Download PDF
Thromb Haemost 2004; 92(03): 509-521
DOI: 10.1160/TH04-03-0144
Blood Coagulation, Fibrinolysis and Cellular Haemostasis
Schattauer GmbH

The catalytic subunit of pseutarin C, a group C prothrombin activator from the venom of Pseudonaja textilis, is structurally similar to mammalian blood coagulation factor Xa

Veena S. Rao
1   Department of Biological Sciences, Faculty of Science, National University of Singapore, Singapore, Republic of Singapore
,
Sanjay Swarup
1   Department of Biological Sciences, Faculty of Science, National University of Singapore, Singapore, Republic of Singapore
,
Manjunatha R. Kini
1   Department of Biological Sciences, Faculty of Science, National University of Singapore, Singapore, Republic of Singapore
2   Department of Biochemistry, Medical College of Virginia, Virginia Commonwealth University, Richmond, Virginia, USA
› Author AffiliationsFinancial support: This work was supported by Academic Research Fund from the National University of Singapore.
Further Information

Publication History

Received 07 March 2004

Accepted after resubmission 10 June 2004

Publication Date:
30 November 2017 (online)

Also available at
    Permissions and Reprints

Summary

Pseutarin C, a group C prothrombin activator from Pseudonaja textilis venom, is a large protein complex consisting of catalytic and nonenzymatic subunits, which are functionally similar to the mammalian FXa-FVa complex. Here, we present the complete cDNA sequence of the catalytic subunit of pseutarin C. The cDNA of the catalytic subunit encodes a protein of 449 amino acids, which includes a 22-residue signal peptide, 18-residue propeptide and a mature protein of 409 amino acids. The deduced amino acid sequence shows 74-83% identity to group D prothrombin activators from snake venom and ∼42% identity to mammalian FX and has identical domain structure. The precursor of the catalytic subunit of pseutarin C has several unique features. The activation peptide of the catalytic subunit of pseutarin C is significantly smaller (27 as compared to 52 residues in mammalian FX) and does not contain any glycosylation sites. Unlike coagulation FXa, Ser52 and Asn45 of the light and heavy chains are O- and N-glycosylated in pseutarin C catalytic subunit. There is a 12-residue insertion in pseutarin C catalytic subunit close to the region that is implicated in binding to FVa. This is the first sequence of the catalytic subunit of a group C prothrombin activator.

Keywords

Prothrombinase complex - factor Xa - procoagulants - snake venom
 

  • References

  • 1 Jackson CM, Nemerson Y. Blood coagulation. Annu Rev Biochem 1980; 49: 765-11.
    CrossrefPubMedGoogle Scholar
  • 2 Fujikawa K, Legaz ME, Davie EW. Bovine factors X 1 and X 2 (Stuart factor). Isolation and characterization. Biochemistry 1972; 19: 4882-91.
    PubMedGoogle Scholar
  • 3 Fujikawa K, Titani K, Davie EW. Activation of bovine factor X (Stuart factor): conversion of factor Xaalpha to factor Xabeta. Proc Natl Acad Sci USA 1975; 72: 3359-63.
    CrossrefPubMedGoogle Scholar
  • 4 Titani K, Fujikawa K, Enfield DL. et al. Bovine factor X1 (Stuart factor): amino acid sequence of heavy chain. Proc Natl Acad Sci USA 1975; 72: 3082-86.
    CrossrefPubMedGoogle Scholar
  • 5 Enfield DL, Ericsson LH, Walsh KA. et al. Bovine factor X1 (Stuart factor). Primary structure of the light chain. Proc Natl Acad Sci USA 1975; 72: 16-19.
    CrossrefPubMedGoogle Scholar
  • 6 Enfield DL, Ericsson LH, Fujikawa K. et al. Amino acid sequence of the light chain of bovine factor X1 (Stuart factor). Biochemistry 1980; 19: 659-67.
    CrossrefPubMedGoogle Scholar
  • 7 Fung MR, Campbell RM, MacGillivray RT. Blood coagulation factor X mRNA encodes a single polypeptide chain containing a prepro leader sequence. Nucleic Acids Res 1984; 12: 4481-92.
    CrossrefPubMedGoogle Scholar
  • 8 Fung MR, Hay CW, MacGillivray RT. Characterization of an almost full-length cDNA coding for human blood coagulation factor X. Proc Natl Acad Sci USA 1985; 82: 3591-95.
    CrossrefPubMedGoogle Scholar
  • 9 Di Scipio RG, Hermodson MA, Davie EW. Activation of human factor X (Stuart factor) by a protease from Russell’s viper venom. Biochemistry 1977; 16: 5253-60.
    CrossrefPubMedGoogle Scholar
  • 10 Suttie JW, Jackson CM. Prothrombin structure, activation, and biosynthesis. Physiol Rev 1977; 57: 1-70.
    CrossrefPubMedGoogle Scholar
  • 11 Rosing J, Tans G, Govers-Riemslag JW. et al. The role of phospholipids and factor Va in the prothrombinase complex. J Biol Chem 1980; 255: 274-83.
    PubMedGoogle Scholar
  • 12 Mann KG, Krishnaswamy S, Lawson JH. Surface-dependent hemostasis. Semin Hematol 1992; 29: 213-26.
    PubMedGoogle Scholar
  • 13 van Rijn JL, Govers-Riemslag JW, Zwaal RF. et al. Kinetic studies of prothrombin activation: effect of factor Va and phospholipids on the formation of the enzyme-substrate complex. Biochemistry 1984; 23: 4557-64.
    CrossrefPubMedGoogle Scholar
  • 14 Nesheim ME, Taswell JB, Mann KG. The contribution of bovine Factor V and Factor Va to the activity of prothrombinase. J Biol Chem 1979; 254: 10952-62.
    PubMedGoogle Scholar
  • 15 Krishnaswamy S, Church WR, Nesheim ME. et al. Activation of human prothrombin by human prothrombinase. Influence of factor Va on the reaction mechanism. J Biol Chem 1987; 262: 3291-99.
    PubMedGoogle Scholar
  • 16 Markland Jr. FS. Snake venoms. Drugs 1997; 54: 1-10.
    PubMedGoogle Scholar
  • 17 Hutton RA, Warrell DA. Action of snake venom components on the haemostatic system. Blood Rev 1993; 07: 176-89.
    CrossrefPubMedGoogle Scholar
  • 18 Rosing J, Tans G. Structural and functional properties of snake venom prothrombin activators. Toxicon 1992; 30: 1515-27.
    CrossrefPubMedGoogle Scholar
  • 19 Gao R, Kini RM, Gopalakrishnakone P. A novel prothrombin activator from the venom of Micropechis ikaheka: isolation and characterization. Arch Biochem Biophys 2002; 408: 87-92.
    CrossrefPubMedGoogle Scholar
  • 20 Silva MB, Schattner M, Ramos CR. et al. A prothrombin activator from Bothrops erythromelas (jararaca-da-seca) snake venom: characterization and molecular cloning. Biochem J 2003; 369: 129-39.
    CrossrefPubMedGoogle Scholar
  • 21 Hasson SS, Theakston RD, Harrison RA. Cloning of a prothrombin activator-like metalloproteinase from the West African saw-scaled viper, Echis ocellatus . Toxicon 2003; 42: 629-34.
    CrossrefPubMedGoogle Scholar
  • 22 Loria GD, Rucavado A, Kamiguti AS. et al. Characterization of ‘basparin A,’ a prothrombin-activating metalloproteinase, from the venom of the snake Bothrops asper that inhibits platelet aggregation and induces defibrination and thrombosis. Arch Biochem Biophys 2003; 418: 13-24.
    CrossrefPubMedGoogle Scholar
  • 23 Kini RM, Morita T, Rosing J. Classification and nomenclature of prothrombin activators isolated from snake venoms. Thromb Haemost 2001; 85: 710-11.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 24 Morita T, Iwanaga S, Suzuki T. The mechanism of activation of bovine prothrombin by an activator isolated from Echis carinatus venom and characterization of the new active intermediates. J Biochem 1976; 79: 1089-108.
    CrossrefPubMedGoogle Scholar
  • 25 Yamada D, Sekiya F, Morita T. Isolation and characterization of carinactivase, a novel prothrombin activator in Echis carinatus venom with a unique catalytic mechanism. J Biol Chem 1996; 271: 5200-7.
    CrossrefPubMedGoogle Scholar
  • 26 Yamada D, Morita T. Purification and characterization of a Ca2+-dependent prothrombin activator, multactivase, from the venom of Echis multisquamatus . J Biochem 1997; 122: 991-7.
    CrossrefPubMedGoogle Scholar
  • 27 Speijer H, Govers-Riemslag JWP, Zwaal RFA. et al. Prothrombin activation by an activator from the venom of Oxyuranus scutellatus (Taipan snake). J Biol Chem 1986; 261: 13258-67.
    PubMedGoogle Scholar
  • 28 Masci PP, Whitaker AN, de Jersey J. Purification and characterization of a prothrombin activator from the venom of the Australian brown snake, Pseudonaja textilis textilis . Biochem Intl 1988; 17: 825-35.
    PubMedGoogle Scholar
  • 29 Tans G, Govers-Riemslag JWP, van Rihn JL. et al. Purification and properties of a prothrombin activator from the venom of Notechis scutatus scutatus . J Biol Chem 1985; 260: 9366-72.
    PubMedGoogle Scholar
  • 30 Rao VS, Kini RM. Pseutarin C, a prothrombin activator from Pseudonaja textilis venom: Its structural and functional similarity to mammalian coagulation factor Xa-Va complex. Thromb Haemost 2002; 88: 611-9.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 31 Kini RM, Rao VS, Joseph JS. Procoagulant proteins from snake venoms. Haemostasis 2001; 31: 218-24.
    PubMedGoogle Scholar
  • 32 Joseph JS, Chung MC, Jeyaseelan K. et al. Amino acid sequence of trocarin, a prothrombin activator from Tropidechis carinatus venom: its structural similarity to coagulation factor Xa. Blood 1999; 94: 621-31.
    PubMedGoogle Scholar
  • 33 Rao VS, Joseph JS, Kini RM. Group D prothrombin activators from snake venom are structural homologues of mammalian blood coagulation factor Xa. Biochem J 2003; 69: 635-42.
    PubMedGoogle Scholar
  • 34 Rao VS, Swarup S, Kini RM. The nonenzymatic subunit of pseutarin C, a prothrombin activator from eastern brown snake (Pseudonaja textilis) venom, shows structural similarity to mammalian coagulation factor V. Blood 2003; 102: 1347-54.
    CrossrefPubMedGoogle Scholar
  • 35 Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970; 227: 680-5.
    CrossrefPubMedGoogle Scholar
  • 36 Joseph JS, Valiyaveettil M, Gowda DC. et al. Occurrence of O-linked Xyl-GlcNac disaccharides in trocarin, a factor Xa homolog from snake venom. J Thromb Haemost 2003; 01: 545-50.
    CrossrefPubMedGoogle Scholar
  • 37 Inoue K, Morita T. Identification of O-linked oligosaccharide chains in the activation peptides of blood coagulation factor X. The role of the carbohydrate moieties in the activation of factor X. Eur J Biochem 1993; 218: 153-63.
    CrossrefPubMedGoogle Scholar
  • 38 Iwanaga S, Nishimura H, Kawabata S. et al. A new trisaccharide sugar chain linked to a serine residue in the first EGF-like domain of clotting factors VII and IX and protein Z. Liu CY, Chien S. (eds): Fibrinogen Thrombosis Coagulation and Fibrinolysis. New York NY: Plenum Press; 1991: 121.
    Google Scholar
  • 39 Bjoern S, Foster DC, Thim L. et al. Human plasma and recombinant factor VII. Characterization of O-glycosylations at serine residues 52 and 60 and effects of site directed mutagenesis at serine 52 to alanine. J Biol Chem 1991; 266: 11051-57.
    PubMedGoogle Scholar
  • 40 Fernlund P, Stenflo J. ß-hydroxyaspartic acid in vitamin K-dependent proteins. J Biol Chem 1983; 258: 12509-12.
    PubMedGoogle Scholar
  • 41 Stenflo J, Lundwal lA, Dahlback B. ß-Hydroxyasparagine in domains homologous to the epidermal growth factor precursor in vitamin K-dependent protein S. Proc Natl Acad Sci USA 1987; 84: 368-72.
    CrossrefPubMedGoogle Scholar
  • 42 Kelly CR, Dickinson CD, Ruf W. Ca2+ binding to the first epidermal growth factor module of coagulation factor VIIa is important for cofactor interaction and proteolytic function. J Biol Chem 1997; 272: 17467-72.
    CrossrefPubMedGoogle Scholar
  • 43 Gierasch LM. Signal sequences. Biochemistry 1989; 28: 923-30.
    CrossrefPubMedGoogle Scholar
  • 44 Rapoport TA. Protein transport across the endoplasmic reticulum membrane: facts, models, mysteries. FASEB J 1991; 05: 2792-98.
    CrossrefPubMedGoogle Scholar
  • 45 von Heijne G. The signal peptide. J Membr Biol 1990; 115: 195-01.
    CrossrefPubMedGoogle Scholar
  • 46 Knobloch JE, Suttie JW. Vitamin-K dependent carboxylase. Control of enzyme activity by the “propeptide” region of factor X. J Biol Chem 1987; 262: 15334-7.
    PubMedGoogle Scholar
  • 47 Suttie JW, Hoskins JA, Engelke J, Hopfgartner A, Ehrlich H, Bang NU, Belagaje RM, Schoner B, Long GL. Vitamin K-dependent carboxylase: possible role of the substrate “propeptide” as an intracellular recognition site. Proc Natl Acad Sci USA 1987; 84: 634-7.
    CrossrefPubMedGoogle Scholar
  • 48 Jorgensen MJ, Cantor AB, Furie BC. et al. Recognition site-directing vitamin K-dependent gamma-carboxylation resides on the propeptide of factor IX. Cell 1987; 48: 185-91.
    CrossrefPubMedGoogle Scholar
  • 49 Foster DC, Rudinski MS, Schach BG. et al. Propeptide of human protein C is necessary for gamma-carboxylation. Biochemistry 1987; 26: 7003-11.
    CrossrefPubMedGoogle Scholar
  • 50 Magnusson S, Sottrup-Jensen L, Petersen TE. et al. Primary structure of the vitamin Kdependent part of prothrombin. FEBS Lett 1974; 44: 189-93.
    CrossrefPubMedGoogle Scholar
  • 51 Nelsestuen GL, Zytkovicz TH, Howard JB. The mode of action of vitamin K. Identification of gamma-carboxyglutamic acid as a component of prothrombin. J Biol Chem 1974; 249: 6347-50.
    PubMedGoogle Scholar
  • 52 Stenflo J, Fernlund P, Egan W. et al. Vitamin K dependent modifications of glutamic acid residues in prothrombin. Proc Natl Acad Sci USA 1974; 71: 2730-33.
    CrossrefPubMedGoogle Scholar
  • 53 Mann KG, Nesheim ME, Church WR. et al. Surface-dependent reactions of the vitamin Kdependent enzyme complexes. Blood 1990; 76: 1-16.
    PubMedGoogle Scholar
  • 54 Sunnerhagen M, Forsen S, Hoffren AM. et al. Structure of the Ca(2+)-free Gla domain sheds light on membrane binding of blood coagulation proteins. Nat Struct Biol 1995; 02: 504-9.
    CrossrefPubMedGoogle Scholar
  • 55 Ratcliffe JV, Furie B, Furie BC. The importance of specific gamma-carboxyglutamic acid residues in prothrombin. Evaluation by site-specific mutagenesis. J Biol Chem 1993; 268: 24339-45.
    PubMedGoogle Scholar
  • 56 Selander-Sunnerhagen M, Ullner M, Persson E. et al. How an epidermal growth factor (EGF)-like domain binds calcium. High resolution NMR structure of the calcium form of the NH2-terminal EGF-like domain in coagulation factor X. J Biol Chem 1992; 267: 19642-9.
    PubMedGoogle Scholar
  • 57 Sunnerhagen M, Olah GA, Stenflo J. et al. The relative orientation of Gla and EGF domains in coagulation factor X is altered by Ca2+ binding to the first EGF domain. A combined NMR-small angle X-ray scattering study. Biochemistry 1996; 35: 11547-59.
    CrossrefPubMedGoogle Scholar
  • 58 Hertzberg MS, Ben-Tal O, Furie B. et al. Construction, expression, and characterization of a chimera of factor IX and factor X. The role of the second epidermal growth factor domain and serine protease domain in factor Va binding. J Biol Chem 1992; 267: 14759-66.
    PubMedGoogle Scholar
  • 59 Husten EJ, Esmon CT, Johnson AE. The active site of blood coagulation factor Xa. Its distance from the phospholipid surface and its conformational sensitivity to components of the prothrombinase complex. J Biol Chem 1987; 262: 12953-61.
    PubMedGoogle Scholar
  • 60 Sinha U, Wolf DL. Carbohydrate residues modulate the activation of coagulation factor X. J Biol Chem 1993; 268: 3048-51.
    PubMedGoogle Scholar
  • 61 Baugh RJ, Krishnaswamy S. Role of the activation peptide domain in human factor X activation by the extrinsic Xase complex. J Biol Chem 1996; 271: 16126-34.
    CrossrefPubMedGoogle Scholar
  • 62 Chattopadhyay A, James HL, Fair DS. Molecular recognition sites on factor Xa, which participate in the prothrombinase complex. J Biol Chem 1992; 267: 12323-9.
    PubMedGoogle Scholar
  • 63 Keogh JS, Shine R, Donnellan S. Phylogenetic relationships of terrestrial Australo-Papuan elapid snakes (subfamily Hydrophiinae) based on cytochrome b and 16S rRNA sequences. Mol Phylogenet Evol 1998; 10: 67-81.
    CrossrefPubMedGoogle Scholar