Natural inhibitors of thrombin

James A. Huntington

doi:10.1160/TH13-10-0811

Thrombosis and Haemostasis, Table of Contents

Thromb Haemost 2014; 111(04): 583-589
DOI: 10.1160/TH13-10-0811

Theme Issue Article

Schattauer GmbH

Natural inhibitors of thrombin

James A. Huntington

¹Department of Haematology, University of Cambridge, Cambridge Institute for Medical Research, Wellcome Trust/MRC Building, Hills Road, Cambridge, UK

› Author Affiliations

Abstract

Summary

The serine protease thrombin is the effector enzyme of blood coagulation. It has many activities critical for the formation of stable clots, including cleavage of fibrinogen to fibrin, activation of platelets and conversion of procofactors to active cofactors. Thrombin carries-out its multiple functions by utilising three special features: a deep active site cleft and two anion binding exosites (exosite I and II). Similarly, thrombin inhibitors have evolved to exploit the unique features of thrombin to achieve rapid and specific inactivation of thrombin. Exogenous thrombin inhibitors come from several different protein families and are generally found in the saliva of haematophagous animals (blood suckers) as part of an anticoagulant cocktail that allows them to feed. Crystal structures of several of these inhibitors reveal how peptides and proteins can be targeted to thrombin in different and interesting ways. Thrombin activity must also be regulated by endogenous inhibitors so that thrombi do not occlude blood flow and cause thrombosis. A single protein family, the serpins, provides all four of the endogenous thrombin inhibitors found in man. The crystal structures of these serpins bound to thrombin have been solved, revealing a similar exosite-dependence on complex formation. In addition to forming the recognition complex, serpins destroy the structure of thrombin, allowing them to be released from cofactors and substrates for clearance. This review examines how the special features of thrombin have been exploited by evolution to achieve inhibition of the ultimate coagulation protease.

Keywords

Exosite - inhibition - protease - regulation - structure

Full Text

References

References
1 Lane DA, Philippou H, Huntington JA. Directing thrombin.. Blood 2005; 106: 2605-2612.
2 Huntington J. Structural Insights into the Life History of Thrombin.. In: Recent Advances in Thrombosis and Hemostasis 2008. Springer; Japan: 2008: 80-106.
3 Macfarlane RG. An Enzyme Cascade in the Blood Clotting Mechanism, and Its Function as a Biochemical Amplifier.. Nature 1964; 202: 498-499.
4 Davie EW, Ratnoff OD. Waterfall Sequence for Intrinsic Blood Clotting.. Science 1964; 145: 1310-1312.
5 Hoffman MM, Monroe DM. Rethinking the coagulation cascade.. Curr Hematol Rep 2005; 4: 391-396.
6 Hoffman M, Monroe DM. 3rd. A cell-based model of hemostasis.. Thromb Hae-most 2001; 85: 958-965.
7 Clemetson KJ. Platelets and primary haemostasis.. Thromb Res 2012; 129: 220-224.
8 Krishnaswamy S, Mann KG, Nesheim ME. The prothrombinase-catalyzed activation of prothrombin proceeds through the intermediate meizothrombin in an ordered, sequential reaction.. J Biol Chem 1986; 261: 8977-8984.
9 Davie EW, Kulman JD. An overview of the structure and function of thrombin.. Semin Thromb Hemost 2006; 32 (Suppl. 01) 3-15.
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 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.
12 Pechik I, Madrazo J, Mosesson MW. 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.
13 Gandhi PS, Chen Z, Di Cera E. Crystal structure of thrombin bound to the un-cleaved extracellular fragment of PAR1.. J Biol Chem 2010; 285: 15393-15398.
14 Myles T, Yun TH, Hall SW. et al. An extensive interaction interface between thrombin and factor V is required for factor V activation.. J Biol Chem 2001; 276: 25143-25149.
15 Myles T, Yun TH, Leung LL. Structural requirements for the activation of human factor VIII by thrombin.. Blood 2002; 100: 2820-2826.
16 Bukys MA, Orban T, Kim PY. et al. The structural integrity of anion binding ex-osite I of thrombin is required and sufficient for timely cleavage and activation of factor V and factor VIII.. J Biol Chem 2006; 281: 18569-18580.
17 Hall SW, Nagashima M, Zhao L. et al. Thrombin interacts with thrombomodu-lin, protein C, and thrombin-activatable fibrinolysis inhibitor via specific and distinct domains.. J Biol Chem 1999; 274: 25510-25516.
18 Janus TJ, Lewis SD, Lorand L. et al. Promotion of thrombin-catalyzed activation of factor XIII by fibrinogen.. Biochemistry 1983; 22: 6269-6272.
19 De Cristofaro R, De Candia E, Landolfi R. et al. Structural and functional mapping of the thrombin domain involved in the binding to the platelet glycoprotein Ib.. Biochemistry 2001; 40: 13268-13273.
20 Celikel R, McClintock RA, Roberts JR. et al. Modulation of alpha-thrombin function by distinct interactions with platelet glycoprotein Ibalpha.. Science 2003; 301: 218-221.
21 Dumas JJ, Kumar R, Seehra J. et al. Crystal structure of the GpIbalpha-thrombin complex essential for platelet aggregation.. Science 2003; 301: 222-226.
22 Nogami K, Zhou Q, Myles T. et al. Exosite-interactive regions in the A1 and A2 domains of factor VIII facilitate thrombin-catalyzed cleavage of heavy chain.. J Biol Chem 2005; 280: 18476-18487.
23 Segers K, Dahlback B, Bock PE. et al. The role of thrombin exosites I and II in the activation of human coagulation factor V.. J Biol Chem 2007; 282: 33915-33924.
24 Koh CY, Kini RM. Molecular diversity of anticoagulants from haematophagous animals.. Thromb Haemost 2009; 102: 437-453.
25 Francischetti IM. Platelet aggregation inhibitors from hematophagous animals.. Toxicon 2010; 56: 1130-1144.
26 Dodt J, Otte M, Strube KH. et al. Thrombin inhibitors of bloodsucking animals.. Semin Thromb Hemost 1996; 22: 203-208.
27 Bode W, Huber R. Structural basis of the endoproteinase-protein inhibitor interaction.. Biochim Biophys Acta 2000; 1477: 241-252.
28 Corral-Rodriguez MA, Macedo-Ribeiro S, Pereira PJ. et al. Leech-derived thrombin inhibitors: from structures to mechanisms to clinical applications.. J Med Chem 2010; 53: 3847-3861.
29 Rydel TJ, Ravichandran KG, Tulinsky A. et al. The structure of a complex of recombinant hirudin and human alpha-thrombin.. Science 1990; 249: 277-280.
30 Rydel TJ, Tulinsky A, Bode W. et al. Refined structure of the hirudin-thrombin complex.. J Mol Biol 1991; 221: 583-601.
31 Grutter MG, Priestle JP, Rahuel J. et al. Crystal structure of the thrombin-hiru-din complex: a novel mode of serine protease inhibition.. EMBO J 1990; 9: 2361-2365.
32 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.
33 van de Locht A, Stubbs MT, Bode W. et al. The ornithodorin-thrombin crystal structure, a key to the TAP enigma?. EMBO J 1996; 15: 6011-6017.
34 Ruhlmann A, Kukla D, Schwager P. et al. Structure of the complex formed by bovine trypsin and bovine pancreatic trypsin inhibitor. Crystal structure determination and stereochemistry of the contact region.. J Mol Biol 1973; 77: 417-436.
35 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.
36 Noeske-Jungblut C, Haendler B, Donner P. et al. Triabin, a highly potent exosite inhibitor of thrombin.. J Biol Chem 1995; 270: 28629-28634.
37 Richardson JL, 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.
38 Martin PD, Malkowski MG, Box J. et al. New insights into the regulation of the blood clotting cascade derived from the X-ray crystal structure of bovine meizo-thrombin des F1 in complex with PPACK.. Structure 1997; 5: 1681-1693.
39 Richardson JL, Fuentes-Prior P, Sadler JE. et al. Characterization of the residues involved in the human alpha-thrombin-haemadin complex: an exosite II-bind-ing inhibitor.. Biochemistry 2002; 41: 2535-2542.
40 Koh CY, Kazimirova M, Trimnell A. et al. Variegin, a novel fast and tight binding thrombin inhibitor from the tropical bont tick.. J Biol Chem 2007; 282: 29101-29113.
41 Koh CY, Kumar S, Kazimirova M. et al. Crystal structure of thrombin in complex with S-variegin: insights of a novel mechanism of inhibition and design of tunable thrombin inhibitors.. PloS one 2011; 6: e26367.
42 Figueiredo AC, de Sanctis D, Gutierrez-Gallego R. et al. Unique thrombin inhibition mechanism by anophelin, an anticoagulant from the malaria vector.. Proc Natl Acad Sci USA 2012; 109: E3649-3658.
43 Huntington JA. Thrombin inhibition by the serpins.. J Thromb Haemost 2013; 11 (Suppl. 01) 254-264.
44 Rau JC, Beaulieu LM, Huntington JA. et al. Serpins in thrombosis, hemostasis and fibrinolysis.. J Thromb Haemost 2007; 5 (Suppl. 01) 102-115.
45 Gettins PG. Serpin structure, mechanism, and function.. Chem Rev 2002; 102: 4751-4804.
46 Huntington JA, Read RJ, Carrell RW. Structure of a serpin-protease complex shows inhibition by deformation.. Nature 2000; 407: 923-926.
47 Li W, Johnson DJ, Esmon CT. et al. Structure of the antithrombin-thrombin-he-parin ternary complex reveals the antithrombotic mechanism of heparin.. Nat Struct Mol Biol 2004; 11: 857-862.
48 Baglin TP, Carrell RW, Church FC. et al. Crystal structures of native and throm-bin-complexed heparin cofactor II reveal a multistep allosteric mechanism.. Proc Natl Acad Sci USA 2002; 99: 11079-11084.
49 Li W, Adams TE, Nangalia J. et al. Molecular basis of thrombin recognition by protein C inhibitor revealed by the 1.6-A structure of the heparin-bridged complex.. Proc Natl Acad Sci USA 2008; 105: 4661-4666.
50 Li W, Huntington JA. Crystal structures of protease nexin-1 in complex with he-parin and thrombin suggest a 2-step recognition mechanism.. Blood 2012; 120: 459-467.
51 Zhou A, Carrell RW, Huntington JA. The serpin inhibitory mechanism is critically dependent on the length of the reactive center loop.. J Biol Chem 2001; 276: 27541-27547.
52 Fredenburgh JC, Stafford AR, Weitz JI. Conformational changes in thrombin when complexed by serpins.. J Biol Chem 2001; 276: 44828-44834.
53 Bock PE, Olson ST, Bjork I. Inactivation of thrombin by antithrombin is accompanied by inactivation of regulatory exosite I.. J Biol Chem 1997; 272: 19837-19845.
54 Li W, Johnson DJ, Adams TE. et al. Thrombin inhibition by serpins disrupts ex-osite II.. J Biol Chem 2010; 285: 38621-38629.
55 Wells MJ, Sheffield WP, Blajchman MA. The clearance of thrombin-antithrom-bin and related serpin-enzyme complexes from the circulation: role of various hepatocyte receptors.. Thromb Haemost 1999; 81: 325-337.
56 Carter WJ, Cama E, Huntington JA. Crystal structure of thrombin bound to he- parin.. J Biol Chem 2005; 280: 2745-2749.