The influence of sodium ion binding on factor IXa activity

Kota Gopalakrishna; Alireza R. Rezaie

doi:10.1160/TH06-03-0156

Thrombosis and Haemostasis, Table of Contents

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

¹Edward A. Doisy Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, St. Louis, Missouri, USA

,

Alireza R. Rezaie

¹Edward A. Doisy Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, St. Louis, Missouri, USA

› Author Affiliations

Abstract

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

Full Text

References

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.
2 Furie B, Furie BC. The molecular basis of blood coagulation. Cell 1988; 53: 505-18.
3 Davie EW, Fujikawa K, Kisiel W. The coagulation cascade: Initiation, maintenance and regulation. Biochemistry 1991; 30: 10363-70.
4 Vehar GA, Keyt B, Eaton D. et al. Structure of human factor VIII. Nature 1984; 312: 337-42.
5 Lollar P, Fay PJ, Fass DN. Factor VIII and factor VIIIa. Methods Enzymol 1993; 222: 128-43.
6 Thompson AR. Structure, function, and molecular defects of factor IX. Blood 1986; 67: 565-72.
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.
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.
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.
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.
11 Hamaguchi N, Charifson PS, Pedersen LG. et al. Expression and characterization of human factor IX. J Biol Chem 1991; 266: 15213-20.
12 Stenflo J. Structure-function relationships of epidermal growth factor modules in vitamin K-dependent clotting factors. Blood 1991; 78: 1637-51.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
24 Rezaie AR, He X. Sodium binding site of factor Xa: Role of sodium in the prothrombinase complex. Biochemistry 2000; 39: 1817-25.
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.
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.
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.
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.
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.
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.
31 Yang L, Manithody C, Rezaie AR. Localization of the heparin binding exosite of factor IXa. J Biol Chem 2002; 277: 50756-60.
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.
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.
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.
35 Di Cera E, Guinto ER, Vindigni A. et al. The Na⁺ binding site of thrombin. J Biol Chem 1995; 270: 22089-92.
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.
37 Esmon CT. Molecular events that control the protein C anticoagulant pathway. Thromb Haemost 1993; 70: 1-5.
38 Wells CM, Di Cera E. Thrombin isa Na⁺-activated enzyme. Biochemistry 1992; 31: 11721-30.
39 He X, Rezaie AR. Identification and characterization of the sodium-binding site of activated protein C. J Biol Chem 1999; 274: 4970-6.
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.
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.
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.
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.