Download PDF
Thromb Haemost 2000; 84(04): 626-634
DOI: 10.1055/s-0037-1614078
Review Article
Schattauer GmbH

Hemophilia B with Mutations at Glycine-48 of Factor IX Exhibited Delayed Activation by the Factor VIIa-tissue Factor Complex

Pai-Chih Wu
1   From the Graduate Institute of Medical Technology, National Taiwan University, College of Medicine, Taiwan, ROC
,
N. Hamaguchi
2   Brandeis University, Boston, USA
,
Yi-Shing Yu
1   From the Graduate Institute of Medical Technology, National Taiwan University, College of Medicine, Taiwan, ROC
,
Ming-Ching Shen
3   Dept. of Laboratory Medicine, National Taiwan University, College of Medicine, Taiwan, ROC
,
Shu-Wha Lin
1   From the Graduate Institute of Medical Technology, National Taiwan University, College of Medicine, Taiwan, ROC
› Author Affiliations
This work was supported by grants to Dr. S. W. Lin from the National Science Council, Republic of China on Taiwan, grant nos. NSC84-2331-B002234, NSC85-2331-B002-213-M02, NSC86-2732-B-002-004, and NSC86-2314-B-002-145.
The authors would like to thank Dr. Johan Stenflo and Dr. Peter Larson for help in the Gla analysis. Also many thanks to Chia-Ni Lin, Chia-Wei Chang, and Chia-Cheng Lee for technical assistance. We are also grateful to Misses Mei-Hua Hung and Yin-Chi Wang at the coagulation laboratory of the National Taiwan University Hospital for preparation of normal pooled plasma and for technical assistance. This work was supported by grants to Dr. S. W. Lin from the National Science Council, Republic of China on Taiwan, grant nos. NSC84-2331-B002-234, NSC85-2331-B002-213-M02, NSC86-2732-B-002-004, and NSC86-2314-B-002-145.
Further Information

Publication History

Received 21 January 2000

Accepted after resubmission 02 May 2000

Publication Date:
11 December 2017 (online)

Also available at
   Permissions and Reprints

Summary

Gly-48 is in the conserved DGDQC sequence (residues 47-51 of human factor IX) of the first EGF (EGF-1)-like domain of factor IX. The importance of the Gly-48 is manifested by two hemophilia B patients; factor IXTainan and factor IX>Malmö27, with Gly-48 replaced by arginine (designated IXG48R) and valine (IXG48V), respectively. Both patients were CRM+ exhibiting mild hemophilic episodes with 25% (former) and 19% (latter) normal clotting activities. We characterize both factor IX variants to show the roles of Gly-48 and the conservation of the DGDQC sequence in factor IX. Purified plasma and recombinant factor IX variants exhibited approximately 26%–27% normal factor IX’s clotting activities with G48R or G48V mutation. Both variants depicted normal quenching of the intrinsic fluorescence by increasing concentrations of calcium ions and Tb3+, indicating that arginine and valine substitution for Gly-48 did not perturb the calcium site in the EGF-1 domain. Activation of both mutants by factor XIa appeared normal. The reduced clotting activity of factors IXG48R and IXG48V was attributed to the failure of both mutants to cleavage factor X; in the presence of only phospholipids and calcium ions, both mutants showed a 4∼7-fold elevation in K m, and by adding factor VIIIa to the system, although factor VIIIa potentiated the activation of factor X by the mutants factor IXaG48R and factor IXaG48V, a 2∼3-fold decrease in the catalytic function was observed with the mutant factor IXa’s, despite that they bound factor VIIIa on the phospholipid vesicles with only slightly reduced affinity when compared to wild-type factor IXa. The apparent K d for factor VIIIa binding was 0.83 nM for normal factor IXa, 1.74 nM for IXaG48R and 1.4 nM for IXaG48V. Strikingly, when interaction with the factor VIIa-TF complex was examined, both mutations were barely activated by the VIIa-TF complex and they also showed abnormal interaction with VIIa-TF in bovine thromboplastinbased PT assays. Taken together, our results suggest that mutations at Gly-48 altered the interaction of factor IX with its extrinsic pathway activator (VIIa-TF complex), its macromolecular substrate (factor X), and its cofactor (factor VIIIa).

Key words

Hemophilia B - factor IX - Gly-48 - mutagenesis
 

  • References

  • 1 McGraw RA, Davis LM, Noyes CM, Lundblad RL, Roberts HR, Graham JB, Stafford DW. Evidence for a prevalent dimorphism in the activation peptide of human factor IX. Proc Natl Acad Sci USA 1985; b; 82: 2847-51.
    CrossrefPubMedSearch in Google Scholar
  • 2 Jesty J, Silverberg S. Kinetics of the tissue factor-dependent activation of coagulation factors IX and X in a bovine plasma system. J Biol Chem 1979; 54: 12337-45.
    PubMedSearch in Google Scholar
  • 3 DiScipio RG, Kurachi K, Davie EW. Activation of human factor IX (Christmas factor). J Clin Invest 1978; 61: 1528-38.
    CrossrefPubMedSearch in Google Scholar
  • 4 Osterud B, Rapaport SI. Activation of factor IX by the reaction product of tissue factor and factor VII: Additional pathway for initiating blood coagulation. Proc Natl Acad Sci USA 1977; 74: 5260-4.
    CrossrefPubMedSearch in Google Scholar
  • 5 Van Diejien G, Tans G, Rosing J, Hemker HC. The role of phospholipid and factor VIIIa in the activation of bovine factor X. J Biol Chem 1981; 256: 3433-42.
    PubMedSearch in Google Scholar
  • 6 Wu Y, Bevilacqua VLH, Berg JM. Fibrillin domain folding and calcium binding significance to Marfan syndrome. Curr Biol 1995; 02: 91-7.
    PubMedSearch in Google Scholar
  • 7 Rebay I, Fleming RJ, Fehon RG, Cherbas L, Cherbas P. Artavanis-Taskonas. Specific EGF repeats to Notch mediate interactions with delta and Serrate: implications for Notch as a multifucntional receptor. Cell 1991; 67: 687-99.
    CrossrefPubMedSearch in Google Scholar
  • 8 Lin SW, Smith KJ, Welch D, Stafford DW. Expression and characterization of human factor IX and factor IX-factor X chimeras in mouse C127 cells. J Biol Chem 1990; 265: 144-50.
    PubMedSearch in Google Scholar
  • 9 Handford PA, Baron M, Mayhew M, Willis A, Beesley T, Brownlee GG, Cambell ID. The first EGF-like domain from human factor IX contain a high-affinity calcium binding site. EMBO J 1990; 09: 475-80.
    PubMedSearch in Google Scholar
  • 10 Handford PA, Mayhew M, Baron M, Winship PR, Cambell ID, Brownlee GG. Key residues involved in calcium binding motifs in EGF-like domains. Nature 1991; 351: 164-7.
    CrossrefPubMedSearch in Google Scholar
  • 11 Huang LH, Ke XH, Sweeney W, Tam JP. Calcium binding and putative activity of the epidermal growth factor domain of blood coagulation factor IX. Biochem. Biophys Res Commu 1989; 160: 133-9.
    CrossrefPubMedSearch in Google Scholar
  • 12 Hughes PE, Morgan G, Rooney EK, Brownlee GG, Handford PA. Tyrosine 69 of the first epidermal growth factor-like domain of human factor IX is essential for clotting activity. J Biol Chem 1993; 268: 17727-33.
    PubMedSearch in Google Scholar
  • 13 Drakenberg T, Fernlund P, Roepstorff P, Stenflo J. Proc Natl Acad Sci USA. 1983; 80: 1802-6.
    PubMedSearch in Google Scholar
  • 14 Rao Z, Handford P, Mayhew M, Knott V, Brownlee GG, Stuart D. The structure of a Ca2+-binding epidermal growth factor-like domain: its role in protein-protein interactions. Cell 1995; 82: 131-41.
    CrossrefPubMedSearch in Google Scholar
  • 15 Zhong D, Smith KJ, Birktoft JJ, Bajaj SP. First epidermal growth factorlike domain of human blood coagulation factor IX is required for its activation by factor VIIa/tissue factor but not by factor XIa. Proc Natl Acad Sci USA 1994; 91: 3574-8.
    CrossrefPubMedSearch in Google Scholar
  • 16 Giannelli F, Green PM, Sommer SS, Poon M-C, Ludwig M, Schwaab R, Reitsma PH, Goossens M, Yoshioka A, Figueiredo MS, Brownlee GG. Haemophilia B:database of point mutations and short additions and deletions, 7th edition. Nucleic Acids Res 1997; 25: 133-5.
    CrossrefPubMedSearch in Google Scholar
  • 17 Chang YJ, Hamaguchi N, Chang SC, Ruf W, Shen MC, Lin SW. Engineered recombinant factor VII Q217 variants with altered inhibitor specificities. Biochemistry 1999; 38: 10940-8.
    CrossrefPubMedSearch in Google Scholar
  • 18 Lin SW, Lin CN, Hamaguchi N, Smith KJ, Shen MC. Characterization of a factor IX variant with a glycine207 to glutamic acid mutation. Blood 1994; 84: 1866-73.
    PubMedSearch in Google Scholar
  • 19 Lin SW, Shen MC. Characterization of genetic defects of Hemophilia B of Chinese origin. Thromb Haemost 1991; 66: 459-63.
    Thieme ConnectPubMedSearch in Google Scholar
  • 20 Hamaguchi N, Roberts H, Stafford DW. Mutations in the catalytic domain of factor IX that are related to the subclass hemophilia Bm. Biochemistry 1993; 32: 6324-9.
    CrossrefPubMedSearch in Google Scholar
  • 21 Cheung WF, Straight DL, Smith KJ, Lin SW, Roberts HR, Stafford DW. The role of the EGF-1 and hydrophobic stack domains of human factor IX in binding to endothelia cells. J Biol Chem 1991; 266: 8797-800.
    PubMedSearch in Google Scholar
  • 22 Smith KJ, Ono K. Monoclonal antibodies to factor IX: characterization and use in immunoassay for factor IX. Thromb Res 1984; 33: 211-24.
    CrossrefPubMedSearch in Google Scholar
  • 23 Smith KJ, Thompson AR, McMullen BA, Frazier D, Lin SW, Stafford DW, Kisiel W, Thibodeau SN, Chen SH, Smith LF. Carrier testing in hemophilia B with an immunoassay which distinguishes a prevalent factor IX dimorphism. Blood 1987; 70: 1006-13.
    PubMedSearch in Google Scholar
  • 24 Frazier DL, Smith KJ, Cheung WF, Ware J, Lin SW, Thompson AR, Reisner H, Bajaj SP, Stafford DW. Mapping of monoclonal antibodies to human factor IX. Blood 1989; 74: 971-7.
    PubMedSearch in Google Scholar
  • 25 Yan SCB, Razzano P, Chao YB, Walls JD, Berg DT, McClure DB, Grinnell BW. Characterization and novel purification of recombinant human protein C from three mammalian cell lines. Biotechnology 1990; 08: 655-61.
    CrossrefPubMedSearch in Google Scholar
  • 26 Nelsestuen GL, Broderius M, Martin G. Role of gamma-carboxyglutamic acid. J Biol Chem 1976; 251: 6886-93.
    PubMedSearch in Google Scholar
  • 27 Prendergast FG, Mann KG. Differentiation of metal ion-induced transitions of prothrombin fragment 1. J Biol Chem 1977; 252: 840-50.
    PubMedSearch in Google Scholar
  • 28 Johnson AE, Esmon NL, Laue TM, Esmon CT. Structural change required for activation of protein C are induced by Ca2+ binding to a high affinity site that does not contain r-carboxyglutamic acid. J Biol Chem 1983; 258: 5554-60.
    PubMedSearch in Google Scholar
  • 29 Morita TM, Isaacs BS, Esmon CT, Johnson AE. Derivatives of blood coagulation factor IX contain a high affinity Ca2+-binding site that lacks γ-carboxyglutamic acid. J Biol Chem 1984; 259: 5698-704.
    PubMedSearch in Google Scholar
  • 31 Lozier JN, Monroe DM, Stanfield-Oakley S, Lin SW, Smith KJ, Roberts HR, High KA. Factor IX New London: Substitution of proline for glutamine at position 50 causes severe hemophilia B. Blood 1990; 75: 1097-104.
    PubMedSearch in Google Scholar
  • 32 Chang J, Jin J, Lollar P, Bode W, Brandstetter Hamaguchi N, Straight DL, Stafford DW. Changing residue 338 in human factor IX from arginine to alanine causes an increase in catalytic activity. J Biol Chen 1998; 273: 12089-94.
    PubMedSearch in Google Scholar
  • 33 Larson PJ, Stanfield-Oakley SA, VanDusen WJ, Kasper CK, Smith KJ, Monroe DM, High KA. Structural integrity of the r-carboxyglutamic acid domain of human blood clotting factor IXa is required for its binding to cofactor VIIIa. J Biol Chem 1996; 271: 3869-76.
    CrossrefPubMedSearch in Google Scholar
  • 34 Bertina RM, Linden IK, Mannuacci PM, Reinalda-Poot HH, Cupers R, Poort SR, Reistma PH. Mutations in hemophilia Bm occur at the Arg 180- Val activation site or in the catalytic domain of factor IX. J Biol Chem 1990; 265: 10876-83.
    PubMedSearch in Google Scholar
  • 35 Selander-Sunnerhagen M, Ullner M, Persson E, Teleman O, Stenflo J, Drakenberg T. How an epidermal growth factor (EGF)-like domain binds calcium. J Biol Chem 1992; 267: 19642-9.
    PubMedSearch in Google Scholar
  • 36 Handford P, Downing AK, Rao Z, Hewett DR, Sykes BC, Kielty CM. The calcium binding properties and molecuar organization of epidermal growth factor-like domains in human fibrillin-1. J Biol Chem 1995; 270: 6751-6.
    CrossrefPubMedSearch in Google Scholar
  • 37 Stenberg Y, Julenius K, Dahlqvist I, Drakenberg T, Stenflo J. Calciumbinding properties of the third and fourth epidermal-growth-factor-like modules in the vitamin K-dependent protein S. Eur J Biochem 1997; b; 248: 163-70.
    CrossrefPubMedSearch in Google Scholar
  • 38 Rand MD, Lindblom A, Carlson J, Villoutreix BO, Stenflo J. Calcium binding of cbEGF-like modules. Expression and characterization of the EGF-like modules of human Notch-1 implicated in receptor-ligand interactions. Protein Sci 1997; 06: 2059-71.
    PubMedSearch in Google Scholar
  • 39 Persson KE, Astermark J, Bjork I, Stenflo J. Calcium binding to the first EGF-like module of human factor IX in a recombinant fragment containing residues 1–85. Mutations V46E and Q50E each manifest anegligible increase in calcium affinity. FEBS Lett 1998; 421: 100-4.
    CrossrefPubMedSearch in Google Scholar
  • 40 Banner DW, D’Arcy A, Chene C, Winkler FK, Guha A, Konigsberg WH, Nemerson Y, Kirchhofer D. The crystal structure of the complex of blood coagulation factor VIIa with soluble tissue factor. Nature 1996; 380: 416.
    CrossrefPubMedSearch in Google Scholar
  • 41 Kelly CR, Dickinson CD, Ruf W. Ca2+ binding to the first epidermal growht factor module of coagulation factor VIIa is important for cofactor interaction and proteolytic function. J Biol Chem 1997; 272: 17467-72.
    CrossrefPubMedSearch in Google Scholar
  • 42 Bajaj SP. Region of factor IXa protease domain that interacts with factor VIIIa: Analysis of selective hemophilia B mutants. Thromb Haemost 1999; 82: 218-25.
    Thieme ConnectPubMedSearch in Google Scholar