Download PDF
Thromb Haemost 2006; 95(06): 936-941
DOI: 10.1160/TH06-03-0156
Blood Coagulation, Fibrinolysis and Cellular Haemostasis
Schattauer GmbH

The influence of sodium ion binding on factor IXa activity

Kota Gopalakrishna
1   Edward A. Doisy Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, St. Louis, Missouri, USA
,
Alireza R. Rezaie
1   Edward A. Doisy Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, St. Louis, Missouri, USA
› Author AffiliationsFinancial support: The research discussed herein was supported by grants awarded by the National Heart, Lung, and Blood Institute of the National Institutes of Health (HL 62565 and HL 68571 to ARR).
Further Information

Publication History

Received 15 March 2006

Accepted after revision 22 April 2006

Publication Date:
30 November 2017 (online)

    Permissions and Reprints

Summary

Sodium plays an important role in modulating both the amidolytic and proteolytic activities of thrombin. By contrast, while the optimal amidolytic activity of factor Xa requires Na+, the proteolytic activity of factor Xa in the prothrombinase complex is minimally affected by the monovalent cation. In this study, we analyzed the effect of Na+ on the amidolytic and proteolytic activity of factor IXa in the absence and presence of factor VIIIa. Factor IXa exhibited normal activity towards a fIXa-specific chromogenic substrate and antithrombin in the presence of physiological concentrations of Ca2+ with no obvious requirement for Na+ in either reaction. Further studies revealed that factor IXa binds to its cofactor factor VIIIa with a normal affinity in the absence of Na+ and that the catalytic function in the intrinsic Xase complex is also independent of Na+ in the presence of physiological concentrations of Ca2+.These results suggest that unlike the important role that Na+ plays in modulating the macromolecular substrate specificity of thrombin, the monovalent cation is not required for the physiological function of factor IXa in the intrinsic Xase complex.

Keywords

Factor IXa - factor X - intrinsic Xase - sodium - calcium
 

  • References

  • 1 Mann KG, Jenny RJ, Krishnaswamy S. Cofactor proteins in the assembly and expression of blood clotting enzyme complexes. Ann Rev Biochem 1988; 57: 915-56.
    CrossrefPubMedGoogle Scholar
  • 2 Furie B, Furie BC. The molecular basis of blood coagulation. Cell 1988; 53: 505-18.
    CrossrefPubMedGoogle Scholar
  • 3 Davie EW, Fujikawa K, Kisiel W. The coagulation cascade: Initiation, maintenance and regulation. Biochemistry 1991; 30: 10363-70.
    CrossrefPubMedGoogle Scholar
  • 4 Vehar GA, Keyt B, Eaton D. et al. Structure of human factor VIII. Nature 1984; 312: 337-42.
    CrossrefPubMedGoogle Scholar
  • 5 Lollar P, Fay PJ, Fass DN. Factor VIII and factor VIIIa. Methods Enzymol 1993; 222: 128-43.
    PubMedGoogle Scholar
  • 6 Thompson AR. Structure, function, and molecular defects of factor IX. Blood 1986; 67: 565-72.
    PubMedGoogle Scholar
  • 7 Yoshitake S, Schach BG, Foster DC. et al. Nucleotide sequence of the gene for human factor IX (antihemophilic factor B). Biochemistry 1985; 24: 3736-50.
    CrossrefPubMedGoogle Scholar
  • 8 Kurachi K, Davie EW. Isolation and characterization of a cDNA coding for human Factor IX. Proc Natl Acad Sci USA 1982; 79: 6461-4.
    CrossrefPubMedGoogle Scholar
  • 9 Osterud B, Rapaport SI. Activation of factor IX by the reaction product of tissue factor and factor VII: an additional pathway for initiating blood coagulation. Proc Natl Acad Sci USA 1977; 74: 5260-4.
    CrossrefPubMedGoogle Scholar
  • 10 Lindquist PA, Fujikawa K, Davie EW. Activation of bovine factor IX (Christmas factor) by factor XIa (activated plasma thromboplastin antecedent and a protease from Russell’s viper venom. J Biol Chem 1978; 253: 1902-9.
    PubMedGoogle Scholar
  • 11 Hamaguchi N, Charifson PS, Pedersen LG. et al. Expression and characterization of human factor IX. J Biol Chem 1991; 266: 15213-20.
    PubMedGoogle Scholar
  • 12 Stenflo J. Structure-function relationships of epidermal growth factor modules in vitamin K-dependent clotting factors. Blood 1991; 78: 1637-51.
    PubMedGoogle Scholar
  • 13 Mertens K, Celie PHN, Kolkman JA. et al. Factor VIII-factor IX interactions: molecular sites involved in enzyme-cofactor complex assembly. Thromb Haemost 1999; 82: 209-17.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 14 Mutucumarana VP, Duffy EJ, Lollar P. et al. The active site of factor IXa is located far above the membrane surface and its conformation is altered upon association with factor VIIIa: A fluorescence study. J Biol Chem 1992; 267: 17012-21.
    PubMedGoogle Scholar
  • 15 Wilkinson FH, Ahmad SS, Walsh PN. The factor IXa second epidermal growth factor (EGF2) domain mediates platelet binding and assembly of the factor X activating complex. J Biol Chem 2002; 277: 5734-41.
    CrossrefPubMedGoogle Scholar
  • 16 Hopfner K-P, Lang A, Karcher A. et al. Coagulation factor IXa: the relaxed conformation of Tyr99 blocks substrate binding. Structure 1999; 07: 989-96.
    CrossrefPubMedGoogle Scholar
  • 17 Blostein MD, Furie BC, Rajotte I. et al. The Gla domain of factor IXa binds to factor VIIIa in the tenase complex. J Biol Chem 2003; 278: 31297-02.
    CrossrefPubMedGoogle Scholar
  • 18 Chang J, Jin J, Lollar P. et al. Changing residue 338 in human factor IX from arginine to alanine causes an increase in catalytic activity. J Biol Chem 1998; 273: 12089-94.
    CrossrefPubMedGoogle Scholar
  • 19 Kolkman JA, Mertens K. Surface-loop residue Lys316 in blood coagulation factor IX is a major determinant for factor X but not antithrombin recognition. Biochem J 2000; 350: 701-7.
    CrossrefPubMedGoogle Scholar
  • 20 Sichler K, Kopetzki E, Huber R. et al. Physiological fIXa activation involves a cooperative conformational rearrangement of the 99-loop. J Biol Chem 2003; 278: 4121-6.
    CrossrefPubMedGoogle Scholar
  • 21 Wilkinson FH, London FS, Walsh PN. Residues 88-109 of factor IXa are important for assembly of factor X activating complex. J Biol Chem 2002; 277: 5725-33.
    CrossrefPubMedGoogle Scholar
  • 22 Dang QD, Vindigni A, Di Cera E. An allosteric switch controls the procoagulant and anticoagulant activities of thrombin. Proc Natl Acad Sci USA 1995; 92: 5977-81.
    CrossrefPubMedGoogle Scholar
  • 23 Dang QD, Di Cera E. Residue 225 determines the Na+-induced allosteric regulation of catalytic activity in serine proteases. Proc Natl Acad Sci USA 1996; 93: 10653-6.
    CrossrefPubMedGoogle Scholar
  • 24 Rezaie AR, He X. Sodium binding site of factor Xa: Role of sodium in the prothrombinase complex. Biochemistry 2000; 39: 1817-25.
    CrossrefPubMedGoogle Scholar
  • 25 Brandstetter H, Bauer M, Huber R. et al. X-ray structure of clotting factor IXa: Active site and module structure related to Xase activity and hemophilia B. Proc Natl Acad Sci USA 1995; 92: 9796-800.
    CrossrefPubMedGoogle Scholar
  • 26 Rezaie AR, Mather T, Sussman F. et al. Mutation of Glu 80 [to] Lys results in a protein C mutant that no longer requires Ca2+ for rapid activation by the thrombin-thrombomodulin complex. J Biol Chem 1994; 269: 3151-4.
    PubMedGoogle Scholar
  • 27 Rezaie AR, Esmon CT. Asp-70 to Lys mutant of factor X lacks the high affinity Ca2+ binding site yet retains function. J Biol Chem 1994; 269: 21495-9.
    PubMedGoogle Scholar
  • 28 Schmidt AE, Stewart JE, Mathur A. et al. Na+ site in blood coagulation factor IXa: Effect on catalysis and factor VIIIa binding. J. Mol Biol 2005; 350: 78-91.
    CrossrefPubMedGoogle Scholar
  • 29 Smirnov MD, Esmon CT. Phosphatidylethanolamine incorporation into vesicles selectively enhances factor Va inactivation by activated protein C. J Biol Chem 1994; 269: 816-9.
    PubMedGoogle Scholar
  • 30 Neuenschwander PF, Bianco-Fisher E, Rezaie AR. et al. Phosphatidylethanolamine augments factor VIIa-tissue factor activity: enhancement of sensitivity to phosphatidylserine. Biochemistry 1995; 34: 13988-93.
    CrossrefPubMedGoogle Scholar
  • 31 Yang L, Manithody C, Rezaie AR. Localization of the heparin binding exosite of factor IXa. J Biol Chem 2002; 277: 50756-60.
    CrossrefPubMedGoogle Scholar
  • 32 Yang L, Manithody C, Olson ST. et al. Contribution of basic residues of the autolysis loop to the substrate and inhibitor specificity of factor IXa. J Biol Chem 2003; 278: 25032-8.
    CrossrefPubMedGoogle Scholar
  • 33 Jenkins PV, Freas J, Schmidt KM. et al. Mutations associated with hemophilia A in the 558-565 loop of the factor VIIIa A2 subunit alter the catalytic activity of the factor Xase complex. Blood 2002; 100: 501-8.
    CrossrefPubMedGoogle Scholar
  • 34 Petrovan RJ, Ruf W. Role of residue Phe225 in the cofactor-mediated, allosteric regulation of the serine protease coagulation factor VIIa. Biochemistry 2000; 39: 14457-63.
    CrossrefPubMedGoogle Scholar
  • 35 Di Cera E, Guinto ER, Vindigni A. et al. The Na+ binding site of thrombin. J Biol Chem 1995; 270: 22089-92.
    CrossrefPubMedGoogle Scholar
  • 36 Guinto ER, Caccia S, Rose T. et al. Unexpected crucial role of residue 225 in serine proteases. Proc Natl Acad Sci USA 1999; 96: 1852-7.
    CrossrefPubMedGoogle Scholar
  • 37 Esmon CT. Molecular events that control the protein C anticoagulant pathway. Thromb Haemost 1993; 70: 1-5.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 38 Wells CM, Di Cera E. Thrombin isa Na+-activated enzyme. Biochemistry 1992; 31: 11721-30.
    CrossrefPubMedGoogle Scholar
  • 39 He X, Rezaie AR. Identification and characterization of the sodium-binding site of activated protein C. J Biol Chem 1999; 274: 4970-6.
    CrossrefPubMedGoogle Scholar
  • 40 Steiner SA, Castellino FJ. Kinetic studies of the role of monovalent cations in the amidolytic activity of activated bovine plasma protein C. Biochemistry 1982; 21: 4609-14.
    CrossrefPubMedGoogle Scholar
  • 41 Camire RM. Prothrombinase assembly and S1 site occupancy restore the catalytic activity of FXa impaired by mutation at the sodium-binding site. J Biol Chem 2002; 277: 37863-70.
    CrossrefPubMedGoogle Scholar
  • 42 Underwood MC, Zhong D, Mathur A. et al. Thermodynamic linkage between the S1 site, the Na+ site, and the Ca2+ site in the protease domain of human factor Xa. J Biol Chem 2000; 275: 36876-84.
    CrossrefPubMedGoogle Scholar
  • 43 Rezaie AR, Kittur FS. The critical role of the 185-189-loop in the factor Xa interaction with Na+ and factor Va in the prothrombinase complex. J Biol Chem 2004; 279: 48262-9.
    CrossrefPubMedGoogle Scholar