Binding of Thrombin to the G-protein-linked Receptor, and Not to Glycoprotein Ib, Precedes Thrombin-mediated Platelet Activation*

(Received for publication, December 22, 1995, and in revised form, September 27, 1996)

Longbin Liu Dagger §, John Freedman par , Adriana Hornstein par , John W. Fenton II **, Yingqi Song § and Frederick A. Ofosu Dagger §Dagger Dagger

From the Dagger  Canadian Red Cross Society, Blood Services, Hamilton, Ontario, L8N 1H8 Canada, the § Department of Pathology, McMaster University, Hamilton, Ontario L8N 3Z5, Canada, the par  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

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES


ABSTRACT

The roles of the G-protein-linked thrombin receptor and platelet glycoprotein Ib (GPIb) as alpha -thrombin-binding sites on platelets remain controversial. alpha -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 alpha -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 alpha -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 alpha -thrombin for 10 s. Parallel binding of alpha -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 alpha -thrombin binding to platelets, cleavage of TR-(1-41), and platelet activation by <= 1.0 nM (but not 10 nM) alpha -thrombin. Proteolysis of platelet GPIb with Serratia marcescens protease or O-sialoglycoprotein endopeptidase had no effect on alpha -thrombin binding to platelets or their subsequent activation. In contrast, chymotrypsin, which cleaves both GPIb and the G-protein-linked receptor, abrogated alpha -thrombin binding to platelets, TR-(1-41) release, and platelet activation. Furthermore, monoclonal antibodies directed against the reported alpha -thrombin-binding site on GPIb inhibited neither alpha -thrombin binding to nor activation of the platelets. Thus, alpha -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 alpha -thrombin activates platelets.


INTRODUCTION

Binding of alpha -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 alpha -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 alpha -thrombin on platelets have been reported (3, 4, 7). GPIb is a disulfide-linked, two-chain protein consisting of a heavy (alpha ) chain (Mr 140,000) and a light (beta ) chain (Mr 24,000). Distinct sites on GPIb for alpha -thrombin and von Willebrand factor binding are located within the Mr 45,000 NH2-terminal domain of GPIbalpha (5, 7, 8, 11, 12). Support for GPIb as a high-affinity binding site for alpha -thrombin arises from observations that Bernard-Soulier platelets (congenitally deficient in platelet GPIb) are poorly activable by alpha -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 alpha -thrombin (13-16). Furthermore, monoclonal antibodies recognizing epitopes in the Mr 45,000 NH2-terminal domain of GPIbalpha inhibit the responses of platelets to <= 1.0 nM alpha -thrombin (17-19).

Another alpha -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). alpha -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 alpha -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 alpha -thrombin with this G-protein-linked thrombin receptor, GPIb, or both are required for platelet activation by alpha -thrombin remains an unresolved question. Some investigators consider the G-protein-linked alpha -thrombin receptor to be the moderate-affinity binding site since there are 1700 copies of this alpha -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 alpha -thrombin (25-28). This level of alpha -thrombin would be expected to bind preferentially to its high-affinity binding sites on platelets. It is possible that GPIb, by initiating alpha -thrombin binding to platelets, could localize alpha -thrombin to sites on platelets where the cleavage of the G-protein-linked alpha -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 alpha -thrombin. Affinity-purified polyclonal antibodies against the 41-mer activation peptide (TR-(1-41)) released from the G-protein-linked thrombin receptor by alpha -thrombin were used to detect cleavage of this receptor and release of the 41-mer activation peptide from platelets incubated with alpha -thrombin. Binding of alpha -thrombin to and activation of the same platelets were assessed by flow cytometry (28). Whether alpha -thrombin binding to GPIb is a prerequisite for platelet activation by alpha -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 alpha -thrombin binding to platelets (11, 17, 18).


EXPERIMENTAL PROCEDURES

Materials

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 alpha -thrombin was isolated using procedures described previously (29). Hirudin was a gift from Dr. R. Wallis (Ciba-Geigy, Horsham, United Kingdom).

Antibodies

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 alpha -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 alpha -thrombin IgG isolated from rabbit anti-alpha -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.

Preparation of Platelets and Platelet-rich and Platelet-poor Plasmas

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.

Treatment of Platelets with Chymotrypsin, S. marcescens Protease, or O-Sialoglycoprotein Endopeptidase

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 by alpha -Thrombin

Washed 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 alpha -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 alpha -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 alpha -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 alpha -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 alpha -thrombin to platelets and the subsequent responsiveness of the platelets to the bound alpha -thrombin.

Immunoblotting Analysis

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 by alpha -Thrombin and Chymotrypsin

The release of TR-(1-41) resulting from the incubation of platelets resuspended in Tyrode's buffer with 1.0, 10.0, and 50 nM alpha -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. alpha -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 alpha -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.

Flow Cytometric Analysis of Platelets

The procedures described previously were used to estimate alpha -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 alpha -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.


RESULTS

Cleavage of the G-protein-linked Thrombin Receptor by alpha -Thrombin

To determine whether activation of platelets by alpha -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 alpha -thrombin for up to 1 min. Both the binding of alpha -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 alpha -thrombin to the platelets and expression of GMP-140 on the activated platelets were observed, beginning 10 s after >= 0.5 nM alpha -thrombin addition. The fluorescence intensity of GMP-140 associated with each concentration of added alpha -thrombin remained unchanged during the next 50 s of incubation. However, both alpha -thrombin binding to platelets and expression of GMP-140 thereon had decreased by ~20% when the incubation of platelets with alpha -thrombin was increased to 30 min (Table I).


Fig. 1. Binding of increasing concentrations of alpha -thrombin to platelets (upper panels) and the resulting platelet activation measured as the expression of GMP-140 (P-selectin) on the platelets (lower panels). Control washed platelets and washed platelets resuspended in the modified Tyrode's buffer and preincubated with O-sialoglycoprotein endopeptidase (OSE), chymotrypsin (CHYMO), or S. marcescens protease (SMP) were incubated with 0.5, 1.0, or 10 nM alpha -thrombin for 10 s. Aliquots were fixed in 1 g/liter paraformaldehyde for 10 min, and alpha -thrombin binding to the platelets and their activation were quantified by flow cytometry. Results are the means ± S.D. (3-5%) of three to four determinations. 4-6% of the platelets not incubated with alpha -thrombin were positive for both alpha -thrombin and GMP-140.
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Table I.

Effects of TM60, LJ-IB10, and ATAP-138 on alpha -thrombin binding to washed platelets and their subsequent activation

The monoclonal antibody or saline was added to washed platelets (5 × 1011/liter) resuspended in Tyrode's buffer, with (first four columns) or without (last four columns) CaC2, for incubation at 37 °C. alpha -Thrombin (0.5 nM) was then added to each platelet suspension. Aliquots were withdrawn at 10 s or 30 min into 1% paraformaldehyde, fixed for 10 min, and subjected to flow cytometry to estimate the percentage of platelets that had bound alpha -thrombin and the percentage of platelets that had become activated (by expressing GMP-140 or P-selectin). The percentage of control platelets not exposed to alpha -thrombin but expressing thrombin at 10 s and 30 min was 5.1%; the percentage of control platelets expressing GMP-140 at 10 s and 30 min were 5.0 and 4.9%, respectively. Each result was obtained after the platelet suspensions from three to four experiments had been pooled prior to centrifugation and flow cytometry.
Antibody Platelets binding alpha -thrombin and expressing GMP-140 after
10 s
30 min
10 s
30 min
Thrombin GMPa Thrombin GMP Thrombin GMP Thrombin GMP

%
None 36.0 38.0 29.6 30.4 29.4 30.0 23.0 24.2
TM60 35.4 36.0 28.7 29.5 24.6 25.5 18.8 19.4
LJ-IB10 35.0 35.5 29.0 28.3 16.2 16.5 9.2 9.7
ATAP-138 4.5 5.0 4.8 6.0 6.0 6.2 6.3 7.1

a  GMP, GMP-140 (P-selectin).

We also explored the response of platelets to a second addition of alpha -thrombin. In these experiments, washed platelets were incubated with 0.5 or 1.0 nM alpha -thrombin for 60 s, followed by the addition of 10 nM alpha -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 alpha -thrombin additions. The addition of 0.5 and 1.0 nM alpha -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 alpha -thrombin to these platelets resulted in >95% of the platelets expressing P-selectin. Thus, platelets unactivated following the addition of suboptimal concentrations of alpha -thrombin respond to a second addition of alpha -thrombin.

Similar dose-dependent expression of CD63 and the activated conformer of GPIIb-IIIa on the platelets was also observed after alpha -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 alpha -thrombin was observed as TR-(1-41) was detected in the supernatants of platelets incubated with >= 0.5 nM alpha -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).


Fig. 2. Release of TR-(1-41) from washed platelets after their incubation at 37 °C for 10 s with alpha -thrombin. The supernatants from control platelets incubated for 10 s at 37 °C with alpha -thrombin (0, 0.5, 1.0, and 10 nM in lanes 1-4, respectively); from platelets preincubated with ATAP-138 (150 g/liter) for 30 min at 37 °C prior to alpha -thrombin (0.5, 1.0, and 10 nM in lanes 5-7, respectively) for 10 s at 37 °C; or from platelets incubated with 50 nM chymotrypsin for 30 s (lane 8), 5 min (lane 9), and 30 min (lane 10) were dot-blotted with biotinylated rabbit anti-human TR-(1-41) IgG.
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The concentrations of TR-(1-41) released from washed platelets incubated with 1.0 or 10 nM alpha -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 alpha -thrombin was observed after incubation for 60 and 10 s, respectively, and 1.0 nM alpha -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 alpha -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.

Table II.

Release of TR-(1-41) from platelets incubated with alpha -thrombin and chymotrypsin

Washed platelets (5 × 1011/liter) isolated from the blood of three healthy volunteers (A, B, and C) were resuspended in the modified Tyrode's buffer containing 2 mM CaC2 and 10 mg/ml bovine serum albumin. Following the addition of alpha -thrombin or chymotryspin, aliquots were withdrawn into 0.02 volume of 50 µM D-Phe-Pro-ArgCH2C (alpha -thrombin) or 50 mM phenylmethylsulfonyl fluoride (chymotrypsin). Following centrifugation at 10,000 × g for 10 min, the concentration of TR-(1-41) released into the supernatant was quantified by ELISA. The concentration of TR-(1-41) measured in each control platelet supernatant was 0.02 nM. Each incubation of platelets with an enzyme was conducted in triplicate, and the platelet supernatants were pooled prior to quantifying the release of TR-(1-41) by ELISA.
Platelets Incubation time(s) [Enzyme] [TR-(1-41)]

min nM
A 10 10 nM alpha -thrombin 0.48
A 60 1 nM alpha -thrombin 0.36
B 10 10 nM alpha -thrombin 0.49
B 60 1 nM alpha -thrombin 0.22
C 10 10 nM alpha -thrombin 0.59
C 60 1 nM alpha -thrombin 0.36
A 10 10 nM chymotrypsin NDa
A 10 50 nM chymotrypsin ND
B 10 50 nM chymotrypsin ND
C 10 10 nM chymotrypsin 0.02
C 10 50 nM chymotrypsin 0.01

ND, none detected.

Effects of ATAP-138 on Thrombin-mediated Platelet Activation

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 alpha -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 alpha -thrombin. ATAP-138 at 150 mg/liter abrogated the binding of both 0.5 and 1.0 nM alpha -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 alpha -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 alpha -thrombin from platelets preincubated with ATAP-138 was similar to that released by 1 nM alpha -thrombin from control platelets. ATAP-138 abrogated the binding of 1 or 10 nM alpha -thrombin to and the subsequent activation of platelets resuspended in pooled normal plasmas (Fig. 4).


Fig. 3. Effects of monoclonal anti-GPIb antibodies TM60, LJ-IB10, and 6D1 and of a monoclonal antibody to the hirudin-like domain of the G-protein-linked thrombin receptor (ATAP-138) on the binding of alpha -thrombin to washed platelets () and their subsequent activation measured as the expression of GMP-140 (square ) on the platelets. Washed platelets resuspended in the modified Tyrode's buffer were preincubated at 37 °C for 10 min with TM60 (100 µg/ml), LJ-IB10 (120 µg/ml), 6D1 (100 µg/ml), or ATAP-138 (150 µg/ml). Following 0.5, 1.0, or 10 nM alpha -thrombin addition to the washed platelets for 10 s at 37 °C, aliquots of the platelet suspensions were fixed in 1% paraformaldehyde for 10 min, and alpha -thrombin binding to the platelets and their activation were estimated by flow cytometry. Results are the means ± S.D. (<= 5%) of four or more determinations. 3-6% of the platelets not incubated with alpha -thrombin were positive for both alpha -thrombin and GMP-140.
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Fig. 4. Effects of monoclonal anti-GPIb antibodies TM60 (100 µg/ml), LJ-IB10 (120 µg/ml), and 6D1 (100 µg/ml) and of ATAP-138 (150 µg/ml; a monoclonal antibody to the hirudin-like domain of the G-protein-linked thrombin receptor) on the binding of alpha -thrombin to platelets resuspended in pooled normal plasmas. Each antibody was added to the platelet-rich plasma for incubation at 37 °C for 10 min. Following the addition of CaCl2 (10 mM final concentration) to enhance alpha -thrombin binding and 1 µM tick anticoagulant peptide r-TAP (to abrogate prothrombin activation), 1.0 or 10 nM alpha -thrombin was then added. Aliquots were withdrawn 10 s later and fixed in 1% paraformaldehyde for 10 min, and alpha -thrombin binding to the platelets () and platelet activation estimated as GMP-140 expression (square ) were quantified by flow cytometry. Results represent the mean ± S.D. (<= 5%) of three or more determinations. ~4% of the platelets resuspended in the recalcified plasma expressed both thrombin and GMP-140 on their surfaces.
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Effects of Platelet GPIb Cleavage on alpha -Thrombin-mediated Platelet Activation

How the enzymatic degradation of GPIb influences alpha -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 alpha -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 alpha -thrombin addition (data not shown). In contrast to platelets incubated with these two proteases, alpha -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 alpha -thrombin.

Effects of Monoclonal Anti-GPIb Antibodies on alpha -Thrombin Binding to Platelets

In additional experiments exploring the role of GPIb in alpha -thrombin-mediated platelet activation, the effects of the three monoclonal anti-GPIb antibodies on alpha -thrombin binding to and activation of platelets were also determined. TM60 and LJ-IB10 are monoclonal antibodies against the high-affinity alpha -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 alpha -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 alpha -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 alpha -thrombin to and the subsequent activation of washed platelets resuspended in Ca2+-free Tyrode's buffer were also investigated using 0.5 nM alpha -thrombin. This concentration of alpha -thrombin was chosen to ensure that only the high-affinity binding sites for alpha -thrombin on platelets would be occupied by the enzyme. As reported previously (28), alpha -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 alpha -thrombin binding to platelets and their activation 10 s and 30 min after 0.5 nM alpha -thrombin had been added to the washed platelets. However, TM60 did not inhibit alpha -thrombin binding to platelets or their activation as effectively as LJ-IB10. Thus, Ca2+ enhances the binding of alpha -thrombin to platelets and in a manner that decreases any requirement for GPIb for directing the initial binding of alpha -thrombin to platelets and their subsequent activation.


DISCUSSION

Platelets have ~25,000 copies of GPIb, the platelet glycoprotein proposed to provide ~50 high-affinity binding sites for alpha -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 alpha -thrombin. Additionally, cleavage of GPIb or occupancy of GPIb by some monoclonal anti-GPIb antibodies inhibits platelet aggregation and release by <= 1.0 nM alpha -thrombin, but not by 10 nM alpha -thrombin (1, 5, 7, 8, 11, 12-16). A G-protein-linked thrombin receptor on platelets to which alpha -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 alpha -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 alpha -thrombin (27, 28, 33). Thus, the primary site on platelets to which alpha -thrombin binds to initiate platelet activation remains unclear.

In this study, cleavage of platelet TR-(1-41) by alpha -thrombin was directly monitored, as were alpha -thrombin binding to platelets and the subsequent activation of the same platelets. No attempt was made in this study to quantify the number of alpha -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 alpha -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 alpha -thrombin to platelets, cleavage and release of TR-(1-41) from the platelets, and activation of the same platelets with each concentration of alpha -thrombin. There was a similar (~1:1) relationship between the binding of alpha -thrombin to platelets and the expression of each of the three markers of platelet activation within 60 s of alpha -thrombin addition. This study also confirmed the observation by Norton et al. (33) that alpha -thrombin releases TR-(1-41) from platelets. It is unclear why 1.0 nM alpha -thrombin did not release TR-(1-41) as effectively as 10 nM alpha -thrombin when both concentrations of the enzyme activated >= 75% of the washed platelets (Figs. 1 and 3). We eliminated the possibility that this alpha -thrombin receptor became inaccessible to alpha -thrombin following the exposure of platelets to suboptimal concentrations of alpha -thrombin. Specifically, we demonstrated that platelets preincubated with 0.5 or 1 nM alpha -thrombin responded appropriately to a subsequent addition of alpha -thrombin. Thus, the fraction of the thrombin receptor not previously occupied by suboptimal concentrations of alpha -thrombin remained accessible to added alpha -thrombin. Since ~2.0 nM TR-(1-41) could be theoretically released from platelets (27), the fact that 10 nM alpha -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 alpha -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 alpha -thrombin to quantitatively cleave all available TR-(1-41) from platelets may reside in the ability of alpha -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 alpha -thrombin to fully cleave the G-protein-linked thrombin receptor and to release TR-(1-41) from platelets parallels the effects alpha -thrombin has on fibrinogen and fibrin has on the enzymatic activity of alpha -thrombin. Similar to the release of TR-(1-41), alpha -thrombin cleaves fibrinogen in a dose-dependent manner, with fibrinopeptide A release proceeding to the maximum extent achievable with each alpha -thrombin concentration within 60 s (37). alpha -Thrombin binding to fibrin also clearly impairs the ability of this enzyme to release fibrinopeptide A from fibrinogen (37). Binding of alpha -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 alpha -thrombin to this site may be important, and one study has reported that continued occupancy of the G-protein-linked receptor by alpha -thrombin is required to propagate tyrosine phosphorylation. Specifically, Lau et al. (38) have reported that addition of hirudin to platelets preincubated with alpha -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 alpha -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 alpha -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 alpha -thrombin binds to and activates washed platelets in the presence of a saturating concentration ATAP-138.

The high-affinity binding sites for alpha -thrombin on GPIb are reportedly located within the Mr 45,000 NH2-terminal domain of GPIbalpha (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 alpha -thrombin (13-16). This study has demonstrated that platelets with this putative high-affinity alpha -thrombin-binding domain on GPIb removed (by protease digestion) bound normally to alpha -thrombin. In further experiments, two monoclonal antibodies against this putative high-affinity alpha -thrombin-binding domain on GPIb (TM60 and LJ-1B10) that inhibit the responses of platelets to <= 1 nM alpha -thrombin (7, 18, 39-42) were used in another attempt to prevent alpha -thrombin binding to platelets via GPIb. In the presence of 2 mM CaCl2, alpha -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 alpha -thrombin with platelets and that cleavage(s) by chymotrypsin additional to GPIb abrogate the responsiveness of platelets to alpha -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 alpha -thrombin, whereas fibrinogen fragment E, thrombomodulin, and hirudin fragment 54-65 did. These results suggest minimal rapid binding interactions between alpha -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 alpha -thrombin binding to platelets in the time required for alpha -thrombin to optimally activate platelets. (i) We used 10-60-s incubations to demonstrate optimal binding of alpha -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 alpha -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 alpha -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 alpha -thrombin to immobilize the enzyme on platelets. Fixation of the platelets also inactivated alpha -thrombin and halted further platelet reactions resulting from alpha -thrombin binding to the platelets. Fixation does not alter the binding of alpha -thrombin to platelets (40). (iii) We also estimated alpha -thrombin binding to platelets resuspended in CaCl2-containing media, while the previous studies were without addition of this salt. CaCl2 enhances the binding of alpha -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 alpha -thrombin binding to washed platelets and their activation, but only in the absence of added CaCl2 (Table I). Inhibition of alpha -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 alpha -thrombin with GPIb localize alpha -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 alpha -thrombin and GPIb to effect alpha -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 alpha -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 alpha -thrombin binding to the platelets (12, 40, 41).


FOOTNOTES

*   This work was supported in part by a grant-in-aid from the Heart and Stroke Foundation of Ontario. 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.
   Recipient of a career development fellowship award from the Canadian Red Cross Society, Blood Services.
Dagger Dagger    To whom correspondence should be addressed: Dept. of Pathology, McMaster University, 1200 Main St. West, Hamilton, Ontario L8N 3Z5, Canada. Tel.: 905-525-9140 (ext. 22263); Fax: 905-521-2613.
1    The abbreviations used are: GP, glycoprotein; TBS, Tris-buffered saline; ELISA, enzyme-linked immunosorbent assay; PBS, phosphate-buffered saline.

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