Die Raumstruktur des Thrombins: ein Wegweiser zu seinen vielfältigen Funktionen

Zusammenfassung

Die durch Röntgenstrukturanalyse des α-Thrombins, seiner Komplexe mit Inhibitoren und Substraten sowie mehrerer Prothrombin-Fragmente gewonnene Strukturinformation gestattet es jetzt, die vielen unterschiedlichen Funktionen des Thrombins durch seine strukturellen Eigenschaften zu erklären. Das α-Thrombin hat eine ungewöhnlich tiefe und enge Activ-site-Spalte, die durch besonders große, typische Peptidschleifen ein canyonartiges Aussehen erhält. Diese Spalte trägt ganz wesentlich zu der engen Substratspezifität des Thrombins bei. Die Thrombinoberfläche läßt sich in eine ganze Reihe strukturell und funktionell unterschiedlicher Regionen einteilen. Wie am Beispiel der Fibrinogenbindung besonders gut gezeigt werden kann, ermöglicht die Nachbarschaft der aktiven Reste zu einer hydrophoben Tasche (der sog. apolaren Bindungsregion) und zu einem alkalischen Bereich (der Fibrinogen-Erkennungsregion) die Feinabstimmung der proteolytischen Aktivität. Das Thrombin benützt diese beiden Kontaktbereiche u. a. auch bei der spezifischen Erkennung und Bindung des Hirudins, eines aus dem medizinischen Egel gewonnenen Thrombin-Inhibitors, sowie des Thrombinrezeptors. Die Verstärkung der Thrombinwechselwirkung mit dem Antithrombin III durch das Heparin läßt sich durch die Verbrückung zweier Heparinbindungsstellen am Thrombin und am Antithrombin III erklären. Die nichtproteolytischen Effekte des Thrombins mit zellulären Strukturen sind dagegen vermutlich im wesentlichen auf eine der beiden die Active-site-Spalte umgebenden Peptidschleifen zurückzuführen.

Schlüsselwörter

Thrombin - Prothrombin - Fibrinogen - Substratspezifität - Inhibition - Hirudin - Antithrombin III - Heparin - Kristallstruktur

• LITERATUR

• 1 Vu TKH, Hung DT, Wheaton VL, Coughlin SR. Molecular cloning of a functional thrombin receptor reveals a novel proteolytic mechanism of receptor activation. Cell 1991; 64: 1057-68.
• 2 Rasmussen UB, Vouret-Craviari V, Jallat S. et al. cDNA cloning and expression of a hamster α-thrombin receptor coupled to Ca²⁺ mobilization. FEBS Lett 1991; 288: 123-8.
• 3 Esmon CT. The roles of protein C and thrombomodulin in the regulation of blood coagulation. J Biol Chem 1989; 264: 4743-6.
• 4 Rosenberg RD, Damus PS. The purification and mechanism of action of human antithrombin-heparin cofactor. J Biol Chem 1973; 248: 6490-505.
• 5 Tollefsen DM, Majerus DW, Blank MK. Heparin cofactor II. Purification and properties of a heparin-dependent inhibitor of thrombin in human plasma. J Biol Chem 1982; 257: 2162-9.
• 6 Cunningham DD, Farrell DH. Thrombin interactions with cultured fibroblasts: relationship to mitogenic stimulation. Ann NY Acad USA 1986; 485: 240-8.
• 7 Björk I, Lindahl V. Mechanism of the anticoagulant action of heparin. Mol Cell Biochem 1982; 48: 161-82.
• 8 Walsmann P, Markwardt F. Biochemische und pharmakologische Aspekte des Thrombininhibitors Hirudin. Pharmazie 1981; 36: 653-60.
• 9 Stone SR, Hofsteenge J. Kinetics of the inhibition of thrombin by hirudin. Biochemistry 1986; 25: 4622-8.
• 10 Dodt J, Köhler S, Baici A. Interaction of site specific hirudin variants with α-thrombin. FEBS Lett 1988; 229: 87-90.
• 11 Stubbs MT, Bode W. A player of many parts: the spotlight falls on thrombin’s structure. Thromb Res 1993; 69: 1-58.
• 12 Cunningham DD, Pulliam L, Vaughan PJ. Protease nexin-1 and thrombin: Injury related processes in the brain. Thromb Haemost 1993; 70: 168-71.
• 13 Bode W, Mayr I, Baumann U. et al. The refined 1.0 Å crystal structure of human athrombin: Interaction with D-Phe-Pro-Argchloromethylketone and significance of the Tyr-Pro-Pro-Trp insertion segment. EMBO J 1989; 08: 3467-75.
• 14 Bode W, Turk D, Karshikov A. The refined 1.9 Â X-ray crystal structure of D-Phe-ProArg chloromethylketone inhibited human α-thrombin. Structure analysis, overall structure, electrostatic properites, detailed active site geometry, structure-function relationships. Protein Sci 1992; 01: 426-71.
• 15 Rydel TJ, Ravichandran KG, Tulinsky A. et al. The structure of a complex of recombinant hirudin and human α-thrombin. Science 1990; 249: 277-80.
• 16 Rydel TJ, Tulinsky A, Bode W, Huber R. Refined structure of the hirudin-thrombin complex. J Mol Biol 1991; 221: 583-601.
• 17 Grtitter MG, Priestle JP, Rahuel J. et al. Crystal structure of the thrombin-hirudin complex: A novel model of serine protease inhibitor. EMBO J 1990; 09: 2361-5.
• 18 Vitali J, Martin PD, Malkowski MG. et al. The structure of a complex of bovine athrombin and recombinant hirudin at 2.8 Â resolution. J Biol Chem 1992; 267: 17670-8.
• 19 Skrzypczak-Jankun E, Carperos V, Ravichandran KG. The structure of the hirugen and hirulog 1 complexes of α-thrombin. J Mol Biol 1991; 221: 1379-93.
• 20 Stubbs MT, Oschkinat H, Mayr I. et al. The interaction of thrombin with fibrinogen a structural basis for its specificity. Eur J Biochem 1992; 206: 187-95.
• 21 Martin PD, Robertson W, Turk D. et al. The structure of residues 7-16 of the Aα-chain of human fibrinogen bound to bovine thrombin at 2.3 Â resolution. J Biol Chem 1992; 267: 7911-20.
• 22 Arni RK, Padmanabhan K, Padmanabhan KP. et al. Structures of the non-covalent complexes of human and bovine prothrombin fragment 2 with human PPACK-thrombin. Biochemistry 1993; 32: 4727-37.
• 23 Padmanabhan K, Padmanabhan KP, Ferrara JD. et al. The structure of α-thrombin inhibited by a 15-mer single stranded DNA aptamer. J Biol Chem 1993; 268: 17651-4.
• 24 Bode W, Turk D, Stürzebecher J. Geometry of binding of the benzamidineand argininebased inhibitors NAPAP and MQPA to human α-thrombin. X-ray crystallographic determination of the NAPAP-trypsin complex and modeling of NAPAP-thrombin and MQPA-thrombin. Eur J Biochem 1990; 193: 175-82.
• 25 Brandstetter H, Turk D, Hoeffken W. et al. Refined 2.3 Å X-ray crystal structure of bovine thrombin complexes formed with the benzamidineand arginine-based inhibitors NAPAP, 4-TAPAP and MQPA: A starting point for improving antithrombotics. J Mol Biol 1992; 226: 1085-99.
• 26 Banner DW, Hadvary P. Crystallographic analysis at 3.0 Å resolution of the binding to human thrombin of four active sitedirected inhibitors. J Biol Chem 1991; 266: 20085-93.
• 27 Qiu X, Yin M, Padmanabhan KP. et al. Structures of thrombin complexes with a designed and a natural exosite peptide inhibitor. J Biol Chem 1993; 268: 20318-26.
• 28 Qiu X, Padmanabhan K, Carperos VE. et al. The structure of the hirulog3-thrombin complex and the nature of the S’ subsites of substrates and inhibitors. Biochemistry 1992; 31: 11689-97.
• 29 Seshadri RP, Tulinsky A, Skrypczak-Jankun E, Park CH. Structure of bovine prothrombin fragment 1 refined at 2.25 Å resolution. J Mol Biol 1991; 220: 481-94.
• 30 Soriano-Garcia M, Padmanabhan K, de Vos AM, Tulinsky A. The CA²⁺ ion and membrane binding structure of the Gla domain of Ca-prothrombin fragment 1. Biochemistry 1992; 31: 2554-66.
• 31 Bode W, Huber R. Natural protein proteinase inhibitors and their interaction with proteinases. Eur J Biochem 1992; 204: 433-52.
• 32 Bode W, Huber R. Proteinase-protein inhibitor interactions. Fibrinolysis 1994; 08: 161-71.
• 33 Stubbs MT, Bode W. The clot thickens: clues provided by thrombin structure. TIBS 1995; 20: 23-8.
• 34 Stubbs MT, Bode W. Coagulation factors and their inhibitors. Curr Opin Struct Biol 1994; 04: 823-32.
• 35 Magnusson S. Thrombin and prothrombin. In: Enzymes. Vol. 3 Boyer PD. (ed). Academic Press; New York, London: 1971: 277-321.
• 36 Degen SJF, MacGillivray RTA, Davie EW. Characterization of the complementary deoxyribonucleic acid and gene coding for human prothrombin. Biochemistry 1983; 87: 2087-97.
• 37 Schechter I, Berger A. On the size of the active site in proteases. I. Papain. Biochem Biophys Res Commun 1967; 27: 157.
• 38 Banfield DK, MacGillivray RTA. Partial characterization of vertebrate prothrombin cDNAs: amplification and sequence analysis of the B chain of thrombin from nine different species. Proc Natl Acad Sci USA 1992; 89: 2779-83.
• 39 LeBonniec BF, Guinto ER, MacGillivray RTA. et al. The role of thrombin’s Tyr-ProPro-Trp motif in the interaction with fibrinogen, thrombomodulin, protein C, antithrombin III, and the Kunitz inhibitors. J Biol Chem 1993; 268: 19055-61.
• 40 Le Bonniec BF, Guinto GR, Esmon CT. Interaction of thrombin des-ETW with antithrombin III, the Kunitz inhibitors, thrombomodulin and protein C. Structural link between the autolysis loop and the Tyr-ProPro-Trp insertion of thrombin. J Biol Chem 1992; 267: 19341-8.
• 41 Karshikov AD, Bode W. Electrostatic properties of thrombin: Importance for structural stabilization and ligand binding. Semin Thromb Hemost 1993; 19: 334-43.
• 42 Berliner LJ, Shen YYL. Physical evidence for an apolar binding site near the catalytic center of human α-thrombin. Biochemistry 1977; 16: 4622-6.
• 43 Ni F, Ripoli DR, Martin PD, Edwards BFP. Solution structure of a platelet receptor peptide bound to bovine α-thrombin. Biochemistry 1992; 31: 11551-7.
• 44 Fenton JWII, Zabinski MP, Hsieh K, Wilner GD. Thrombin non-covalent protein binding and fibrin(ogen) recognition. Thromb Haemost 1981; 46: 177.
• 45 Bode W, Schwager P. The refined control structure of bovine β-trypsin at 1.8 Å resolution. J Mol Biol 1975; 198: 693-717.
• 46 Folkers PJM, Clore GM, Driscoll PC. et al. Solution structure of recombinant hirudin and the Lys-47-Glu mutant: A nuclear magnetic resonance and hybrid distance geometry-dynamical simulated annealing study. Biochemistry 1989; 28: 2601-17.
• 47 Haruyama H, Wüthrich K. The conformation of recombinant disulfatohirudin in aqueous solution determined by nuclear magnetic resonance. Biochemistry 1989; 28: 4301-12.
• 48 Karshikov A, Bode W, Tulinsky A, Stone SR. Electrostatic interactions in the association of proteins: an analysis of the thrombinhirudin complex. Protein Science 1992; 01: 727-35.
• 49 Betz A, Hofsteenge J, Stone SR. Ionic interactions in the formation of the thrombinhirudin complex. Biochem J 1991; 275: 801-3.
• 50 Church FC, Pratt CW, Noyes CM. et al. Structural and functional properties of human α-thrombin, phosphopyridoxylated α-thrombin and γ_T-thrombin. Identification of lysyl residues in α-thrombin that are critical for heparin and fibrin(ogen) interactions. J Biol Chem 1989; 264: 18419-25.
• 51 Gan ZR, Li Y, Chen Z. et al. Identification of basic amino acid residues in thrombin essential for heparin-catalyzed inactivation by antithrombin. J Biol Chem. in press.
• 52 Bourin M-C, Lindahl U. Glycosaminoglycans and the regulation of blood coagulation. Biochem J 1993; 289: 313-30.
• 53 Bourin M-C, Öhlin A-K, Lane DA. et al. Relationship between anticoagulant activities and polyanionic properties of rabbit thrombomodulin. J Biol Chem 1988; 263: 8044-52.
• 54 Olson TA, Sonder SA, Wilner GD, Fenton II JW. Heparin binding in proximity to the catalytic site of human α-thrombin. Ann NY Acad Sci 1986; 485: 96-103.
• 55 Hageman TC, Scheraga HA. Mechanism of action of thrombin on fibrinogen; Reaction of the N-terminal CNBR fragment from the Aa chain of human fibrinogen with bovine thrombin. Arch Biochem Biophys 1974; 164: 707-15.
• 56 Hogg DH, Blombäck B. The specificity of the fibrinogen-thrombin interaction. Thromb Res 1974; 05: 685-93.
• 57 Lord ST, Byrd PA, Hede KL. et al. Analysis of fibrinogen Aα-fusion proteins. Mutants which inhibit thrombin equivalently are not equally good substrates. J Biol Chem 1990; 265: 838-43.
• 58 Fischer E. Einfluß der Konfiguration auf die Wirkung der Enzyme. Ber Dtsch Chem Ges 1894; 27: 2992.
• 59 Huber R, Carrell RW. Implications of the three-dimensional structure of α₁antitrypsin for structure and function of serpins. Biochemistry 1989; 28: 8951-66.
• 60 Engh RA, Schulze AJ, Huber R, Bode W. Serpin structures. Behring Inst. Mitt 1993; 93.
• 61 VanDeerlin VMD, Tollefsen DM. The Nterminal acidic domain of heparin cofactor II mediates the inhibition of α-thrombin in the presence of glycosaminoglycans. J Biol Chem 1991; 266: 20223-31.
• 62 Rogers SJ, Pratt CW, Whinna HC, Church FC. Role of thrombin exosites in inhibition by heparin cofactor II. J Biol Chem 1992; 267: 3613-7.
• 63 Pomerantz MW, Owen WG. A catalytic role for heparin. Evidence for a ternary complex of heparin cofactor thrombin and heparin. Biochim Biophys Acta 1978; 535: 66-77.
• 64 Griffith MJ. Kinetics of the heparin-enhanced antithrombin Ill/thrombin reaction. Evidence for a template model for the mechanism of action of heparin. J Biol Chem 1982; 257: 7360-5.
• 65 Nesheim ME. A simple rate law that describes the kinetics of the heparin-catalyzed reaction between antithrombin III and thrombin. J Biol Chem 1983; 258: 14708-17.
• 66 Stubbs MT, Bode W. Crystal structures of thrombin and thrombin complexes as a framework for antithrombotic drug design. Perspect Drug Discov Des 1993; 01: 431-52.
• 67 Krishnaswamy S, Mann KG, Nesheim ME. The prothrombinase-catalyzed activation of prothrombin proceeds through the intermediate mezothrombin in an ordered sequential reaction. J Biol Chem 1986; 261: 8977-84.
• 68 Kalafatis M, Swords NA, Rand MD, Mann KDG. Membrane dependent reactions in blood coagulation: role of the vitamin K-dependent enzyme complexes. Biochem Biophys Acta 1994; 1227: 113-29.
• 69 Krishnaswami S, Jones KC, Mann KG. Prothrombinase complex assembly. Kinetic mechanism of enzyme assembly on phospholipid vesicles. J Biol Chem 1988; 263: 3823-34.
• 70 Tulinsky A. The structure of domains of blood proteins. Thromb Haemost 1991; 66: 16-31.
• 71 Myrmel KH, Lundblad RD, Mann KG. Characteristics of the association between prothrombin fragment 2 and α-thrombin. Biochemistry 1976; 15: 1767-73.
• 72 Walker FJ, Esmon CT. The effect of prothrombin fragment 2 on the inhibition of thrombin by antithrombin III. J Biol Chem 1979; 254: 5618-22.
• 73 Jakubowski HV, Owen WG. Macromolecular specificity determinants on thrombin for fibrinogen and thrombomodulin. J Biol Chem 1989; 264: 11117-21.
• 74 Turk D. PhD. Thesis, Technische Universität München, 1992
• 75 Kraulis P. MOLSCRIPT: A program to produce both detailed and schematic plots of protein structures. J Appl Cryst 1991; 24: 946-50.
• 76 Jones TA, Zou J-Y, Cowan SW, Kjeldgaard M. Improved methods for building protein models in electron density maps and location of errors in these models. Acta Crystallogr 1978; 15: 23-31.