Prothrombin Structure: Unanticipated Features and Opportunities

Editorial

Prothrombin structure: unanticipated features and opportunities

Expert Rev. Proteomics 11(6), 000–000 (2014)

Nicola Pozzi The structure of prothrombin has eluded investigators for decades but recent efforts 5 Edward A. Doisy Department of have succeeded in revealing the architecture of this important clotting factor. Biochemistry and Molecular Unanticipated features have emerged outlining the significant flexibility of the Biology, Saint Louis University zymogen due to linker regions connecting the g carboxyglutamic domain, kringles and AQ2 School of Medicine, St. Louis, protease domain. A new, structure-based framework helps in defining a molecular MO 63104, USA mechanism of prothrombin activation, rationalizes the severe bleeding phenotypes of several naturally occurring mutations and identifies targets for drug design. 10

Prothrombin, or coagulation factor II, is Prothrombin is a vitamin K-depen- Enrico Di Cera abundantly present in the blood where it dent zymogen composed of a gamma Author for correspondence: circulates at a concentration of 0.1 mg/ml carboxyglutamic (Gla) domain (residues 15 Edward A. Doisy Department of and a half-life of about 60 h [1]. In the 1–46), kringle-1 (residues 65–143), Biochemistry and Molecular Biology, Saint Louis University penultimate step of the coagulation cas- kringle-2 (residues 170–248) and the School of Medicine, St. Louis, cade, prothrombin is proteolytically con- protease domain with the A chain MO 63104, USA verted to the active protease thrombin (residues 285–320) and the catalytic B Tel.: +1 314 977 9201 20 Fax: +1 314 977 9206 by the prothrombinase complex com- chain (residues 321–579). The pro- Email: [email protected] prising the protease factor Xa and cofac- thrombinase complex converts pro- tor Va assembled on a phospholipid thrombin to thrombin by cleaving at membrane in the presence of Ca2+. R271 and R320, generating the inter- Thrombin catalyzes the conversion of mediates prethrombin-2 and meizo- fibrinogen to an insoluble fibrin clot, thrombin, respectively. Under conditions 25 the activation of platelets via the pro- most relevant to physiology, that is, on AQ teinase-activated receptor 1 and the feed- the surface of platelets, prothrombinase back control of the coagulation response activates prothrombin along the pre- via the thrombomodulin-dependent prothrombin-2 pathway [7]. On non-platelet tein C pathway. Because of the crucial surfaces or synthetic phospholipids, acti- 30 roles of thrombin, the body has an abso- vation proceeds along the meizothrom- lute requirement for its zymogen precur- bin pathway [8]. The mechanism of how sor and no living patient has ever been prothrombinase directs cleavage at reported with undetectable levels of pro- R271 or R320 remains poorly under- thrombin in the blood [2]. However, the stood at the molecular level and highly 35 importance of prothrombin extends to controversial [9–11]. Cofactor Va and other physiological processes. Prothrom- phospholipids control both the rate and bin is involved in embryonic develop- pathway of prothrombin activation, ment [3,4], is elevated in the cerebrospinal although the cofactor has the greatest fluid of patients suffering from progres- effect in enhancing kcat by almost 40 sive neurodegenerative diseases [5] and 2000-fold [12], but the molecular origin becomes the target of antibodies in the of this large effect remains unknown. antiphospholipids syndrome [6]. Studies Notwithstanding decades of intense on the structure and function of pro- investigation, our information on the thrombin are therefore crucial to the factors that control the rate and pathway 45 understanding of processes that are essen- of prothrombin activation is largely phe- tial for life and constitute a focus of nomenological. For example, perturba- therapeutic intervention. tions of the Gla domain anchoring

KEYWORDS: blood coagulation • drug design • protease • prothrombin • structural biology

informahealthcare.com 10.1586/14789450.2014.971763 Ó 2014 Informa UK Ltd ISSN 1478-9450 1 Editorial Pozzi & Di Cera

prothrombin to the membrane switch the pathway of activation prothrombin activation by prothrombinase [25], which implies 50 from meizothrombin to prethrombin-2 [13,14], but how this that the recent structure of prothrombin devoid of Lnk2 is an 105 domain communicates with the sites of cleavage >80 A˚ away to intriguing representation of the conformation of prothrombin inform the pathway of activation is not known. Active site optimized for catalysis by binding of cofactor Va in the pro- occupancy of prothrombin also switches the pathway of activa- thrombinase complex. A molecular picture of prothrombin tion from meizothrombin to prethrombin-2 [15], but again no activation begins to emerge from structural biology [24,25]. Fac- 55 compelling structural explanation has been offered. Kringle-1 tor Xa and prothrombin anchored to the membrane by their 110 and kringle-2 interact with cofactor Va [16,17] and so do residues Gla domain need to align properly for efficient thrombin gen- in the autolysis loop [18] and exosite I [19] of the B chain. Fac- eration. The flexibility of Lnk2 enables prothrombin to sample tor Xa interacts with kringle-2 [20] and residues near exosite II multiple conformations, and this may constitute an entropic of the B chain [21] as well as with the Gla domain [22]. Knowl- barrier to optimal interaction with factor Xa that itself under- 60 edge from these epitopes is most useful when complemented goes conformational transitions on the plane of the membrane. 115 by structural information on the prothrombin–prothrombinase Binding of cofactor Va, acting in concert with the membrane, complex. The structure of a surrogate prothrombinase has been relieves this entropy cost by providing a scaffold that aligns solved recently [23]. More relevant to human biology is the both factor Xa and prothrombin for optimal catalysis. A key structure of prothrombin itself [24,25]. component of this action is to ‘compress’ prothrombin in a 65 Three linkers connect kringle-1 to the Gla domain (Lnk1, way that is mimicked by the structure devoid of Lnk2 [25]. 120 residues 47–64), the two kringles (Lnk2, residues 144–169) This model offers a testable framework for studies of the struc- and kringle-2 to the mature A chain (Lnk3, residues 249–284), ture and dynamics of the zymogen free and bound to compo- respectively. The linkers are encoded by separate exons (IV, VII nents of the prothrombinase complex using single molecule and VIII), but Lnk2 is unique insofar as it shares exon VII detection. The discovery of prothrombin plasticity due to its 70 with kringle-2 in the longest coding region of the gene [26]. linker regions has also pointed to effective strategies for crystal- 125 The architecture of the prothrombin gene supports an impor- lization of the zymogen under conditions most relevant to tant role for the linkers, yet these structural elements have not physiology. attracted attention until recently [24,25]. Prothrombin has a The crystal structure of prothrombin enables a rational highly plastic arrangement with the Gla domain, kringles and approach to the identification of epitopes for factor Xa and 75 protease domain not vertically stacked and Lnk2 and Lnk3 cofactor Va binding and reveals docking sites for the design 130 highly disordered. Luminescence resonance energy transfer of small molecules aimed at interfering with the interaction measurements in solution document the flexibility of Lnk2 that of prothrombin with prothrombinase. Charged and aromatic functions like a spring connecting the kringle-2/protease residues >70% exposed to solvent become prime candidates domain pair on the C-terminal side to the Gla domain/kringle- for mutagenesis and offer a critical assessment of targets iden- 80 1 pair on the N-terminal side [24]. tified in previous studies [16–22].Additionaltargetsarepro- 135 The unanticipated plasticity of prothrombin due to its flexi- vided by naturally occurring mutations of prothrombin ble linkers influences both the rate and pathway of activation. interspersed with residues singled out by the solvent exposure Deletion of Lnk1 perturbs Ca2+ binding to the Gla domain criterion. Some of these mutations are buried but point to and the level of g-carboxylation and results in a switch of the important determinants of recognition. For example, the 85 pathway of prothrombin activation from meizothrombin to severe bleeding reported in prothrombin Puerto Rico 140 prethrombin-2. Lnk1 directs prothrombinase to cleave at (T122M/R457Q) is associated with normal antigen levels [28] R320 by channeling information from the Gla domain to the and may be due to mutation of R457, a residue in the B activation domain. The crystal structure of prothrombin sug- chain making a strong ionic interaction with D306 in the A gests how this communication is established at the molecular chain [28]. The A chain contains four residues arranged in an 90 level [24,25]. Lnk1 comprises two helices connected by the C47– ionquartetwhereR296iscagedbythesidechainsofE300, 145 C60 disulfide bond [25]. Through this hinge region, the Gla D306 and E309. D306 also ion-pairs with R457. Each of domain anchored to the membrane affects the position of the four Ala mutants of the quartet has been reported to acti- kringle-1 and propagates conformational changes to the rest of vate poorly due to the lack of cleavage by prothrombinase at the molecule using the flexible Lnk2, thereby influencing long- R271 [29]. Remarkably, prothrombin Denver (E300K and 95 range the directionality of cleavage. The importance of this E309K) causes severe bleeding because of direct perturbation 150 region of Lnk1 is further demonstrated by the naturally occur- of prothrombin activation [30], and so does prothrombin ring mutant prothrombin Mumbai (C47S) that is associated Puerto Rico (T122M/R457Q) [28]. Through the structure of with profoundly altered coagulation parameters and intracranial prothrombin, a molecular understanding of the bleeding phe- bleeding [27]. Lnk2 also contributes to direct prothrombinase at notype associated with naturally occurring mutations becomes 100 R320. Deletion of Lnk2 reduces the preference for the meizo- possible. 155 thrombin pathway and drastically increases the rate of activa- A new, structure-based framework has emerged for the study tion in the absence but not the presence of cofactor Va. Most of prothrombin. The unanticipated important role of the link- of Lnk2 can be deleted with a small perturbation of the rate of ers and the flexibility of the zymogen have a direct bearing on

2 Expert Rev. Proteomics 11(6), (2014) Prothrombin structure Editorial

the mechanism of activation and warrant proper attention in supported in part by the National Institutes of Health Research Grants 160 future studies. The structure of prothrombin also offers a HL049413, HL073813 and HL112303. molecular interpretation of the phenotypes associated with naturally occurring mutations and points to new targets for Financial & competing interests disclosure 170 drug design. The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict Acknowledgements with the subject matter or materials discussed in the manuscript. This 165 Several important contributions to the field could not be discussed due to includes employment, consultancies, honoraria, stock ownership or options, space limitations imposed by editorial guidelines, and the authors sincerely expert testimony, grants or patents received or pending, or royalties. 175 apologize to investigators not mentioned in the article. This study was No writing assistance was utilized in the production of this manuscript.

References 11. Orcutt SJ, Krishnaswamy S. Binding of linear sequence of the kringle 2 domain of substrate in two conformations to human prothrombin. J Biochem 1994;116:589-97 1. Butenas S, van’t Veer C, Mann KG. prothrombinase drives consecutive cleavage “Normal” thrombin generation. Blood 21. Yegneswaran S, Mesters RM, Griffin JH. at two sites in prothrombin. J Biol Chem 1999;94:2169-78 Identification of distinct sequences in 2004;279:54927-36 human blood coagulation factor Xa and 2. Lancellotti S, Basso M, De Cristofaro R. 12. Rosing J, Tans G, Govers-Riemslag JW, prothrombin essential for substrate and Congenital prothrombin deficiency: et al. The role of phospholipids and factor cofactor recognition in the prothrombinase an update. Semin Thromb Hemost Va in the prothrombinase complex. J Biol complex. J Biol Chem 2003;278:33312-18 2013;39:596-606 Chem 1980;255:274-83 22. Harlos K, Holland SK, Boys CW, et al. 3. Sun WY, Witte DP, Degen JL, et al. 13. Bukys MA, Orban T, Kim PY, et al. The Vitamin K-dependent blood coagulation Prothrombin deficiency results in embryonic interaction of fragment 1 of prothrombin proteins form hetero-dimers. Nature and neonatal lethality in mice. Proc Natl with the membrane surface is a prerequisite 1987;330:82-4 Acad Sci USA 1998;95:7597-602 for optimum expression of factor Va 23. Lechtenberg BC, Murray-Rust TA, 4. Xue J, Wu Q, Westfield LA, et al. cofactor activity within prothrombinase. Johnson DJ, et al. Crystal structure of the Incomplete embryonic lethality and fatal Thromb Haemost 2008;99:511-22 prothrombinase complex from the venom of neonatal hemorrhage caused by 14. Bradford HN, Orcutt SJ, Krishnaswamy S. Pseudonaja textilis. Blood 2013;122:2777-83 prothrombin deficiency in mice. Proc Natl Membrane binding by prothrombin Acad Sci USA 1998;95:7603-7 24. Pozzi N, Chen Z, Gohara DW, et al. mediates its constrained presentation to Crystal structure of prothrombin reveals 5. Huang YC, Wu YR, Tseng MY, et al. prothrombinase for cleavage. J Biol Chem conformational flexibility and mechanism of Increased prothrombin, apolipoprotein 2013;288:27789-800 activation. J Biol Chem 2013;288:22734-44 A-IV, and haptoglobin in the cerebrospinal 15. Bianchini EP, Orcutt SJ, Panizzi P, et al. fluid of patients with Huntington’s disease. 25. Pozzi N, Chen Z, Pelc LA, et al. The linker Ratcheting of the substrate from the PLoS One 2011;6:e15809 connecting the two kringles plays a key role zymogen to proteinase conformations directs in prothrombin activation. Proc Natl Acad 6. Sciascia S, Sanna G, Murru V, et al. the sequential cleavage of prothrombin by Sci USA 2014;111:7630-5 Anti-prothrombin (aPT) and anti- prothrombinase. Proc Natl Acad Sci USA 26. Degen SJ, Davie EW. Nucleotide sequence phosphatidylserine/prothrombin (aPS/PT) 2005;102:10099-104 antibodies and the risk of thrombosis in the of the gene for human prothrombin. 16. Deguchi H, Takeya H, Gabazza EC, et al. antiphospholipid syndrome. A systematic Biochemistry 1987;26:6165-77 Prothrombin kringle 1 domain interacts review. Thromb Haemost 2014;111:354-64 27. Kulkarni B, Kanakia S, Ghosh K, et al. with factor Va during the assembly of Prothrombin Mumbai causes severe 7. Haynes LM, Bouchard BA, Tracy PB, et al. prothrombinase complex. Biochem J 1997; prothrombin deficiency due to a novel Prothrombin activation by platelet-associated 321(Pt 3):729-35 prothrombinase proceeds through the Cys90Ser mutation. Ann Hematol 2012;91: 17. Kotkow KJ, Deitcher SR, Furie B, et al. prethrombin-2 pathway via a concerted 1667-8 The second kringle domain of prothrombin mechanism. J Biol Chem 2012;287:38647-55 28. Lefkowitz JB, Weller A, Nuss R, et al. promotes factor Va-mediated prothrombin A common mutation, Arg457–Gln, links 8. Tans G, Janssen-Claessen T, Hemker HC, activation by prothrombinase. J Biol Chem prothrombin deficiencies in the Puerto et al. Meizothrombin formation during 1995;270:4551-7 factor Xa-catalyzed prothrombin activation. Rican population. J Thromb Haemost 18. Yegneswaran S, Mesters RM, Fernandez JA, Formation in a purified system and in 2003;1:2381-8 et al. Prothrombin residues 473-487 plasma. J Biol Chem 1991;266:21864-73 29. Papaconstantinou ME, Bah A, Di Cera E. contribute to factor Va binding in the Role of the chain in thrombin function. 9. Brufatto N, Nesheim ME. Analysis of the prothrombinase complex. J Biol Chem Cell Mol Life Sci 2008;65:1943-7 kinetics of prothrombin activation and 2004;279:49019-25 evidence that two equilibrating forms of 30. LefkowitzJB,HaverT,ClarkeS,etal.The 19. Chen L, Yang L, Rezaie AR. Proexosite-1 prothrombinase are involved in the process. prothrombin Denver patient has two different on prothrombin is a factor Va-dependent J Biol Chem 2003;278:6755-64 prothrombin point mutations resulting in recognition site for the prothrombinase Glu-300-Lys and Glu-309-Lys substitutions. 10. Lee CJ, Wu S, Eun C, et al. A revisit to the complex. J Biol Chem 2003;278:27564-9 one form kinetic model of prothrombinase. Br J Haematol 2000;108:182-7 20. Taneda H, Andoh K, Nishioka J, et al. Biophys Chem 2010;149:28-33 Blood coagulation factor Xa interacts with a

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