The Structure of Thrombin: A Janus-Headed Proteinase

ABSTRACT

Through a series of successive, cascade-like proteinase activation and amplification steps, any vascular injury triggers a rapid burst of α-thrombin, a trypsin-like serine proteinase. Thrombin, the main executioner of the coagulation cascade, has procoagulant as well as anticoagulant and antifibrinolytic properties. It exhibits quite diverse physiological functions, but also gives rise to several thrombotic disorders, such as thromboembolism, myocardial infarction, and stroke, thus making it an attractive target for antithrombotic agents. Thrombin interacts specifically with several protein substrates, receptors, cofactors, inhibitors, carbohydrates, and modulators. It cleaves fibrinogen, factors XI (FXI) and FXIII, cofactors V and VIII, and the thrombin receptors; uses thrombomodulin to activate protein C and thrombin-activatable-fibrinolysis inhibitor; is inhibited by heparin cofactor II and antithrombin III with the help of acidic carbohydrates; and its activity/specificity is modulated by sodium ions. A large number of crystal structures of α-thrombin in complexes with synthetic polypeptides and protein inhibitors, substrate fragments, cofactors, and carbohydrates have displayed extended recognition sites on the thrombin surface, reflecting the versatility and multifunctional specificity of this remarkable proteinase. These structures essentially show that the thrombin surface can be subdivided into several functional regions, which recognize different chemical moieties. By using different combinations of these surface elements, thrombin can interact with a variety of molecules with high specificity, accounting for its multifunctional properties.

KEYWORDS

Coagulation - thrombin - anion binding exosites - cofactors - crystal structures

REFERENCES

• 1 Davie E W. A brief historical review of the waterfall/cascade of blood coagulation.  J Biol Chem. 2003;  278 50819-50832
• 2 Hougie C. The waterfall-cascade and autoprothrombin hypotheses of blood coagulation: personal reflections from an observer.  J Thromb Haemost. 2004;  2 1225-1233
• 3 Wells C M, Di Cera E. Thrombin is a Na(+)-activated enzyme.  Biochemistry. 1992;  31 11721-11730
• 4 Di Cera E. Thrombin: a paradigm for enzymes allosterically activated by monovalent cations.  C R Biol. 2004;  327 1065-1076
• 5 Coughlin S R. Protease-activated receptors in hemostasis, thrombosis and vascular biology.  J Thromb Haemost. 2005;  3 1800-1814
• 6 Esmon C T. The roles of protein C and thrombomodulin in the regulation of blood coagulation.  J Biol Chem. 1989;  264 4743-4746
• 7 Bajzar L, Morser J, Nesheim M. TAFI, or plasma procarboxypeptidase B, couples the coagulation and fibrinolytic cascades through the thrombin-thrombomodulin complex.  J Biol Chem. 1996;  271 16603-16608
• 8 Bar-Shavit R, Benezra M, Sabbah V, Bode W, Vlodavsky I. Thrombin as a multifunctional protein: induction of cell adhesion and proliferation.  Am J Respir Cell Mol Biol. 1992;  6 123-130
• 9 Bode W, Mayr I, Baumann U et al.. The refined 1.9 A crystal structure of human alpha-thrombin: interaction with D-Phe-Pro-Arg chloromethylketone and significance of the Tyr-Pro-Pro-Trp insertion segment.  EMBO J. 1989;  8 3467-3475
• 10 Bode W, Turk D, Karshikov A. The refined 1.9-A X-ray crystal structure of D-Phe-Pro-Arg chloromethylketone-inhibited human alpha-thrombin: structure analysis, overall structure, electrostatic properties, detailed active-site geometry, and structure-function relationships.  Protein Sci. 1992;  1 426-471
• 11 Bode W, Brandstetter H, Mather T, Stubbs M T. Comparative analysis of haemostatic proteinases: structural aspects of thrombin, factor Xa, factor IXa and protein C.  Thromb Haemost. 1997;  78 501-511
• 12 Stubbs M T, Bode W. A player of many parts: the spotlight falls on thrombin's structure.  Thromb Res. 1993;  69 1-58
• 13 Stubbs M T, Bode W. The clot thickens: clues provided by thrombin structure.  Trends Biochem Sci. 1995;  20 23-28
• 14 Huntington J A. Molecular recognition mechanisms of thrombin.  J Thromb Haemost. 2005;  3 1861-1872
• 15 Schechter I, Berger A. On the size of the active site in proteases. I. Papain.  Biochem Biophys Res Commun. 1967;  27 157-162
• 16 Bode W. The transition of bovine trypsinogen to a trypsin-like state upon strong ligand binding. II. The binding of the pancreatic trypsin inhibitor and of isoleucine-valine and of sequentially related peptides to trypsinogen and to p-guanidinobenzoate-trypsinogen.  J Mol Biol. 1979;  127 357-374
• 17 Vijayalakshmi J, Padmanabhan K P, Mann K G, Tulinsky A. The isomorphous structures of prethrombin2, hirugen-, and PPACK-thrombin: changes accompanying activation and exosite binding to thrombin.  Protein Sci. 1994;  3 2254-2271
• 18 Rawlings N D, Morton F R, Barrett A J. MEROPS: the peptidase database.  Nucleic Acids Res. 2006;  34 D270-D272
• 19 Fredenburgh J C, Stafford A R, Weitz J I. Evidence for allosteric linkage between exosites 1 and 2 of thrombin.  J Biol Chem. 1997;  272 25493-25499
• 20 Guinto E R, Vindigni A, Ayala Y M, Dang Q D, Di Cera E. Identification of residues linked to the slow→fast transition of thrombin.  Proc Natl Acad Sci USA. 1995;  92 11185-11189
• 21 Le Bonniec B F, Myles T, Johnson T et al.. Characterization of the P2′ and P3′ specificities of thrombin using fluorescence-quenched substrates and mapping of the subsites by mutagenesis.  Biochemistry. 1996;  35 7114-7122
• 22 Banner D W, Hadvary P. Crystallographic analysis at 3.0-A resolution of the binding to human thrombin of four active site-directed inhibitors.  J Biol Chem. 1991;  266 20085-20093
• 23 Brandstetter H, Turk D, Hoeffken H W et al.. Refined 2.3 A X-ray crystal structure of bovine thrombin complexes formed with the benzamidine and arginine-based thrombin inhibitors NAPAP, 4-TAPAP and MQPA. A starting point for improving antithrombotics.  J Mol Biol. 1992;  226 1085-1099
• 24 Baglin T P, Carrell R W, Church F C, Esmon C T, Huntington J A. Crystal structures of native and thrombin-complexed heparin cofactor II reveal a multistep allosteric mechanism.  Proc Natl Acad Sci USA. 2002;  99 11079-11084
• 25 Stubbs M T, Oschkinat H, Mayr I et al.. The interaction of thrombin with fibrinogen. A structural basis for its specificity.  Eur J Biochem. 1992;  206 187-195
• 26 Martin P D, Robertson W, Turk D et al.. The structure of residues 7-16 of the A alpha-chain of human fibrinogen bound to bovine thrombin at 2.3-A resolution.  J Biol Chem. 1992;  267 7911-7920
• 27 Malkowski M G, Martin P D, Lord S T, Edwards B F. Crystal structure of fibrinogen-A alpha peptide 1-23 (F8Y) bound to bovine thrombin explains why the mutation of Phe-8 to tyrosine strongly inhibits normal cleavage at Arg-16.  Biochem J. 1997;  326(pt 3) 815-822
• 28 Sadasivan C, Yee V C. Interaction of the factor XIII activation peptide with alpha -thrombin. Crystal structure of its enzyme-substrate analog complex.  J Biol Chem. 2000;  275 36942-36948
• 29 Janus T J, Lewis S D, Lorand L, Shafer J A. Promotion of thrombin-catalyzed activation of factor XIII by fibrinogen.  Biochemistry. 1983;  22 6269-6272
• 30 Le Bonniec B F, Esmon C T. Glu-192-Gln substitution in thrombin mimics the catalytic switch induced by thrombomodulin.  Proc Natl Acad Sci USA. 1991;  88 7371-7375
• 31 van de Locht A, Bode W, Huber R et al.. The thrombin E192Q-BPTI complex reveals gross structural rearrangements: implications for the interaction with antithrombin and thrombomodulin.  EMBO J. 1997;  16 2977-2984
• 32 Ayala Y, Di Cera E. Molecular recognition by thrombin. Role of the slow→fast transition, site-specific ion binding energetics and thermodynamic mapping of structural components.  J Mol Biol. 1994;  235 733-746
• 33 Di Cera E, Guinto E R, Vindigni A et al.. The Na+ binding site of thrombin.  J Biol Chem. 1995;  270 22089-22092
• 34 Pineda A O, Carrell C J, Bush L A et al.. Molecular dissection of Na+ binding to thrombin.  J Biol Chem. 2004;  279 31842-31853
• 35 Huntington J A, Esmon C T. The molecular basis of thrombin allostery revealed by a 1.8 A structure of the “slow” form.  Structure. 2003;  1 469-479
• 36 Carter W J, Myles T, Gibbs C S, Leung L L, Huntington J A. Crystal structure of anticoagulant thrombin variant E217K provides insights into thrombin allostery.  J Biol Chem. 2004;  279 26387-26394
• 37 De Filippis V, De Dea E, Lucatello F, Frasson R. Effect of Na+ binding on the conformation, stability and molecular recognition properties of thrombin.  Biochem J. 2005;  390 485-492
• 38 Rydel T J, Tulinsky A, Bode W, Huber R. Refined structure of the hirudin-thrombin complex.  J Mol Biol. 1991;  221 583-601
• 39 Grutter M G, Priestle J P, Rahuel J et al.. Crystal structure of the thrombin-hirudin complex: a novel mode of serine protease inhibition.  EMBO J. 1990;  9 2361-2365
• 40 Skrzypczak-Jankun E, Carperos V E, Ravichandran K G et al.. Structure of the hirugen and hirulog 1 complexes of alpha-thrombin.  J Mol Biol. 1991;  221 1379-1393
• 41 Mengwasser K E, Bush L A, Shih P, Cantwell A M, Di Cera E. Hirudin binding reveals key determinants of thrombin allostery.  J Biol Chem. 2005;  280 26997-27003
• 42 van de Locht A, Lamba D, Bauer M et al.. Two heads are better than one: crystal structure of the insect derived double domain Kazal inhibitor rhodniin in complex with thrombin.  EMBO J. 1995;  14 5149-5157
• 43 van de Locht A, Stubbs M T, Bode W et al.. The ornithodorin-thrombin crystal structure, a key to the TAP enigma?.  EMBO J. 1996;  15 6011-6017
• 44 Fuentes-Prior P, Noeske-Jungblut C, Donner P et al.. Structure of the thrombin complex with triabin, a lipocalin-like exosite-binding inhibitor derived from a triatomine bug.  Proc Natl Acad Sci USA. 1997;  94 11845-11850
• 45 Pechik I, Madrazo J, Mosesson M W et al.. Crystal structure of the complex between thrombin and the central “E” region of fibrin.  Proc Natl Acad Sci USA. 2004;  101 2718-2723
• 46 Rose T, Di Cera E. Substrate recognition drives the evolution of serine proteases.  J Biol Chem. 2002;  277 19243-19246
• 47 Martin P D, Malkowski M G, DiMaio J et al.. Bovine thrombin complexed with an uncleavable analog of residues 7-19 of fibrinogen A alpha: geometry of the catalytic triad and interactions of the P1′, P2′, and P3′ substrate residues.  Biochemistry. 1996;  35 13030-13039
• 48 Hofsteenge J, Stone S R. The effect of thrombomodulin on the cleavage of fibrinogen and fibrinogen fragments by thrombin.  Eur J Biochem. 1987;  168 49-56
• 49 Binnie C G, Lord S T. A synthetic analog of fibrinogen alpha 27-50 is an inhibitor of thrombin.  Thromb Haemost. 1991;  65 165-168
• 50 Mosesson M W. Fibrinogen and fibrin structure and functions.  J Thromb Haemost. 2005;  3 1894-1904
• 51 Vu T K, Wheaton V I, Hung D T, Charo I, Coughlin S R. Domains specifying thrombin-receptor interaction.  Nature. 1991;  353 674-677
• 52 Ayala Y M, Cantwell A M, Rose T et al.. Molecular mapping of thrombin-receptor interactions.  Proteins. 2001;  45 107-116
• 53 Mathews I I, Padmanabhan K P, Ganesh V et al.. Crystallographic structures of thrombin complexed with thrombin receptor peptides: existence of expected and novel binding modes.  Biochemistry. 1994;  33 3266-3279
• 54 De Cristofaro R, De Candia E, Landolfi R, Rutella S, Hall S W. Structural and functional mapping of the thrombin domain involved in the binding to the platelet glycoprotein Ib.  Biochemistry. 2001;  40 13268-13273
• 55 Huntington J A, Read R J, Carrell R W. Structure of a serpin-protease complex shows inhibition by deformation.  Nature. 2000;  407 923-926
• 56 Li W, Johnson D J, Esmon C T, Huntington J A. Structure of the antithrombin-thrombin-heparin ternary complex reveals the antithrombotic mechanism of heparin.  Nat Struct Mol Biol. 2004;  11 857-862
• 57 Panizzi P, Friedrich R, Fuentes-Prior P, Bode W, Bock P E. The staphylocoagulase family of zymogen activator and adhesion proteins.  Cell Mol Life Sci. 2004;  61 2793-2798
• 58 Friedrich R, Panizzi P, Kawabata S et al.. Structural basis for reduced staphylocoagulase-mediated bovine prothrombin activation.  J Biol Cell. 2006;  281 1188-1195
• 59 Friedrich R, Panizzi P, Fuentes-Prior P et al.. Staphylocoagulase is a prototype for the mechanism of cofactor-induced zymogen activation.  Nature. 2003;  425 535-539
• 60 Panizzi P, Friedrich R, Fuentes-Prior P et al.. Fibrinogen substrate recognition by staphylocoagulate-(pro)thrombin complexes.  JBC. 2005;  , In press
• 61 Church F C, Meade J B, Treanor R E, Whinna H C. Antithrombin activity of fucoidan. The interaction of fucoidan with heparin cofactor II, antithrombin III, and thrombin.  J Biol Chem. 1989;  264 3618-3623
• 62 Gan Z R, Li Y, Chen Z, Lewis S D, Shafer J A. Identification of basic amino acid residues in thrombin essential for heparin-catalyzed inactivation by antithrombin III.  J Biol Chem. 1994;  269 1301-1305
• 63 Carter W J, Cama E, Huntington J A. Crystal structure of thrombin bound to heparin.  J Biol Chem. 2005;  280 2745-2749
• 64 Olson S T, Halvorson H R, Bjork I. Quantitative characterization of the thrombin-heparin interaction. Discrimination between specific and nonspecific binding models.  J Biol Chem. 1991;  266 6342-6352
• 65 Bray B, Lane D A, Freyssinet J M, Pejler G, Lindahl U. Anti-thrombin activities of heparin. Effect of saccharide chain length on thrombin inhibition by heparin cofactor II and by antithrombin.  Biochem J. 1989;  262 225-232
• 66 Gettins P G. Serpin structure, mechanism, and function.  Chem Rev. 2002;  102 4751-4804
• 67 Dementiev A, Petitou M, Herbert J M, Gettins P G. The ternary complex of antithrombin-anhydrothrombin-heparin reveals the basis of inhibitor specificity.  Nat Struct Mol Biol. 2004;  11 863-867
• 68 Sheehan J P, Sadler J E. Molecular mapping of the heparin-binding exosite of thrombin.  Proc Natl Acad Sci USA. 1994;  91 5518-5522
• 69 Arni R K, Padmanabhan K, Padmanabhan K P, Wu T P, Tulinsky A. Structures of the noncovalent complexes of human and bovine prothrombin fragment 2 with human PPACK-thrombin.  Biochemistry. 1993;  32 4727-4737
• 70 Martin P D, Malkowski M G, Box J, Esmon C T, Edwards B F. New insights into the regulation of the blood clotting cascade derived from the X-ray crystal structure of bovine meizothrombin des F1 in complex with PPACK.  Structure. 1997;  5 1681-1693
• 71 Richardson J L, Kroger B, Hoeffken W et al.. Crystal structure of the human alpha-thrombin-haemadin complex: an exosite II-binding inhibitor.  EMBO J. 2000;  19 5650-5660
• 72 Richardson J L, Fuentes-Prior P, Sadler J E, Huber R, Bode W. Characterization of the residues involved in the human alpha-thrombin-haemadin complex: an exosite II-binding inhibitor.  Biochemistry. 2002;  41 2535-2542
• 73 Soslau G, Class R, Morgan D A et al.. Unique pathway of thrombin-induced platelet aggregation mediated by glycoprotein Ib.  J Biol Chem. 2001;  276 21173-21183
• 74 Ramakrishnan V, DeGuzman F, Bao M et al.. A thrombin receptor function for platelet glycoprotein Ib-IX unmasked by cleavage of glycoprotein V.  Proc Natl Acad Sci USA. 2001;  98 1823-1828
• 75 Vanhoorelbeke K, Ulrichts H, Romijn R A, Huizinga E G, Deckmyn H. The GPIbalpha-thrombin interaction: far from crystal clear.  Trends Mol Med. 2004;  10 33-39
• 76 Dumas J J, Kumar R, Seehra J, Somers W S, Mosyak L. Crystal structure of the GpIbalpha-thrombin complex essential for platelet aggregation.  Science. 2003;  301 222-226
• 77 Celikel R, McClintock R A, Roberts J R et al.. Modulation of alpha-thrombin function by distinct interactions with platelet glycoprotein Ibalpha.  Science. 2003;  301 218-221
• 78 Fay P J. Activation of factor VIII and mechanisms of cofactor action.  Blood Rev. 2004;  18 1-15
• 79 Adams T E, Hockin M F, Mann K G, Everse S J. The crystal structure of activated protein C-inactivated bovine factor Va: implications for cofactor function.  Proc Natl Acad Sci USA. 2004;  101 8918-8923
• 80 Esmon C T, Lollar P. Involvement of thrombin anion-binding exosites 1 and 2 in the activation of factor V and factor VIII.  J Biol Chem. 1996;  271 13882-13887
• 81 Myles T, Yun T H, Hall S W, Leung L L. An extensive interaction interface between thrombin and factor V is required for factor V activation.  J Biol Chem. 2001;  276 25143-25149
• 82 Myles T, Yun T H, Leung L L. Structural requirements for the activation of human factor VIII by thrombin.  Blood. 2002;  100 2820-2826
• 83 Walsh P N. Roles of factor XI, platelets and tissue factor-initiated blood coagulation.  J Thromb Haemost. 2003;  1 2081-2086
• 84 Baglia F A, Walsh P N. A binding site for thrombin in the apple 1 domain of factor XI.  J Biol Chem. 1996;  271 3652-3658
• 85 Yun T H, Baglia F A, Myles T et al.. Thrombin activation of factor XI on activated platelets requires the interaction of factor XI and platelet glycoprotein Ib alpha with thrombin anion-binding exosites I and II, respectively.  J Biol Chem. 2003;  278 48112-48119
• 86 Esmon C T. Thrombomodulin as a model of molecular mechanisms that modulate protease specificity and function at the vessel surface.  FASEB J. 1995;  9 946-955
• 87 Fuentes-Prior P, Iwanaga Y, Huber R et al.. Structural basis for the anticoagulant activity of the thrombin-thrombomodulin complex.  Nature. 2000;  404 518-525
• 88 Wang W, Nagashima M, Schneider M, Morser J, Nesheim M. Elements of the primary structure of thrombomodulin required for efficient thrombin-activable fibrinolysis inhibitor activation.  J Biol Chem. 2000;  275 22942-22947
• 89 Wood M J, Sampoli Benitez B A, Komives E A. Solution structure of the smallest cofactor-active fragment of thrombomodulin.  Nat Struct Biol. 2000;  7 200-204
• 90 Ye J, Rezaie A R, Esmon C T. Glycosaminoglycan contributions to both protein C activation and thrombin inhibition involve a common arginine-rich site in thrombin that includes residues arginine 93, 97, and 101.  J Biol Chem. 1994;  269 17965-17970
• 91 Nagashima M, Lundh E, Leonard J C, Morser J, Parkinson J F. Alanine-scanning mutagenesis of the epidermal growth factor-like domains of human thrombomodulin identifies critical residues for its cofactor activity.  J Biol Chem. 1993;  268 2888-2892
• 92 Gerlitz B, Grinnell B W. Mutation of protease domain residues Lys37-39 in human protein C inhibits activation by the thrombomodulin-thrombin complex without affecting activation by free thrombin.  J Biol Chem. 1996;  271 22285-22288
• 93 Vincenot A, Gaussem P, Pittet J L, Debost S, Aiach M. Amino acids 225-235** of the protein C serine-protease domain are important for the interaction with the thrombin-thrombomodulin complex.  FEBS Lett. 1995;  367 153-157
• 94 Yang L, Rezaie A R. The fourth epidermal growth factor-like domain of thrombomodulin interacts with the basic exosite of protein C.  J Biol Chem. 2003;  278 10484-10490
• 95 Xu H, Bush L A, Pineda A O, Caccia S, Di Cera E. Thrombomodulin changes the molecular surface of interaction and the rate of complex formation between thrombin and protein C.  J Biol Chem. 2005;  280 7956-7961
• 96 Rezaie A R, Yang L. Mutagenesis studies toward understanding the mechanism of the cofactor function of thrombomodulin.  Biophys Chem. 2005;  117 255-261
• 97 Lu G, Chhum S, Krishnaswamy S. The affinity of protein C for the thrombin-thrombomodulin complex is determined in a primary way by active site-dependent interactions.  J Biol Chem. 2005;  280 15471-15478
• 98 Di Cera E. Thrombin interactions.  Chest. 2003;  124 11S-17S
• 99 Fukudome K, Kurosawa S, Stearns-Kurosawa D J et al.. The endothelial cell protein C receptor. Cell surface expression and direct ligand binding by the soluble receptor.  J Biol Chem. 1996;  271 17491-17498
• 100 Coll M, Guasch A, Aviles F X, Huber R. Three-dimensional structure of porcine procarboxypeptidase B: a structural basis of its inactivity.  EMBO J. 1991;  10 1-9
• 101 Schneider M, Nagashima M, Knappe S et al.. Amino acid residues in the P6-P'3 region of thrombin-activable fibrinolysis inhibitor (TAFI) do not determine the thrombomodulin dependence of TAFI activation.  J Biol Chem. 2002;  277 9944-9951
• 102 Overduin M, de Beer T. The plot thickens: how thrombin modulates blood clotting.  Nat Struct Biol. 2000;  7 267-269
• 103 Rydel T J, Ravichandran K G, Tulinsky A et al.. The structure of a complex of recombinant hirudin and human alpha-thrombin.  Science. 1990;  249 277-280
• 104 Huntington J A, Baglin T P. Targeting thrombin-rational drug design from natural mechanisms.  Trends Pharmacol Sci. 2003;  24 589-595

1 Protein Data Bank [PDB] accession code 1PPB.

2 EC 3.4.21.5; MEROPS classification peptidase S01.217 of family S1A, clan PA(S).[18]

3 PDB code 1JMO.

4 PDB codes 1FPH and 1BBR.

5 PDB code 1YCP.

6 PDB code 1DE7.

7 PDB code 1BTH.

8 PDB codes 1SGI, 1SG8, 1SHH, and 1SFQ.

9 PDB code 1JOU_EF.

10 PDB code 1RD3.

11 PDB code 4HTC.

12 PDB code 1HGT.

13 PDB codes 1TBR and 1TOC.

14 PDB code 1AVG.

15 PDB code 1QVH.

16 PDB code 1UCY.

17 PDB code 1NRS.

18 PDB code 1NRN.

19 PDB code 1JMO.

20 PDB codes 1NU7 and 2A1D.

21 PDB code 1NU9.

22 PDB code 1XMN.

23 PDB code 1TB6.

24 PDB code 1SR5.

25 PDB codes 2HPQ and 2HPP.

26 PDB code 1A0H.

27 PDB code 1E0F.

28 PDB code 1DX5.

29 PDB code 1P8V and 100K.