From the Department of Biochemistry, University of Vermont College of Medicine, Burlington, Vermont 05405
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ABSTRACT |
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The platelet high affinity binding site for
thrombin appears to be described by a classical receptor-ligand
interaction that is distinct from the platelet thrombin
receptor/substrate, PAR-1. However, the identification and function of
the high affinity binding site with respect to its physiological
importance have continued to elude investigators. Prior studies using
two mutant thrombins suggested that thrombin interaction with the
platelet high affinity binding site is mediated through an extensive
portion of the thrombin molecule involving residues within the
substrate binding pocket and the anion binding exosite (Leong, L.,
Henriksen, R. A., Kermode, J. C., Rittenhouse, S. E., and Tracy,
P. B. (1992) Biochemistry 31, 2567-2575) and may mimic a
thrombin-hirudin interaction. To test this hypothesis, an anti-hirudin
peptide antibody (anti-hirpeptide Ab) was raised against a peptide
mimicking the COOH terminus of hirudin. The Ab recognized adherent
platelets and those in suspension as determined by enzyme-linked
immunosorbent assay and immunofluorescence microscopy, respectively.
125I-Thrombin binding to platelets was inhibited in the
presence of the anti-hirpeptide Ab in a dose-dependent
manner with maximal inhibition >90%. Analyses of data from binding
studies of 125I-thrombin to platelets at a fixed Ab
concentration indicated that the anti-hirpeptide Ab inhibited the high
affinity binding interaction exclusively. In addition, thrombin-induced
increases in platelet [Ca2+]i
were enhanced by blocking the high affinity binding site with the Ab
due to redistribution of the agonist to PAR-1. Thrombin Quick I-induced
platelet calcium mobilization was unaffected by the presence of the Ab,
consistent with the inability of thrombin Quick I to bind to the high
affinity site. Even though glycoprotein (GP) Ib contains a hirudin-like
region within the High affinity, dissociable binding of thrombin to human platelets
has been demonstrated and described by several laboratories (1-5).
Data from several investigators have led to the postulate that the
glycoprotein (GP)1Ib-IX-V
complex is a high affinity binding site for thrombin on the platelet
surface which may facilitate platelet activation (6-8). In addition,
it has been proposed that kininogen binding to GP Ib-IX-V modulates
thrombin binding to platelets and subsequent aggregation (9). To date,
definitive isolation of the high affinity binding site for thrombin on
platelets has not been accomplished. The focus of this investigation is
to characterize the identity and function of the platelet high affinity
binding site for thrombin.
Thrombin interaction with the high affinity binding site on platelets
is governed by a Kd The amino acid substitution of Val for Gly at position 558 [226]2in thrombin Quick II
falls within the primary substrate binding pocket (13). Thrombin Quick
II has decreased binding affinity for hirudin, suggesting that the
conformation of the active site cleft is not optimal for binding
hirudin (10). Decreased affinity of thrombin Quick II for a hirudin
mutant with substitutions in the amino terminus implies that this
region of hirudin interacts near the active site of thrombin (10).
Interestingly, thrombin Quick II binding to platelets
(Kd Thrombin Quick I, containing an Arg382 The platelet high affinity binding site for thrombin is distinct from
the seven-transmembrane domain platelet thrombin receptor/substrate, PAR-1, which mediates thrombin-induced platelet activation events described by the formation of a Michaelis complex (14, 15). We have
shown previously that in the presence of anti-TR32-46, a
polyclonal antibody raised against a peptide spanning the thrombin cleavage site within PAR-1, the platelet intracellular calcium mobilization induced by thrombin and thrombin Quick I is inhibited >90% (14). Platelet activation induced by thrombin Quick I proceeds through PAR-1 and requires 10-fold more enzyme to yield responses equal
in magnitude to thrombin stimulation (1, 14). In addition, platelet
activation does not require the high affinity binding site because
saturation of the high affinity binding site with FPR-thrombin,
although not inducing platelet activation, enhances the activation
response to thrombin stimulation (1, 14). It is important to note that
thrombin Quick I does not demonstrate high affinity binding to the
platelet surface at concentrations that effect platelet activation, nor
does active site blocked thrombin increase its agonist activity
(1).
Based on these combined observations, we hypothesized that the platelet
high affinity binding site for thrombin mimics the conformation of the
COOH terminus of hirudin. We generated an anti-hirudin peptide antibody
(anti-hirpeptide Ab) and demonstrated that it recognizes human
platelets. The anti-hirpeptide Ab was used as a tool to characterize
the interaction between thrombin and its high affinity binding site on
the platelet surface. We further described the functional role of the
high affinity binding site with respect to thrombin-induced platelet
activation responses. In addition, we explored the relationship of the
high affinity binding site with GP Ib and PAR-1.
Materials--
Taipan snake venom, potato apyrase, Sephadex
G-25-150, prostaglandin E1, o-phenylenediamine
dihydrochloride, 1,3,4,6-tetrachloro-3 Proteins--
Human coagulation proteins were obtained from
fresh-frozen plasma obtained from the American Red Cross or from
volunteer donors collected at the Clinical Research Center, Fletcher
Allen Health Care, Burlington, VT. Prothrombin was isolated as
described previously (16). Thrombin was prepared from human prothrombin
by activation with Taipan snake venom (17). Thrombin Quick I, a well
characterized mutant thrombin defective within the anion binding
exosite, was generously provided by Dr. Ruth Ann Henriksen (18). Active
site-blocked thrombin (FPR-thrombin) was prepared using
D-Phe-Pro-Arg chloromethyl ketone as described
previously (1, 14). Protein purity was assessed by
SDS-polyacrylamide gel electrophoresis before and after disulfide bond
reduction according to the method of Laemmli (19). Proteins were
visualized by Coomassie Brilliant Blue staining. Molecular weights and
extinction coefficients,
E280 nm, 1 cm[1%],
used were taken as follows: prothrombin, 72,000, 14.2 (20); thrombin,
37,000, 17.4 (21).
Thrombin and active site-blocked thrombin were radioiodinated using a
modified IODO-GEN transfer technique as described previously (1). The
labeled thrombins were >95% precipitable with 10% trichloroacetic
acid, with specific radioactivities of 2,200-2,500 cpm/ng.
Radioiodinated thrombin retained full clotting activity (~3,000 NIH
units/mg).
Platelet Preparations--
Washed platelets were prepared as
described previously (14). For most experiments, washed platelets were
suspended in 5 mM HEPES-buffered Tyrode's solution (0.137 M NaCl, 2.7 mM KCl, 12 mM
NaHCO2, 0.36 mM
NaH2PO4, 1 mM MgCl2, 2 mM CaCl2, 5 mM dextrose), pH 7.4, with 0.35% bovine serum albumin (HEPES Tyrode's albumin) at 37 °C.
CaCl2 was omitted from the wash solutions for experiments measuring intraplatelet [Ca2+]. In some experiments,
platelets were pretreated with 1 mM acetylsalicylic acid
and 0.5 µM prostaglandin E1 in platelet-rich
plasma for 30 min before washing.
Peptide Synthesis and Preparation of Antibodies Directed against
the COOH-terminal Region of Hirudin--
Based on binding studies with
the mutant thrombins, the most significant interactions involved in
thrombin binding to hirudin occur between its anion binding exosite and
the COOH-terminal region of hirudin. Therefore, a peptide mimicking the
COOH-terminal region of full-length hirudin was designed. The peptide
RNPNDGDFEEIPEEYLQNE was synthesized manually as a peptide amide using
4-methylbenzhydralamine and t-butyloxycarbonyl-blocked amino
acids with an additional COOH-terminal cysteine for coupling as
described previously (22). In addition, peptides representing the
hirudin-like regions found in PAR-1 and platelet GP Ib
Three rabbit anti-peptide antisera were prepared by Cocalico
Biologicals (Reamstown, PA) according to our supplied protocol (14).
Immunoglobulins, both nonimmune and specific, were purified using
protein A immobilized to Sepharose 4B. Protein purity was assessed by
SDS-polyacrylamide gel electrophoresis. Molecular weight and the
extinction coefficient,
E280 nm, 1 cm[1%],
for IgG were 150,000 and 14.0, respectively.
Specificity of the anti-peptide Abs was assessed by solid phase
enzyme-linked immunoassay (ELISA) against antigen peptides and an
irrelevant peptide, CVPDRGQQYQGR, as described previously (22). The
anti-hirpeptide, anti-TR52-69, and anti-GP
Ib269-287 Abs recognized their respective antigen
peptides. Binding to the irrelevant peptide was not observed.
Extensive Characterization of the Anti-hirpeptide
Immunoglobulin--
Having demonstrated that the anti-hirpeptide Ab
recognized the COOH terminus of hirudin, we evaluated its ability to
recognize full-length hirudin. Hirudin inhibition of thrombin-catalyzed chromogenic substrate hydrolysis was examined in the presence of
anti-hirpeptide Ab. Hirudin (6 nM) was preincubated with
0-500 nM anti-hirpeptide Ab for 3 min followed by the
addition of 5 nM thrombin in 20 mM HEPES, 150 mM NaCl, 0.01% Tween 80, pH 7.4. Thrombin activity was
monitored by hydrolysis of 0.25 mM Spectrozyme TH over 15 min. Hirudin inhibition of thrombin proteolytic activity was
reversed
Based on our hypothesis that the conformation of the high affinity
binding site resembles hirudin, we anticipated that the anti-hirpeptide
Ab would bind to platelets. To test this prediction, we examined
anti-hirpeptide Ab reactivity for platelets by ELISA and
immunofluorescence microscopy using techniques described previously (14, 22). The anti-hirpeptide Ab reacted positively with platelets in a
dose-dependent manner compared with nonimmune Ig using both techniques, indicating that the anti-hirpeptide Ab recognizes adherent
(Fig. 1) and suspended platelets (data
not shown). In control experiments, platelets showed no reactivity
toward secondary Ab alone in the immunofluorescent protocols (data not
shown).
Platelet Responses in the Presence of Anti-hirpeptide
Ab--
For studies that examined the effect of the anti-hirpeptide Ab
on thrombin interaction with platelets, the platelets (2-4 × 108/ml) were preincubated with anti-hirpeptide Ab or
nonimmune IgG for 10 min at ambient temperature. Measurements of
dissociable, equilibrium binding of 125I-thrombin to
platelets and thrombin-induced platelet intracellular calcium flux were
performed as described previously (14).
Thrombin Binding to GP Ib-IX-V-expressing Cell Lines--
Cell
lines, CHO and fibroblast-like L cells, transfected with GP Ib
Direct binding of 125I-thrombin to the CHO and L cell lines
was performed as described above with minor modifications. Cells were
lifted at confluence with 0.54 mM EDTA. Cells were washed three times in phosphate-buffered saline, 2 mM EDTA, 1%
BSA. Cells were counted and adjusted to 1 × 106/ml.
125I-Thrombin (1 nM) was added to the cell
suspensions, and aliquots were removed over time (0-30 min). Bound and
free fractions were separated by centrifugation over oil as described
previously (1). In other experiments, the binding was determined over a
range of thrombin concentrations (0-25 nM). Nonspecific
binding was quantified by the addition of unlabeled FPR-thrombin (50 nM) to the 125I-thrombin-cell suspension.
Immunofluorescence and Confocal Microscopy--
Washed platelets
(1 × 107/ml) were triple labeled with Ab directed
against platelet proteins as described previously (14). The Ab directed
toward PAR-1 and GP Ib were chosen as such because they recognize
epitopes other than the hirudin-like regions found within these
platelet membrane proteins. To platelets suspended in HEPES-Tyrode's
buffer with 20 mM EDTA and 5 µM prostaglandin E1, the following antibodies were added: 2 µM
rabbit anti-hirpeptide Ab followed by Texas Red-conjugated donkey
anti-rabbit IgG (Jackson Immunologicals, Inc.); Cy5-conjugated
monoclonal anti-TR32-46 (2 µM), specific for
PAR-1 (14); and FITC-conjugated monoclonal AN51 (10 µM),
specific for platelet GP Ib (Dako). Control platelet samples were
labeled with FITC or Cy5-conjugated nonspecific mouse IgG and Texas
Red-conjugated donkey anti-rabbit IgG. Platelets were visualized using
the Bio-Rad MRC 1000 confocal system connected to a Olympus BX50
upright microscope in the Cell Imaging Facility, University of Vermont
College of Medicine. The FITC, Texas Red, and Cy5 fluorophores were
excited at 488, 568, and 647 nm, respectively, with a krypton/argon
laser. Image analysis was performed using Adobe Photoshop software.
Effect of Anti-hirpeptide Ab on High Affinity Binding of Thrombin
to Platelets--
The anti-hirpeptide Ab was generated to test the
hypothesis that the high affinity binding site for thrombin mimics the
conformation of hirudin. Having shown that the anti-hirpeptide Ab
recognized platelets, we examined its ability to inhibit the
dissociable equilibrium and high affinity binding of
125I-thrombin to platelets. 125I-Thrombin (1 nM) binding to platelets (3 × 108/ml was
assessed over a range of anti-hirpeptide Ab concentrations (0-2
µM) (Fig. 2). Specific
binding was quantified and expressed as the percent of binding in the
presence of anti-hirpeptide Ab compared with the binding in the
presence of nonimmune rabbit IgG, which was without effect on
125I-thrombin binding to platelets. The anti-hirpeptide
Ab inhibited the specific binding of 125I-thrombin to
platelets in a dose-dependent manner with maximal inhibition >90% at 2 µM Ab.
125I-FPR-thrombin binding to platelets was inhibited by
anti-hirpeptide Ab to the same extent as native thrombin (data not
shown).
To determine whether the inhibitory activity of anti-hirpeptide Ab was
specific for the high affinity binding site, thrombin binding studies
were performed in the presence of a fixed Ab concentration. The
anti-hirpeptide Ab (2 µM) was preincubated with platelets (3 × 108/ml) for 10 min followed by the addition of
125I-thrombin (0.025-50 nM) for 2 min.
Thrombin binding to platelets was reduced substantially in the presence
of the anti-hirpeptide Ab (Fig.
3A). LIGAND analyses of the
binding data obtained in the absence of the anti-hirpeptide Ab
indicated that thrombin binding to the platelet surface was best
described by a one-site model with a nonsaturable component. Thrombin
interaction with this high affinity site was characterized by a
Kd = 0.34 ± 0.23 × 10 Effect of Anti-hirpeptide Ab on Agonist-induced Calcium
Responses--
The contribution of high affinity, dissociable binding
to agonist-induced platelet activation has been characterized
previously in studies from our laboratory (1). We demonstrated that in the presence of FPR-thrombin, at concentrations that saturate the high
affinity binding site, thrombin-induced platelet activation results in
an enhanced response compared with the response in the absence of
FPR-thrombin. These data were interpreted to indicate that the high
affinity binding site sequesters low concentrations of thrombin at the
platelet surface, thereby regulating the effective thrombin
concentration available for platelet activation. Because the
anti-hirpeptide Ab recognized the high affinity site, it should function in a similar manner when present during agonist-induced platelet activation.
Platelet intracellular calcium mobilization induced by thrombin was
determined in the presence of anti-hirpeptide Ab. Platelets (2 × 108/ml) were incubated with anti-hirpeptide Ab (0-500
nM) for 5 min before the addition of 0.5 nM
thrombin and monitored for changes in intracellular calcium
mobilization (14). In the presence of anti-hirpeptide Ab,
thrombin-induced platelet calcium mobilization was increased
substantially compared with the response in the presence of nonimmune
Ig (Fig. 4). Similar platelet activation profiles were observed for all seven donors; however, the maximal enhancement of the calcium response was donor-dependent
(range 120-240%).
To characterize further the role of the high affinity binding site with
respect to platelet activation, we utilized the mutant thrombin,
thrombin Quick I, which also mediates platelet activation through
cleavage of PAR-1 (14). However, thrombin Quick I does not demonstrate
high affinity, dissociable binding and therefore would not distribute
between the high affinity binding site and PAR-1 (1). Thus, we
anticipate that thrombin Quick I-induced platelet calcium mobilization
would be unaffected by the anti-hirpeptide Ab. Platelet intracellular
calcium flux was assessed using thrombin Quick I or thrombin as the
agonist. Platelets, washed with acetylsalicylic acid and prostaglandin
E1 (2 × 108/ml), were preincubated with
anti-hirpeptide Ab (5-100 nM) followed by stimulation
with 2 nM thrombin Quick I or 0.2 nM thrombin, concentrations that will induce near equal activation responses. The
presence of anti-hirpeptide Ab did not enhance the thrombin Quick
I-induced platelet calcium flux compared with that induced by thrombin
(Fig. 5). These data demonstrate further
that the anti-hirpeptide Ab recognizes the high affinity binding site. Interestingly, thrombin- and thrombin Quick I-induced intraplatelet calcium mobilization could be inhibited by higher anti-hirpeptide Ab
concentrations (Fig. 5), consistent with previous results using FPR-thrombin (1). These data suggest the anti-hirpeptide Ab may
recognize the hirudin-like region of PAR-1 in addition to the
hypothesized hirudin-like region in the high affinity binding site.
The Anti-hirpeptide Ab Recognizes Hirudin-like Regions within PAR-1
and GP Ib--
The identification of the platelet high affinity
binding site for thrombin has eluded investigators. Studies have
demonstrated that thrombin binding to platelets is inhibited in the
presence of an anti-GP Ib Ab (6). The amino-terminal domain of the GP Ib
Briefly, anti-hirpeptide Ab was incubated with increasing
concentrations of the HPLC-purified peptides (10 GP Ib Is Not the High Affinity Binding Site for Thrombin--
To
investigate the possibility that GP Ib may be the high affinity binding
site for thrombin found on platelets, we characterized thrombin binding
to CHO and L cell lines expressing GP Ib-IX-V complexes using
125I-thrombin. By examining thrombin binding in a
transfected cell system, we can define the surface expression of the
protein of interest in the absence of other platelet proteins that may
confound the assay system. We verified that all cell lines were
expressing the appropriate GP subunits using flow cytometric
analyses as described previously (24).
Radiolabeled thrombin binding to the CHO and L cell lines expressing
the GP Ib
The argument could be made that the GP Ib-IX-V complex expressed on
these cells does not mimic that expressed by platelets. If the
hirudin-like region found within GP Ib The Platelet High Affinity Binding Site Is Distinct from GP
Ib--
These collective data suggest that the high affinity binding
site for thrombin is a unique platelet protein resembling hirudin. To
establish the unique character of this platelet membrane protein, we
utilized confocal microscopy and three fluorescent-tagged antibodies to
label surface-expressed high affinity binding sites, GP Ib and PAR-1.
We chose Ab to GP Ib and PAR-1 which do not recognize the hirudin-like
regions found within these two platelet proteins. Thus, platelets were
labeled with rabbit anti-hirpeptide Ab followed by Texas Red-conjugated
donkey anti-rabbit IgG; Cy5-conjugated monoclonal
anti-TR32-46, specific for PAR-1 (14); and FITC-conjugated
monoclonal AN51, specific for platelet GP Ib. Control platelet samples
were labeled with Texas Red-conjugated donkey anti-rabbit IgG alone,
Cy5-nonimmune mouse IgG, or FITC-nonimmune mouse IgG. The
anti-hirpeptide Ab recognized a population of membrane sites (Fig.
8, A and D, shown
in red) which is distinct from PAR-1 (Fig. 8, B
and D, shown in blue) and from platelet GP Ib
(Fig. 8, C and D, shown in green).
There is some colocalization of the anti-hirpeptide Ab with the anti-GP
Ib Ab and the anti-TR32-46 Ab as anticipated because of
the ability of the anti-hirpeptide Ab to recognize the hirudin-like
regions of GP Ib and PAR-1. Control platelet samples stained negatively
(data not shown). These data are consistent with the results obtained
from the platelet activation (Figs. 5 & 7) and ELISA (Fig. 6)
experiments. The data clearly indicate that the anti-hirpeptide Ab
recognizes a unique platelet membrane protein.
The current investigation characterizes and clarifies the
functional role of the platelet high affinity binding site for
thrombin. In this study, we have presented data that demonstrate that
the platelet high affinity binding site is a unique platelet membrane protein. The anti-hirudin peptide Ab, generated to the
carboxyl-terminal domain of hirudin, recognizes platelets and competes
with thrombin for hirudin. In addition, we demonstrate that the
anti-hirpeptide Ab recognizes a specific platelet protein by confocal
microscopy (Fig. 8). Together with the functional studies, these
results indicate that the protein identified with the anti-hirpeptide Ab is distinct from PAR-1 and GP Ib.
The anti-hirpeptide Ab also provided a means for characterizing the
high affinity binding site with respect to platelet function. The
processes of binding to and activation of platelets are distinct events
occurring at unique sites on the platelet surface. Whereas dissociable,
equilibrium binding of thrombin to the platelet surface is inhibited
when the high affinity binding site is occupied by the anti-hirpeptide
Ab, thrombin-induced platelet activation is enhanced. As the effective
concentration of anti-hirpeptide Ab is increased, thrombin-induced
platelet calcium mobilization is inhibited, indicating the presence of
anti-hirpeptide Ab binding to PAR-1. Based on these results, we
hypothesize that the high affinity binding site sequesters low
concentrations of thrombin at the platelet surface thereby decreasing
the local thrombin concentration available for interaction with
PAR-1.
PAR-1 and GP Ib contain amino acid sequences similar to that found in
the COOH-terminal region of hirudin, and they are postulated to
interact with thrombin (25, 26). Peptides mimicking the hirudin-like
region of PAR-1 bind to thrombin and inhibit thrombin hydrolytic
activity toward chromogenic substrates (26, 27). Crystal structure data
and platelet studies have demonstrated that this region of PAR-1
interacts with the anion binding exosite of thrombin (15, 28, 29).
Based on the observations that the anion binding exosite of thrombin
binds to the hirudin-like region of PAR-1 and recognizes the COOH
terminus of hirudin, we hypothesized that the anti-hirpeptide Ab would
recognize the hirudin-like domain of PAR-1. The anti-hirpeptide Ab was
8-fold less reactive toward the TR52-69 peptide compared
with the COOH-terminal hirudin peptide in a solution phase ELISA. These
data support our observation that higher concentrations of the
anti-hirpeptide Ab inhibit thrombin- and thrombin Quick I-induced
platelet calcium mobilization mediated by PAR-1.
GP Ib contains a hirudin-like sequence within the amino-terminal domain
of the The functional role of glycoprotein Ib in thrombin high affinity
binding to platelets and thrombin-induced platelet activation remains
unclear. Surface expression of GP Ib molecules on platelets, Thrombin binding to the platelet high affinity site can be compared
with the thrombin-hirudin binding interaction, both of which are
governed by high affinity dissociation constants (1, 32, 33). The
carboxyl-terminal domain of hirudin binds the anion binding exosite of
thrombin, an extension of the active site cleft dominated by positively
charged side chains (34). The importance of the anion binding exosite
of thrombin in high affinity, dissociable binding to platelets can be
demonstrated from studies with thrombin Quick I.
Thrombin Quick I contains an Arg to Cys substitution within the anion
binding exosite which alters the function and conformation of this
thrombin molecule (10, 13). Thrombin Quick I demonstrates decreased
fibrinogen clotting activity and platelet aggregation ability but
nearly normal activity toward chromogenic substrates (18, 35). Thrombin
Quick I equilibrium binding to the platelet surface is undetectable and
competes weakly with thrombin, highlighting the importance of the anion
binding exosite in high affinity binding to platelets. Consistent with
this observation, thrombin Quick I binding to hirudin is 4 orders of
magnitude less than native thrombin-hirudin binding (10). Thrombin
Quick I induces increases in intraplatelet [Ca2+] less
effectively than thrombin at equivalent concentrations, such that
10-fold more thrombin Quick I is required to produce a response equal
to that of thrombin (1). Thrombin Quick I-induced platelet
intracellular [Ca2+] mobilization was unaffected by low
concentrations of anti-hirpeptide Ab, consistent with previous studies
from our laboratory in which the presence of low concentrations of
FPR-thrombin had no effect on thrombin Quick I-induced intraplatelet
[Ca2+] (1). In contrast, thrombin-induced platelet
activation was enhanced by saturation of the high affinity binding site
with FPR-thrombin, suggesting that the high affinity binding site is regulating the effective concentration of thrombin available at the
platelet surface (1).
In conclusion, we propose that the platelet high affinity binding site
for thrombin allows for the regulation of local thrombin concentrations
at the platelet surface thus modulating platelet activation. The
importance of this regulatory mechanism is yet to be explored fully.
Thrombin sequestration at the high affinity binding site on platelets
may provide an essential regulatory mechanism for thrombin-induced
platelet activation in vivo such that a threshold level of
thrombin is required to initiate explosive platelet activation
responses via cleavage of PAR-1 and subsequent assembly of
prothrombinase, generation of thrombin, and platelet aggregation. The
platelet high affinity binding site for thrombin may be most effective
in preventing platelet activation and thrombus formation at low
thrombin concentrations. By sequestering low levels of thrombin at the
platelet surface, the high affinity binding site can prevent unwanted
cleavage of PAR-1 and subsequent platelet desensitization. By
down-regulating the effective thrombin concentration, platelets
modulate the coagulation response within the vasculature, both arterial
and venous circulation, at sites of endothelial injury or developing
atherosclerotic plaque formation. In effect, the platelet high affinity
binding site for thrombin may delay or prevent thrombus formation in
pathological conditions such as thrombotic stroke or coronary artery occlusion.
subunit, the postulated high affinity binding
site, direct binding of 125I-thrombin could not be
demonstrated to transfected Chinese hamster ovary and L cells
expressing the GP Ib-IX-V complex. Furthermore, an anti-GP Ib Ab,
raised to the peptide region proposed as the thrombin high affinity
site, did not enhance thrombin-induced platelet calcium mobilization.
The anti-hirpeptide Ab recognized a population of platelet membrane
proteins distinct from PAR-1 and GP Ib by three-color
immunofluorescence using confocal microscopy. These combined studies
demonstrate that the high affinity binding site for thrombin is a
unique platelet protein distinct from GP Ib which modulates the
effective thrombin concentration localized at the human platelet surface.
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
0.1-4.0 nM at
50-1,200 sites as determined by dissociable, equilibrium binding
techniques (1, 3, 4). Additional studies from our laboratory with native human thrombin and two mutant thrombins, thrombin Quick I and
thrombin Quick II, demonstrate that thrombin binding to the platelet
surface is mediated through an extensive portion of the thrombin
surface, involving residues near the substrate binding pocket and the
anion binding exosite (1). Studies characterizing thrombin Quick I and
thrombin Quick II binding to platelets (1) together with studies
examining the binding of hirudin to these mutant thrombins (10) led to
our hypothesis that the conformation of the platelet high affinity
binding site for thrombin resembles hirudin.
3.3-9.5 nM) is decreased by 1 order of magnitude compared with thrombin (1). We postulated that the
conformational changes in Quick II which limit access of hirudin to the
active site must influence interactions between platelets and the anion
binding exosite, the primary site of dissociable equilibrium binding.
Cys [67] amino
acid substitution within the anion binding exosite, does not
demonstrate high affinity binding to the platelet surface
(Kd
0.17-0.27 µM) (1). The
affinity of thrombin Quick I for full-length hirudin is
104-fold less compared with thrombin (10). Truncated
hirudin, lacking the COOH-terminal tail region, is a competitive
inhibitor of thrombin Quick I, suggesting that the interactions that
occur between thrombin and the COOH-terminal extension of hirudin are
completely disrupted in the mutant thrombin (10). The decreased
affinity of thrombin Quick I for platelets is comparable in magnitude
to its decreased affinity for hirudin. Taken together with the studies
using Quick II, we hypothesized that the interaction of thrombin with
the high affinity binding site on platelets occurs such that the active site of thrombin is directed toward the platelet surface and involves regions of the anion binding exosite as well as those near the active site.
EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
,6
-diphenylglycouril, and
digitonin (50% purity) were purchased from Sigma. Digitonin was
purified further by recrystallization from 100% ethanol. Fura 2-AM was
obtained from Molecular Probes, Inc. Spectrozyme TH was purchased from
American Diagnostica. Hirudin was purchased from Genentech.
Crystallized bovine serum albumin (BSA) was purchased from ICN
Immunobiologicals. Na125I was purchased from Amersham
Pharmacia Biotech. D-Phe-Pro-Arg chloromethyl ketone was
obtained from Calbiochem. Amino acid derivatives for peptide synthesis
were purchased from Peninsula Laboratories Inc. 4-Methylbenzhydrylamine
resin was purchased from Advanced ChemTech. Anisole,
1,3-diisopropylcarbodiimide, diisopropylethylamine, and dimethyl
sulfide were obtained from Aldrich and used without further
purification. Fluorescein isothiocyanate (FITC) goat anti-rabbit IgG
(heavy and light chains) was purchased from Vector Laboratories, and
goat anti-rabbit IgG horseradish peroxidase was obtained from Southern
Biotechnology Associates. Apiezon A was purchased from James G. Biddle,
and n-butyl phthalate was from Fisher. Dulbecco's modified
minimal essential medium (MEM),
-MEM, low endotoxin fetal bovine
serum, G418, HAT supplement, and methotrexate were obtained from Life
Technologies, Inc.
were
synthesized according to the same procedure. The PAR-1 peptide sequence
was YEPFWEDEEKNESGLTEY, representing residues 52-69, whereas the GP
Ib
peptide sequence was DEGDTDLYDYYPEEDTEGD, representing residues
269-287. All peptides were purified by HPLC. Peptide compositions and
concentrations were determined by amino acid analysis performed by the
Protein Chemistry Laboratory at the University of Texas Medical Branch Cancer Center. The peptides are designated as follows: the
COOH-terminal hirudin peptide, hirpeptide; the PAR-1 peptide,
TR52-69; and the GP Ib
peptide, GP
Ib269-287.
50% by the presence of the anti-hirpeptide Ab (500 nM) compared with inhibition in the absence of
anti-hirpeptide Ab (data not shown). These data indicate that the
anti-hirpeptide Ab recognizes the COOH-terminal region of hirudin
within the full-length protein.
View larger version (13K):
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Fig. 1.
Anti-hirpeptide Ab recognizes human
platelets. Platelets (1 × 106/ml) were adhered
to microtiter plates in phosphate-buffered saline, 0.3% BSA. Wells
were blocked with 3% BSA. Anti-hirpeptide Ab (0-200 µg/ml) was
added to the adhered platelets. Horseradish peroxidase-coupled goat
anti-rabbit IgG was added as secondary Ab. Reactivity was determined at
490 nm. Anti-hirpeptide Ab ( ) recognized platelets compared with
nonimmune control IgG (
). Results are the mean ± S.E. and are
representative of three donors.
,
Ib
, IX, and/or V subunits, were a generous gift from Dr. Jose Lopez,
Baylor College of Medicine. The stable CHO and L cell lines have been
characterized extensively (23). Untransfected CHO cells were maintained
in
-MEM containing 10% heat-inactivated fetal bovine serum (FBS).
CHO
IX cells expressing GP Ib
and IX subunits were maintained in
-MEM supplemented with 10% FBS and 400 µg/ml G418. CHO
IX
cells, expressing Ib
, Ib
, and IX subunits, were maintained in
-MEM with 10% FBS, 400 µg/ml G418, and 80 µM
methotrexate (23). The L cell lines were grown in Dulbecco's modified
MEM supplemented with 10% FBS. L2H cells expressing Ib
, Ib
, and
IX were maintained in Dulbecco's modified MEM supplemented with 10%
FBS, 1 mM sodium hypoxanthine, 4 µM
aminopterin, and 160 µM thymidine. L2H cells expressing
GP V were supplemented further with 500 µg/ml hygromycin B. All cells
were maintained at 37 °C in an atmosphere of 5% CO2 and
99% humidity. Expression of the GP Ib/IX/V protein subunits was
verified using specific monoclonal Ab for each glycoprotein by flow
cytometric techniques as described previously (24). Subunits of the
complex were labeled with the following antibodies: GP Ib
-AN51 (Dako
Corp.), GP IX-FMC-25 (provided by Dr. Jose Lopez), and GP V-CD 42b (Dako).
RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References
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Fig. 2.
Inhibition of dissociable equilibrium binding
of thrombin to platelets by anti-hirpeptide Ab. Platelets (3 × 108/ml) were preincubated with varying amounts of
anti-hirpeptide Ab or nonimmune rabbit Ig (0-2 µM) in
HEPES Tyrode's buffer, 0.35% BSA, pH 7.4, followed by the addition of
1 nM 125I-thrombin for 2 min. Specific binding
is expressed as the percent of binding in the presence of
anti-hirpeptide Ab compared with binding in the presence of nonimmune
rabbit Ig. All binding data were corrected for nonspecific binding
determined in the presence of excess unlabeled FPR-thrombin (50 nM). Data represent the mean ± S.E. of three
different donors.
9
M with 70 ± 38 binding sites (five donors). These
binding parameters are consistent with values reported previously from
our laboratory and others (1, 3, 4). There was no improvement in the fit when a two-site model for thrombin binding was employed, consistent with our previous studies (1). Scatchard analyses of the binding data
clearly demonstrate that the high affinity binding interaction between
thrombin and platelets is completely inhibited by the presence of the
anti-hirpeptide Ab (Fig. 3B). Furthermore, the analyses of
the binding data obtained in the presence of the anti-hirpeptide Ab
demonstrated a slight decrease in the apparent binding affinity (Kd = 0.53 ± 0.10 × 10
9
M, five donors), suggesting that the anti-hirpeptide Ab is
a competitive inhibitor of thrombin.
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Fig. 3.
Inhibition of thrombin binding to the
platelet high affinity binding site. Platelets (3 × 108/ml) were incubated with a fixed amount of
anti-hirpeptide Ab or nonimmune Ig (2 µM) in HEPES
Tyrode's buffer, 0.35% BSA, pH 7.4, for 10 min.
125I-Thrombin (0.05-50 nM) was added to the
platelet suspension for 2 min. Nonspecific binding was determined at
each thrombin concentration by the addition of unlabeled FPR-thrombin
(50-fold molar excess). Panel A, thrombin binding to
platelets in the presence of nonimmune Ig ( ) or anti-hirpeptide Ab
(
) is expressed as molecules of thrombin bound per platelet as a
function of labeled ligand added. The inset is an
enlargement of the graph at the lower thrombin concentrations. Data are
representative of five experiments using different donors. Panel
B, Scatchard plot of the data presented in panel
A.
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Fig. 4.
Effect of anti-hirpeptide Ab on
thrombin-induced platelet calcium response. Platelets (2 × 108/ml) were incubated with varying concentrations of
anti-hirpeptide Ab (0-500 nM) before stimulation by
thrombin. Intraplatelet calcium mobilization in response to thrombin
(0.5 nM, ) is shown. The change in intraplatelet
[Ca2+] is expressed as a percent of the response induced
by thrombin alone. Results are the mean ± range from duplicate
determinations. Some error bars fall within the limits of the symbol.
Data are representative platelet activation responses from 12 individual donors.
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Fig. 5.
Platelet activation in the presence of
increased anti-hirpeptide Ab. Platelets (2 × 108/ml) were incubated with increasing concentrations of
anti-hirpeptide Ab (0-2 µM) for 10 min at ambient
temperature. Platelets were activated with either thrombin (0.2 nM, ) or thrombin Quick I (2 nM,
). The
change in intracellular [Ca2+] is expressed as a percent
of the response induced by the agonist in the presence of nonimmune
rabbit Ig, which had no effect on that observed with thrombin alone.
Results are the mean ± range from duplicate determinations. Data
are representative of four different donors.
chain is postulated to contain a high affinity binding site for
thrombin. This amino acid domain, comprised of several negatively charged amino acids, has been compared with similar domains within PAR-1 and the carboxyl-terminal region of hirudin. Because PAR-1, GP
Ib, and the high affinity binding site are all expressed on the
platelet surface, we hypothesized that an Ab directed against a peptide
rich in anionic amino acids could potentially recognize any or all of
these sites. To address this hypothesis, we characterized the
specificity of the anti-hirpeptide Ab for its antigen peptide compared
with synthetic peptides of hirudin-like regions found in PAR-1 (peptide
TR52-69) and GP Ib (peptide GPIb269-287) in a solution phase ELISA.
8 to
10
4 M) for 1 h in solution at ambient
temperature. After the solution phase incubation, aliquots of each
reaction were transferred onto hirpeptide-coated ELISA plates. Any
anti-hirpeptide Ab unbound to peptide from the competitive solution
phase reaction could then react with hirpeptide coated on the plate.
The results indicate that the anti-hirpeptide Ab is more reactive for
the hirpeptide, EC50
2 × 10
6
M, compared with TR52-69, EC50
6 × 10
5 M; or GPIb269-287,
EC50
7 × 10
5 M (Fig.
6). EC50 is defined in this
context as the concentration of peptide required to bind 50% of the
anti-hirpeptide Ab from the solution phase. These data indicate that
the anti-hirpeptide Ab interacts with the TR52-69,
consistent with its ability to inhibit thrombin-induced intraplatelet
[Ca2+] mobilization at high Ab concentrations. The
anti-hirpeptide Ab also interacts with GPIb269-287,
consistent with the presence of a hirudin-like domain in GP Ib
.
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Fig. 6.
Reactivity of anti-hirpeptide Ab for
hirudin-like peptides. Anti-hirpeptide Ab (66 µM)
was incubated with various concentrations of each of the following
peptides: hirpeptide ( ), peptide TR52-69 (
), peptide
GPIb269-287 (
), or buffer alone (
) for 1 h.
Aliquots of the anti-hirpeptide Ab/peptide suspensions were added to
4.14 mM hirpeptide-coated 96-well plates. By ELISA, the
remaining unbound anti-hirpeptide Ab reactivity was assessed. The
anti-hirpeptide Ab demonstrated greater reactivity for hirpeptide
compared with peptide TR52-69 or peptide
GPIb269-287. Data are expressed as mean ± S.D. All
error bars fall within the limits of the symbol.
-Ib
-IX or Ib
-IX or Ib
-Ib
-IX-V subunit was performed as described previously for platelets (14). Cells (1 × 106/ml) were incubated with 125I-thrombin (1 nM) for 0-30 min or with varying concentrations of
thrombin (0-25 nM) for 2 min. There was no detectable
time- or concentration-dependent binding of
125I-thrombin to any of the cell lines (data not shown).
The CHO and L cell lines expressing the Ib
-IX, Ib
-IX, or
Ib
-IX-V complex demonstrated no specific thrombin binding
compared with nontransfected CHO or L cells. Nonspecific binding was
measured in the presence of excess unlabeled FPR-thrombin (50-fold
molar excess). Because nonspecific binding was = 5% of the added
125I-thrombin, specific binding, if present, could have
been quantified easily. These data suggest that the glycoprotein
Ib-IX-V complex is not the high affinity binding site for thrombin
found on platelets.
expressed at the platelet
surface is a high affinity binding site for thrombin, then an Ab raised
against this region should induce an enhanced platelet activation
response in a manner identical to that observed with the
anti-hirpeptide Ab. We examined thrombin-induced platelet calcium
mobilization in the presence of anti-GP Ib269-287 Ab.
Thrombin-induced platelet calcium mobilization was not enhanced in the
presence of the anti-GP Ib269-287 in contrast to the
enhancement seen in the presence of the anti-hirpeptide Ab (Fig.
7). Presumably, the inhibition of
platelet calcium flux by the anti-GP Ib269-287 Ab can be
explained by its reactivity for the hirudin-like region of PAR-1
(peptide TR52-69). We determined that the reactivity
(EC50 as described above) of anti-GP Ib269-287
Ab for peptide GP Ib269-287
0.45 µM and
for peptide TR52-69
2.11 µM by ELISA.
The inability of the anti- GP Ib269-287 Ab to enhance
thrombin-induced platelet calcium mobilization coupled with our
inability to demonstrate direct thrombin binding to the cell lines
strongly argues against the concept that GP Ib is the high affinity
binding site for thrombin.
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Fig. 7.
Anti-GP Ib Ab does not enhance
thrombin-induced platelet activation. Platelets (2 × 108/ml) were incubated with increasing concentrations of
either anti-hirpeptide Ab or anti-GP Ib269-287 (0-2
µM) for 10 min at ambient temperature. 0.2 nM
thrombin-induced platelet calcium mobilization in the presence of
anti-hirpeptide Ab ( ) or anti-GP Ib269-287 (
) is
expressed as a percent of the activation response in the presence of
control nonimmune rabbit Ig. Data are the mean ± range from
duplicate determinations. All error bars fall within the limits of the
symbol. The results are representative of two individual donors.
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Fig. 8.
Demonstration that GP Ib and the high
affinity binding site are distinct membrane proteins by confocal
microscopy. Platelets (1 × 107/ml) were labeled
with rabbit anti-hirpeptide Ab (2 µM) followed by Texas
Red-conjugated donkey anti-rabbit IgG, Cy5-conjugated monoclonal
anti-TR32-46 (2 µM), and FITC-conjugated
monoclonal AN51 (10 µM). Platelets stained positive with
anti-hirpeptide Ab (panel A), anti-TR32-46
(panel B), and AN51 (panel C). Panel D
shows the collective labeling from the three fluorescent-tagged
antibodies. Platelets labeled with control antibodies were negative.
Data are representative of results obtained from four experiments with
individual donors.
DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
chain which interacts with thrombin under select conditions
(6, 25). Synthetic mimetic peptides to the hirudin-like region within
GP Ib inhibited thrombin binding to purified GP Ib and platelets,
suggesting that this region of GP Ib may be a binding site for thrombin
(25). If a hirudin peptide motif is present in platelet membrane
proteins, it is possible an Ab against the hirudin-like peptide in GP
Ib
would also recognize PAR-1. We demonstrate an
anti-GP269-287 Ab, directed against the hirudin-like
peptide in GP Ib
, inhibits thrombin-induced calcium mobilization
(Fig. 7). There is no enhanced platelet activation response, suggesting
that this Ab does not recognize the high affinity binding site for
thrombin which we hypothesize also contains a hirudin-like peptide.
Based on studies of thrombin interactions with platelets and GP Ib
using anti-GP Ib
antibodies, several investigators propose that GP
Ib is a high affinity binding site for thrombin on platelets (25, 30, 31). An alternative explanation for these data includes the possibility
that the anti-GP Ib Ab used in these experiments cross-reacts with
hirudin-like regions found on other platelet membrane proteins including PAR-1 and the unique high affinity binding site identified in
this study.
25,000,
far outnumbers high affinity binding sites,
50-1,200 (1, 3, 4, 25).
If GP Ib were the high affinity binding site, then only a subset of the
GP Ib molecules would be involved in thrombin binding, and such a
subset has not been identified. Furthermore, we are unable to
demonstrate dissociable, equilibrium thrombin binding to CHO and L cell
lines expressing GP Ib
-IX-V complexes. These results contradict
those obtained by Lopez and colleagues whereby they demonstrate
thrombin (1 nM) binding to L2H/V cells that express the GP
Ib-IX-V complex by flow cytometry. An explanation for the discrepancy
between our results includes the different techniques used: direct
radiolabeled thrombin binding versus indirect
immunofluorescence detection of thrombin. Based on studies with the
transfected cell lines, Lopez has proposed that a macromolecular
complex of glycoproteins Ib, IX, and V forms a high affinity binding
site for thrombin (8).
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ACKNOWLEDGEMENTS |
---|
We acknowledge the Clinical Research Center at Fletcher Allen Health Care for blood drawing services, Dr. William Church for peptide synthesis, Dr. John Kermode for assistance with binding data analyses, and Dr. Jose Lopez for providing the GP Ib-IX-V transfected cell lines and antibodies to the GP Ib-IX-V subunits. We also acknowledge Dr. Douglas Taatjes and the Cell Imaging Facility at the University of Vermont College of Medicine for assistance with confocal microscopy studies. We thank Dr. Ruth Ann Henriksen for providing the thrombin Quick I.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant HL-46703 (Project 4) (to P. B. T.) and by National Institutes of Health National Research Service Award F32 HL-09376 (to K. L. H.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Biochemistry,
College of Medicine, University of Vermont, Given Bldg. C409,
Burlington, VT 05405. Tel.: 802-656-1995; FAX: 802-862-8229; E-mail:
ptracy{at}salus.med.umv.edu.
The abbreviations used are: GP, glycoprotein; Ab, antibody(ies); BSA, bovine serum albumin; FITC, fluorescein isothiocyanate; MEM, minimal essential medium; HPLC, high performance liquid chromatography; ELISA, enzyme-linked immunosorbent assay; CHO, Chinese hamster ovary; FBS, fetal bovine serum; PAR, protease-activated receptor; FPR, D-Phe-Pro-Arg chloromethyl ketone.
2 Human thrombin residues are numbered according to the human prothrombin sequence (11). The corresponding chymotrypsin-based numbering is given in square brackets (12).
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REFERENCES |
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