Download PDF
Thromb Haemost 2005; 93(03): 420-429
DOI: 10.1160/TH04-11-0707
Blood Coagulation, Fibrinolysis and Cellular Haemostasis
Schattauer GmbH

Gene duplication of coagulation factor V and origin of venom prothrombin activator in Pseudonaja textilis snake

Thi Nguyet Minh Le
1   Department of Biological Sciences, Faculty of Science, National University of Singapore, Singapore, Republic of Singapore
,
Md Abu Reza
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
,
R. Manjunatha Kini
1   Department of Biological Sciences, Faculty of Science, National University of Singapore, Singapore, Republic of Singapore
2   Department of Biochemistry and Molecular Biophysics, Medical College of Virginia, Virginia Commonwealth University, Richmond, Virginia, USA
› Author AffiliationsFinancial support: This work was supported by Academic research grants from the National University of Singapore, Singapore.
Further Information

Publication History

Received 02 November 2004

Accepted after revision 10 February 2004

Publication Date:
14 December 2017 (online)

Also available at
    Permissions and Reprints

Summary

The origin and evolution of venom toxins is a mystery that has evoked much interest. We have recently shown that pseutarin C, a prothrombin activator from Pseudonaja textilis venom, is structurally and functionally similar to mammalian coagulation factor Xa – factor Va complex. Its catalytic subunit is homologous to factor Xa while the nonenzymatic subunit is homologous to factor Va. P.textilis therefore has two parallel prothrombin activator systems: one expressed in its venom gland as a toxin and the other expressed in its liver and released into its plasma as a haemostatic factor. Here we report the complete amino acid sequence of factor V (FV) from its liver determined by cDNA cloning and sequencing. The liver FV shows 96% identity to pseutarin C nonenzymatic subunit. Most of the functional sites involved in its interaction with factor Xa and prothrombin are conserved. However, many potential sites of post-translational modifications and one critical cleavage site for activated protein C are different. The absence of the latter cleavage site makes pseutarin C nonenzymatic subunit resistant to inactivation and enhances its potential as an excellent toxin. By PCR and real-time quantitative analysis, we show that pseutarin C nonenzymatic subunit gene is expressed specifically in the venom gland at ~280 fold higher than that of FV gene in liver. These two are thus encoded by two separate genes that express in a highly tissue-specific manner. Our results imply that the gene encoding pseutarin C nonenzymatic subunit was derived by the duplication of plasma FV gene and they have evolved to perform distinct functions.

Keywords

Coagulation factor V - gene duplication - Pseudonaja textilis - venom toxin evolution
 

  • References

  • 1 Suttie JW, Jackson CM. Prothrombin structure, activation, and biosynthesis. Physiol Rev 1977; 57: 1-70.
    CrossrefPubMedGoogle Scholar
  • 2 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
  • 3 van Rijn JL. 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
  • 4 Mann KG. et al. Cofactor proteins in the assembly and expression of blood clotting enzyme complexes. Annu Rev Biochem 1988; 57: 915-56.
    CrossrefPubMedGoogle Scholar
  • 5 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
  • 6 Boskovic DS, Giles AR, Nesheim ME. Studies of the role of factor Va in the factor Xa-catalyzed activation of prothrombin, fragment 1.2-prethrombin-2, and dansyl-L-glutamyl-glycyl-L-arginine-meizothrombin in the absence of phospholipid. J Biol Chem 1990; 265: 10497-505.
    PubMedGoogle Scholar
  • 7 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: 3291-9.
    CrossrefPubMedGoogle Scholar
  • 8 Boskovic DS, Bajzar LS, Nesheim ME. Channeling during prothrombin activation. J Biol Chem 2001; 276: 28686-93.
    CrossrefPubMedGoogle Scholar
  • 9 Hutton RA, Warrell DA. Action of snake venom components on the haemostatic system. Blood Rev 1993; 7: 176-89.
    CrossrefPubMedGoogle Scholar
  • 10 Markland , Jr FS. Snake venoms. Drugs 1997; 54 (Suppl. 03) 1-10.
    PubMedGoogle Scholar
  • 11 Kini RM, Morita T, Rosing J. Classification and nomenclature of prothrombin activators isolated from snake venoms. Thromb Haemost 2001; 86: 710-1.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 12 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 Int 1988; 17: 825-35.
    PubMedGoogle Scholar
  • 13 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
  • 14 Speijer H. et al. Prothrombin activation by an activator from the venom of Oxyuranus scutellatus (Taipan snake). J Biol Chem 1986; 261: 13258-67.
    PubMedGoogle Scholar
  • 15 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
  • 16 Joseph JS. et al. Effect of snake venom procoagulants on snake plasma: implications for the coagulation cascade of snakes. Toxicon 2002; 40: 175-83.
    CrossrefPubMedGoogle Scholar
  • 17 Kane WH, Majerus PW. Purification and characterization of human coagulation factor V. J Biol Chem 1981; 256: 1002-7.
    PubMedGoogle Scholar
  • 18 Takahashi N, Bauman RA, Ortel TL. et al. Internal triplication in the structure of human ceruloplasmin. Proc Natl Acad Sci U S A 1983; 80: 115-9.
    CrossrefPubMedGoogle Scholar
  • 19 Adams TE, Hockin MF, Mann KG. et al. The crystal structure of activated protein C-inactivated bovine factor Va: Implications for cofactor function. Proc Natl Acad Sci U S A 2004; 101: 8918-23.
    CrossrefPubMedGoogle Scholar
  • 20 Zeibdawi AR. et al. Coagulation factor Va Glu- 96-Asp-111: a chelator-sensitive site involved in function and subunit association. Biochem J 2004; 377: 141-8.
    CrossrefPubMedGoogle Scholar
  • 21 Kalafatis M, Mann KG. The role of the membrane in the inactivation of factor va by plasmin. Amino acid region 307–348 of factor V plays a critical role in factor Va cofactor function. J Biol Chem 2001; 276: 18614-23.
    CrossrefPubMedGoogle Scholar
  • 22 Kalafatis M, Beck DO. Identification of a binding site for blood coagulation factor Xa on the heavy chain of factor Va. Amino acid residues 323–331 of factor V represent an interactive site for activated factor X. Biochemistry 2002; 41: 12715-28.
    CrossrefPubMedGoogle Scholar
  • 23 Poole S, Firtel RA, Lamar E. et al. Sequence and expression of the discoidin I gene family in Dictyostelium discoideum. J Mol Biol 1981; 153: 273-89.
    CrossrefPubMedGoogle Scholar
  • 24 Nicolaes GA, Villoutreix BO, Dahlback B. Mutations in a potential phospholipid binding loop in the C2 domain of factor V affecting the assembly of the prothrombinase complex. Blood Coagul Fibrinolysis 2000; 11: 89-100.
    CrossrefPubMedGoogle Scholar
  • 25 Guinto ER, Esmon CT, Mann KG. et al. The complete cDNA sequence of bovine coagulation factor V. J Biol Chem 1992; 267: 2971-8.
    PubMedGoogle Scholar
  • 26 Jenny RJ, Pittman DD, Toole JJ. et al. Complete cDNA and derived amino acid sequence of human factor V. Proc Natl Acad Sci U S A 1987; 84: 4846-50.
    CrossrefPubMedGoogle Scholar
  • 27 Yang TL, Cui J, Rehumtulla A. et al. The structure and function of murine factor V and its inactivation by protein C. Blood 1998; 91: 4593-9.
    PubMedGoogle Scholar
  • 28 Nazarenko IA, Bhatnagar SK, Hohman RJ. A closed tube format for amplification and detection of DNA based on energy transfer. Nucleic Acids Res 1997; 25: 2516-21.
    CrossrefPubMedGoogle Scholar
  • 29 Nesheim ME, Mann KG. Thrombin-catalyzed activation of single chain bovine factor V. J Biol Chem 1979; 254: 1326-34.
    PubMedGoogle Scholar
  • 30 Thorelli E, Kaufman RJ, Dahlback B. Cleavage requirements for activation of factor V by factor Xa. Eur J Biochem 1997; 247: 12-20.
    CrossrefPubMedGoogle Scholar
  • 31 Steen M, Dahlback B. Thrombin-mediated proteolysis of factor V resulting in gradual B-domain release and exposure of the factor Xa-binding site. J Biol Chem 2002; 277: 38424-30.
    CrossrefPubMedGoogle Scholar
  • 32 Kalafatis M, Beck DO, Mann KG. Structural requirements for expression of factor Va activity. J Biol Chem 2003; 278: 33550-61.
    CrossrefPubMedGoogle Scholar
  • 33 Orfeo T, Brufatto N, Nesheim ME. et al. The factor v activation paradox. J Biol Chem 2004; 279: 19580-91.
    CrossrefPubMedGoogle Scholar
  • 34 Kalafatis M, Rand MD, Mann KG. The mechanism of inactivation of human factor V and human factor Va by activated protein C. J Biol Chem 1994; 269: 31869-80.
    PubMedGoogle Scholar
  • 35 Mann KG, Hockin MF, Begin KJ. et al. Activated protein C cleavage of factor Va leads to dissociation of the A2 domain. J Biol Chem 1997; 272: 20678-83.
    CrossrefPubMedGoogle Scholar
  • 36 Esmon CT. Role of coagulation inhibitors in inflammation. Thromb Haemost 2001; 86: 51-6.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 37 Nicolaes GA. et al. Peptide bond cleavages and loss of functional activity during inactivation of factor Va and factor VaR506Q by activated protein C. J Biol Chem 1995; 270: 21158-66.
    CrossrefPubMedGoogle Scholar
  • 38 Kalafatis M, Mann KG. Role of the membrane in the inactivation of factor Va by activated protein C. J Biol Chem 1993; 268: 27246-57.
    PubMedGoogle Scholar
  • 39 van der Neut KM, Dirven RJ, Vos HL. et al. Factor Va is inactivated by activated protein C in the absence of cleavage sites at Arg-306, Arg-506, and Arg-679. J Biol Chem 2004; 279: 6567-75.
    CrossrefPubMedGoogle Scholar
  • 40 Nesheim ME, Canfield WM, Kisiel W. et al. Studies of the capacity of factor Xa to protect factor Va from inactivation by activated protein C. J Biol Chem 1982; 257: 1443-7.
    PubMedGoogle Scholar
  • 41 Xue J, Kalafatis M, Mann KG. Determination of the disulfide bridges in factor Va light chain. Biochemistry 1993; 32: 5917-23.
    CrossrefPubMedGoogle Scholar
  • 42 Xue J, Kalafatis M, Silveira JR, Kung C, Mann KG. Determination of the disulfide bridges in factor Va heavy chain. Biochemistry 1994; 33: 13109-16.
    CrossrefPubMedGoogle Scholar
  • 43 Bruin T, Sturk A, ten Cate JW. et al. The function of the human factor V carbohydrate moiety in blood coagulation. Eur J Biochem 1987; 170: 305-10.
    CrossrefPubMedGoogle Scholar
  • 44 Thorelli E, Kaufman RJ, Dahlback B. The C-terminal region of the factor V B-domain is crucial for the anticoagulant activity of factor V. J Biol Chem 1998; 273: 16140-5.
    CrossrefPubMedGoogle Scholar
  • 45 Kalafatis M. Identification and partial characterization of factor Va heavy chain kinase from human platelets. J Biol Chem 1998; 273: 8459-66.
    CrossrefPubMedGoogle Scholar
  • 46 Hortin GL. Sulfation of tyrosine residues in coagulation factor V. Blood 1990; 76: 946-52.
    PubMedGoogle Scholar
  • 47 Pittman DD, Tomkinson KN, Michnick D. et al. Posttranslational sulfation of factor V is required for efficient thrombin cleavage and activation and for full procoagulant activity. Biochemistry 1994; 33: 6952-9.
    CrossrefPubMedGoogle Scholar
  • 48 Rao VS, Swarup S, Manjunatha KR. 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. Thromb Haemost 2004; 92: 509-21.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 49 Ogawa T, Onoue H, Nakagawa K. et al. Localization and expression of phospholipases A2 in Trimeresurus flavoviridis (habu snake) venom gland. Toxicon 1995; 33: 1645-52.
    CrossrefPubMedGoogle Scholar
  • 50 Davidson CJ, Hirt RP, Lal K. et al. Molecular evolution of the vertebrate blood coagulation network. Thromb Haemost 2003; 89: 420-8.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 51 Toso R, Camire RM. Removal of B-domain sequences from factor V rather than specific proteolysis underlies the mechanism by which cofactor function is realized. J Biol Chem 2004; 279: 21643-50.
    CrossrefPubMedGoogle Scholar
  • 52 Ohno M. et al. Molecular evolution of snake toxins: is the functional diversity of snake toxins associated with a mechanism of accelerated evolution?. Prog Nucleic Acid Res Mol Biol 1998; 59: 307-64.
    PubMedGoogle Scholar