Platelet Glycoprotein Ibalpha Binds to Thrombin Anion-binding Exosite II Inducing Allosteric Changes in the Activity of Thrombin*

Chester Q. LiDagger , Alessandro VindigniDagger , J. Evan Sadler§, and Mark R. WardellDagger §||

From the Dagger  Department of Biochemistry & Molecular Biophysics, the § Department of Medicine, and the  Howard Hughes Medical Institute, Washington University School of Medicine, St. Louis, Missouri 63110

Received for publication, May 16, 2000, and in revised form, October 6, 2000



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The glycoprotein (GP) Ib-IX complex is a platelet surface receptor that binds thrombin as one of its ligands, although the biological significance of thrombin interaction remains unclear. In this study we have used several approaches to investigate the GPIbalpha -thrombin interaction in more detail and to study its effect on the thrombin-induced elaboration of fibrin. We found that both glycocalicin and the amino-terminal fragment of GPIbalpha reduced the release of fibrinopeptide A from fibrinogen by about 50% by a noncompetitive allosteric mechanism. Similarly, GPIbalpha caused in thrombin an allosteric reduction in the rate of turnover of the small peptide substrate D-Phe-Pro-Arg-pNA. The Kd for the glycocalicin-thrombin interaction was 1 µM at physiological ionic strength but was highly salt-dependent, decreasing to 0.19 µM at 100 mM NaCl (Gamma salt = -4.2). The salt dependence was characteristic of other thrombin ligands that bind to exosite II of this enzyme, and we confirmed this as the GPIbalpha -binding site on thrombin by using thrombin mutants and by competition binding studies. R68E or R70E mutations in exosite I of thrombin had little effect on its interaction with GPIbalpha . Both the allosteric inhibition of fibrinogen turnover caused by GPIbalpha binding to these mutants, and the Kd values for their interactions with GPIbalpha were similar to those of wild-type thrombin. In contrast, R89E and K248E mutations in exosite II of thrombin markedly increased the Kd values for the interactions of these thrombin mutants with GPIbalpha by 10- and 25-fold, respectively. Finally, we demonstrated that low molecular weight heparin (which binds to thrombin exosite II) but not hirugen (residues 54-65 of hirudin, which binds to exosite I of thrombin) inhibited thrombin binding to GPIbalpha . These data demonstrate that GPIbalpha binds to thrombin exosite II and in so doing causes a conformational change in the active site of thrombin by an allosteric mechanism that alters the accessibility of both its natural substrate, fibrinogen, and the small peptidyl substrate D-Phe-Pro-Arg-pNA.



    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Thrombin is one of the most potent physiological agonists of platelets, inducing activation responses such as cytokinesis, aggregation, secretion, and associated metabolic changes (1, 2). The first receptor for thrombin identified on the platelet surface was the glycoprotein (GP)1 Ib-IX complex (3-6), although the biological significance of this interaction remains unknown 20 years later. In the interim, other thrombin receptors have also been identified on the platelet surface including three members of the seven transmembrane domain receptor superfamily known as proteolytically activated receptor-1 (PAR-1), PAR-3, and PAR-4 (7-10). PAR-1 was shown to mediate platelet activation (7) and took much attention away from GPIb-IX as a thrombin receptor until it was found that PAR-1 knockout mice had no demonstrable bleeding disorder and that their platelets retained normal responses to alpha -thrombin (11). Furthermore, synthetic peptides of PAR-3 and PAR-4 that mimic the sequences of the potentially activating tethered ligands, either failed to show (PAR-3) or showed very low (PAR-4) activation effects on platelets (9, 10). Therefore, attention has again turned to the GPIb-IX complex to understand, for example, why the platelets from patients with Bernard-Soulier syndrome, which lack or have dysfunctional GPIb-IX complexes on their surfaces fail to respond to low doses of thrombin (12, 13).

Glycoprotein Ib-IX consists of three polypeptides (GPIbalpha , GPIbbeta , and GPIX) that are each transmembrane proteins that span the platelet membrane once. They are also each members of the leucine-rich repeat superfamily (14). Glycoprotein Ibalpha and GPIbbeta are linked together via a disulfide bond that is situated in the extracellular space very close to the platelet membrane, and GPIX is noncovalently associated with these two. On the platelet surface, a fourth leucine-rich repeat protein, GPV, appears to link two GPIb-IX trimers together and may be responsible for even more complex aggregation states (14-16). Of this multimeric complex it is the extracellular portion of GPIbalpha , known as glycocalicin, that forms the site of ligand interaction. Glycocalicin can be released from the surface of platelets by proteolysis near the platelet membrane and consists of two subregions known as the macroglycopeptide and the amino-terminal domain (14). It is the amino-terminal domain, which consists of about 300 amino acids, that provides the sites within glycocalicin for ligand interaction (3), and within this the thrombin-binding site has been localized between residues 269 and 287 (17). The site on thrombin, however, where GPIbalpha binds is more controversial. Within the structure of thrombin four prominent regions have been identified: its active site, a sodium ion-binding site (18, 19), and two surface electropositive patches known as anion binding exosites I and II (20) that have both been implicated in thrombin binding. Although some studies have indicated that GPIbalpha binds to anion-binding exosite I (also known as the fibrinogen recognition site) of thrombin (21-24), the studies of De Cristofaro and colleagues (25-27) have implicated anion-binding exosite II (also known as the heparin binding site) of thrombin as the site of GPIbalpha interaction.

Thrombin is an allosteric serine protease (28) with changes in the conformation of its active site being induced by the binding of ligands at the other sites of the enzyme. For example, the binding of ligands at exosite I changes the rate of turnover of small synthetic peptidyl and natural substrates (29, 30), whereas the binding of heparin at exosite II alters the kinetics of the inhibition of thrombin by hirudin (31) consistent with allosteric linkage between exosites I and II of thrombin (32). We wondered whether the binding of GPIbalpha to thrombin also produced an allosteric response in the enzyme. Furthermore, we sought to clarify which of the exosites of thrombin was the GPIbalpha -binding site in the expectation that such knowledge would lead to the formulation of new hypotheses about the biological role of GPIbalpha -thrombin interaction, because the exosite involved should be directly related to the consequence of this binding.

In this study, we have used four approaches to investigate the binding interaction of GPIbalpha with thrombin: 1) an HPLC method measuring the effect of ligands on thrombin-induced fibrinogen turnover that has been well characterized in the study of thrombin-fibrinogen and thrombin-thrombomodulin interactions (33, 34), 2) mutant thrombins with single amino acid substitutions in either exosite I or II (35-37), 3) a resin-based assay for studying the direct binding of thrombin to GPIbalpha , and 4) competition binding assays using small ligands of known exosite interaction as competitors of the GPIbalpha -thrombin interaction. Our results clearly indicated that the binding site for GPIbalpha is located in thrombin anion-binding exosite II and that the binding of GPIbalpha to thrombin induces conformational changes at the active site of thrombin by an allosteric mechanism that alters the activity of thrombin toward both physiological and small substrates.


    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Preparation of Glycocalicin and the Amino-terminal Fragment of GPIbalpha from Human Platelets

Glycocalicin was isolated from outdated human platelets using a modification of methods previously reported (38, 39). Briefly, after glycocalicin was cleaved from the surface of platelets by calpain released as a result of their sonication, it was isolated by wheat germ agglutinin Sepharose 4B and Q-Sepharose anion exchange chromatography. Purified glycocalicin was dialyzed into 20 mM Tris-HCl, pH 8.0, and concentrated to 1 mg/ml, and aliquots were snap frozen in liquid nitrogen and stored at -80 °C until use. The amino-terminal fragment of GPIbalpha was generated from purified glycocalicin by cleavage with porcine pancreatic elastase at an enzyme to substrate ratio of 1:250 (w/w). Porcine pancreatic elastase cleaves glycocalicin after residue Val289 yielding the amino-terminal fragment and a macroglycopeptide, which are well separated by Q-Sepharose. Both fragments were concentrated, dialyzed, and stored as described for glycocalicin above.

Recombinant Expression of the Amino-terminal Fragment of GPIbalpha

The amino-terminal region of GPIbalpha was expressed from baculovirus-infected insect cells (40) and purified as described previously (41). Briefly, the cDNA coding for the signal peptide and amino-terminal domain of GPIbalpha (residues -16 to 289) followed by the calmodulin (CaM) gene was inserted into the baculovirus expression vector pAcSG2 (Pharmingen, San Diego, CA). The resultant plasmid, pWIbalpha wt, was then recombined with BacVector-3000 triple cut virus DNA (Novagen, Madison, WI) to produce infective virus. A high titer baculovirus stock was obtained from a single plaque by repeated infection of Sf9 insect cells. Recombinant virus was then used to infect High Five insect cells for expression of the GPIbalpha -CaM fusion protein. The calmodulin moiety of the fusion protein was then exploited in the first stage of recombinant protein isolation since it binds to N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7)-agarose (prepared as described previously (42)). Calmodulin could be released from the GPIbalpha () fragment by digestion with porcine pancretaic elastase (1:100 w/w E:S ratio), and the GPIbalpha () could be retrieved in pure form after passage over Q-Sepharose anion exchange resin.

Recombinant Prothrombin Expression

Recombinant mutant prothrombins R68E, R70E (exosite I mutants), R89E, and K248E (exosite II mutants) were expressed and characterized as described previously (36, 37). Amino acid residues are numbered from the first residue of the human thrombin B chain. Purified prothrombins were activated with Echis carinatus snake venom, and the thrombin products were further purified to homogeneity by ion exchange chromatography on Amberlite CG-50. The purity of each thrombin was confirmed by SDS-polyacrylamide gel electrophoresis and silver staining. The activities of recombinant mutant and wild-type thrombins were tested as described (43, 44).

Characterization of the Thrombin-GPIbalpha Interaction

Effect of Glycocalicin on the Thrombin-Fibrinogen Interaction-- The binding of glycocalicin to thrombin was determined by modifying an HPLC-based assay that has been well characterized for the study of fibrinogen binding to thrombin (33). First, thrombin was incubated with fibrinogen under the desired solution conditions (5 mM Tris-HCl, 0.1% PEG-8000, pH 8.0 at 25 °C) and the progress curves for fibrinopeptide A (FpA) release were measured to determine the values of the specificity constant kcat/Km. The fibrinogen concentration was 0.2 µM, thrombin concentrations varied according to their specific activities in the range from 0.08-2 nM, and the ionic strength was kept constant with NaCl. The reaction was initiated by addition of thrombin to the fibrinogen solution (or to the fibrinogen and glycocalicin solution) and quenched at different time intervals with 3 M perchloric acid. The sample was then centrifuged, and the amount of FpA in the supernatant was determined by HPLC, as described previously (33). Next, glycocalicin at varying concentrations was added to the assay system, and the equilibrium dissociation constants (Kd) for glycocalicin binding to thrombin were determined by analysis of the inhibition of FpA release as a function of glycocalicin concentration according to the following equation.


[<UP>FpA</UP>]=[<UP>FpA</UP>]<SUB>∞</SUB>{1−<UP>exp</UP>(−se<SUB>⊤</SUB>t<SUB>c</SUB>)} (Eq. 1)
where
s=<FR><NU><FENCE><FR><NU>k<SUB><UP>cat</UP></SUB></NU><DE>K<SUB>m</SUB></DE></FR></FENCE><SUB>0</SUB>+<FENCE><FR><NU>k<SUB><UP>cat</UP></SUB></NU><DE>K<SUB>m</SUB></DE></FR></FENCE><SUB>1</SUB><FR><NU>x</NU><DE>K<SUB><UP>d</UP></SUB></DE></FR></NU><DE>1+<FR><NU>x</NU><DE>K<SUB>d</SUB></DE></FR></DE></FR> (1a)
and x is the GPIbalpha concentration, eT is the active thrombin concentration, (kcat/Km)0 and (kcat/Km)1 are the specificity constants for FpA release in the absence of and at saturating concentration of GPIbalpha , respectively, and tc = Km/eTkcat corresponds to the point in the progress curve that gives the greatest change in the amount of FpA released as a function of the inhibitor concentration. The exosite mutant thrombins were substituted for wild-type thrombin in this assay to investigate the effect that GPIbalpha had on their cleavage reactions of fibrinogen. Before each assay the concentration of thrombin was adjusted to ensure the same thrombin activity was present in each reaction.

The same assay was used for investigating the salt dependence of the thrombin-glycocalicin interaction in the range of 100-150 mM NaCl. The data plotted as log Kd versus log [salt] were fitted to a straight line according to the following expression (34).
<UP>−ln</UP> K<SUB>d</SUB>=A<SUB>0</SUB>+&Ggr;<SUB><UP>salt</UP></SUB><UP>ln</UP>[<UP>salt</UP>] (Eq. 2)
Kd is a dissociation constant. The slope of this line yields Gamma salt, which represents a thermodynamic measure of the effect of salt concentration on binding equilibria (28, 34).

Direct Thrombin Binding Assay-- The direct binding of thrombin to GPIbalpha was investigated using the recombinant GPIbalpha -CaM fusion protein expressed in insect cells. 50 µl of W-7-agarose solution containing 25 µl of packed resin was first added to a 500-µl Eppendorf tube. The resin beads were then washed with washing buffer (100 mM NaCl, 50 mM Tris-HCl, pH 7.4) three times. 1 nmol of recombinant GPIbalpha -CaM fusion protein in 100 µl of binding solution (100 mM NaCl, 50 mM Tris-HCl, pH 7.4, 20 mM CaCl2) was added to the 25 µl of washed W-7-agarose beads and incubated for 2 h at 25 °C. The beads were then washed once with binding solution and blocked with 3% bovine serum albumin, 0.1% PEG-8000 in binding solution for another 2 h. After blocking, duplicates of increasing quantities (2.5, 5.0, 7.5, 10, or 12.5 pmol) of thrombin (wild type or mutants) were added to separate tubes in binding solution in a total volume of 70 µl and incubated for 2 h at 25 °C. The total volume of each reaction, including W-7-agarose, was therefore 95 µl containing final concentrations of thrombin of 26, 52, 79, 105, and 131 nM, respectively. As a negative control, W-7 beads were incubated for the indicated time in the absence of GPIbalpha -CaM. The beads were kept suspended during each incubation step by inverting the tubes every 10 min. At the end of the incubation, the beads were pelleted by centrifugation at 600 × g for 1 min. 50 µl of each supernatant was transferred to a 96-well microtiter plate and incubated with 50 µl of 2.5 mM thrombin substrate D-Phe-Pro-Arg-pNA. The release of p-nitroaniline was monitored by spectrophotometry at 405 nm using a THERMOmax microplate reader (Molecular Devices Sunnyvale, CA) at 5-min intervals from 5 to 60 min.

Competition of GPIbalpha -Thrombin Binding Using Known Exosite Ligands-- These assays were performed essentially as for the direct thrombin binding assay described above but with the addition of either hirugen (residues 54-65 of hirudin with Tyr63 sulfated; Sigma) or low molecular weight (LMW) heparin (Rhône-Poulenc Rorer Pharmaceuticals Inc., Collegeville, PA) added as inhibitors of exosites I and II, respectively. 50-µl aliquots of W-7-agarose solution containing 25 µl of packed beads were added to tubes and washed as above. 1 nmol of recombinant GPIbalpha -CaM fusion protein in binding solution was added to the beads and incubated for 2 h at 25 °C in constant suspension. After blocking for 2 h as above, 12.5 pmol of thrombin and various quantities (0, 50, 100, 250, 500, 750, 1000, 1500, and 2000 pmol) of hirugen or LMW heparin were added to duplicate tubes in a volume of 70 µl making final concentrations of 0, 0.53, 1.05, 2.63, 5.26, 7.89, 10.53, 15.79, and 21.05 µM, respectively, each in a total volume of 95 µl. After 2 h incubation at 25 °C, 50 µl of each supernatant was transferred to a 96-well microtiter plate and incubated with 50 µl of 2.5 mM thrombin substrate D-Phe-Pro-Arg-pNA. The release of p-nitroaniline was monitored spectrophotometrically at 405 nm using a THERMOmax microplate reader, as above.

Allosteric Effect of Thrombin Exosite Ligands on the Amidolytic Activity of Thrombin

12.5 pmol of human alpha -thrombin was preincubated for 10 min in a total volume of 70 µl as above with various concentrations (between 0 and 14.29 µM) of glycocalicin, hirugen (Sigma), or LMW heparin (Rhône-Poulenc Rorer Pharmaceuticals Inc., Collegeville, PA). After incubation, 50 µl of each solution was transferred to a 96-well microtiter plate and incubated with 50 µl of 2.5 mM thrombin substrate D-Phe-Pro-Arg-pNA. The rate of hydrolysis of the chromogenic substrate was determined spectrophotometrically at 405 nm by a THERMOmax microplate reader as above.


    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Noncompetitive Inhibition of Fibrinopeptide A Release-- We began our studies by a detailed examination of the influence that glycocalicin had on the fibrinogen-thrombin interaction. Fibrinogen interacts with thrombin exosite I and is thereafter cleaved in its Aalpha chain at Arg16 and in its Bbeta chain at Arg14, releasing FpA and FpB, respectively. We utilized a well characterized HPLC-based assay that quantitatively measures the release of FpA after thrombin-catalyzed hydrolysis of fibrinogen (33). Various concentrations of glycocalicin were added to solutions containing both thrombin and fibrinogen to determine whether the glycocalicin could inhibit the thrombin-mediated cleavage of fibrinogen. As shown in Fig. 1, the addition of glycocalicin decreased the amount of FpA released from the fibrinogen. The FpA release could not be totally inhibited, even by a large glycocalicin excess, however, indicating that glycocalicin was not acting in a competitive manner and was not binding to thrombin exosite I. The curve in Fig. 1 was best fit by Equation 1, which describes a noncompetitive mode of inhibition, and because thrombin is an allosteric enzyme (28), we concluded that the inhibitory effect of glycocalicin occurred by an allosteric mechanism. The equilibrium dissociation constant (Kd) for the thrombin-glycocalicin interaction derived from this equation was 1.04 ± 0.008 µM at 150 mM NaCl. Identical results were obtained when the amino-terminal domain of GPIbalpha (residues 1-289), whether derived from human platelets or recombinantly expressed, was substituted for glycocalicin (data not shown).



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Fig. 1.   The amount of FpA released at time tc = Km/eTkcat by thrombin-catalyzed proteolysis of fibrinogen as a function of GPIbalpha concentration. Experimental conditions are: 5 mM Tris-HCl, 0.15 M NaCl, 0.1% PEG-8000 at 25 °C. The data were fit by the continuous line according to Equation 1 with the best fit parameters: Kd = 1.04 ± 0.08 µM, kcat/Km = 24.4 µM-1 s-1. The concentration of thrombin was 0.1 nM, and the fibrinogen concentration was 0.2 µM.

The Effect of Salt on the Glycocalicin-Thrombin Interaction-- The binding affinities of two other ligands that interact with thrombin exosite II, heparin (45) and the chondroitin sulfate moiety of thrombomodulin (34) have been shown to be very sensitive to salt concentration, whereas the affinities of ligands binding to thrombin exosite I are not (33, 34, 46). Fig. 2 shows that the influence of glycocalicin on the thrombin-induced FpA release from fibrinogen was salt-dependent, with the derived equilibrium dissociation constants for the glycocalicin-thrombin interaction varying nearly an order of magnitude between the NaCl concentrations of 100 and 150 mm (K100 mM NaCl = 0.187 ± 0.009 µM; Kd125 mM NaCl = 0.41 ± 0.03 µM; Kd150 mM NaCl = 1.04 ± 0.008 µM). The thrombin-fibrinogen interaction is minimally affected within this range of salt concentration (33). At higher salt concentrations than reported here, the affinity of glycocalicin for thrombin dropped considerably, whereas at lower concentrations the Kd could not be determined accurately because of changes in the kinetic mechanism for the release of fibrinopeptides.



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Fig. 2.   The effect of NaCl on the Kd of the GPIbalpha -thrombin interaction. The experimental conditions were the same as for Fig. 1 except that each curve was generated at a different concentration of NaCl as indicated in the inset. Each point represents the amount of FpA released by thrombin-catalyzed cleavage of fibrinogen at time tc = Km/eTkcat, which is the time along the progress curve most sensitive to inhibition by glycocalicin. The data at each NaCl concentration were fit by Equation 1 with the best fit parameters for 0.15 M NaCl () the same as given in the legend to Fig. 1; for 0.125 M NaCl (black-square), Kd = 0.41 ± 0.03 µM, kcat/Km = 32.5 µM-1 s-1; for 0.1 M NaCl (black-triangle), Kd = 0.187 ± 0.009 µM, kcat/Km = 49.2 µM-1 s-1. The concentration of thrombin eT was 0.1 nM, and the fibrinogen concentration was 0.2 µM.

The effect of salt on the interaction of glycocalicin with thrombin can be further quantified by plotting the data as log Kd verses log [salt] on a straight line according to Equation 2 (33, 34). When the log of the equilibrium dissociation constants derived for the glycocalicin-thrombin interaction at different concentrations of NaCl was plotted against the log of their respective [Na+], the straight line in Fig. 3 was obtained. The value of Gamma salt for this interaction was calculated from the slope of the line as -4.2. For comparison, the equivalent plots reported by other investigators for the thrombin-heparin (45) and thrombin-hirugen (34) interactions are also shown in Fig. 3 as representatives of thrombin exosite II and exosite I interactions, respectively. The value of Gamma salt for the glycocalicin-thrombin interaction is compared in Table I with the Gamma salt values obtained for thrombin and other exosite I and II ligands obtained from the literature. As can be seen, thrombin ligands that bind at its exosite I and show little salt dependence (fibrinogen and hirudin) have values of Gamma salt around 1.0, whereas known exosite II-binding ligands (heparin and the chondroitin sulfate moiety of thrombomodulin) are characterized by Gamma salt values around 4-5. In this respect, glycocalicin is behaving as a thrombin exosite II-binding ligand.



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Fig. 3.   Comparison of the NaCl concentration dependence of the GPIbalpha -thrombin interaction with those of the heparin-thrombin and hirugen-thrombin interactions. The dissociation constants for the glycocalicin-thrombin interaction () derived from the curves in Fig. 2 were plotted as a function of the respective Na+ ion concentration on a log-log scale and fitted by linear regression according to Equation 2. For comparison, the equivalent data previously reported for the thrombin exosite II-heparin (45) (black-square) and thrombin exosite I-hirugen (34) (black-triangle) interactions are shown. The slope of the line for the glycocalicin-thrombin interaction, Gamma salt, is listed in Table I along side the Gamma salt values previously reported by other investigators for other thrombin exosite-ligand interactions.


                              
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Table I
Comparison of Gamma salt for the GPIbalpha -thrombin interaction with those from the literature for the interaction of thrombin with other exosite ligands

Interaction of Thrombin Exosite Mutants with Glycocalicin-- To address more directly the exosite on thrombin that mediates GPIbalpha interaction, four thrombin exosite mutants were employed in binding studies. Previous investigators showed that mutations in the anion-binding exosite II of thrombin (R89E, R245E, K248E, and K252E) greatly reduced its affinity for heparin (35, 36) and that mutations in exosite I (R68E and R70E) reduced its affinity for fibrinogen (37). We first determined the equilibrium dissociation constants for the interaction between each of these four mutants and glycocalicin in the fibrinogen assay utilized above. The Kd values derived from the curves in Fig. 4 for the interaction of glycocalicin with the exosite I mutants were comparable with that of wild-type thrombin (KdR68E = 0.1 µM; KdR70E = 0.034 µM at 100 mM NaCl, although because thrombin exosite I mutants interfere with the thrombin-fibrinogen interaction, the concentration of exosite I mutants used in the assay was 4-40-fold more than that of wild-type thrombin). The exosite II mutants, however, interacted very poorly with glycocalicin having 10- and 25-fold higher Kd values than wild-type thrombin (KdR89E = 1.4 µM; KdK248E = 4.9 µM at 100 mM NaCl) and showed almost no inhibitory effect on FpA release.



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Fig. 4.   The effect of exosite mutations in thrombin on the GPIbalpha -mediated modulation of FpA release from fibrinogen. Experiments were performed in the same manner as for Fig. 1, except the NaCl concentration was 0.1 M, and thrombin mutants containing single amino acid substitutions of alanine for basic residues within either exosite I or II, as indicated in the inset, were used. The concentration of fibrinogen was 0.2 µM, and the concentration of each thrombin preparation was adjusted to ensure the same thrombin activity was present in each reaction. The continuous lines were drawn to fit the data according to Equation 1 for wild type and exosite I mutants or by linear regression for exosite II mutants with the best fit parameters: for wild-type thrombin (), Kd = 0.187 ± 0.009 µM, kcat/Km = 49.2 µM-1 s-1; for R68E (black-diamond ), Kd = 0.1 ± 0.001 µM, kcat/Km = 1.0 µM-1 s-1; for R70E (black-down-triangle ), Kd = 0.0335 ± 0.001 µM, kcat/Km = 34 µM-1 s-1; for K248E (black-square), Kd = 4.9 ± 0.001 µM, kcat/Km = 27 µM-1 s-1; and for R89E (black-triangle), Kd = 1.4 ± 0.7 µM, kcat/Km = 13.67 µM-1 s-1.

Direct Binding of Thrombin Exosite Mutants to Recombinant GPIbalpha -- This assay utilized the properties of CaM to attach the recombinant GPIbalpha -CaM fusion protein to W-7-agarose beads. The thrombin-binding properties of this GPIbalpha -CaM fusion protein were indistinguishable from either glycocalicin or the amino-terminal domain of GPIbalpha (data not shown). The thrombin that bound to the immobilized GPIbalpha pelleted with the W-7-agarose beads and was quantified from the difference between the total amount of thrombin activity added to the tube and that remaining in the supernatant. In this way it was found that the thrombin exosite I mutants interacted with the GPIbalpha () attached to the resin in an identical way as did wild-type thrombin (Fig. 5). In contrast, the thrombin exosite II mutants showed minimal binding activity toward the resin-associated GPIbalpha () and had similar binding curves to the negative control in which no recombinant GPIbalpha () was attached to the resin (Fig. 5). Taken together, these studies also support the concept that the GPIbalpha binding-site on thrombin is within anion-binding exosite II.



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Fig. 5.   The effect of thrombin exosite mutations on the direct binding of thrombin to GPIbalpha . Recombinant GPIbalpha () was attached to agarose beads via its fusion partner, calmodulin, and wild-type () or mutant thrombin variants with single amino acid substitutions in either exosite I, R68E (black-diamond ) and R70E (black-down-triangle ), or exosite II, R89E (black-triangle) and K248E (black-square), were added. Thrombin that bound to the immobilized GPIbalpha () was determined from the difference between the total amidolytic activity of thrombin added to each tube and that remaining in the supernatant after pelleting the beads, as determined by its hydrolysis of substrate D-Phe-Pro-Arg-pNA. The negative control (open circle ) had no recombinant GPIbalpha -CaM fusion protein attached to the agarose beads to provide a measure of the total amount of thrombin that was available to bind to the GPIbalpha .

Thrombin Exosite I and II Ligands as Inhibitors of Glycocalicin Binding-- We next investigated the abilities of hirugen (a thrombin exosite I ligand) and LMW heparin (a thrombin exosite II ligand) to inhibit thrombin from binding to the GPIbalpha attached to the W-7-agarose beads. Again we derived the difference between the total thrombin activity added to controls with no GPIbalpha -CaM fusion protein attached to the W-7 beads, and the residual thrombin activity left in the supernatant after pelleting the thrombin that had bound to the GPIbalpha () attached to the resin particles. During these experiments we observed that the activity of the unbound thrombin toward the peptidyl substrate D-Phe-Pro-Arg-pNA was itself influenced by the binding of hirugen and LMW heparin. This is shown in Fig. 6 together with the allosteric effect of glycocalicin on the amidolytic activity of thrombin toward this small synthetic tripeptidyl substrate. Whereas the binding of hirugen resulted in allosteric enhancement of the amidolytic activity of thrombin, the binding of LMW heparin and glycocalicin resulted in allosteric inhibition of the amidolytic activity of thrombin.



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Fig. 6.   The allosteric effects of GPIbalpha and other exosite ligands on the amidolytic activity of thrombin toward the small peptidyl substrate D-Phe-Pro-Arg-pNA. Wild-type alpha -thrombin (12.5 pmol) was incubated for 10 min with the indicated concentrations of glycocalicin (), hirugen (black-triangle), and LMW heparin (black-square), and the rate of hydrolysis of the chromogenic tripeptidyl substrate, D-Phe-Pro-Arg-pNA (2.5 mM), was then determined spectrophotometrically in a microplate reader as described under "Experimental Procedures."

It can be seen from Fig. 7A that at a concentration of 21 µM, LMW heparin completely inhibited thrombin binding to the GPIbalpha () on the resin, because all the available thrombin activity remained unbound in the supernatant. In contrast, hirugen had no effect on the thrombin-GPIbalpha () interaction, as determined by the absence of convergence of the two curves in Fig. 7B. In the presence of all concentrations of hirugen, most of the added thrombin binds to the GPIbalpha () attached to the resin and is removed from solution resulting in a marked and constant reduction in the unbound thrombin activity in the supernatants in each tube. These results indicate that LMW heparin, which binds to thrombin exosite II, can fully inhibit GPIbalpha binding to thrombin, whereas the exosite I ligand, hirugen, is not an inhibitor of this interaction.



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Fig. 7.   The effect of LMW heparin (an exosite II ligand) and hirugen (an exosite I ligand) on the GPIbalpha -thrombin interaction. Recombinant GPIbalpha attached to W-7-agarose via its calmodulin fusion moiety was incubated with thrombin (12.5 pmol) in 50 mM Tris-HCl, 0.1 M NaCl, pH 7.4, for 2 h in the presence of the indicated concentrations of either LMW heparin (A) or hirugen (B). The dotted lines in A and B show the allosteric effects on the amidolytic activity of thrombin toward the peptidyl substrate D-Phe-Pro-Arg-pNA caused by adding just LMW heparin or hirugen, respectively, to thrombin in a solution of W-7-agarose with no GPIbalpha attached to the beads. The solid lines in each panel indicate the amount of thrombin that had bound to the GPIbalpha immobilized on the W-7-agarose in the presence of increasing concentrations of either LMW heparin (A) or hirugen (B) as competitors. Bound thrombin that had been removed from the solution by pelleting the W-7-agarose beads was calculated from the difference between the total thrombin activity added to the reaction tubes minus that remaining in the supernatant after pelleting the beads (unbound thrombin activity). The convergence of the solid curve with the dotted curve in A indicates that LMW heparin inhibited the binding of thrombin to the immobilized GPIbalpha so that all of the thrombin added to the reaction remained unbound by the addition of 20 µM LMW heparin. Conversely, the addition of hirugen did not inhibit thrombin from binding to the immobilized GPIbalpha .



    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Evidence has been accumulating for some time that thrombin undergoes changes in the conformation of its active site as an allosteric response to ligands binding at other sites of the enzyme. For example, the binding of proteins or peptides corresponding to segments of natural inhibitors and substrates that bind to exosite I (30) and the binding of the chondroitin sulfate moiety of thrombomodulin to exosite II (34) have been shown to alter the amidolytic activity of thrombin toward small synthetic substrates. Thrombomodulin binding to exosite I of thrombin enhances the cleavage of protein C at least 500-fold. Most of this effect has been attributed to the influence of thrombomodulin on the conformation of protein C in the thrombin-thrombomodulin-protein C ternary complex. However, an approximately 15-fold rate enhancement appears to be due to the influence of thrombomodulin on the active site architecture of thrombin as measured by turnover of small substrates that mimic the sequence cut by thrombin in protein C (28). Another notable example of allostery involves the sodium-binding site the occupancy of which triggers the transition of thrombin between the slow and the fast forms (28) that are primarily associated with the anticoagulant and procoagulant functions of thrombin, respectively (47).

Our present studies provide an additional example of the allosteric nature of thrombin. This became evident when we investigated the detailed effect of GPIbalpha on the thrombin-fibrinogen interaction. The results showed that glycocalicin inhibited the cleavage of fibrinogen by thrombin, confirming the findings of previous investigators (23, 24, 48). The inhibition was not, however, of a competitive nature because it was not possible to completely inhibit FpA release by increasing the concentration of glycocalicin. Instead, the equation that best fit the inhibition curve of Fig. 1 described a noncompetitive mode of inhibition that would be consistent with the operation of an allosteric mechanism. Therefore, unlike the previous investigators (23, 24, 48), we concluded that glycocalicin did not bind to thrombin exosite I. Although our conclusions differed, the experimental findings reported here are similar to those of Jandrot-Perrus et al. (23, 24) and De Marco et al. (48). The key to understanding the different interpretations of the findings resides in consideration of the magnitude of the inhibition caused by GPIbalpha on the thrombin-fibrinogen interaction. In no study did GPIbalpha completely inhibit thrombin-fibrinogen interaction. Our present data now indicate that this is because GPIbalpha does not compete for the same binding site on thrombin as fibrinogen but rather induces a conformational change at the active site through an allosterical mechanism that slows the rate of fibrinogen cleavage. As shown in Fig. 6, the alteration in the architecture of the active site of thrombin induced allosterically by the binding of GPIbalpha to its exosite II also decreases the amidolytic activity of thrombin toward the small synthetic tripeptidyl substrate D-Phe-Pro-Arg-pNA.

The binding of glycocalicin to thrombin was highly salt-dependent. This finding was reminiscent of ligands that bind to thrombin exosite II such as heparin and the chondroitin sulfate moiety attached to thrombomodulin and implicated GPIbalpha as a thrombin exosite II ligand. This would make GPIbalpha the first protein to directly bind to thrombin exosite II through protein-protein interactions. In terms of negative charge density, however, the thrombin-binding site on GPIbalpha (residues 269-287 (17)) could be said to resemble that of heparin. Of the 19 residues in this region of GPIbalpha , 13 are negatively charged, including three sulfated tyrosines (residues 276, 278, and 279 (49, 50)), which themselves give this region of GPIbalpha even more resemblance to the polysulfated glycosaminoglycan, heparin. The value of Gamma salt is a quantitative measure of the salt dependence of a binding interaction and for the GPIbalpha -thrombin interaction was determined to be -4.2 (51), in close agreement with that recently reported by others (52) (Table I). This value of Gamma salt far exceeds the values reported for fibrinogen and hirudin binding (Table I) and signals a much larger electrostatic contribution to the binding of GPIbalpha to thrombin. Consistent with this is the similarity of Gamma salt for GPIbalpha -thrombin with that of the heparin-thrombin interaction that has been characterized as a predominantly nonspecific electrostatic interaction (45). If it is assumed that the salt-dependent interactions between thrombin and its exosite II ligands are solely due to electrostatic association, then the values of Gamma salt will also indicate the minimum number of ionic bonds involved in the binding (45). Thus, it might be expected that a minimum of four ionic bonds contribute to the association of GPIbalpha with thrombin.

More direct evidence that GPIbalpha binds to exosite II of thrombin was derived from our studies using mutant thrombins with single amino acid substitutions in either exosite I or II. The thrombin mutants with either R89E or K248E substitutions in exosite II both displayed dramatically decreased interactions with GPIbalpha (Figs. 4 and 5). In contrast, the exosite I mutations of R68E and R70E had little effect on GPIbalpha binding (Figs. 4 and 5). The mutant thrombins were employed here in two different assays. The first was in the same HPLC assay used above to investigate the effect that glycocalicin had on fibrinogen turnover by thrombin. Because, however, the thrombins with exosite I mutations themselves had reduced interactions with fibrinogen, potentially complicating the interpretation of the results of this assay, we also utilized an assay we had developed to study the direct binding of thrombin to a recombinant portion of GPIbalpha containing the thrombin-binding site (51). The results from both assays indicated that exosite II mutations, but not exosite I mutations, drastically reduced the binding of thrombin to GPIbalpha .

The final way in which we investigated the GPIbalpha interaction site on thrombin was to employ hirugen and LMW heparin as inhibitors of binding to exosites I and II, respectively. As seen in Fig. 6 and by the dashed curves in Fig. 7, both of these small molecules caused allosteric conformational changes in the active site of thrombin as determined by changes in the amidolytic activity of thrombin toward the peptidyl substrate D-Phe-Pro-Arg-pNA. The solid lines in panels A and B of Fig. 7 represent how much thrombin was inhibited from binding to GPIbalpha immobilized on W-7-agarose beads by LMW heparin and hirugen, respectively. The convergence of the two curves in Fig. 7A indicate that at a concentration of 21 µM, LMW heparin had inhibited all of the thrombin from binding to the immobilized GPIbalpha because all of the available thrombin amidolytic activity remained in the supernatant. Conversely, hirugen caused no detectable increase in the activity of unbound thrombin in the supernatant indicating that the binding of thrombin to the immobilized GPIbalpha was not being inhibited by hirugen. This again shows that GPIbalpha is interacting with thrombin exosite II.

In the present work we have determined the affinity for thrombin binding to glycocalicin as an inhibitor of the thrombin-fibrinogen interaction in an assay system that has been well characterized in the study of other inhibitors of this reaction (33, 34). Under these conditions, glycocalicin behaved as a classical noncompetitive inhibitor of the thrombin-fibrinogen interaction with a Kd value of 1 µM for binding to thrombin in the presence of 150 mM NaCl. Thus, thrombin binds more avidly to glycocalicin than to the extracellular amino-terminal fragment of the classical thrombin receptor, PAR-1, for which the Km is 15-30 µM, also at 150 mM NaCl (53). If thrombin binding to the platelet surface involved the formation of a GPIbalpha -thrombin-PAR-1 ternary complex, then thermodynamic considerations dictate that the 1 µM thrombin-glycocalicin interaction added to that of the 15-30 µM thrombin-PAR-1 interaction could reduce the Kd of thrombin binding to as low as 15-30 × 10-12 M, which would be compatible with the Kd values of 10-8-10-10 M reported for thrombin binding to platelets (48, 54). The finding that glycocalicin binds to thrombin exosite II, whereas PAR-1 binds at thrombin exosite I, would allow such a ternary complex. Alternatively, GPIbalpha might act as a "ligand-passing" receptor, initially trapping thrombin at the platelet surface to make it available to the PAR receptors, in a manner analogous to that proposed for the passing of tumor necrosis factor from tumor necrosis factor receptor-2 to tumor necrosis factor receptor-1 (55). In this role it would be advantageous for the association and dissociation of thrombin to and from GPIbalpha to be rapid, and a micromolar dissociation constant for their interaction would be consistent with this.

A further hypothesis, again involving a ternary complex mechanism, would describe a functional role for GPIbalpha binding to thrombin to retain and localize the enzyme at sites where fibrin generation is needed for the maturation and stabilization of blood clots (56). The platelet surface provides a major site for thrombin generation (57) through the clotting sequence. Like other factors involved in this pathway, the precursor of thrombin, prothrombin, is anchored to the platelet phosopholipid membrane by gamma -carboxylated glutamate residues (58-61), which do not form part of the active alpha -thrombin enzyme when it is released from prothrombin by proteolysis. Perhaps at this time thrombin binds via its anion binding exosite II to GPIbalpha to be retained in the locality where fibrin generation is required. Fibrinogen would subsequently bind to thrombin exosite I and be cleaved to the products utilized for cross-linking into the insoluble fibrin matrix found in mature thrombi. Binding to GPIbalpha through exosite II would also prevent the inhibition of thrombin by antithrombin, as recently shown (52), because the inhibitory mechanism requires heparin binding (62) to thrombin exosite II. Obviously, it would be desirable to only temporarily prevent thrombin inhibition by antithrombin until the fibrin clot became large enough to stop further blood loss but not so large as to totally occlude blood flow through the vessel. The relatively weak affinity between thrombin and GPIbalpha described by a micromolar dissociation rate would be consistent with this, facilitating the ready release of thrombin from GPIbalpha for its subsequent inhibition by antithrombin. Furthermore, the 50% allosteric reduction in the rate of fibrinogen turnover, caused by the binding of GPIbalpha to thrombin at physiological ionic strength (Fig. 1), may be an additional mechanism to regulate blood coagulation in vivo, similar to the allosteric "switch" mechanism described recently for exosite inhibitors of factor VIIa (63). The localization of thrombin-GPIb complexes on the platelet surface might also recruit and activate additional platelets during the thrombus formation.

Although the precise role for thrombin binding to GPIbalpha is not known, several observations suggest that this interaction is physiologically important. For example, selective inhibition of thrombin binding to GPIbalpha shifts the dose-response curve of platelets induced by low doses of thrombin (21), and the thrombin-GPIbalpha interaction is necessary for thrombin-induced platelet procoagulant activity (64). Therefore, the thrombin-GPIb interaction may contribute to the initiation and maintenance of platelet responses during hemostasis.


    ACKNOWLEDGEMENTS

We thank Pei Ye, Dr. Huifang Wang, Dr. J. Justin Robert, Linda A. Wardell, and Milan Kapadia for technical assistance and Dr. Enrico Di Cera for critical reading of the manuscript.


    FOOTNOTES

* This work was supported in part by National Institutes of Health Grant HL60617 (to M. R. W.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

|| To whom correspondence should be addressed: New Century Pharmaceuticals, Inc., 895 Martin Rd., Huntsville, AL 35824. E-mail: mwardell@newcenturypharm.com.

Published, JBC Papers in Press, October 9, 2000, DOI 10.1074/jbc.M004164200


    ABBREVIATIONS

The abbreviations used are: GP, glycoprotein; CaM, calmodulin; FpA, fibrinopeptide A; LMW, low molecular weight; W-7, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide; PAR, proteolytically activated receptor; HPLC, high pressure liquid chromatography.


    REFERENCES
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ABSTRACT
INTRODUCTION
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RESULTS
DISCUSSION
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