Characterization of the Initial alpha -Thrombin Interaction with Glycoprotein Ibalpha in Relation to Platelet Activation*

Mario Mazzucato, Luigi De MarcoDagger , Adriana Masotti, Paola Pradella, Wadie F. Bahou§, and Zaverio M. Ruggeripar

From the Servizio Immunotrasfusionale e Analisi Cliniche, Centro di Riferimento Oncologico, 33081 Aviano, PN, Italy, the § Department of Medicine, State University of New York, Stony Brook, New York 11794, and the  Roon Research Center for Arteriosclerosis and Thrombosis, Division of Experimental Hemostasis and Thrombosis, Departments of Molecular and Experimental Medicine and of Vascular Biology, Scripps Research Institute, La Jolla, California 92037

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

We have evaluated the properties of alpha -thrombin interaction with platelets within 1 min from exposure to the agonist, a time frame during which most induced activation responses are initiated and completed. Binding at 37 °C was rapidly reversible and completely blocked by a monoclonal antibody, LJ-Ib10, previously shown to be directed against the alpha -thrombin interaction site on glycoprotein (GP) Ibalpha . By 2-5 min, however, binding was no longer fully reversible and was only partially inhibited by the anti-GP Ibalpha antibody. Results were similar at room temperature (22-25 °C), whereas the initial characteristics of alpha -thrombin interaction with platelets were preserved for at least 20 min at 4 °C. Equilibrium binding isotherms obtained at the latter temperature were compatible with a two-site model, but the component ascribed to GP Ibalpha , completely inhibited by LJ-Ib10, had "moderate" affinity (kd on the order of 10-8 M) and relatively high capacity, rather than "high" affinity (kd on the order of 10-10 M) and low capacity as currently thought. The parameters of alpha -thrombin binding to intact GP Ibalpha on platelets at 4 °C corresponded closely to those measured with isolated GP Ibalpha fragments regardless of temperature. Blocking the alpha -thrombin-GP Ibalpha interaction caused partial inhibition of ATP release and prevented the association with platelets of measurable proteolytic activity. These results support the concept that GP Ibalpha contributes to the thrombogenic potential of alpha -thrombin.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

Platelet deposition at sites of vascular injury is thought to be enhanced by alpha -thrombin during normal hemostasis as well as pathological arterial thrombosis (1-3), but the mechanisms responsible for this effect have yet to be fully understood. There is evidence that alpha -thrombin-induced platelet activation is initiated by coupling of the agonist to specific receptors (4-6), whose nature is still a topic of debate. In this context, it is recognized that glycoprotein (GP)1 Ibalpha , a component of the GP Ib-IX-V complex (7, 8), binds alpha -thrombin possibly with affinity higher than other sites on platelets (6, 9, 10). This interaction has been variably judged as functionally relevant (10-12) or irrelevant (13) or even as a negative regulator acting through sequestration of the enzyme (14). It is also known that alpha -thrombin cleaves GP V (15, 16) but with no apparent relation to platelet activation (17, 18).

The agonist activity of alpha -thrombin depends on proteolysis, a fact explained with the identification of a seven-transmembrane domain receptor stimulated by a tethered ligand sequence exposed as the new amino terminus of the molecule after cleavage of an internal Arg-Ser bond (19). This protease-activated receptor, PAR1, exemplifies an effector mechanism common to a family of related proteins exhibiting distinct specificities as substrates for different proteases (20). Because the function of PAR1 seemed to explain many of alpha -thrombin effects on platelets, it was surprising that deletion of the homologous mouse gene, albeit lethal in many homozygous embryos, failed to result in decreased thrombogenic potential in the animals born alive (21). The subsequent demonstration on platelets of PAR3, another member of the family with specificity similar to PAR1 (22), provided a reasonable solution to the puzzle and reinforced the concept that a protease-activated receptor pathway is crucially involved in mediating responses to alpha -thrombin. Yet the participation of GP Ibalpha in these processes remains a possibility that must be addressed conclusively.

There are apparent contradictions in the reported characteristics of alpha -thrombin binding to platelets. Only few hundred high affinity sites have been ascribed to GP Ibalpha (6, 10), but the latter is present in greater number on the membrane (23). Moreover, a specific anti-GP Ibalpha antibody has been shown to block the interaction of approximately 5,000 alpha -thrombin molecules with platelets, abolishing higher affinity sites but also decreasing markedly the moderate affinity ones (10). Finally, the apparent kd of the highest affinity sites on platelets, 0.25-1.3 nM (10), is substantially lower than that reported for alpha -thrombin interaction with the isolated amino-terminal domain of GP Ibalpha , approximately 20 nM (24). Platelet binding parameters have been typically deduced from experiments with relatively long incubation times, in contrast with the rapidity of platelet responses to alpha -thrombin stimulation (25-27), and may reflect events not relevant for activation. Indeed, the results presented here indicate that GP Ibalpha accounts for most of the initial alpha -thrombin binding to platelets but apparently with the same "moderate" affinity assigned to the corresponding isolated functional domain (24). Different conclusions in this regard may be explained by time- and temperature-dependent deviations from equilibrium conditions. The fully reversible alpha -thrombin interaction with GP Ibalpha supports the association with platelets of a proteolytically active enzyme that may contribute to activation.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Purification and Iodination of alpha -Thrombin-- Purified human alpha -thrombin with specific clotting activity between 2,180 and 2,800 NIH units/mg (28) (a gift of Dr. John W. Fenton II, Wadsworth Center for Laboratories and Research, New York Department of Health, Albany) was radiolabeled with 125I (Amersham Corp.) using IODO-GEN (Pierce) (29). The radiolabeled ligand, with specific radioactivity between 5 and 7 mCi/mg, was characterized and stored as described (10) and was used within 2 weeks of iodination.

Preparation of Washed Platelets-- Blood was drawn from normal volunteers, who had denied ingestion of drugs known to interfere with platelet function for at least 1 week and given their informed consent to these experimental studies according to the declaration of Helsinki, and was collected into one-sixth final volume of citric acid/citrate/dextrose, pH 4.5, containing 25 nM prostaglandin E1 (Sigma). Platelets were washed free of plasma constituents using the albumin density gradient method (30), with modifications previously described (31).

Antibodies for Inhibition Studies-- The epitope of LJ-Ib10 lies between residues 238 and 290 of the amino-terminal domain of GP Ibalpha . This monoclonal antibody inhibits alpha -thrombin binding to platelets (10, 32) without effect on von Willebrand factor binding (33, 34). The rabbit polyclonal antibody, anti-TR1-160, binds to one or more epitopes within the 160 amino-terminal residues of PAR1 and abolishes platelet activation induced by low doses of alpha -thrombin and by the SFLL peptide ligand (35). Purified IgG and divalent F(ab')2 fragments, prepared as reported (36), were stored at -80 °C in 20 mM Tris, 150 mM NaCl, pH 7.4, until used.

Binding of alpha -Thrombin to Platelets-- Binding was measured according to a procedure described previously in detail (6, 9), in the presence of a binding buffer composed of 25 mM Tris-HCl and 136 mM CH3CO2Na, pH 7.3, containing 0.6% polyethylene glycol (average molecular weight 6,000-7,000; Serva, Heidelberg, Germany) and 1% bovine serum albumin (Sigma). Washed platelets were kept at the temperature selected for any given experiment for 15 min before use. Binding as a function of ligand concentration was measured by adding platelets (2.8 × 108/ml, final count) to a mixture composed of a constant concentration (0.1 nM) of 125I-labeled alpha -thrombin and increasing concentrations (0.1-1000 nM) of nonlabeled alpha -thrombin. Each experimental mixture had a total volume of 125 µl. After incubation, platelet-bound and free alpha -thrombin were separated by centrifugation through a 20% sucrose layer at 12,000 × g for 4 min. Binding isotherms, each consisting of 20 experimental points, were analyzed using the COLD option of the computer-assisted program LIGAND, calculating nonsaturable binding as a fitted parameter (37, 38). A concentration of 1 nM 125I-alpha -thrombin was employed in time course and dissociation assays. In the latter, a 1,000-fold excess of nonlabeled alpha -thrombin was added to platelets after incubation with the labeled ligand.

Binding of alpha -Thrombin to Immobilized Glycocalicin-- The extracytoplasmic domain of GP Ibalpha was purified from fresh platelet concentrates as reported (39). The glycoprotein was immobilized onto Sepharose CL 4B beads (Sigma) bearing covalently bound anti-GP Ibalpha monoclonal antibody, LJ-P3 (33), by incubating 800 µl of packed beads with 2 ml of glycocalicin solution (0.5-2 mg/ml) for 1 h at 22-25 °C with constant mixing. The beads were then washed twice with a buffer composed of 100 mM Tris, 500 mM LiCl2, 1 mM EDTA, pH 7.4, and 1 volume of packed beads was resuspended into 6 volumes of binding buffer containing 0.6% polyethylene glycol and 4.1% bovine serum albumin. This suspension was used immediately. The presence of purified glycocalicin on the beads was confirmed by SDS-polyacrylamide gel electrophoresis of protein eluted at pH 2.9 (39) and by measuring the binding of two different 125I-labeled anti-GP Ibalpha monoclonal antibodies, LJ-P19 and LJ-Ib10 (24). The binding of 125I-labeled alpha -thrombin was evaluated by mixing 20 µl of bead suspension (corresponding to 3 µl of packed beads) with 65 µl of binding buffer, or other appropriate reagent, and 40 µl of the desired ligand concentration. After incubation at the desired temperature, the radiolabeled ligand bound to the beads was separated from free ligand by centrifugation at 12,000 × g for 4 min through a 20% sucrose layer. Binding isotherms were analyzed with the computer-assisted program LIGAND (37, 38).

Flow Cytometric Analysis-- Surface membrane expression of P-selectin (40, 41) and GP Ibalpha was determined by using, respectively, an anti-CD62P monoclonal antibody (Becton-Dickinson) labeled with phycoerythrin (PE) and the anti-GP Ibalpha monoclonal antibody, LJ-Ib1 (33), labeled with fluorescein isothiocyanate (42). Washed platelets were stimulated with increasing concentrations of alpha -thrombin (0.01-100 nM) at a desired temperature, until recombinant hirudin (Iketon, Milan, Italy) was added at the final concentration of 200 NIH units/ml to neutralize the proteolytic activity. Platelets were then fixed with 1% paraformaldehyde for 30 min at 4 °C, washed twice in Tris buffer, incubated for 15 min at 22-25 °C with the specific antibodies or, in control experiments, with mouse IgG-labeled with the same fluorochromes (43), and analyzed in a flow cytometer (Becton-Dickinson). Fluorescence intensity was measured on an arbitrary scale, and platelets were considered positive for a given marker when their level of fluorescence was at least twice that of background or control platelets.

Measurement of Platelet ATP Secretion-- The release of ATP from the dense granules of platelets was measured by the luciferin-luciferase assay (44). Washed platelets were resuspended in calcium-free Tyrode buffer (31) at a count of 2 × 108/ml, and 0.4 ml were mixed with 150 µg/ml F(ab')2 fragment of the anti-GP Ibalpha monoclonal antibody, LJ-Ib10, or the rabbit polyclonal anti-TR1-160 antibody. In control experiments, the same F(ab')2 fragment concentration of the anti-GP Ibalpha monoclonal antibody, LJ-Ib1, and of preimmune rabbit IgG was used as control. The mixtures were placed in a glass cuvette and stirred at 1,200 revolutions per min (rpm) with a Teflon-coated magnetic bar for 5 min at 37 °C in a lumiaggregometer (Chrono-log Corp.). At the end of the incubation, 50 µl of luciferin-luciferase (Chrono-lume reagent, Chrono-log Corp.) was added, and platelet release was induced by the addition of alpha -thrombin at final concentration between 0.5 and 3 nM. Luminescence was recorded to monitor ATP release, measured by comparing peak height with that generated by known standard amounts of ATP.

Amidolytic Activity of alpha -Thrombin-- Washed platelets at a count of 2.8 × 108/ml, treated with control buffer or test antibodies, were mixed with 3 nM alpha -thrombin and binding buffer to a volume of 0.6 ml. After incubation for 1 min at 37 °C, platelets were sedimented by centrifugation through a 20% sucrose layer at 12,000 × g for 2 min. The supernatant containing free alpha -thrombin was removed, the sucrose layer was discarded, and the platelet pellet was resuspended with 600 µl of binding buffer. The chromogenic substrate S-2238 (Kabi) (45) was added into the supernatant as well as the resuspended platelets at the concentration of 0.4 mM, and the incubation was continued for 5 min at 37 °C. The hydrolysis reaction was then stopped with 4% acetic acid, and the release of p-nitroaniline was measured at 405 nm in a spectrophotometer (Beckman DU-65) after removing the platelets by centrifugation at 12,000 × g for 2 min.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

Time Course and Reversibility of alpha -Thrombin Binding to Platelets-- The binding of 125I-alpha -thrombin to washed platelets reached a maximum in 5 min at 37 °C but required 10 min and was about 30% lower at 4 °C (Fig. 1). Concurrent addition of 1000-fold excess of nonlabeled alpha -thrombin inhibited the binding of labeled ligand by greater than 90% at either temperature (not shown). In contrast, addition of nonlabeled ligand 20 min after 125I-alpha -thrombin, when binding of the latter was maximum, resulted in 80-95% dissociation of bound ligand at 4 °C but only about 50-60% at room temperature (22-25 °C) and 25-30% at 37 °C (Fig. 2). At the latter temperature, dissociation was approximately 70% when nonlabeled ligand was added 1 min after 125I-alpha -thrombin, 60% when it was added after 2 min, and 45% when it was added after 10 min (Fig. 2). The dissociation of bound ligand at 4 °C was not only greater in extent but occurred more rapidly than at higher temperatures, being almost maximal in 1 min as opposed to 5 min (Fig. 2).


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Fig. 1.   Time course of alpha -thrombin binding to platelets. Washed platelets, 2.8 × 108/ml final count, were incubated at 37 °C (filled circles) or 4 °C (open circles) with a constant concentration (1 nM) of 125I-labeled alpha -thrombin. At each indicated time point, the reaction was terminated by centrifuging the platelets through 20% sucrose in modified Tyrode buffer (31) for 4 min at 12,000 × g, and the radioactivity in the pellet was measured in a gamma  counter. Nonspecific binding, determined by mixing a 1000-fold excess of nonlabeled alpha -thrombin with the labeled ligand, was less than 0.02% of the total counts added and 10% or less of total bound ligand both at 37 and 4 °C and was subtracted from total binding to give the values shown in the figure. The number of 125I-alpha -thrombin molecules bound per platelet was calculated assuming a molecular mass of 37 kDa. Each point represents the mean ± S.E. of three experiments.


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Fig. 2.   Dissociation of bound alpha -thrombin from platelets. a-c, washed platelets were mixed with 1 nM 125I-labeled alpha -thrombin, as indicated in the legend to Fig. 1. After 20 min at either 4 °C (a), 22-25 °C (room temperature; (b) or 37 °C (c), 1 µM nonlabeled alpha -thrombin (filled circles) or the same volume of binding buffer (open circles) was added, and the incubation was continued. All indicated concentrations were in the final mixture. Bound 125I-alpha -thrombin was calculated (see legend to Fig. 1) immediately before and at selected time intervals after addition of the nonlabeled ligand. Each point represents the mean ± S.E. of three experiments at 4 °C or two each at 37 °C and room temperature. d, washed platelets were incubated at 37 °C with 125I-alpha -thrombin; nonlabeled ligand was added after 1, 2, or 10 min (arrows). Binding was measured immediately before and at selected time points after addition of the nonlabeled ligand. Similar results were obtained in two experiments.

Inhibitory Effect of Antibody LJ-Ib10 on alpha -Thrombin Binding to Platelets and Purified Glycocalicin as a Function of Incubation Time and Temperature-- The anti-GP Ibalpha monoclonal antibody, LJ-Ib10, inhibited the maximum binding of 125I-alpha -thrombin to platelets, measured after incubation of 20 min, by approximately 75% at 4 °C but only 60% at 22-25 °C and less than 50% at 37 °C (Fig. 3). The same antibody inhibited the maximum binding to glycocalicin, the isolated extracytoplasmic domain of GP Ibalpha , by at least 90% at all temperatures tested (Fig. 3). In the latter case, the degree of inhibition was equivalent to that produced by a 1000-fold excess of nonlabeled ligand added concurrently with 125I-alpha -thrombin (not shown). At 37 °C, the time of incubation between 125I-alpha -thrombin and platelets influenced the inhibitory effect of LJ-Ib10 on the interaction. Inhibition was essentially complete, i.e. equivalent to that caused by a 1000-fold excess of unlabeled ligand, during the first 2 min of incubation, when binding reached approximately 50% of maximum (Fig. 4). With continuing incubation, however, the inhibitory effect of the antibody progressively decreased as compared with that seen with excess unlabeled ligand (Fig. 4). Altogether, the results shown in Figs. 1-4 are compatible with the hypothesis that alpha -thrombin binding to GP Ibalpha on platelets becomes progressively less reversible as a consequence of changes related to activation, as they occur better at 37 °C than at lower temperatures. The fact that alpha -thrombin binding to isolated glycocalicin was inhibited by the antibody LJ-Ib10 in identical manner at all the temperatures tested is in agreement with such a concept.


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Fig. 3.   Effects of temperature on the inhibition of alpha -thrombin binding to platelets or purified glycocalicin by the anti-GP Ibalpha antibody LJ-Ib10. Twenty µl of washed platelets (shaded bars) or beads with immobilized glycocalicin (solid bars) were incubated for 10 min at room temperature with 65 µl of F(ab')2 fragment of the anti-GP Ibalpha antibody, LJ-Ib10 (150 µg/ml concentration in the final mixture), or binding buffer. The samples were placed at either 4, 22-25, or 37 °C for 10 min, as indicated, and then mixed with 40 µl of 125I-labeled alpha -thrombin (1 nM concentration in the final mixture). After an additional incubation of 20 min at the indicated temperatures, bound and free ligand were separated as indicated in the legend to Fig. 1. Residual binding in the presence of LJ-Ib10 was expressed as percentage of that measured in control mixtures without antibody. Results shown are the mean ± 95% confidence limits of six experiments with platelets (shaded bars) or four with glycocalicin (solid bars).


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Fig. 4.   Time course of alpha -thrombin binding to platelets at 37 °C in the presence of the anti-GP Ibalpha antibody LJ-Ib10. Washed platelets (2.8 × 108/ml final count) were incubated for 10 min at room temperature, followed by 10 min at 37 °C, with either binding buffer (circles), or 150 µg/ml F(ab')2 fragment of the anti-GP Ibalpha antibody LJ-Ib10 (squares), or 1 µM nonlabeled alpha -thrombin (triangles). A constant concentration (1 nM) of 125I-labeled alpha -thrombin was then added to all the samples, and the incubation was continued at 37 °C. All indicated concentrations were in the final mixture. At selected time points (indicated on the abscissa), bound alpha -thrombin was measured as described in the legend to Fig. 1. Upper panel, results obtained in the 1st min after addition of labeled ligand. Lower panel, results obtained between 1 and 30 min after addition of labeled ligand. Similar findings were observed in three separate experiments.

Markers of alpha -Thrombin-induced Platelet Activation-- The following experiments were performed to evaluate the time course of platelet stimulation by alpha -thrombin and correlate the membrane expression of an activation marker, P-selectin, with changes in the accessibility of GP Ibalpha to antibody probes. Greater than 50% of platelets incubated with alpha -thrombin concentrations as low as 0.1 nM for 20 min at 37 °C exhibited increased P-selectin membrane expression, and greater than 80% was positive when the agonist concentration was in the range of 1-10 nM; in contrast, at 4 °C there was no significant change relative to nonstimulated platelets even with concentrations as high as 100 nM (Fig. 5). The number of platelets displaying surface expression of P-selectin increased rapidly after stimulation with alpha -thrombin, reaching a maximum in 20-40 s at 37 °C (Fig. 5).


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Fig. 5.   Platelet membrane expression of P-selectin. Upper panel, washed platelets (final count 2.8 × 108/ml) were incubated for 20 min at 37 °C (filled circles) or 4 °C (open circles) with increasing concentrations of alpha -thrombin (0.01-100 nM, corresponding approximately to 0.001-10 NIH units/ml); at the end of the incubation, hirudin was added at the final concentration of 200 units/ml. After 2 min, platelets were fixed with 1% paraformaldehyde, washed twice with Tris buffer, resuspended in 100 µl of the same buffer to the initial count of 2.8 × 108/ml, and then mixed with 5 µl of PE-labeled anti-CD62P (P-selectin) monoclonal antibody (corresponding to a final concentration of 10 µg/ml purified IgG). After 30 min at 22-25 °C, positive (fluorescent) platelets that had bound the anti-P-selectin antibody were detected by flow cytometric analysis. Background fluorescence was measured by adding the same concentration of PE-labeled nonspecific mouse IgG and corresponded to that measured with the anti-CD62P antibody in the absence of alpha -thrombin stimulation. At either temperature less than 10% of untreated washed platelets reacted with the anti-P-selectin antibody. Each point represents the mean ± S.E. of the percent of fluorescence-positive platelets (see "Experimental Procedures") measured in two experiments at 4 °C and six at 37 °C. Lower panel, these experiments were performed at 37 °C as described above, except that a constant concentration of 1 nM alpha -thrombin was incubated with platelets for the indicated periods before adding hirudin. The results shown represent the mean ± S.E. of three separate experiments.

Exposure of platelets to alpha -thrombin at 4 °C had no significant effect on the binding of an anti-GP Ibalpha antibody, whereas progressively lower binding as a function of agonist concentration was seen at 37 °C (Fig. 6). Identical results were observed whether or not 2 mM Ca2+ and/or 1 mM Mg2+ was present in the incubation mixtures (data not shown). The observed changes in anti-GP Ibalpha antibody binding started after a time interval approximately 10-fold longer (Fig. 6) than required for increase in P-selectin surface expression (Fig. 5) following alpha -thrombin stimulation.


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Fig. 6.   Effect of alpha -thrombin on anti-GP Ibalpha antibody binding to platelets. a and b, these experiments were performed as described in the legend to Fig. 5, upper panel, except that 100 µg/ml of fluorescein isothiocyanate-labeled anti-GP Ibalpha antibody, LJ-Ib1, was used instead of the anti-CD62P antibody. a, platelets stimulated at 37 °C; b, platelets stimulated at 4 °C. Curve 1, platelets stimulated with 1 nM alpha -thrombin; curve 2, platelets stimulated with 100 nM alpha -thrombin. The curves obtained at other thrombin concentrations have been omitted for graphical clarity. Curve NS, fluorescence distribution of nonstimulated platelets. The curve at the extreme left corresponds to background fluorescence. Similar results were obtained in three separate experiments. c, platelets were incubated with 1 nM alpha -thrombin at 37 °C for the indicated periods before measuring anti-GP Ibalpha antibody binding (see above). Each point is the mean ± S.E. of three experiments in which the median fluorescence intensity of stimulated platelets was expressed as percentage of that of nonstimulated ones.

Effects of Temperature on alpha -Thrombin Binding to Platelets and Immobilized Glycocalicin-- The concentration-dependent binding of alpha -thrombin to platelets was different at 4 °C as compared with 37 °C (Fig. 7). Scatchard-type analysis of data generated at 37 °C, performed for comparative purposes even though binding was not at equilibrium (see above), resulted in a plot with downward concavity in the range of ligand concentrations between 0.1 and 3.5 nM and upward concavity at higher concentrations. The data generated at 4 °C, on the other hand, yielded an upward concave Scatchard plot (Fig. 7). The results obtained at 4 °C could be represent by a two-site model, with fitted nonsaturable binding in good agreement with experimentally determined values on the order of 2 × 10-2 (Table I). In contrast to the results with platelets, the binding of 125I-labeled alpha -thrombin to immobilized glycocalicin was the same at 4 and 37 °C, yielding a linear Scatchard plot indicative of a single class of binding sites (Fig. 7) with a kd of 4 ± 1 × 10-8 M (mean ± S.E. of four experiments at each temperature). The latter corresponded closely to the kd of the lower affinity sites detected on platelets at 4 °C (Table I). Binding isotherms generated at 4 °C in the presence of the antibody LJ-Ib10 (Fig. 8) revealed selective and complete inhibition of the lower affinity sites but no effect on the higher affinity ones (Table I). Of note, the function blocking anti-PAR1 rabbit polyclonal antibody, anti-TR1-160, did not interfere with alpha -thrombin binding to platelets at 4 °C (Table I).


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Fig. 7.   Isotherms of alpha -thrombin binding to platelets or purified glycocalicin. Upper panel, washed platelets (final count 2.8 × 108/ml) were incubated for 20 min at 37 °C (filled circles) or 4 °C (open circles) with a constant concentration of 125I-labeled alpha -thrombin (0.1 nM, corresponding to 0.0125 pmol in each binding mixture) mixed with increasing concentrations of nonlabeled alpha -thrombin (0.1-1000 nM, corresponding to 0.0125-125 pmol in the binding mixtures). At the end of the incubation, platelet-bound radioactivity was determined as described in the legend to Fig. 1. The COLD option of the computer-assisted program LIGAND was used to construct binding isotherms representing total bound as a function of added ligand as shown. The inset contains the Scatchard-type plot of the data (bound/free versus bound ligand). Similar results were obtained in four separate experiments. Lower panel, 20 µl of a suspension of beads bearing immobilized glycocalicin (see "Experimental Procedures") was mixed with 65 µl of binding buffer and 40 µl of increasing concentrations of 125I-alpha -thrombin, as indicated. After 30 min at 37 °C (filled circles) or 4 °C (open circles), the amount of bound ligand was calculated on the basis on its specific activity, and expressed as a function of added ligand. The inset contains the Scatchard-type plot of the data. Similar results were obtained in four experiments.

                              
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Table I
Parameters of alpha -thrombin binding to platelets
Control indicates normal platelets; LJ-Ib10 and anti-TR1-160 indicate normal platelets treated with saturating amounts (150 µg/ml) of F(ab')2 fragment of the anti-GP Ibalpha or anti-PAR1 antibody, respectively. All parameters and the corresponding standard error of estimated values were calculated with LIGAND (37, 38).


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Fig. 8.   Effect of antibody LJ-Ib10 on alpha -thrombin binding to platelets at 4 °C. Washed platelets (final count 2.8 × 108/ml) were incubated for 10 min at room temperature with 150 µg/ml F(ab')2 fragment of the anti-GP Ibalpha antibody LJ-Ib10 (open circles) or binding buffer (filled circles). The samples were kept for 10 min at 4 °C before adding a constant concentration of 125I-labeled alpha -thrombin (0.1 nM) mixed with increasing concentrations (0.1-500 nM) of nonlabeled alpha -thrombin. After additional incubation for 20 min at 4 °C, platelet-bound alpha -thrombin was separated from free ligand and measured as described in the legend to Fig. 1. Binding isotherms were generated as described in the legend to Fig. 7. Upper panel, bound alpha -thrombin as a function of added ligand. The inset shows an enlargement of the curves representing the results obtained at the lower ligand concentrations. Lower panel, Scatchard-type plot of the data evidencing complete inhibition of the lower affinity binding sites by the antibody LJ-Ib10. The inset shows the plot obtained in the presence of LJ-Ib10 on an expanded scale.

Inhibition of alpha -Thrombin-induced ATP Secretion by Anti-GP Ibalpha and Anti-PAR1 Antibodies-- Washed platelets stimulated with 3 nM alpha -thrombin at 37 °C exhibited rapid ATP release. This was inhibited approximately 50% by LJ-Ib10 and 80% by anti-TR1-160 but greater than 90% when the two were used together (Fig. 9). The difference between the effect of anti-TR1-160 alone and in combination with LJ-Ib10 was significant (t test: p = 0.01). Since release of ATP always occurs in parallel with that of the platelet agonist, ADP (46), these results indicate that alpha -thrombin binding to GP Ibalpha is associated with a response capable of enhancing platelet pro-thrombotic functions.


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Fig. 9.   Inhibition of alpha -thrombin-induced ATP secretion by anti-GP Ibalpha and anti-TR1-160 antibodies. Washed platelets (2.8 × 108/ml final count) were incubated for 5 min at 22-25 °C with either a mixture of monoclonal anti-GP Ibalpha antibody, LJ-Ib1, and preimmune rabbit IgG as control; or monoclonal anti-GP Ibalpha antibody, LJ-Ib10; or rabbit polyclonal anti-TR1-160 antibody directed against epitopes within the 160 amino-terminal residues of the thrombin receptor PAR-1; or a mixture of the latter two antibodies, as indicated. All antibodies were F(ab')2 fragments at the final concentration of 150 µg/ml. At the end of the incubation, each sample was placed in a lumiaggregometer cuvette, at 37 °C, and stirred at 1,200 rpm. After the addition of 50 µl of luciferin-luciferase reagent and 3 nM alpha -thrombin (equivalent to 0.3 NIH units/ml), ATP secretion was recorded continuously. A known amount of ATP (2 nmol) was used to calibrate the instrument and obtain a quantitative estimate of that released from platelets. The values shown are the mean of three experiments with 95% confidence limits.

Amidolytic Activity of alpha -Thrombin Associated with GP Ibalpha -- Measurable amidolytic activity could be partitioned with platelets after incubation with alpha -thrombin, and this association was selectively inhibited by LJ-Ib10 (Fig. 10). The appearance of platelet-associated alpha -thrombin activity corresponded to an equivalent decrease measured in the suspension medium after platelet separation, and such a decrease was also blocked selectively by LJ-Ib10 (Fig. 10). Thus, alpha -thrombin that had become associated with GP Ibalpha retained similar amidolytic activity as the free enzyme.


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Fig. 10.   Amidolytic activity of alpha -thrombin associated with platelet GP Ibalpha . Washed platelets at the final count of 2.8 × 108/ml were incubated for 10 min at room temperature with either binding buffer; or 150 µg/ml F(ab')2 fragment of the monoclonal anti-GP Ibalpha antibody LJ-Ib10; or 150 µg/ml F(ab')2 fragment of the monoclonal anti-GP Ibalpha antibody LJ-Ib1 as control; or 20 units/ml hirudin, as indicated. alpha -Thrombin at the final concentration of 0.3 NIH units/ml (3 nM) was then added into each mixture and incubated for 1 min at 37 °C. At this point, part of each mixture was kept intact and part was rapidly centrifuged to separate the platelet pellet. The chromogenic substrate S-2238 (0.4 mM) was then added either into the total mixture (left bar), or into the platelet pellet resuspended in buffer to the original volume (middle bar), or into the supernatant separated from the platelet pellet (right bar). The amidolytic activity of alpha -thrombin was measured after 5 min at 37 °C. The activity shown here was calculated as mean percentage ± S.E. (two experiments) of that obtained in control mixtures without platelets.

    DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References

The effects of alpha -thrombin on platelets are rapidly manifest (27), and most of the induced responses may reach completion in a time frame of seconds (25, 26). Here we show that within 1 min from exposure to the agonist at 37 °C, a time sufficient for maximal activation as judged by surface expression of P-selectin, the initial alpha -thrombin interaction with platelets is rapidly reversible and completely blocked by a specific anti-GP Ibalpha antibody. By 5-10 min, however, alpha -thrombin binding to platelets is less promptly reversible and only partially blocked by the anti-GP Ibalpha antibody. These changes occur in temporal relationship with decreased accessibility of GP Ibalpha to antibody probes (43, 47-49), an event that is also known to correlate with decreased von Willebrand factor binding (50), and both are prevented at 4 °C. The latter temperature, therefore, appears to preserve in time the characteristics of alpha -thrombin binding to GP Ibalpha as during the initial interaction with platelets, thus providing appropriate conditions to obtain equilibrium binding isotherms. Accordingly, in the time frame relevant for agonist-induced activation, GP Ibalpha may bind alpha -thrombin with "intermediate" or moderate affinity (kd on the order of 10-8 M), rather than being the "highest" affinity site (kd on the order of 10-10 M) as currently thought (6, 10, 12). Our findings also indicate that selective blockade of alpha -thrombin interaction with GP Ibalpha dampens responses to the agonist and prevents the association with platelets of a proteolytically active, thus potentially procoagulant, enzyme. Such conclusions are in agreement with recent evidence showing that kininogens inhibit alpha -thrombin-induced platelet aggregation because they interfere with agonist binding to GP Ibalpha (51). Indeed, owing to its relatively high membrane density, the function of GP Ibalpha may be relevant in localizing alpha -thrombin at sites of vascular injury, thus facilitating its action on specific substrates.

Application of Scatchard-type analysis to alpha -thrombin-platelet binding isotherms has usually resulted in curvilinear, upwardly concave plots indicative of a deviation from the simplest model of reversible ligand interaction with a homogeneous class of noninteracting receptors (52). This lack of uniformity has been interpreted as evidence for the presence of more than one type of receptor (6, 10, 12), leading to the proposed existence of three alpha -thrombin binding sites with high, intermediate, and low affinity (kd of approximately 0.3, 30, and 3000 nM, respectively) without nonspecific binding (6). Even considering the more likely possibility that the low affinity site represents nonspecific binding with respect to physiologic significance (32), accepting the existence of the other two sites with the reported characteristics requires prior exclusion of alternative explanations for the observed curvilinear Scatchard plots and more direct experimental evidence. Regardless of the method used for analysis of experimental results, the validity of estimated binding parameters, such as dissociation constant and receptor density, depends on the assumption that ideal thermodynamic conditions, including reversibility of ligand-receptor coupling (37, 38), are satisfied in the assay. We show here that, using intact platelets and active alpha -thrombin, this condition is met at 4 °C but not at 22-25 or 37 °C, owing to partly irreversible ligand binding at 37 °C as well as room temperature (usually 22-25 °C). Notably, previous studies proposing the concept that GP Ibalpha is the high affinity alpha -thrombin binding site were performed at room temperature (6, 10, 12) and, thus, may have resulted in incorrect estimates for the parameters of interaction.

The results obtained with platelets at 4 °C are still best fitted with a two-site model represented by an upwardly concave Scatchard plot but are compatible with the conclusion that the binding of alpha -thrombin to GP Ibalpha occurs with a kd of between 4 and 9 × 10-8 M, as shown by obliteration of this class of sites by the monoclonal antibody LJ-Ib10 without any influence on the putative higher affinity sites. The latter observation is relevant, since previous evaluation of the effects of this antibody at room temperature had shown apparent inhibition of both high and intermediate affinity receptors, as well as appearance of a new class of binding sites, not present on control platelets, with affinity halfway between high and intermediate (10). This finding, later confirmed independently (12), could be taken to reflect the existence of negative cooperativity between two distinct receptors but, in view of the above considerations on equilibrium binding, is more likely to indicate that the parameters estimated at 22-25 °C were erroneous. It is also relevant to note that the sum of presumed high and intermediate affinity sites inhibited by LJ-Ib10 at room temperature (10, 32) is of the same order of magnitude as the number of homogeneously intermediate affinity sites inhibited at 4 °C, in agreement with the notion that the sites may be the same and the estimated affinities at room temperature may be misleading.

The conclusion that alpha -thrombin interaction with GP Ibalpha on platelets has a kd on the order of 10-8 M is substantiated both by results obtained with isolated glycocalicin, as shown here and in agreement with independent data reported elsewhere (24, 32), and by previous findings with the recombinant amino-terminal domain of GP Ibalpha (24). In the case of isolated receptor fragments containing the alpha -thrombin binding site, interactions with the ligand are fully reversible at 4 °C as well as 37 °C and occur with kd between 1 and 5 × 10-8 M, reflecting the initial attributes of alpha -thrombin pairing with GP Ibalpha on platelets. These results cannot support the proposed alternative possibility that high and intermediate affinity sites are both expressed on GP Ibalpha (9). They also indicate that other components of the GP Ib-IX-V complex have no direct influence on the function of the alpha -thrombin binding site on GP Ibalpha , suggesting that data to the contrary obtained in heterologous expression systems (53) may depend on specific experimental conditions. The nature and physiologic significance of the alpha -thrombin binding sites not inhibited by LJ-Ib10, of higher affinity than GP Ibalpha sites on the basis of the results obtained at 4 °C, remain undetermined at present. Candidates for their identification may include PAR1 (13, 19), PAR3 (22), and protease nexin 1 (54).

Despite evidence to the contrary from independently performed experiments (10, 12), others have reached the conclusion that antibodies against the proposed alpha -thrombin binding site on GP Ibalpha , such as LJ-Ib10, have no inhibitory effect on platelet interaction with the agonist nor on activation (13). As in the case of studies aimed at determining binding characteristics, the methodology used may have influenced the conclusions reached. Lack of inhibitory effect by LJ-Ib10 was reported in experiments in which platelets were fixed after incubation with alpha -thrombin, then processed for indirect detection of bound ligand after repeated washing steps. It may be that, after such a procedure, the alpha -thrombin remaining associated with platelets, not defined in terms of quantity or binding characteristics, is interacting with PAR1 rather than GP Ibalpha , as suggested (13); such a conclusion is compatible with the two-site model discussed here. On the other hand, the present studies provide direct evidence that the reversible alpha -thrombin binding to platelets at 37 °C can be blocked by LJ-Ib10 within the first 60 s of incubation. The same antibody reduces dense granule ATP release, acting with an anti-PAR1 antibody to yield more efficient inhibition. Moreover, it is apparent that the alpha -thrombin associated with platelets through a GP Ibalpha -dependent mechanism remains available as an active enzyme. One function of this relatively high capacity site, therefore, may be that of increasing the concentration of alpha -thrombin onto or in proximity of the platelet membrane for subsequent proteolytic cleavage of appropriate substrates. This latter event may take place not while the enzyme is bound to GP Ibalpha , assuming that the association prevents catalytic function (14), but after dissociation from the receptor that may occur rapidly during the time frame of interaction relevant for platelet activation and clotting. Therefore, even without considering the possibility of coupling to a distinct signaling pathway that remains to be proven directly (12), our present findings add evidence to the previously proposed concept (10, 12) that alpha -thrombin interaction with GP Ibalpha has a net prothrombotic effect.

    ACKNOWLEDGEMENTS

We thank Dr. John W. Fenton II for the generous gift of human alpha -thrombin, James R. Roberts and Benjamin Gutierrez for help in the preparation of monoclonal antibodies, the late Faye Miller and Julie Kopjoe and Ellye Lukaschewsky for secretarial assistance.

    FOOTNOTES

* This work was supported in part by National Institutes of Health Grant HL-42846 and by funds from the Italian Ministry of Health, Ricerca Finalizzata 1992-1994.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.

Dagger To whom correspondence should be addressed: Servizio Immunotrasfusionale e Analisi Cliniche, Centro di Riferimento Oncologico, Via Pedemontana Occidentale, 12, 33081 Aviano, Italy. Tel.: 39-434-659 360; Fax: 39-434-659 427.

par To whom correspondence should be addressed: Dept. of Molecular and Experimental Medicine, SBR-8, Scripps Research Institute, 10550 North Torrey Pines Rd., La Jolla, CA 92037. Tel.: 619-784-8950; Fax: 619-784-2026; E-mail: ruggeri{at}scripps.edu.

1 The abbreviations used are: GP, glycoprotein; PAR, protease-activated receptor; PE, phycoerythrin.

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Discussion
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