Journal Mobile Options
Table of Contents
Vol. 34, No. 4-5, 2005
Issue release date: May 2006
Pathophysiol Haemos Thromb 2005;34:184–187
(DOI:10.1159/000092420)

Hemextin AB Complex – A Snake Venom Anticoagulant Protein Complex That Inhibits Factor VIIa Activity

Banerjee Y.a · Mizuguchi J.b · Iwanaga S.b · Kini R.M.a, c
aProtein Science Laboratory, Department of Biological Sciences, Faculty of Science, National University of Singapore, Singapore; bBlood Products Research Department, The Chemo-Sero-Therapeutic Research Institute, Kumamoto, Japan; cDepartment of Biochemistry, VCU Medical Center, Medical College of Virginia, Virginia Commonwealth University, Richmond, Va., USA
email Corresponding Author

Abstract

Snake venom is a veritable gold mine of bioactive molecules, capable of binding to a wide variety of pharmacological targets, including the blood coagulation cascade. Here, we report the isolation and characterization of two synergistically acting anticoagulant three-finger proteins, hemextin A and hemextin B, from the venom of Hemachatus haemachatus (African Ringhals cobra). Hemextin A but not hemextin B exhibits mild anticoagulant activity. However, hemextin B interacts with hemextin A and forms a complex (hemextin AB complex), and synergistically enhances its anticoagulant potency. Prothrombin time assay showed that these two proteins form a 1:1 complex. Using a ‘a dissection approach’, we found that hemextins A and AB complex prolong clotting by inhibiting extrinsic tenase activity. Further studies showed that hemextin AB complex potently inhibits the proteolytic activity of factor VIIa (FVIIa) and its complexes. Kinetic studies showed that hemextin AB complex is a non-competitive inhibitor of FVIIa-soluble tissue factor proteolytic activity with a Ki of 25 nM. Hemextin AB complex is the first reported natural inhibitor of FVIIa that does not require either tissue factor or factor Xa scaffold to mediate its inhibitory activity. Molecular interactions of hemextin AB complex with FVIIa/tissue factor-FVIIa may provide a new paradigm in the search for anticoagulants inhibiting the initiation of blood coagulation.


 goto top of outline Key Words

  • Anticoagulant
  • Synergism
  • Extrinsic tenase complex
  • Factor VIIa
  • Tissue factor
  • Three-finger toxin
  • Snake venom

 goto top of outline Abstract

Snake venom is a veritable gold mine of bioactive molecules, capable of binding to a wide variety of pharmacological targets, including the blood coagulation cascade. Here, we report the isolation and characterization of two synergistically acting anticoagulant three-finger proteins, hemextin A and hemextin B, from the venom of Hemachatus haemachatus (African Ringhals cobra). Hemextin A but not hemextin B exhibits mild anticoagulant activity. However, hemextin B interacts with hemextin A and forms a complex (hemextin AB complex), and synergistically enhances its anticoagulant potency. Prothrombin time assay showed that these two proteins form a 1:1 complex. Using a ‘a dissection approach’, we found that hemextins A and AB complex prolong clotting by inhibiting extrinsic tenase activity. Further studies showed that hemextin AB complex potently inhibits the proteolytic activity of factor VIIa (FVIIa) and its complexes. Kinetic studies showed that hemextin AB complex is a non-competitive inhibitor of FVIIa-soluble tissue factor proteolytic activity with a Ki of 25 nM. Hemextin AB complex is the first reported natural inhibitor of FVIIa that does not require either tissue factor or factor Xa scaffold to mediate its inhibitory activity. Molecular interactions of hemextin AB complex with FVIIa/tissue factor-FVIIa may provide a new paradigm in the search for anticoagulants inhibiting the initiation of blood coagulation.

Copyright © 2005 S. Karger AG, Basel


 goto top of outline References
  1. Hutton RA, Warrell DA: Action of snake venom components on the haemostatic system. Blood Rev 1993;7:176–189.
  2. Markland FS: Snake venoms and the hemostatic system. Toxicon 1998;36:1749–1800.
  3. Langdell RD, Wagner RH, Brinkhous KM: Effect of antihemophilic factor on one-stage clotting tests; a presumptive test for hemophilia and a simple one-stage antihemophilic factor assy procedure. J Lab Clin Med 1953;41:637–647.
  4. Quick AJ: The prothrombin time in haemophilia and in obstructive jaudice. J Biol Chem 1935;109:73–74.
  5. Hougie C: Effect of Russell’s viper venom (stypven) on Stuart clotting defect. Proc Soc Exp Biol Med 1956;98:570–573.
  6. Jim R: A study of the plasma thrombin time. J Lab Clin Med 1957;50:45–60.
  7. Condrea E, Fletcher JE, Rapuano BE, Yang CC, Rosenberg P: Effect of modification of one histidine residue on the enzymatic and pharmacological properties of a toxic phospholipase A2 from Naja nigricollis snake venom and less toxic phospholipases A2 from Hemachatus haemachatus and Naja atra snake venoms. Toxicon 1981;19:61–71.
  8. Kini RM, Banerjee Y: Dissection approach: a simple strategy for the identification of the step of action of anticoagulant agents in the blood coagulation cascade. J Thromb Haemost 2005;3:170–171.
  9. Stefansson S, Kini RM, Evans HJ: The inhibition of clotting complexes of the extrinsic coagulation cascade by the phospholipase A2 isoenzymes from Naja nigricollis venom. Thromb Res 1989;55:481–491.
  10. Kini RM, Evans HJ: The role of enzymatic activity in inhibition of the extrinsic tenase complex by phospholipase A2 isoenzymes from Naja nigricollis venom. Toxicon 1995;33:1585–1590.
  11. Doorty KB, Bevan S, Wadsworth JD, Strong PN: A novel small conductance Ca2+-activated K+ channel blocker from Oxyuranus scutellatus taipan venom. Re-evaluation of taicatoxin as a selective Ca2+ channel probe. J Biol Chem 1997;272:19925–19930.
  12. Habermann E, Breithaupt H: Mini-review. The crotoxin complex – an example of biochemical and pharmacological protein complementation. Toxicon 1978;16:19–30.
  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;88:611–619.
  14. Wang R, Kini RM, Chung MC: Rhodocetin, a novel platelet aggregation inhibitor from the venom of Calloselasma rhodostoma (Malayan pit viper): synergistic and noncovalent interaction between its subunits. Biochemistry 1999;38:7584–7593.
  15. Lindhout T, Franssen J, Willems G: Kinetics of the inhibition of tissue factor-factor VIIa by tissue factor pathway inhibitor. Thromb Haemost 1995;74:910–915.
  16. Lee AY, Vlasuk GP: Recombinant nematode anticoagulant protein c2 and other inhibitors targeting blood coagulation factor VIIa/tissue factor. J Intern Med 2003;254:313–321.

 goto top of outline Author Contacts

R. Manjunatha Kini
Protein Science Laboratory, Department of Biological Sciences, Faculty of Science
National University of Singapore
117 543 (Singapore)
Tel. +65 6874 5235, Fax +65 6779 2486, E-Mail dbskinim@nus.edu.sg


 goto top of outline Article Information

This work is supported by a Program grant from Biomedical Research Council, Singapore.

Number of Print Pages : 4
Number of Figures : 2, Number of Tables : 0, Number of References : 16


 goto top of outline Publication Details

Pathophysiology of Haemostasis and Thrombosis

Vol. 34, No. 4-5, Year 2005 (Cover Date: May 2006)

Journal Editor: Rosing, J. (Maastricht)
ISSN: 1424–8832 (print), 1424–8840 (Online)

For additional information: http://www.karger.com/PHT


Copyright / Drug Dosage / Disclaimer

Copyright: All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher or, in the case of photocopying, direct payment of a specified fee to the Copyright Clearance Center.
Drug Dosage: The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in goverment regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any changes in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug.
Disclaimer: The statements, opinions and data contained in this publication are solely those of the individual authors and contributors and not of the publishers and the editor(s). The appearance of advertisements or/and product references in the publication is not a warranty, endorsement, or approval of the products or services advertised or of their effectiveness, quality or safety. The publisher and the editor(s) disclaim responsibility for any injury to persons or property resulting from any ideas, methods, instructions or products referred to in the content or advertisements.

Abstract

Snake venom is a veritable gold mine of bioactive molecules, capable of binding to a wide variety of pharmacological targets, including the blood coagulation cascade. Here, we report the isolation and characterization of two synergistically acting anticoagulant three-finger proteins, hemextin A and hemextin B, from the venom of Hemachatus haemachatus (African Ringhals cobra). Hemextin A but not hemextin B exhibits mild anticoagulant activity. However, hemextin B interacts with hemextin A and forms a complex (hemextin AB complex), and synergistically enhances its anticoagulant potency. Prothrombin time assay showed that these two proteins form a 1:1 complex. Using a ‘a dissection approach’, we found that hemextins A and AB complex prolong clotting by inhibiting extrinsic tenase activity. Further studies showed that hemextin AB complex potently inhibits the proteolytic activity of factor VIIa (FVIIa) and its complexes. Kinetic studies showed that hemextin AB complex is a non-competitive inhibitor of FVIIa-soluble tissue factor proteolytic activity with a Ki of 25 nM. Hemextin AB complex is the first reported natural inhibitor of FVIIa that does not require either tissue factor or factor Xa scaffold to mediate its inhibitory activity. Molecular interactions of hemextin AB complex with FVIIa/tissue factor-FVIIa may provide a new paradigm in the search for anticoagulants inhibiting the initiation of blood coagulation.



 goto top of outline Author Contacts

R. Manjunatha Kini
Protein Science Laboratory, Department of Biological Sciences, Faculty of Science
National University of Singapore
117 543 (Singapore)
Tel. +65 6874 5235, Fax +65 6779 2486, E-Mail dbskinim@nus.edu.sg


 goto top of outline Article Information

This work is supported by a Program grant from Biomedical Research Council, Singapore.

Number of Print Pages : 4
Number of Figures : 2, Number of Tables : 0, Number of References : 16


 goto top of outline Publication Details

Pathophysiology of Haemostasis and Thrombosis

Vol. 34, No. 4-5, Year 2005 (Cover Date: May 2006)

Journal Editor: Rosing, J. (Maastricht)
ISSN: 1424–8832 (print), 1424–8840 (Online)

For additional information: http://www.karger.com/PHT


Copyright / Drug Dosage

Copyright: All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher or, in the case of photocopying, direct payment of a specified fee to the Copyright Clearance Center.
Drug Dosage: The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in goverment regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any changes in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug.
Disclaimer: The statements, opinions and data contained in this publication are solely those of the individual authors and contributors and not of the publishers and the editor(s). The appearance of advertisements or/and product references in the publication is not a warranty, endorsement, or approval of the products or services advertised or of their effectiveness, quality or safety. The publisher and the editor(s) disclaim responsibility for any injury to persons or property resulting from any ideas, methods, instructions or products referred to in the content or advertisements.

References

  1. Hutton RA, Warrell DA: Action of snake venom components on the haemostatic system. Blood Rev 1993;7:176–189.
  2. Markland FS: Snake venoms and the hemostatic system. Toxicon 1998;36:1749–1800.
  3. Langdell RD, Wagner RH, Brinkhous KM: Effect of antihemophilic factor on one-stage clotting tests; a presumptive test for hemophilia and a simple one-stage antihemophilic factor assy procedure. J Lab Clin Med 1953;41:637–647.
  4. Quick AJ: The prothrombin time in haemophilia and in obstructive jaudice. J Biol Chem 1935;109:73–74.
  5. Hougie C: Effect of Russell’s viper venom (stypven) on Stuart clotting defect. Proc Soc Exp Biol Med 1956;98:570–573.
  6. Jim R: A study of the plasma thrombin time. J Lab Clin Med 1957;50:45–60.
  7. Condrea E, Fletcher JE, Rapuano BE, Yang CC, Rosenberg P: Effect of modification of one histidine residue on the enzymatic and pharmacological properties of a toxic phospholipase A2 from Naja nigricollis snake venom and less toxic phospholipases A2 from Hemachatus haemachatus and Naja atra snake venoms. Toxicon 1981;19:61–71.
  8. Kini RM, Banerjee Y: Dissection approach: a simple strategy for the identification of the step of action of anticoagulant agents in the blood coagulation cascade. J Thromb Haemost 2005;3:170–171.
  9. Stefansson S, Kini RM, Evans HJ: The inhibition of clotting complexes of the extrinsic coagulation cascade by the phospholipase A2 isoenzymes from Naja nigricollis venom. Thromb Res 1989;55:481–491.
  10. Kini RM, Evans HJ: The role of enzymatic activity in inhibition of the extrinsic tenase complex by phospholipase A2 isoenzymes from Naja nigricollis venom. Toxicon 1995;33:1585–1590.
  11. Doorty KB, Bevan S, Wadsworth JD, Strong PN: A novel small conductance Ca2+-activated K+ channel blocker from Oxyuranus scutellatus taipan venom. Re-evaluation of taicatoxin as a selective Ca2+ channel probe. J Biol Chem 1997;272:19925–19930.
  12. Habermann E, Breithaupt H: Mini-review. The crotoxin complex – an example of biochemical and pharmacological protein complementation. Toxicon 1978;16:19–30.
  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;88:611–619.
  14. Wang R, Kini RM, Chung MC: Rhodocetin, a novel platelet aggregation inhibitor from the venom of Calloselasma rhodostoma (Malayan pit viper): synergistic and noncovalent interaction between its subunits. Biochemistry 1999;38:7584–7593.
  15. Lindhout T, Franssen J, Willems G: Kinetics of the inhibition of tissue factor-factor VIIa by tissue factor pathway inhibitor. Thromb Haemost 1995;74:910–915.
  16. Lee AY, Vlasuk GP: Recombinant nematode anticoagulant protein c2 and other inhibitors targeting blood coagulation factor VIIa/tissue factor. J Intern Med 2003;254:313–321.