SEMINARS IN THROMBOSIS AND HEMOSTASIS—VOLUME 18, NO. 2, 1992
Thrombin Binding to Platelet Membrane Glycoprotein Ib
The serine protease thrombin is the most potent physiologic platelet agonist, and thrombin-induced platelet activation is critical for hemostasis and thrombosis.1 However, despite considerable effort by a number of laboratories, the mechanisms by which thrombin activates platelets have not yet been fully elucidated. A variety of evidence has indicated that platelet activation by thrombin requires both thrombin binding to the cell membrane and expression of the enzyme proteolytic activity. 1-3 The proteolytic step of platelet stimulation was first ascribed to the thrombin-induced hydrolysis of platelet membrane glycoprotein V (GFV).4,5 However, the lack of relationship between the rates of platelet activation and GPV hydrolysis has raised doubt concerning the relevance of GPV hydrolysis as a crucial step in the platelet activation process.6-8 Furthermore, anti-GPV antibodies that inhibited GPV hydrolysis failed to block thrombin-induced platelet activation.9 Therefore, the role of thrombin-catalyzed GPV hydrolysis in platelet activation remains to be elucidated. More recently, previously unrecognized thrombin binding proteins have been identified through direct expression cloning in Xenopus oocytes.10,12 mRNA encoding these proteins were detected in human platelets and endothelial cells and in hamster fibroblasts. They are novel members of the superfamily of seven transmembrane domain proteins. In platelets, the newly discovered thrombin binding protein behaves both as a substrate and a receptor for thrombin at the cell surface, and contains
From the Laboratoire de Recherche sur l'Hémostase et la Thrombose, Faculté de Médecine Xavier Bichat, Paris, France. Reprint requests: Dr. Jandrot-Perrus, Laboratoire de Recherche sur l'Hemostase et la Thrombose, Faculté de Médecine Xavier Bichat, 16, rue Henri Huchard, 75018 Paris, France.
its own ligand, which is unmasked on thrombin-catalyzed hydrolysis.12 Glycoprotein Ib (GPIb) is the major sialoglycoprotein at the platelet surface.13 Besides its role as a binding site for von Willebrand factor,14 GPIb functions as a binding site for α-thrombin. Indeed, thrombin binds to immobilized glycocalicin, the soluble fragment obtained through proteolysis of GPIb,15 glycocalicin behaves as a competitive inhibitor of thrombin binding to plate lets, 16,17 and α-thrombin can be chemically cross-linked to GPIb in intact platelets.18-21 However, the role of GPIb-thrombin interaction in the activation process is questionable, since platelets that are devoid of GPIb, such as platelets from patients with the Bernard-Soulier syndrome22'23 or platelets that have been depleted in GPIb by proteolytic treatment with chymotrypsin,24,25 elastase,26,27 or Serratia marcescens protease,28'29 ex hibit a lower sensitivity to thrombin, but do respond to higher doses of thrombin, although after a prolonged lag phase. Altogether, these observations argue against the concept that GPIb may have a crucial role as a true platelet receptor, but rather indicate that GPIb serves to accelerate reactions triggered through binding to the newly discovered thrombin binding protein.13 When compared with the other serine proteases, thrombin is unique in that its specificity may be attributed not only to regions surrounding the catalytic site, but also to exosites and other distinct structural features.30 In this report, we demonstrate that thrombin binds to platelet GPIb via an exosite that is masked or disrupted by α-thrombin conversion to β-thrombin, and blocked by the C-terminal hirudin peptide 54-65, or by chemical modification of lysyl residue or residues located on the 18-73 sequence (the sequence numbering is that of the human thrombin B chain) of the human thrombin B chain.
Copyright © 1992 by Thieme Medical Publishers, Inc., 381 Park Avenue South, New York, NY 10016. All rights reserved.
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MARTINE JANDROT-PERRUS, M.D., MARIE-GENEVIÈVE HUISSE, M.D., CATHERINE TERNISIEN, M.D., ANNIE BEZEAUD, M.D., Ph.D., and MARIE-CLAUDE GUILLIN, M.D., Ph.D.
SEMINARS IN THROMBOSIS AND HEMOSTASIS—VOLUME 18, NO. 2, 1992
FIG. 1. Cross-linking of iodinated thrombin to platelets. Washed platelets were incubated for 3 minutes at 37°C with 125I-labeled thrombin. Platelets were then incubated with 0.1 mM bis-sulfosuccinimidylsuberate, for 5 minutes at room temperature. After washing, platelets were solubilized in 2% sodium dodecyl sulfate (SDS), and proteins were analyzed by SDS-polyarcylamide gel electrophoresis (PAGE) on 7 to 12% acrylamide slab gels. 125I-labeled proteins or complexes were located by autoradiography. Different forms of iodinated thrombin were used: a: α-thrombin (5 nM); b: γ-thrombin (80 nM); c: β-thrombin (25 nM); d: α-thrombin (5 nM) in presence of 2 U/ml hirudin; e: α-thrombin (5 nM) in presence of 20 µM hirudin 54-65; f: pyridoxylated thrombin (100 nM); g: heparin protected pyridoxylated thrombin (10 nM).
MATERIAL AND METHODS
RESULTS AND DISCUSSION
Human α-, β- and γ-thrombins were isolated as previously described.31,32 Chemical modification of lysyl residues in α-thrombin by pyridoxal-5'-phosphate was performed essentially as described by White et al, 33 resulting in the incorporation of 2 mol of pyridoxal phosphate/1 mol of α-thrombin. Phosphopyridoxylation was also performed in the presence of heparin, resulting in the incorporation of 1 mol of pyridoxal phosphate/1 mol of α-thrombin.34 Thrombin binding to GPIb was studied by chemicalcross-linking of iodinated α-thrombin and thrombin de rivatives to human washed platelets with bis-sulfosuccin imidylsuberate.19 After platelet solubilization by sodium dodecyl sulfate, proteins were separated on 7 to 12% acrylamide slab gels35 and thrombin-containing com plexes were identified by direct autoradiography of the dried gels. Hirudin was from Diagnostica Stago and the C-terminal hirudin peptide 54-65 was from Bachem.
α-Thrombin cross-linked to GPIb forms a well-de fined 235 kd complex (Fig. 1a). Thrombin active site is not required, for tosyl lysine chloromethyl ketone-inactivated thrombin forms complexes with GPIb as well as active α-thrombin (not shown). The amount of GPIbbound 125I-α-thrombin, estimated by counting the radio activity associated with the 235 kd band, increased lin early with the amount of thrombin bound to platelets (Fig. 2). The minimal dose of 125I-α-thrombin required to observe a complex with GPIb was 0.25 to 0.5 nM, which correlates well with the dose of thrombin required for platelet activation. In these conditions, about 15% of the labeled thrombin associated with platelets was found complexed to GPIb. Chemical cross-linking of 125I-γ-thrombin to plate lets has been performed using a large range of concentra tions (25 to 180 nM) known to induce platelet aggrega tion.7 125I-γ-thrombin was identified as labeled bands of M r 9000, 10,000 and 12,000, corresponding to its three
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FIG. 2. Relation between thrombin binding to platelets and to glycoprotein lb (GPIb). Various amounts of α-thrombin were incubated with 109 platelets/ml before cross-linking. Platelets were isolated by centrifugation. The total amount of thrombin bound to platelets was calculated from the radioactivity associated with the pellet. After solubilization of the pellet in sodium dodecyl sulfate (SDS) and SDS-polyacrylamide gel electrophoresis (PAGE) separation of the platelet proteins, 125I-labeled α-thrombin cross-linked to GPIb was quantitated by counting the radioactivity associated with the band corresponding to the 235 kd thrombin-GPIb complex. Results presented are from one significant experiment of three performed on different donors.
noncovalently associated subunits (Fig. 1b). A faint band of Mr 25,000 corresponding to contaminating β-thrombin was observed, but no α-thrombin was detected. Whatever the concentration used, γ-thrombin did not form complexes of less than 400 kd with platelets and, in particular, there were no labeled bands in the positions expected for complexes formed between GPIb and one or more of the 7-thrombin subunits. Human γ-thrombin isolated as previously de scribed32 consisted of less than 1% α-thrombin, 90% β-thrombin, and 9% 7-thrombin. However, due to the instability of β-thrombin during the labeling procedure, 125 I-labeled β-thrombin preparations were shown by densitometric scanning to be converted to less than 1% α-thrombin, 50% β-thrombin, and 49% 7-thrombin. Cross-linking of this preparation to the platelets resulted in the formation of species of 400, 235, 215, and 64 kd (Fig. 1c). Since γ-thrombin does not bind to GPIb, the 215 kd species must result from the binding of β-throm bin to GPIb. The equal repartition between the 235 and 215 kd complexes contrasted with the much greater con centration of β-compared to α-thrombin in the thrombin preparation used, indicating that the affinity of β-throm bin for GPIb is greatly reduced compared with α-throm bin.
Human β-thrombin, obtained upon controlled trypsin-catalyzed hydrolysis, results from a single cleav age between Arg 73 and Asn 74 on the B chain, whereas conversion of β- to γ-thrombin is the consequence of additional cleavages at Lys 154-Gly 155 and preceding positions.36 Our data indicate that altogether the β/γ cleavages either disrupt or mask the thrombin binding sites for GPIb. The single β cleavage is responsible for a dramatic reduction in thrombin affinity for GPIb. How ever, β- to 7-thrombin conversion further increases the defect, resulting in a complete loss of affinity for GPIb. This indicates that both the β- and γ-cleavage domains may play a role in GPIb recognition. The crystallographic structure determination of a recombinant hirudin-α-thrombin complex has allowed the identification of most of the thrombin residues in volved in thrombin-hirudin interactions.37,38 Thus, hiru din and hirudin-derived peptides are now very useful tools for the identification of functional domains in thrombin. Therefore, we have tested the ability of throm bin complexed to hirudin or its C-terminal tail to bind to GPIb. Both hirudin and its carboxy-terminal derived pep tide 54-65 inhibit the binding of thrombin to GPIb (Fig. lc,d). The carboxy-terminal tail of hirudin has been shown to bind to the thrombin B chain, in close proximity
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SEMINARS IN THROMBOSIS AND HEMOSTASIS—VOLUME 18, NO. 2, 1992 TABLE 1. Sequence Homologies Between Glycoprotein lb and the C-Terminal Segment of Hirudin
Hirudin
269 D
E
G
D
T
D
L
Y
D
Y
Y
P
E
E
G
D
—
F
E
E
I
P
E
65 E
to the β-cleavage site, in a long groove in which numer ous electrostatic and apolar interactions are possible.37'38 Our results indicate that the thrombin structures involved in the interaction with the carboxy-terminal hirudin pep tide are also involved in GPIb recognition. Interestingly, inspection of the amino acid sequence of the hydrophilic region 215-299 of the GPIb a chain, which has been proposed to contain the binding site for thrombin, re vealed an intriguing structural similarity between the se quence 269-287 of GPIb a and the carboxy-terminal hir udin peptide 54-65, with an unusually high content of negatively charged amino acids (Table I). We have used the selective chemical modification of lysyl residues in thrombin as another approach to identify the key structures involved in thrombin-GPIb interac tions. As a preliminary, we have confirmed that, as pre viously reported by others, 33,39 modification of lysyl res idues by pyridoxal-5'-phosphate affects two different sites on thrombin and induces the loss of clotting and platelet stimulating activities, with little effects toward small substrates. When the modification was performed in the presence of heparin, one site of thrombin was protected and the enzymic activities were partly pre served. Thus, both sites of modifications appear to be involved in fibrinogen and platelet recognition. We show in Figure 1 that phosphopyridoxylation of thrombin blocks its specific binding to platelet membrane GPIb (Fig. 1f), whereas heparin-protected modified thrombin retains most of its ability to bind to GPIb (Fig. 1g). This observation indicates that lysyl residues located in the heparin-protected domain are critical for thrombin bind ing to platelet GPIb, either through ionic interactions, or by maintaining an appropriate conformation. Since our previous observations suggested that the thrombin do mains involved in binding to GPIb and to hirudin C-terminal segment might share common structures, we have tested the ability of pyridoxylated thrombin to bind hiru din or the carboxy-terminal hirudin peptide 54-65. Mod ification of lysyl residues in the absence of heparin was shown to induce a dramatic decrease in the ability of thrombin to interact with hirudin.34 In contrast, heparin-
D
T
E
Y SO3-
L
Q
G
287 D
protected modified thrombin retained its ability to bind the carboxy-terminal hirudin peptide 54-65. Our results, thus, indicate that the lysyl residues protected from phos phopyridoxylation by heparin are critical for thrombin binding to GPIb and are located within the thrombin binding site for the carboxy-terminal domain of hirudin. The lysyl residues in positions 21, 52, 65, 77, 106, and 107 have been implicated in ionic interactions with the carboxy-terminal tail of hirudin.40 When we degraded 3 H-labeled modified thrombin by either trypsin or cyano gen bromide, we were able to show that, in our experi mental conditions the lysyl residues protected by heparin from the modification were located on the thrombin B chain sequence extending from Leu 18 to Arg 73. 3 4 In conclusion, our results indicate that residues lo cated within the 18-73 segment of the human thrombin B chain contribute to the recognition of platelet GPIb. Inhi bition of thrombin-GPIb interaction by the hirudin C-terminal peptide 54-65 further suggests that these residues lie in the long groove extending from Arg 73 and designated as the "anion binding exosite." 37,38 The recognition of fibrin(ogen) also requires contacts with the anion binding exosite. 30,41 These observations are consistent with the proposal that GPIb and fibrin(ogen) bind to a common site on the thrombin B chain. How ever, subtle differences in GPIb and fibrin(ogen) recog nition have been observed. In particular, thrombin bind ing to fibrin(ogen) is almost entirely abolished on conversion of α-to β-thrombin, whereas both (3- and γ-cleavages are required before a complete loss of throm bin affinity for GPIb is observed. Thus, we propose that GPIb and fibrin(ogen) bind to overlapping but nonidentical sites.
Acknowledgments. The authors gratefully acknowledge Laurence Venisse for technical assistance and Micheline Besnard and Patricia Castex for secretarial help. This work was supported by grants from the Institut National de la Santé et de la Recherche Médicale (CRE 89 50 08), and from the Faculté Xavier Bichat, Université Paris VII.
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GPIb
REFERENCES 1. Detwiler TC: Hypothetical models for the thrombin-platelet inter action. Ann NY Acad Sci 370:67-71, 1981. 2. Tollefsen DM, JR Feagler, PW Majerus: The binding of thrombin to the surface of human platelets. J Biol Chem 249:2646-2651, 1976. 3. Workmann EF, GC White, RY Lundblad: Structure-function rela tionship in the interaction of α-thrombin with blood platelets. J Biol Chem 252:7118-7123, 1977. 4. Mosher DF, A Yaheri, JJ Chaote, GG Gahmberg: Action of throm bin on surface glycoproteins of human platelets. Blood 53:437445, 1979. 5. Phillips DR, PP Agin: Platelet plasma membrane glycoproteins. Identification of a proteolytic substrate for thrombin. Biochim Biophys Res Commun 75:437-445, 1977. 6. Jandrot-Perrus M, MC Guillin, AT Nurden: Human gammathrombin: Lack of correlation between a platelet functional re sponse and glycoprotein V hydrolysis. Thromb Haemost 58:915920,1987. 7. McGowan EB, AM Ding, TC Detwiler: Correlation of thrombininduced glycoprotein V hydrolysis and platelet activation. J Biol Chem 258:11243-11248, 1983. 8. White GC II, CL Knupp: Glycoprotein V hydrolysis by thrombins. Lack of correlation with secretion. Thromb Res 38:643-648, 1985. 9. Bienz D, W Schnippering, KJ Clemetson: Glycoprotein V is not the thrombin activation receptor on human blood platelets. Blood 68:720-725, 1986. 10. Pipili-Synetos E, ML Gershengorn, EA Jaffe: Expression of func tional thrombin receptors in Xenopus oocytes injected with human endothelial cell mRNA. Biochem Biophys Res Commun 111:913917, 1991. 11. Van-Obberghen-Schilling E, JL Chambard, P Lory, J Nargeot, J Pouyssegur: Functional expression of Ca + mobilizing alphathrombin receptors in mRNA-injected Xenopus oocytes. FEBS Lett 262:330-334, 1990. 12. Vu TKH, DT Hund, VI Wheaton, SH Coughlin: Molecular clon ing of a functional thrombin receptor reveals a novel proteolytic mechanism of receptor activation. Cell 64:1057-1068, 1991. 13. Phillips DR, PP Agin: Platelet plasma membrane glycoproteins. J Biol Chem 252:2121-2126, 1977. 14. Ruggeri ZM, TS Zimmerman: Von Willebrand factor and von Willebrand disease. Blood 70:895-904, 1987. 15. Okumura T, M Hasitz, GA Jamieson: Platelet glycocalicin. Inter action with thrombin and role as thrombin receptor of the platelet surface. J Biol Chem 253:3435-3443, 1978. 16. Ganguly P, ML Gould: Thrombin receptors of platelets: Thrombin binding and antithrombin properties of glycoprotein I. Br J Haema tol 42:137-145, 1979. 17. Harmon JT, GA Jamieson: The glycocalicin portion of platelet glycoprotein Ib expresses both high and moderate affinity receptor sites for thrombin. J Biol Chem 261:13224-13229, 1986. 18. Jandrot-Perrus M, D Didry, MC Guillin, AT Nurden: Cross-link ing of alpha- and gamma-thrombin to distinct binding sites on human platelets. Eur J Biochem 174:359-367, 1988. 19. Jung SM, M Moroi: Cross-linking of platelet glycoprotein Ib by N-succinimidyl (4-azidophenyldithio) propionate and 3,3dithiobis(sulfosuccinimidyl) propionate. Biochim Biophys Acta 761:152-162, 1983. 20. Larsen NE, ER Simons: Preparation and application of a photoreactive thrombin analogue: Binding to human platelets. Biochemis try 20:4141-4147, 1981.
265 21. Takamatsu J, MK Home, MR Gralnick: Identification of the thrombin receptor on human platelets by chemical cross-linking. J Clin Invest 77:362-368, 1986. 22. Jamieson GA, T Okumura: Reduced thrombin binding and aggre gation in Bernard-Soulier platelets. J Clin Invest 61:861-864, 1978. 23. Jandrot-Perrus M, F Rendu, JP Caen, S Levy-Toledano, MC Guil lin: The common pathway for a and 7-thrombin induced platelet activation is independent of GPIb: A study of Bernard-Soulier platelets. Br J Haematol 74:385-392, 1990. 24. McGowan EB, TC Detwiler: Modified platelet responses to throm bin. Evidence for two types of receptors or coupling mechanisms. J Biol Chem 261:739-746, 1986. 25. Tarn SW, JW Fenton, TC Detwiler: Platelet thrombin recep tors. Binding of thrombin is coupled to signal generation by a chymotrypsin sensitive mechanism. J Biol Chem 255:6626-6632, 1980. 26. Brower MS, RI Levin, KE Garry: Human neutrophil elastase mod ulates platelet function by limited proteolysis of membrane glyco proteins. J Clin Invest 75:657-666, 1985. 27. Wicki AN, KJ Clemetson: Structure and function of platelet mem brane glycoproteins Ib and V. Effects of leukocyte elastase and other proteases on platelet response to von Willebrand factor and thrombin. Eur J Biochem 153:1-11, 1985. 28. Cooper HA, WP Bennet, GC White II, RH Wagner: Hydrolysis of platelet membrane glycoprotein with a Serratia marcescens metalloprotease. Effect on response to thrombin and von Willebrand factor. Proc Natl Acad Sci USA 79:1433-1438, 1982. 29. Yamamoto N, NJ Greco, MR Barnard, K Tanoue, H Yamazaki, GA Jamieson, AD Michelson: Glycoprotein Ib (GPIb) dependent and GPIb independent pathways of thrombin-induced platelet acti vation. Blood 77:1740-1748, 1991. 30. Fenton JW II, A Ofusu, DG Moon, JM Maraganore: Thrombin structure and function: Why thrombin is the primary target for antithrombotics. Blood Coag Fibrinolysis 2:69-75, 1991. 31. Bezeaud A, MH Denninger, MC Guillin: Interaction of human alpha-thrombin and gamma-thrombin with antithrombin III, pro tein C and thrombomodulin. Eur J Biochem 153:491-496, 1985. 32. Bezeaud A, MC Guillin: Enzymic and non-enzymic properties of human β-thrombin. J Biol Chem 263:3576-3581, 1988. 33. White GC, RL Lundblad, MJ Griffith: Structure-function relations in platelet-thrombin reactions. Inhibition of platelet-thrombin in teractions by lysine modification. J Biol Chem 256:1763-1766, 1981. 34. Ternisien C, M Jandrot-Perrus, MG Huisse, MC Guillin: Effect of phosphopyridoxylation on thrombin interaction with platelet gly coprotein Ib. Blood Coag Fibrinolysis 2:521-528, 1991. 35. Nurden AT, D Dupuis, TJ Kunicki, JP Caen: Analysis of the glycoprotein and protein composition of Bernard-Soulier platelets by single and two-dimensional gel electrophoresis. J Clin Invest 67:1431-1440, 1981. 36. Hofsteenge J, PJ Braun, SR Stone: Enzymatic properties of prote olytic derivatives of human α-thrombin. Biochemistry 27:21442151, 1988. 37. Grutter MG, JP Priestle, J Rahuel, H Grossenbacher, W Bode, J Hofsteenge, SR Stone: Crystal structure of the thrombin-hirudin complex: A novel mode of serine protease inhibition. EMBO J 9:2361-2365, 1990. 38. Rydel JR, KG Ravichandran, A Tulinsky, W Bode, R Huber, C Roitsch, JW Fenton: The structure of a complex of recombinant hirudin and human α-thrombin. Science 249:277-280, 1990. 39. Church FL, CW Pratt, CM Noyes, T Kalayanamit, GB Sherrill,
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RB Tobin, JB Meade: Structural and functional properties of hu man α-thrombin, pyridoxylated α-thrombin and γT-thrombin. J Biol Chem 264:18419-18425, 1989. 40. Chang JY: The hirudin binding site of human α-thrombin. Identifi cation of lysyl residues which participate in the combining site of hirudin-thrombin complex. J Biol Chem 264:7141-7146, 1989.
41. Fenton JW, TA Olson, MP Zabinski, GD Wilner: Anion-binding exosite of human α-thrombin and fibrin(ogen) recognition. Bio chemistry 27:7106-7112, 1988.
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Thrombin Binding to Platelet Membrane Glycoprotein Ib
The serine protease thrombin is the most potent physiologic platelet agonist, and thrombin-induced platelet activation is critical for hemostasis and thrombosis.1 However, despite considerable effort by a number of laboratories, the mechanisms by which thrombin activates platelets have not yet been fully elucidated. A variety of evidence has indicated that platelet activation by thrombin requires both thrombin binding to the cell membrane and expression of the enzyme proteolytic activity. 1-3 The proteolytic step of platelet stimulation was first ascribed to the thrombin-induced hydrolysis of platelet membrane glycoprotein V (GFV).4,5 However, the lack of relationship between the rates of platelet activation and GPV hydrolysis has raised doubt concerning the relevance of GPV hydrolysis as a crucial step in the platelet activation process.6-8 Furthermore, anti-GPV antibodies that inhibited GPV hydrolysis failed to block thrombin-induced platelet activation.9 Therefore, the role of thrombin-catalyzed GPV hydrolysis in platelet activation remains to be elucidated. More recently, previously unrecognized thrombin binding proteins have been identified through direct expression cloning in Xenopus oocytes.10,12 mRNA encoding these proteins were detected in human platelets and endothelial cells and in hamster fibroblasts. They are novel members of the superfamily of seven transmembrane domain proteins. In platelets, the newly discovered thrombin binding protein behaves both as a substrate and a receptor for thrombin at the cell surface, and contains
From the Laboratoire de Recherche sur l'Hémostase et la Thrombose, Faculté de Médecine Xavier Bichat, Paris, France. Reprint requests: Dr. Jandrot-Perrus, Laboratoire de Recherche sur l'Hemostase et la Thrombose, Faculté de Médecine Xavier Bichat, 16, rue Henri Huchard, 75018 Paris, France.
its own ligand, which is unmasked on thrombin-catalyzed hydrolysis.12 Glycoprotein Ib (GPIb) is the major sialoglycoprotein at the platelet surface.13 Besides its role as a binding site for von Willebrand factor,14 GPIb functions as a binding site for α-thrombin. Indeed, thrombin binds to immobilized glycocalicin, the soluble fragment obtained through proteolysis of GPIb,15 glycocalicin behaves as a competitive inhibitor of thrombin binding to plate lets, 16,17 and α-thrombin can be chemically cross-linked to GPIb in intact platelets.18-21 However, the role of GPIb-thrombin interaction in the activation process is questionable, since platelets that are devoid of GPIb, such as platelets from patients with the Bernard-Soulier syndrome22'23 or platelets that have been depleted in GPIb by proteolytic treatment with chymotrypsin,24,25 elastase,26,27 or Serratia marcescens protease,28'29 ex hibit a lower sensitivity to thrombin, but do respond to higher doses of thrombin, although after a prolonged lag phase. Altogether, these observations argue against the concept that GPIb may have a crucial role as a true platelet receptor, but rather indicate that GPIb serves to accelerate reactions triggered through binding to the newly discovered thrombin binding protein.13 When compared with the other serine proteases, thrombin is unique in that its specificity may be attributed not only to regions surrounding the catalytic site, but also to exosites and other distinct structural features.30 In this report, we demonstrate that thrombin binds to platelet GPIb via an exosite that is masked or disrupted by α-thrombin conversion to β-thrombin, and blocked by the C-terminal hirudin peptide 54-65, or by chemical modification of lysyl residue or residues located on the 18-73 sequence (the sequence numbering is that of the human thrombin B chain) of the human thrombin B chain.
Copyright © 1992 by Thieme Medical Publishers, Inc., 381 Park Avenue South, New York, NY 10016. All rights reserved.
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MARTINE JANDROT-PERRUS, M.D., MARIE-GENEVIÈVE HUISSE, M.D., CATHERINE TERNISIEN, M.D., ANNIE BEZEAUD, M.D., Ph.D., and MARIE-CLAUDE GUILLIN, M.D., Ph.D.
SEMINARS IN THROMBOSIS AND HEMOSTASIS—VOLUME 18, NO. 2, 1992
FIG. 1. Cross-linking of iodinated thrombin to platelets. Washed platelets were incubated for 3 minutes at 37°C with 125I-labeled thrombin. Platelets were then incubated with 0.1 mM bis-sulfosuccinimidylsuberate, for 5 minutes at room temperature. After washing, platelets were solubilized in 2% sodium dodecyl sulfate (SDS), and proteins were analyzed by SDS-polyarcylamide gel electrophoresis (PAGE) on 7 to 12% acrylamide slab gels. 125I-labeled proteins or complexes were located by autoradiography. Different forms of iodinated thrombin were used: a: α-thrombin (5 nM); b: γ-thrombin (80 nM); c: β-thrombin (25 nM); d: α-thrombin (5 nM) in presence of 2 U/ml hirudin; e: α-thrombin (5 nM) in presence of 20 µM hirudin 54-65; f: pyridoxylated thrombin (100 nM); g: heparin protected pyridoxylated thrombin (10 nM).
MATERIAL AND METHODS
RESULTS AND DISCUSSION
Human α-, β- and γ-thrombins were isolated as previously described.31,32 Chemical modification of lysyl residues in α-thrombin by pyridoxal-5'-phosphate was performed essentially as described by White et al, 33 resulting in the incorporation of 2 mol of pyridoxal phosphate/1 mol of α-thrombin. Phosphopyridoxylation was also performed in the presence of heparin, resulting in the incorporation of 1 mol of pyridoxal phosphate/1 mol of α-thrombin.34 Thrombin binding to GPIb was studied by chemicalcross-linking of iodinated α-thrombin and thrombin de rivatives to human washed platelets with bis-sulfosuccin imidylsuberate.19 After platelet solubilization by sodium dodecyl sulfate, proteins were separated on 7 to 12% acrylamide slab gels35 and thrombin-containing com plexes were identified by direct autoradiography of the dried gels. Hirudin was from Diagnostica Stago and the C-terminal hirudin peptide 54-65 was from Bachem.
α-Thrombin cross-linked to GPIb forms a well-de fined 235 kd complex (Fig. 1a). Thrombin active site is not required, for tosyl lysine chloromethyl ketone-inactivated thrombin forms complexes with GPIb as well as active α-thrombin (not shown). The amount of GPIbbound 125I-α-thrombin, estimated by counting the radio activity associated with the 235 kd band, increased lin early with the amount of thrombin bound to platelets (Fig. 2). The minimal dose of 125I-α-thrombin required to observe a complex with GPIb was 0.25 to 0.5 nM, which correlates well with the dose of thrombin required for platelet activation. In these conditions, about 15% of the labeled thrombin associated with platelets was found complexed to GPIb. Chemical cross-linking of 125I-γ-thrombin to plate lets has been performed using a large range of concentra tions (25 to 180 nM) known to induce platelet aggrega tion.7 125I-γ-thrombin was identified as labeled bands of M r 9000, 10,000 and 12,000, corresponding to its three
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FIG. 2. Relation between thrombin binding to platelets and to glycoprotein lb (GPIb). Various amounts of α-thrombin were incubated with 109 platelets/ml before cross-linking. Platelets were isolated by centrifugation. The total amount of thrombin bound to platelets was calculated from the radioactivity associated with the pellet. After solubilization of the pellet in sodium dodecyl sulfate (SDS) and SDS-polyacrylamide gel electrophoresis (PAGE) separation of the platelet proteins, 125I-labeled α-thrombin cross-linked to GPIb was quantitated by counting the radioactivity associated with the band corresponding to the 235 kd thrombin-GPIb complex. Results presented are from one significant experiment of three performed on different donors.
noncovalently associated subunits (Fig. 1b). A faint band of Mr 25,000 corresponding to contaminating β-thrombin was observed, but no α-thrombin was detected. Whatever the concentration used, γ-thrombin did not form complexes of less than 400 kd with platelets and, in particular, there were no labeled bands in the positions expected for complexes formed between GPIb and one or more of the 7-thrombin subunits. Human γ-thrombin isolated as previously de scribed32 consisted of less than 1% α-thrombin, 90% β-thrombin, and 9% 7-thrombin. However, due to the instability of β-thrombin during the labeling procedure, 125 I-labeled β-thrombin preparations were shown by densitometric scanning to be converted to less than 1% α-thrombin, 50% β-thrombin, and 49% 7-thrombin. Cross-linking of this preparation to the platelets resulted in the formation of species of 400, 235, 215, and 64 kd (Fig. 1c). Since γ-thrombin does not bind to GPIb, the 215 kd species must result from the binding of β-throm bin to GPIb. The equal repartition between the 235 and 215 kd complexes contrasted with the much greater con centration of β-compared to α-thrombin in the thrombin preparation used, indicating that the affinity of β-throm bin for GPIb is greatly reduced compared with α-throm bin.
Human β-thrombin, obtained upon controlled trypsin-catalyzed hydrolysis, results from a single cleav age between Arg 73 and Asn 74 on the B chain, whereas conversion of β- to γ-thrombin is the consequence of additional cleavages at Lys 154-Gly 155 and preceding positions.36 Our data indicate that altogether the β/γ cleavages either disrupt or mask the thrombin binding sites for GPIb. The single β cleavage is responsible for a dramatic reduction in thrombin affinity for GPIb. How ever, β- to 7-thrombin conversion further increases the defect, resulting in a complete loss of affinity for GPIb. This indicates that both the β- and γ-cleavage domains may play a role in GPIb recognition. The crystallographic structure determination of a recombinant hirudin-α-thrombin complex has allowed the identification of most of the thrombin residues in volved in thrombin-hirudin interactions.37,38 Thus, hiru din and hirudin-derived peptides are now very useful tools for the identification of functional domains in thrombin. Therefore, we have tested the ability of throm bin complexed to hirudin or its C-terminal tail to bind to GPIb. Both hirudin and its carboxy-terminal derived pep tide 54-65 inhibit the binding of thrombin to GPIb (Fig. lc,d). The carboxy-terminal tail of hirudin has been shown to bind to the thrombin B chain, in close proximity
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SEMINARS IN THROMBOSIS AND HEMOSTASIS—VOLUME 18, NO. 2, 1992 TABLE 1. Sequence Homologies Between Glycoprotein lb and the C-Terminal Segment of Hirudin
Hirudin
269 D
E
G
D
T
D
L
Y
D
Y
Y
P
E
E
G
D
—
F
E
E
I
P
E
65 E
to the β-cleavage site, in a long groove in which numer ous electrostatic and apolar interactions are possible.37'38 Our results indicate that the thrombin structures involved in the interaction with the carboxy-terminal hirudin pep tide are also involved in GPIb recognition. Interestingly, inspection of the amino acid sequence of the hydrophilic region 215-299 of the GPIb a chain, which has been proposed to contain the binding site for thrombin, re vealed an intriguing structural similarity between the se quence 269-287 of GPIb a and the carboxy-terminal hir udin peptide 54-65, with an unusually high content of negatively charged amino acids (Table I). We have used the selective chemical modification of lysyl residues in thrombin as another approach to identify the key structures involved in thrombin-GPIb interac tions. As a preliminary, we have confirmed that, as pre viously reported by others, 33,39 modification of lysyl res idues by pyridoxal-5'-phosphate affects two different sites on thrombin and induces the loss of clotting and platelet stimulating activities, with little effects toward small substrates. When the modification was performed in the presence of heparin, one site of thrombin was protected and the enzymic activities were partly pre served. Thus, both sites of modifications appear to be involved in fibrinogen and platelet recognition. We show in Figure 1 that phosphopyridoxylation of thrombin blocks its specific binding to platelet membrane GPIb (Fig. 1f), whereas heparin-protected modified thrombin retains most of its ability to bind to GPIb (Fig. 1g). This observation indicates that lysyl residues located in the heparin-protected domain are critical for thrombin bind ing to platelet GPIb, either through ionic interactions, or by maintaining an appropriate conformation. Since our previous observations suggested that the thrombin do mains involved in binding to GPIb and to hirudin C-terminal segment might share common structures, we have tested the ability of pyridoxylated thrombin to bind hiru din or the carboxy-terminal hirudin peptide 54-65. Mod ification of lysyl residues in the absence of heparin was shown to induce a dramatic decrease in the ability of thrombin to interact with hirudin.34 In contrast, heparin-
D
T
E
Y SO3-
L
Q
G
287 D
protected modified thrombin retained its ability to bind the carboxy-terminal hirudin peptide 54-65. Our results, thus, indicate that the lysyl residues protected from phos phopyridoxylation by heparin are critical for thrombin binding to GPIb and are located within the thrombin binding site for the carboxy-terminal domain of hirudin. The lysyl residues in positions 21, 52, 65, 77, 106, and 107 have been implicated in ionic interactions with the carboxy-terminal tail of hirudin.40 When we degraded 3 H-labeled modified thrombin by either trypsin or cyano gen bromide, we were able to show that, in our experi mental conditions the lysyl residues protected by heparin from the modification were located on the thrombin B chain sequence extending from Leu 18 to Arg 73. 3 4 In conclusion, our results indicate that residues lo cated within the 18-73 segment of the human thrombin B chain contribute to the recognition of platelet GPIb. Inhi bition of thrombin-GPIb interaction by the hirudin C-terminal peptide 54-65 further suggests that these residues lie in the long groove extending from Arg 73 and designated as the "anion binding exosite." 37,38 The recognition of fibrin(ogen) also requires contacts with the anion binding exosite. 30,41 These observations are consistent with the proposal that GPIb and fibrin(ogen) bind to a common site on the thrombin B chain. How ever, subtle differences in GPIb and fibrin(ogen) recog nition have been observed. In particular, thrombin bind ing to fibrin(ogen) is almost entirely abolished on conversion of α-to β-thrombin, whereas both (3- and γ-cleavages are required before a complete loss of throm bin affinity for GPIb is observed. Thus, we propose that GPIb and fibrin(ogen) bind to overlapping but nonidentical sites.
Acknowledgments. The authors gratefully acknowledge Laurence Venisse for technical assistance and Micheline Besnard and Patricia Castex for secretarial help. This work was supported by grants from the Institut National de la Santé et de la Recherche Médicale (CRE 89 50 08), and from the Faculté Xavier Bichat, Université Paris VII.
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GPIb
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