(Received for publication, December 22, 1995, and in revised form, September 27, 1996)
From the Canadian Red Cross Society, Blood Services,
Hamilton, Ontario, L8N 1H8 Canada, the § Department of
Pathology, McMaster University, Hamilton, Ontario L8N 3Z5, Canada, the
Department of Medicine, St. Michael's Hospital, University of
Toronto, Toronto, M5B 1W8 Ontario, Canada, and the ** New York
Department of Health, Albany, New York 12201-0509
The roles of the G-protein-linked thrombin
receptor and platelet glycoprotein Ib (GPIb) as -thrombin-binding
sites on platelets remain controversial.
-Thrombin has been proposed
to bind to both GPIb and the hirudin-like domain of the
G-protein-linked receptor (from which it cleaves the
NH2-terminal extracellular domain to release a 41-mer
peptide (TR-(1-41), where TR is
-thrombin receptor)) to initiate
platelet activation. Using affinity-purified rabbit anti-human
TR-(1-41) IgG and immunoblotting, we demonstrated TR-(1-41) release
from platelets suspended in Tyrode's buffer containing 2 mM CaCl2 and incubated with
0.5
nM
-thrombin for 10-60 s at 37 °C. As quantified by
enzyme-linked immunosorbent assay, 0.32-0.59 nM TR-(1-41)
was released from washed platelets (5 × 1011
platelets/liter) after their incubation with 10 nM
-thrombin for 10 s. Parallel binding of
-thrombin to and
activation of the platelets were confirmed by flow cytometry. A
monoclonal antibody against the hirudin-like domain of the
G-protein-linked receptor abrogated
-thrombin binding to platelets,
cleavage of TR-(1-41), and platelet activation by
1.0 nM
(but not 10 nM)
-thrombin. Proteolysis of platelet GPIb
with Serratia marcescens protease or
O-sialoglycoprotein endopeptidase had no effect on
-thrombin binding to platelets or their subsequent activation. In
contrast, chymotrypsin, which cleaves both GPIb and the
G-protein-linked receptor, abrogated
-thrombin binding to platelets,
TR-(1-41) release, and platelet activation. Furthermore, monoclonal
antibodies directed against the reported
-thrombin-binding site on
GPIb inhibited neither
-thrombin binding to nor activation of the platelets. Thus,
-thrombin binds to and cleaves the G-protein-linked receptor when it activates platelets, and GPIb does not appear to serve
as an important binding site when
-thrombin activates platelets.
Binding of -thrombin to platelets precedes platelet activation
by this enzyme and two platelet membrane glycoproteins have been
identified as thrombin-binding sites (1-12). Based on the results of
studies estimating
-125I-thrombin binding to platelets,
~50 high-affinity sites (Kd ~ 1 nM)
involving GPIb1 and ~2000
GPIb-independent binding sites with moderate affinity (Kd ~ 10 nM) for
-thrombin on
platelets have been reported (3, 4, 7). GPIb is a disulfide-linked,
two-chain protein consisting of a heavy (
) chain
(Mr 140,000) and a light (
) chain (Mr 24,000). Distinct sites on GPIb for
-thrombin and von Willebrand factor binding are located within the
Mr 45,000 NH2-terminal domain of
GPIb
(5, 7, 8, 11, 12). Support for GPIb as a high-affinity binding
site for
-thrombin arises from observations that Bernard-Soulier
platelets (congenitally deficient in platelet GPIb) are poorly
activable by
-thrombin (1). Additionally, cleavage of GPIb by
chymotrypsin, elastase, or Serratia marcescens protease
impairs the responses of platelets to subnanomolar (but not higher)
concentrations of
-thrombin (13-16). Furthermore, monoclonal
antibodies recognizing epitopes in the Mr 45,000 NH2-terminal domain of GPIb
inhibit the responses of
platelets to
1.0 nM
-thrombin (17-19).
Another -thrombin receptor on platelets, a member of the superfamily
of G-protein-linked receptors and also found on endothelial cells,
smooth muscle cells, and fibroblasts, has been cloned (9, 17, 20-22).
-Thrombin binds to and cleaves this receptor at Arg-41/Ser-42,
releasing a 41-mer activation peptide (called TR-(1-41) in this study,
where TR is
-thrombin receptor) and exposing a new
NH2-terminal domain, which then binds to an undefined part of the same receptor to activate the platelets (9, 20, 21). Whether
interactions of
-thrombin with this G-protein-linked thrombin
receptor, GPIb, or both are required for platelet activation by
-thrombin remains an unresolved question. Some investigators consider the G-protein-linked
-thrombin receptor to be the
moderate-affinity binding site since there are 1700 copies of this
-thrombin receptor/platelet (23, 24), and Bernard-Soulier platelets
have normal numbers of this receptor (24). However, monoclonal
antibodies that bind to the hirudin-like domain of the G-protein-linked
thrombin receptor abrogate the responses of platelets to
1.0
nM
-thrombin (25-28). This level of
-thrombin would
be expected to bind preferentially to its high-affinity binding sites
on platelets. It is possible that GPIb, by initiating
-thrombin
binding to platelets, could localize
-thrombin to sites on platelets
where the cleavage of the G-protein-linked
-thrombin receptor would
be facilitated to cause platelet activation (19, 23).
This study examined whether the cleavage of the G-protein-linked
thrombin receptor necessarily occurs when platelets are activated with
0.5, 1.0, and 10 nM -thrombin. Affinity-purified
polyclonal antibodies against the 41-mer activation peptide
(TR-(1-41)) released from the G-protein-linked thrombin receptor by
-thrombin were used to detect cleavage of this receptor and release
of the 41-mer activation peptide from platelets incubated with
-thrombin. Binding of
-thrombin to and activation of the same
platelets were assessed by flow cytometry (28). Whether
-thrombin
binding to GPIb is a prerequisite for platelet activation by
-thrombin was also explored by cleaving GPIb from platelets with
three proteases known to cleave this platelet glycoprotein (13-16) and
by using a panel of monoclonal anti-GPIb antibodies previously reported to inhibit
-thrombin binding to platelets (11, 17, 18).
Chymotrypsin, S. marcescens protease,
Gly-Pro-Arg-Pro (GPRP), and other chemicals were obtained from
Sigma. O-Sialoglycoprotein endopeptidase
was obtained from Cedarlane Laboratories Ltd. (Hornby, Canada). Protein
G-Sepharose 4B and CNBr-Sepharose 4B were obtained from Pharmacia
Canada (Montreal). Reagents for biotinylating IgG and alkaline
phosphatase-conjugated streptavidin were obtained from Amersham Canada
(Oakville, Canada). Human -thrombin was isolated using procedures
described previously (29). Hirudin was a gift from Dr. R. Wallis
(Ciba-Geigy, Horsham, United Kingdom).
The following monoclonal antibodies were gifts:
TM60 from Dr. Naomasa Yamamoto (Tokyo Metropolitan Institute of Medical
Science) (18), LJ-IB10 from Dr. Zaverio M. Ruggeri (Scripps Research Institute) (11), 6D1 from Dr. Barry Coller (Mount Sinai Medical Center)
(30), and ATAP-138 from Dr. Lawrence F. Brass (University of
Pennsylvania) (27). Polyclonal antibodies against the 41-amino acid
peptide released from platelets after -thrombin cleaves the
G-protein-linked thrombin receptor (and subsequently called TR-(1-41)
in this study) were raised by immunizing rabbits and chickens with 20 µg of the synthetic peptide corresponding to the first 41 amino acid
residues of the G-protein-linked thrombin receptor at biweekly
intervals. The IgG fraction was isolated from the rabbit antisera by
chromatography on a protein G-Sepharose 4B column, and specific rabbit
anti-human TR-(1-41) IgG was isolated by immunoaffinity chromatography
of the IgG on a TR-(1-41)-Sepharose 4B column. IgG isolated from egg
yolk (30) was also subjected to affinity chromatography on a
TR-(1-41)-Sepharose 4B column to isolate specific chicken anti-human
TR-(1-41) IgG. The other antibodies used in the flow cytometric
studies were phycoerythrin-conjugated monoclonal anti-GMP-140 IgG
(Becton Dickinson Advanced Cellular Biology, San Jose, CA) and
polyclonal rabbit anti-human
-thrombin IgG isolated from rabbit
anti-
-thrombin serum and biotinylated as described previously (28).
Fluorescein isothiocyanate-labeled monoclonal anti-native GPIIb-IIIa
and anti-activated GPIIb-IIIa antibodies were also obtained from Becton
Dickinson Advanced Cellular Biology.
Venous blood of healthy volunteers who had not taken any
medication for 7 days was collected into 38 g/liter sodium citrate (9 parts blood to 1 part sodium citrate). Platelet-poor plasmas were
isolated by centrifugation at 1500 × g for 20 min at
4 °C. Pooled normal plasma was obtained by adding equal volumes of
platelet-poor plasmas from at least 20 normal healthy subjects and was
stored at 50 °C. Washed platelets were prepared using the
procedures of Mustard et al. (32). Briefly, blood from
healthy volunteers not on any medication was collected into ACD
anticoagulant solution containing 5 mM citric acid, 85 mM trisodium citrate, and 111 mM glucose at a
ratio of 6 volumes of blood and 1 volume of ACD. Platelet-rich plasma
was isolated by centrifuging blood at 190 × g for 15 min at ~23 °C, followed by a second centrifugation at 2500 × g for 15 min at ~23 °C. After discarding the plasma, the platelet pellet was washed twice in a modified Tyrode's buffer containing 3.5 g/liter bovine serum albumin, 5 mM HEPES, 2 mM CaCl2, 1 mM MgCl2, 1 g/liter glucose, and apyrase adjusted to pH 7.35 at 37 °C. The
washed platelets were resuspended in the modified Tyrode's buffer
(1 × 1012 platelets/liter) as the stock platelet
suspension for use in all experiments requiring platelets resuspended
in buffer or plasma. Platelet-rich plasmas were made by resuspending
the washed platelets in pooled normal plasma to a final platelet count
of 2 × 1011/liter. To delay any fibrin formed from
polymerizing, 5 nM GPRP (final concentration) was added to
the platelet-rich plasmas (28). All experiments were performed at
37 °C.
Washed platelets resuspended in the modified Tyrode's buffer (2 × 1011 platelets/liter) were incubated at 37 °C with 50 nM chymotrypsin for 30 min, with 25 mg/liter S. marcescens protease for 30 min, or with 10 mg/liter O-sialoglycoprotein endopeptidase for 60 min. Aliquots of the platelet suspensions were fixed in 1% paraformaldehyde for 10 min at ~23 °C and subsequently analyzed by flow cytometry to estimate markers of platelet activation and alterations in platelet glycoproteins. Other periodic aliquots were also taken into the modified Tyrode's buffer containing 1 µM hirudin and the supernatants to estimate the cleavage of TR-(1-41) from platelets by dot blotting as detailed below.
Platelet Activation byWashed control
platelets in the modified Tyrode's buffer or platelets preincubated
with a protease (2 × 1011 platelets/liter) were
incubated with 0, 0.5, 1, or 10 nM -thrombin at 37 °C
for up to 30 min. In some experiments, the washed platelets were
resuspended in the modified Tyrode's buffer without 2 mM CaCl2 and incubated with
-thrombin for up to 30 min at
37 °C. Periodic aliquots were fixed in 10 g/liter paraformaldehyde
for flow cytometric analysis or were added to the modified Tyrode's buffer containing 1 µM hirudin to inactivate the added
-thrombin, followed by detection of TR-(1-41) release from the
platelets as detailed below. In other experiments, the following
monoclonal anti-platelet glycoproteins (at the final concentrations
shown in parentheses) were added to control washed platelets
resuspended in the modified Tyrode's buffer for 10 min at 37 °C
prior to the addition of
-thrombin and flow cytometric analysis:
TM60 (100 mg/liter), LJ-IB10 (120 mg/liter), 6D1 (100 mg/liter), and
ATAP-138 (150 mg/liter). These experiments determined how each
monoclonal antibody influenced the binding of
-thrombin to platelets
and the subsequent responsiveness of the platelets to the bound
-thrombin.
Washed platelets preincubated with a protease were centrifuged at 15,500 × g for 1 min at 37 °C to determine the cleavage and release of TR-(1-41) from the extracellular domain of the G-protein-linked thrombin receptor into the supernatants. The supernatants were recovered and subjected to dot blotting by loading 10 µl of each supernatant onto strips of nitrocellulose membrane, which were then air-dried at room temperature and incubated in 10 g/liter gelatin dissolved in a buffer containing 20 mM Tris, 500 mM NaCl, 0.2 g/liter trisodium azide, and 0.5 g/liter Tween 20, pH 7.4 (TBS-Tween) overnight. After washing twice with TBS-Tween, the membranes were incubated with 2 mg/liter biotinylated rabbit anti-human TR-(1-41) IgG (in TBS-Tween containing 1 g/liter gelatin) for 2 h. After washing the membrane four times with the above buffer, blots containing TR-(1-41) were identified using alkaline phosphatase-conjugated streptavidin, followed by color development with 5-bromo-4-chloro-3-indolyl phosphate, p-toluidine, and nitro blue tetrazolium.
Quantification of TR-(1-41) Release byThe release of TR-(1-41) resulting from the
incubation of platelets resuspended in Tyrode's buffer with 1.0, 10.0, and 50 nM -thrombin or with 10 and 50 nM
chymotrypsin was quantified by an ELISA for TR-(1-41). In this ELISA,
200 µl of affinity-purified chicken anti-human TR-(1-41) dissolved
in 0.1 M NaHCO3, pH 9.6 to a concentration of
10 µg of IgG/liter was added to each well of microtiter platelets and
incubated at 4 °C for 16 h. The free IgG was removed by
suction, and free sites on the wells of the microtiter plates were
blocked with 1 g/liter fatty acid-free bovine serum albumin in a buffer
containing 0.01 M Tris-HCl, 0.15 M NaCl, and
0.5 g/liter Tween 20, pH 8.0 (TBS-T). After four washes with a buffer
containing 0.01 M Na2HPO4, 0.145 M NaCl, and 0.5 g/liter Tween 20, pH 7.4 (PBS-T), a
standard curve for estimating the concentration of TR-(1-41) was
constructed as follows. 100 µl of increasing concentrations of
TR-(1-41) (20 pM to 5 nM) in TBS-T containing
10 g/liter bovine serum albumin were added to microtiter wells and
incubated at 37 °C for 60 min. Each well then received four washes
with PBS-T, followed by the addition of 100 µl of biotinylated
chicken anti-human TR-(1-41) (100 µg/liter) for a 60-min incubation
at 37 °C. After four washes with PBS-T, 100 µl of alkaline
phosphate-conjugated streptavidin diluted 1:10,000 were then added, and
the plates were incubated at 37 °C for 1 h. After another four
washes with PBS-T, 100 µl of 1 g/liter p-nitrophenyl phosphate were added for a 40-min incubation at 37 °C, and the color
yield at 405 nM was quantified.
To quantify the release of TR-(1-41) from platelets, washed platelets
(5 × 1011/liter) were resuspended in the modified
Tyrode's buffer supplemented with bovine serum albumin to a final
concentration of 10 g/liter. -Thrombin (1.0 or 10.0 nM)
or chymotrypsin (10 or 50 nM) was then added to the
platelet suspensions. Periodic aliquots were removed and added to 0.02 volume of 50 µM D-Phe-Pro-ArgCH2Cl
(for platelets incubated with
-thrombin) or 1.5 mM
phenylmethylsulfonyl fluoride (for platelets incubated with
chymotrypsin). The concentrations of TR-(1-41) released from the
platelets were estimated by ELISA immediately after the platelets had
been centrifuged at 10,000 × g for 10 min at
22 °C.
The procedures
described previously were used to estimate -thrombin binding to
platelets (28). Briefly, platelets fixed in 10 g/liter paraformaldehyde
for 10 min at 22 °C were centrifuged at 1175 × g
for 15 min and resuspended in 154 mM NaCl. The fixed platelets were next incubated with biotinylated rabbit anti-human thrombin IgG (at a concentration of 1 g/liter) for 30 min at 22 °C and then washed twice with 154 mM NaCl containing 1 g/liter
bovine serum albumin. After resuspension in FACSFlow fluid (Becton
Dickinson, Mississauga, Canada), the platelets were incubated with
phycoerythrin-conjugated Z-avidin for 30 min, washed twice with 154 mM NaCl containing 1 g/liter bovine serum albumin, and
finally resuspended in FACSFlow fluid. The percentage of 10,000 platelets that had bound
-thrombin and the associated fluorescence
intensity were determined using a FACScan argon ion flow cytometer
operating at 488 nm and at 15-milliwatt power using LysisII software.
The instrument was set up to measure the size (forward scatter),
granularity (side scatter), and platelet fluorescence. All parameters
were collected using a 4-decade logarithmic amplification. The data are
reported as thrombin fluorescence intensity on the platelets (mean
channel fluorescence in arbitrary units).
Similar procedures were used to quantify the expression of GMP-140 (P-selectin), CD63, and the resting and activated conformers of GPIIb-IIIa on platelets using the appropriate monoclonal antibodies, except that the data are reported as the percentage of platelets expressing the marker under study. The panel of monoclonal anti-GPIb antibodies (TM60, LJ-IB10, and 6D1) was also used to detect GPIb on control washed platelets and washed platelets preincubated with chymotrypsin, S. marcescens protease, or O-sialoglycoprotein endopeptidase.
To determine whether activation of platelets by
-thrombin necessarily coincided with cleavage of the
G-protein-linked thrombin receptor, washed platelets resuspended in the
modified Tyrode's buffer were incubated with up to 10 nM
-thrombin for up to 1 min. Both the binding of
-thrombin to the
platelets and activation of the same platelets were estimated. Platelet
activation was estimated by quantifying GMP-140 (Fig.
1), CD63, and the activated conformer of GPIIb-IIIa
expression on platelets by flow cytometry. As shown in Fig. 1,
dose-dependent binding of
-thrombin to the platelets and
expression of GMP-140 on the activated platelets were observed,
beginning 10 s after
0.5 nM
-thrombin addition. The fluorescence intensity of GMP-140 associated with each
concentration of added
-thrombin remained unchanged during the next
50 s of incubation. However, both
-thrombin binding to
platelets and expression of GMP-140 thereon had decreased by ~20%
when the incubation of platelets with
-thrombin was increased to 30 min (Table I).
|
We also explored the response of platelets to a second addition of
-thrombin. In these experiments, washed platelets were incubated
with 0.5 or 1.0 nM
-thrombin for 60 s, followed by the addition of 10 nM
-thrombin for a 10-s incubation.
The percentage of platelets expressing P-selectin was used to estimate
the responses of the platelets to the first and subsequent
-thrombin
additions. The addition of 0.5 and 1.0 nM
-thrombin to
these platelets for 60 s resulted in 31 and 76% of the platelets,
respectively, expressing P-selectin. The subsequent addition of 10 nM
-thrombin to these platelets resulted in >95% of
the platelets expressing P-selectin. Thus, platelets unactivated
following the addition of suboptimal concentrations of
-thrombin
respond to a second addition of
-thrombin.
Similar dose-dependent expression of CD63 and the activated
conformer of GPIIb-IIIa on the platelets was also observed after -thrombin addition (data not shown). Using affinity-purified rabbit
anti-human TR-(1-41) IgG and dot blotting, release of TR-(1-41) from
platelets incubated with the three concentrations of
-thrombin was
observed as TR-(1-41) was detected in the supernatants of platelets
incubated with
0.5 nM
-thrombin for 10 s (Fig.
2). The staining intensity of TR-(1-41) seen at 10 s remained unchanged for the next 50 s (data not shown).
The concentrations of TR-(1-41) released from washed platelets
incubated with 1.0 or 10 nM -thrombin or with 10 or 50 nM chymotrypsin were quantified by ELISA, and the results
are summarized in Table II. This ELISA could quantify
20 pM synthetic TR-(1-41) with an intra-assay
variability of ±7% (data not shown). Maximum release of TR-(1-41) by
1.0 and 10.0 nM
-thrombin was observed after incubation
for 60 and 10 s, respectively, and 1.0 nM
-thrombin released 0.36, 0.22, and 0.37 nM TR-(1-41) from the
platelets isolated from the blood of the three individuals studied. The concentrations of TR-(1-41) released by 10 nM
-thrombin
from the three platelet preparations were 0.48, 0.49, and 0.59 nM, respectively. The fragment cleaved from the
seven-transmembrane receptor by up to 50 nM chymotrypsin
(and detected by dot blotting; see Fig. 2) could not be accurately
quantified by ELISA for intact TR-(1-41) as the maximum concentration
of TR-(1-41) detected in the platelet supernatants was <30
pM.
ATAP-138, a monoclonal antibody against the
hirudin-like domain of the G-protein-linked thrombin receptor (27),
abrogates thrombin-mediated activation of platelets suspended in
buffers or plasma by preventing the binding of -thrombin to
platelets (27, 28). We explored whether abrogation of platelet
activation by ATAP-138 was associated with the inhibition of the
release of TR-(1-41) from the platelets by
-thrombin. ATAP-138 at
150 mg/liter abrogated the binding of both 0.5 and 1.0 nM
-thrombin to and the associated activation of washed platelets (Fig.
3), TR-(1-41) release from the platelets (Fig. 2), and
the expression of CD63 and the activated GPIIb-IIIa conformer on the
platelets (data not shown). However, 10 nM
-thrombin
normally bound to and activated washed platelets preincubated with 150 mg/liter ATAP-138 (Fig. 3), and both events in this case were
associated with cleavage of this thrombin receptor and release of
TR-(1-41) into the supernatant (Fig. 2). The concentration of
TR-(1-41) released by 10 nM
-thrombin from platelets
preincubated with ATAP-138 was similar to that released by 1 nM
-thrombin from control platelets. ATAP-138 abrogated
the binding of 1 or 10 nM
-thrombin to and the
subsequent activation of platelets resuspended in pooled normal plasmas
(Fig. 4).
Effects of Platelet GPIb Cleavage on
How the enzymatic degradation of GPIb influences
-thrombin binding to and activation of platelets was next
determined. O-Sialoglycoprotein endopeptidase and S. marcescens protease specifically cleave GPIb, while chymotrypsin
probably cleaves other platelet glycoproteins (13-16). None of these
three proteases directly activated the platelets or altered the initial
fluorescence of the resting and activated GPIIb-IIIa conformers on
platelets (data not shown). However, each protease completely removed
GPIb from the platelets or markedly altered the tertiary structure of
GPIb since none of the monoclonal anti-GPIb antibodies (TM60, LJ-IB10,
or 6D1) bound to platelets preincubated with any of these three
proteases (data not shown).
In spite of this observation, 0.5, 1, or 10 nM -thrombin
normally bound to and activated platelets preincubated with S. marcescens protease and O-sialoglycoprotein
endopeptidase (Fig. 1). Additionally, neither protease inhibited the
expression of CD63 or the activated GPIIb-IIIa conformer on platelets
following 0.5, 1.0, or 10 nM
-thrombin addition (data
not shown). In contrast to platelets incubated with these two
proteases,
-thrombin neither bound to nor activated platelets
preincubated with chymotrypsin (Fig. 1). Immunoblotting confirmed the
release of fragment(s) of the G-protein-linked thrombin receptor that
reacted with anti-human TR-(1-41) IgG from platelets preincubated with
chymotrypsin for 30 s (Fig. 2). Cleavage of this receptor by
chymotrypsin abrogated the activation of the platelets by
-thrombin.
In additional experiments exploring the role of GPIb
in -thrombin-mediated platelet activation, the effects of the three monoclonal anti-GPIb antibodies on
-thrombin binding to and
activation of platelets were also determined. TM60 and LJ-IB10 are
monoclonal antibodies against the high-affinity
-thrombin-binding
domain on GPIb (7, 18), while 6D1 is directed against the von
Willebrand factor-binding domain of GPIb (11, 12). Thus, unlike TM60 and LJ-IB10, 6D1 was not expected to inhibit the interactions of
-thrombin with platelets. The binding of each monoclonal antibody to
the platelets was verified by the positive and maximal staining of the
platelets with either fluorescein isothiocyanate- or
phycoerythrin-conjugated goat anti-mouse antibodies (data not shown).
In spite of the above observation, none of the three anti-GPIb
antibodies inhibited
-thrombin binding to or the subsequent
activation of washed platelets (Fig. 3) or washed platelets resuspended
in pooled normal plasma (Fig. 4).
The binding of -thrombin to and the subsequent activation of washed
platelets resuspended in Ca2+-free Tyrode's buffer were
also investigated using 0.5 nM
-thrombin. This
concentration of
-thrombin was chosen to ensure that only the
high-affinity binding sites for
-thrombin on platelets would be
occupied by the enzyme. As reported previously (28),
-thrombin bound
to ~20% fewer platelets in the absence than in the presence of
Ca2+ (Table I). In the absence of Ca2+, LJ-IB10
significantly inhibited
-thrombin binding to platelets and their
activation 10 s and 30 min after 0.5 nM
-thrombin
had been added to the washed platelets. However, TM60 did not inhibit
-thrombin binding to platelets or their activation as effectively as
LJ-IB10. Thus, Ca2+ enhances the binding of
-thrombin to
platelets and in a manner that decreases any requirement for GPIb for
directing the initial binding of
-thrombin to platelets and their
subsequent activation.
Platelets have ~25,000 copies of GPIb, the platelet glycoprotein
proposed to provide ~50 high-affinity binding sites for -thrombin (Kd ~ 1 nM) since platelets of
Bernard-Soulier patients (and thus congenitally deficient in GPIb)
aggregate slowly, but demonstrate normal dense body release in response
to subnanomolar
-thrombin. Additionally, cleavage of GPIb or
occupancy of GPIb by some monoclonal anti-GPIb antibodies inhibits
platelet aggregation and release by
1.0 nM
-thrombin,
but not by 10 nM
-thrombin (1, 5, 7, 8, 11, 12-16). A
G-protein-linked thrombin receptor on platelets to which
-thrombin
binds (probably via the hirudin-like domain of this receptor) and
cleaves off the first 41 amino acid residues (called TR-(1-41) in this
study) has been described (9, 10, 19-21). There are ~1700 copies of
this receptor/platelet (27), and some investigators have assigned the
moderate-affinity
-thrombin-binding sites (Kd ~ 10 nM) on platelets to this receptor (11, 23, 24).
Antibodies against the hirudin-like domain of this G-protein-linked
receptor inhibit the responsiveness of platelets to
-thrombin (27,
28, 33). Thus, the primary site on platelets to which
-thrombin binds to initiate platelet activation remains unclear.
In this study, cleavage of platelet TR-(1-41) by -thrombin was
directly monitored, as were
-thrombin binding to platelets and the
subsequent activation of the same platelets. No attempt was made in
this study to quantify the number of
-thrombin molecules/platelet or
the concentrations of markers of platelet activation that became expressed on activated platelets. Rather, the percentages of platelets that rapidly bound
-thrombin and subsequently expressed surface P-selection, CD63, and the activated conformer of GPIIb-IIIa for each
concentration of the enzyme were quantified. We have presented data
demonstrating the parallel binding of
-thrombin to platelets, cleavage and release of TR-(1-41) from the platelets, and activation of the same platelets with each concentration of
-thrombin. There was a similar (~1:1) relationship between the binding of
-thrombin to platelets and the expression of each of the three markers of platelet activation within 60 s of
-thrombin addition. This
study also confirmed the observation by Norton et al. (33)
that
-thrombin releases TR-(1-41) from platelets. It is unclear why
1.0 nM
-thrombin did not release TR-(1-41) as
effectively as 10 nM
-thrombin when both concentrations
of the enzyme activated
75% of the washed platelets (Figs. 1 and 3).
We eliminated the possibility that this
-thrombin receptor became
inaccessible to
-thrombin following the exposure of platelets to
suboptimal concentrations of
-thrombin. Specifically, we
demonstrated that platelets preincubated with 0.5 or 1 nM
-thrombin responded appropriately to a subsequent addition of
-thrombin. Thus, the fraction of the thrombin receptor not
previously occupied by suboptimal concentrations of
-thrombin remained accessible to added
-thrombin. Since ~2.0 nM
TR-(1-41) could be theoretically released from platelets (27), the
fact that 10 nM
-thrombin fully activated the platelets
but released only
0.6 nM TR-(1-41) suggests that
complete cleavage of the receptor is not required for maximum platelet
activation. Nonetheless, partial cleavage of this
-thrombin receptor
is required to initiate platelet activation since abrogation of
thrombin-mediated cleavage of this receptor by ATAP-138 also abrogated
platelet activation.
A likely reason for the failure of -thrombin to quantitatively
cleave all available TR-(1-41) from platelets may reside in the
ability of
-thrombin to induce endocytosis of this receptor, as
demonstrated for two megaloblastic cell lines, namely human erythroleukemia cells and Children's Hospital Research Foundation cell
line 288 (34-36). This failure of up to 10 nM
-thrombin
to fully cleave the G-protein-linked thrombin receptor and to release TR-(1-41) from platelets parallels the effects
-thrombin has on
fibrinogen and fibrin has on the enzymatic activity of
-thrombin. Similar to the release of TR-(1-41),
-thrombin cleaves fibrinogen in a dose-dependent manner, with fibrinopeptide A release
proceeding to the maximum extent achievable with each
-thrombin
concentration within 60 s (37).
-Thrombin binding to fibrin
also clearly impairs the ability of this enzyme to release
fibrinopeptide A from fibrinogen (37). Binding of
-thrombin to the
cleaved receptor (which then becomes phosphorylated (17, 38)) may
similarly impair the ability of the bound enzyme to cleave nearby
receptors. Continued tight binding of
-thrombin to this site may be
important, and one study has reported that continued occupancy of the
G-protein-linked receptor by
-thrombin is required to propagate
tyrosine phosphorylation. Specifically, Lau et al. (38) have
reported that addition of hirudin to platelets preincubated with
-thrombin for 60 s does not deaggregate the platelets, but
inhibits specific tyrosine phosphorylation and simultaneously
accelerates specific tyrosine dephosphorylation. Occupancy of this
receptor by
-thrombin at the hirudin-like domain of the receptor is
clearly crucial for platelet activation since ATAP-138 abrogates the
binding of 0.5 or 1 nM
-thrombin to platelets, release
of TR-(1-41) from the platelets, and activation of the platelets. As
previously reported by Brass et al. (27), we found that 10 nM
-thrombin binds to and activates washed platelets in
the presence of a saturating concentration ATAP-138.
The high-affinity binding sites for -thrombin on GPIb are reportedly
located within the Mr 45,000 NH2-terminal domain of GPIb
(3, 5, 7, 18, 39-42), and
removal of GPIb from platelets by chymotrypsin, S. marcescens protease, or elastase yields platelets with a lower
sensitivity to
1.0 nM
-thrombin (13-16). This study
has demonstrated that platelets with this putative high-affinity
-thrombin-binding domain on GPIb removed (by protease digestion)
bound normally to
-thrombin. In further experiments, two monoclonal
antibodies against this putative high-affinity
-thrombin-binding
domain on GPIb (TM60 and LJ-1B10) that inhibit the responses of
platelets to
1 nM
-thrombin (7, 18, 39-42) were used
in another attempt to prevent
-thrombin binding to platelets via
GPIb. In the presence of 2 mM CaCl2,
-thrombin bound normally to and activated platelets that had been
preincubated with either monoclonal anti-GPIb antibody.
Therefore, we conclude that GPIb does not normally participate in the
initial interactions of -thrombin with platelets and that
cleavage(s) by chymotrypsin additional to GPIb abrogate the responsiveness of platelets to
-thrombin. Chymotrypsin cleaves the
G-protein thrombin-linked receptor at a point distal to Arg-41/Ser-42 (43, 44). This cleavage may explain why only <30 pM
TR-(1-41) was detected by the ELISA for TR-(1-41). Using a chimeric
fusion protein consisting of glutathione S-transferase and
residues 25-97 corresponding to the NH2-terminal
extracellular domain of the G-protein-linked thrombin as the substrate,
Bouton et al. (44) reported that the glycocalicin portion of
GPIb did not alter the kinetics describing the cleavage of this fusion
protein by
-thrombin, whereas fibrinogen fragment E, thrombomodulin,
and hirudin fragment 54-65 did. These results suggest minimal rapid
binding interactions between
-thrombin and the extracellular domain
of GPIb when the enzyme normally cleaves the G-protein-linked thrombin
receptor.
There are three reasons why we could not ascribe a critical role to
GPIb for mediating -thrombin binding to platelets in the time
required for
-thrombin to optimally activate platelets. (i) We used
10-60-s incubations to demonstrate optimal binding of
-thrombin to
platelets, compared with
30-min incubations used in some of the
previous studies (7, 18, 24). The incubation times of 10 and 60 s
were chosen as activation of platelets by
-thrombin proceeds to the
maximum extent achievable with each concentration of thrombin in
60 s
(28). This choice was also justified by the demonstration of decreased
binding of
-thrombin to platelets after the enzyme was incubated
with platelets for 30 min (compared with 10 s), as shown in Table
I. (ii) The platelets used in this study were fixed with 10 g/liter
paraformaldehyde after their incubation with
-thrombin to immobilize
the enzyme on platelets. Fixation of the platelets also inactivated
-thrombin and halted further platelet reactions resulting from
-thrombin binding to the platelets. Fixation does not alter the
binding of
-thrombin to platelets (40). (iii) We also estimated
-thrombin binding to platelets resuspended in
CaCl2-containing media, while the previous studies were
without addition of this salt. CaCl2 enhances the binding
of
-thrombin to platelets and stabilizes the expression of
P-selectin on the activated platelets (28), as was confirmed in this
study. Additionally, two monoclonal anti-GPIb antibodies (LJ-IB10 and
TM60) inhibited
-thrombin binding to washed platelets and their
activation, but only in the absence of added CaCl2 (Table
I). Inhibition of
-thrombin binding to platelets by these two
monoclonal anti-GPIb antibodies (in the absence of Ca2+)
has been reported by many other investigators (11, 12, 39-42).
Previous reports have hypothesized that GPIb and the G-protein-linked
thrombin receptor form a functional complex on platelets. Specifically,
interactions of -thrombin with GPIb localize
-thrombin to sites
that facilitate cleavage of nearby G-protein-linked thrombin receptors
during the activation process (18, 23). Our results do not support
significant interactions between
-thrombin and GPIb to effect
-thrombin binding to platelets, in the presence of Ca2+,
to initiate platelet activation. The G-protein-linked thrombin receptor
appears to be the primary site to which
-thrombin binds to initiate
platelet activation. Our observations, however, do not exclude GPIb
modulating additional signaling events, including changes in
extracellular Ca2+ and aggregation resulting from
-thrombin binding to the platelets (12, 40, 41).