocked by LJ-Ib10
within the first 60 s of incubation. The same antibody reduces
dense granule ATP release, acting with an anti-PAR1 antibody to yield more efficient inhibition. Moreover, it is apparent that the
-thrombin associated with platelets through a GP
Ib
-dependent mechanism remains available as an active
enzyme. One function of this relatively high capacity site, therefore,
may be that of increasing the concentration of
-thrombin onto or in
proximity of the platelet membrane for subsequent proteolytic cleavage
of appropriate substrates. This latter event may take place not while
the enzyme is bound to GP Ib
, assuming that the association prevents
catalytic function (14), but after dissociation from the receptor that
may occur rapidly during the time frame of interaction relevant for
platelet activation and clotting. Therefore, even without considering
the possibility of coupling to a distinct signaling pathway that
remains to be proven directly (12), our present findings add evidence to the previously proposed concept (10, 12) that
-thrombin interaction with GP Ib
has a net prothrombotic effect.
We thank Dr. John W. Fenton II for the
generous gift of human
-thrombin, James R. Roberts and Benjamin
Gutierrez for help in the preparation of monoclonal antibodies, the
late Faye Miller and Julie Kopjoe and Ellye Lukaschewsky for
secretarial assistance.