Pseutarin C, a Prothrombin Activator from Pseudonaja textilis Venom: Its Structural and Functional Similarity to Mammalian Coagulation Factor Xa-Va Complex

Veena S. Rao; R. Manjunatha Kini

doi:10.1055/s-0037-1613264

Thrombosis and Haemostasis, Table of Contents

Thromb Haemost 2002; 88(04): 611-619
DOI: 10.1055/s-0037-1613264

Review Article

Schattauer GmbH

Pseutarin C, a Prothrombin Activator from Pseudonaja textilis Venom: Its Structural and Functional Similarity to Mammalian Coagulation Factor Xa-Va Complex

Veena S. Rao

¹Department of Biological Sciences, Faculty of Science, National University of Singapore, Singapore, Republic of Singapore

,

R. Manjunatha Kini

¹Department of Biological Sciences, Faculty of Science, National University of Singapore, Singapore, Republic of Singapore

› Author Affiliations

Abstract

Summary

Several snake venoms contain procoagulant proteins that can activate prothrombin. We have purified pseutarin C, a prothrombin activator from the venom of the Australian brown snake (Pseudonaja textilis). It converts prothrombin to thrombin by cleaving both the peptide bonds Arg₂₇₄ – Thr₂₇₅ and Arg₃₂₃ – Ile_324, similar to mammalian factor Xa. It is a protein complex (∼250 Kd) consisting of an enzymatic and a nonenzymatic subunit. These subunits were separated by reverse phase HPLC and their interactions with bovine factor Xa and factor Va were studied. The enzymatic subunit of pseutarin C has a ∼13 fold higher affinity for bovine factor Va (K _d of 11.4 nM for pseutarin C enzymatic subunit – bovine factor Va interaction as compared to a K _d of 147.4 nM for the bovine factor Xa-Va interaction). The non-enzymatic component, however, was unable to activate bovine factor Xa. N-terminal sequence analysis of the catalytic subunit of pseutarin C showed ∼ 60% homology to mammalian factor Xa and ∼78% homology to trocarin, a group D prothrombin activator from Tropidechis carinatus venom. Structural information for the non-enzymatic subunit of pseutarin C was obtained by amino terminal sequencing of several internal peptides. The sequence data obtained indicates that the non-enzymatic subunit of pseutarin C has similar domain architecture like the mammalian factor Va and the overall homology is ∼55%. Thus pseutarin C is the first venom procoagulant protein that is structurally and functionally similar to mammalian factor Xa-Va complex.

Keywords

Prothrombinase complex - eastern brown snake - blood coagulation - thrombin formation - procoagulant toxin

Full Text

References

References
1 Suttie JW, Jackson CM. Prothrombin structure, activation, and biosynthesis Physiol Rev. 1977; 57: 1-70.
2 Rosing J, Tans G, Govers-Riemslag JWP, Zwaal RFA, Hemker HC. The role of phospholipids and factor Va in the prothrombinase complex. J Biol Chem 1980; 255: 274-83.
3 Mann KG, Krishnaswamy S, Lawson JH. Surface-dependent hemostasis. Semin Hematol 1992; 29: 213-26.
4 van Rijn JL, Govers-Riemslag JWP, Zwaal RFA, Rosing J. Kinetic studies of prothrombin activation: effect of factor Va and phospholipids on the formation of the enzyme-substrate complex. Biochemistry 1984; 23: 557-64.
5 Rosing J, Tans G. Inventory of exogenous prothrombin activators For the Subcommittee on Nomenclature of Exogenous Hemostatic Factors of the Scientific and Standardization Committee of the International Society on Thrombosis and Haemostasis. Thromb Haemost 1991; 65: 627-30.
6 Markland Jr FS. Snake venoms. Drugs 1997; 54: 1-10.
7 Hutton RA, Warrell DA. Action of snake venom components on the haemostatic system. Blood Rev 1993; 07: 176-89.
8 Rosing J, Tans G. Structural and functional properties of snake venom prothrombin activators. Toxicon 1992; 30: 1515-27.
9 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.
10 Kini RM, Morita T, Rosing J. Classification and nomenclature of prothrombin activators isolated from snake venoms. Thromb Haemost 2001; 85: 710-1.
11 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.
12 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.
13 Tans G, Govers-Riemslag JWP, van Rihn JLML, Rosing J. Purification and properties of a prothrombin activator from the venom of Notechis scutatus scutatus . J Biol Chem 1985; 260: 9366-72.
14 Joseph JS, Chung MC, Jeyaseelan K, Kini RM. Amino acid sequence of trocarin, a prothrombin activator from Tropidechis carinatus venom: its structural similarity to coagulation factor Xa. Blood 1999; 94: 621-31.
15 Speijer H, Govers-Riemslag JWP, Zwaal RFA, Rosing J. Prothrombin activation by an activator from the venom of Oxyuranus scutellatus (Taipan snake). J Biol Chem 1986; 261: 13258-67.
16 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.
17 Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970; 227: 680-5.
18 Towbin H, Staehelin T, Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications Biotechnology. 1992; 24: 145-9.
19 Inglis AS. Cleavage at aspartic acid. Meth Enzymol 1983; 91: 324-32.
20 Pirkle H, Marsh N. Nomenclature of exogenous hemostatic factors. Thromb Haemost 1991; 66: 264.
21 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.
22 Pryzdial EL, Mann KG. The association of coagulation factor Xa and factor Va. J Biol Chem 1991; 266: 8969-77.
23 Kerns RT, Kini RM, Stefansson S, Evans HJ. Targeting of venom phospholipases: the strongly anticoagulant phospholipase A₂ from Naja nigricollis venom binds to coagulation factor Xa to inhibit the prothrombinase complex. Arch Biochem Biophys 1999; 369: 107-13.
24 Kane WH, Davie EW. Cloning of a cDNA coding for human factor V, a blood coagulation factor homologous to factor VIII and ceruloplasmin. Proc Natl Acad Sci USA 1986; 83: 6800-4.
25 Guinto ER, Esmon CT, Mann KG, MacGillivray RT. The complete cDNA sequence of bovine coagulation factor V. J Biol Chem 1992; 267: 2971-8.
26 Jenny RJ, Pittman DD, Toole JJ, Kriz RW, Aldape RA, Hewick RM, Kaufman RJ, Mann KG. Complete cDNA and derived amino acid sequence of human factor V. Proc Natl Acad Sci USA 1987; 84: 4846-50.
27 Joseph JS, Chung MC, Mirtschin PJ, Kini RM. Effect of snake venom procoagulants on snake plasma: implications for the coagulation cascade of snakes. Toxicon 2002; 40: 175-83.