Download PDF
Thromb Haemost 1997; 78(03): 1049-1054
DOI: 10.1055/s-0038-1657685
Rapid Communication
Schattauer GmbH Stuttgart

Isolation and Characterization of Mouse Coagulation Factor X – Biophysical and Enzymological Properties

H L James
1   The Department of Biochemistry, The University of Texas Health Center at Tyler, Tyler, Texas, USA
,
P J Larson
2   The Division of Hematology, Department of Pediatrics, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, USA
,
Y B Chao
3   The Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, Indiana, USA
,
S B Yan
3   The Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, Indiana, USA
,
D J Kim
1   The Department of Biochemistry, The University of Texas Health Center at Tyler, Tyler, Texas, USA
› Author Affiliations
Further Information

Publication History

Received 01 1996

Accepted 05 May 1997

Publication Date:
12 July 2018 (online)

Also available at
  PDF Download  Permissions and Reprints

Summary

Mouse factor X was highly purified from plasma using barium ion precipitation and chromatography on anion-exchange and heparin- agarose affinity chromatography columns. Intact and reduced patterns of mouse factor X in SDS-PAGE were similar to those of human factor X. The specific absorption E 1%/1 cm at 280 nm of mouse factor X was found to be 11.2. Content of carbohydrate moieties of mouse factor X, determined to be 10% by weight, differs both quantitatively and quantitatively from that of human factor X, while the γ-carboxy- glutamic acid (Gla) and β-hydroxy-aspartic acid (β-OH-Asp) content were essentially the same as for human factor X. The amino-terminal amino acid sequences of the light and heavy chains of mouse factor X separated by SDS-PAGE were ANSFF--FKK and SVALXTSDSE, respectively. Underlined residues are non-identical with those of human factor X. Clotting time-based assays using human factor X- deficient plasma as substrate exhibited the following apparent extents of activation of factor X in mouse plasma, using human plasma as the standard: 195% (intrinsic); 200% (extrinsic); and 190% (RVV-X). Using the purified proteins in the same assay systems, the following apparent activation of mouse factor X was demonstrated, compared with human factor X: 195% (intrinsic); 27% (extrinsic); and 41% (RVV-X). These activity profiles suggest that the human extrinsic coagulation pathway functions less efficiently than the corresponding mouse pathway in the activation of mouse factor X. Furthermore, mouse brain thromboplastin satisfactorily replaced rabbit brain thromboplastin in extrinsic activation of factor X in mouse plasma, but not of human plasma or purified mouse or human factor X, in line with studies by others suggesting that human factor Vila poorly activates factor X in the presence of mouse tissue factor. While fully RVV-X-activated mouse factor X activated human prothrombin at a rate equal to about 117% of that for human factor X, it hydrolyzed the synthetic substrate, S-2222, at a rate of only about 18% of that for human factor X. These results are expected to be useful in making the mouse suitable for study of the mammalian blood coagulation pathways.

 

  • References

  • 1 James HL, Factor X. In: Haemostasis and Thrombosis Vol 1. Bloom AL, Forbes CD, Thomas DP, Tuddenham EGD. eds. Churchill Livingstone, Edinburgh; 1994. pp 439-464
  • 2 Pfeiffer RA, Ott R, Gilgenkrantz S, Alexandre P. Deficiency of coagulation factors VII and X associated with deletions of a chromosome 13 (q 34). Evidence from two cases with 46, XY, t (13; y) (q 11; q 34). Hum Genet 1982; 62: 358-360
    CrossrefPubMedGoogle Scholar
  • 3 Leytus SP, Foster DC, Kurachi K, Davie EW. Gene for factor X: a blood coagulation factor whose gene organization is essentially identical with that of factor IX and protein C. Biochemistry 1986; 1986: 5098-5102
    PubMedGoogle Scholar
  • 4 Zhang L, Castellino FJ. Influence of specific 7-carboxyglutamic acid residues on the integrity of the calcium-dependent conformation of human protein C. J Biol Chem 1992; 267: 26078-26084
    PubMedGoogle Scholar
  • 5 Ratcliffe JV, Furie B, Furie BC. The importance of specific γ-carboxyglutamic acid residues in prothrombin. Evaluation by site-specific mutagenesis. J Biol Chem 1993; 1993: 24339-45
    PubMedGoogle Scholar
  • 6 Fujikawa K, Titani K, Davie EW. Activation of bovine factor X (Stuart factor): conversion of activation of factor Xaa to Xaβ. Proc Natl Acad Sci USA 1975; 1975: 3359-3363
    PubMedGoogle Scholar
  • 7 Hemker HC, Esnouf MP, Hemker PW, Swart ACW, Macfarlane RG. Formation of prothrombin converting activity. Nature 1967; 1967: 248-251
    PubMedGoogle Scholar
  • 8 Nesheim ME, Taswell JB, Mann KG. The contribution of bovine factor V and factor Va to the activity of prothrombinase. J Biol Chem 1979; 1979: 10952-10962
    PubMedGoogle Scholar
  • 9 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; 1980: 274-283
    PubMedGoogle Scholar
  • 10 Abildgaard CF, Lewis JP, Harrison J. Quantitative coagulation studies in mice. Lab Anim Sci 1972; 1972: 099-101
    PubMedGoogle Scholar
  • 11 Kase F, Pospísil J. Comparison of blood clotting mechanisms in man and some common laboratory animals. Acta Univ Carol [Med Monogr] (Praha) 1987; 1987: 11-22
    PubMedGoogle Scholar
  • 12 Fair DS, Plow EF, Edington TS. Combined functional and immunochemical analysis of normal and abnormal human factor X. J Clin Invest 1979; 1979: 884-894
    PubMedGoogle Scholar
  • 13 Nesheim ME, Myrmel KH, Hibbard L, Mann KG. Isolation and characterization of single chain bovine factor V. J Biol Chem 1979; 1979: 508-517
    PubMedGoogle Scholar
  • 14 Schwartz BS, Levy GA, Curtiss LK, Fair DS, Edgington TS. Plasma lipoprotein induction and suppression of the generation of cellular procoagulant activity in vitro. I. Two activities are produced by peripheral blood mononuclear cells. J Clin Invest 1981; 67: 1650-1658
    CrossrefPubMedGoogle Scholar
  • 15 Bajaj SP, Birktoft JJ. Human factor IX and factor IXa. Meth Enzymol 1993; 222: 096-128
    PubMedGoogle Scholar
  • 16 Di Scipio RG, Hermodson MA, Yates SG, Davie EW. A comparison of human prothrombin, factor IX (Christmas factor), factor X (Stuart factor) and protein S. Biochemistry 1977; 1977: 698-706
    PubMedGoogle Scholar
  • 17 Yan SB, Razzano PY, Chao YB, Wall JD, Berg DT, McClure DB, Grinnell BW. Characterization and novel purification of recombinant human protein C from three mammalian cell lines. Bio/Technology 1990; 08: 655-661
    CrossrefPubMedGoogle Scholar
  • 18 Przysiecki CT, Staggers JE, Ramit HG, Musson DG, Stem AM, Bennett CD, Friedman PA. Occurrence of p-hydroxylated asparagine residues in non-vitamin K-dependent proteins containing epidermal growth factor-like domains. Proc Natl Acad Sci USA 1987; 1987: 7856-7860
    PubMedGoogle Scholar
  • 19 Matsudaira P. Sequence from picomole quantities of proteins electroblotted onto polyvinylidene difluoride membranes. J Biol Chem 1987; 1987: 10035-10038
    PubMedGoogle Scholar
  • 20 Fair DS, Revak DJ, Hubbard JG, Girolami A. Isolation and characterization of the factor X Friuli variant. Blood 1989; 73: 2108-2116
    PubMedGoogle Scholar
  • 21 Chattopadyay A, Fair DS. Molecular recognition in the activation of human blood coagulation factor X. J Biol Chem 1989; 264: 11035-11043
    CrossrefPubMedGoogle Scholar
  • 22 Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic local alignment search tool. J Mol Biol 1990; 215: 403-410
    CrossrefPubMedGoogle Scholar
  • 23 DiScipio RG, Hermodson MA, Davie EW. Activation of human factor X (Stuart factor) by a protease from Russell’s viper venom. Biochemistry 1977; 16: 5253-5260
    CrossrefPubMedGoogle Scholar
  • 24 Inoue K, Morita T. Identification of O-linked oligosaccharide chains in the activation peptide of blood coagulation factor X. Eur J Biochem 1993; 218: 153-163
    CrossrefPubMedGoogle Scholar
  • 25 Fang CH, Lin T-C, Guha A, Nemerson Y, Konigsberg WH. Activation of factor X by factor Vila complexed with human-mouse tissue factor chimeras requires human exon 3. Thromb Haemost 1996; 76: 361-368
    Article in Thieme ConnectPubMedGoogle Scholar
  • 26 Aronson DL, Mustafa AJ, Mushinski JF. Purification of human factor X and comparison of peptide maps of human factor X and prothrombin. Biochim Biophys Acta 1969; 188: 25-30
    CrossrefPubMedGoogle Scholar
  • 27 Femlund P, Stenflo J. β-hydroxyaspartic acid in vitamin K-dependent proteins. J Biol Chem 1983; 258: 12509-12512
    PubMedGoogle Scholar
  • 28 Fujikawa K, Lepaz ME, Davie EW. Bovine factorsXx and X2 (Stuart factor), isolation and characterization. Biochemistry 1972; 11: 4882-4891
    CrossrefPubMedGoogle Scholar
  • 29 Weinstein RE, Rickies FR, Walker FJ. Purification and preliminary characterization of rabbit vitamin K-dependent coagulation proteins. Thromb Res 1990; 1990: 759-772
    PubMedGoogle Scholar
  • 30 Graves CB, Munns TW, Willingham AK, Strauss AW. Rat factor X is synthesized as a single chain precursor inducible by prothrombin fragments. J Biol Chem 1982; 257: 13108-13113
    PubMedGoogle Scholar
  • 31 Yan SB, Chao YB, van Halbeek H. Novel Asn-linked oligosaccharides terminating in GalNAc(3 (1 4) [Fuca (1 =^> 3)] GlcNAcp (1 =* ·) are present in recombinant human protein C expressed in human kidney 293 cells. Glycobiology 1993; 1993: 597-608
    PubMedGoogle Scholar
  • 32 Nakagawa H, Takahashi N, Fujikawa K, Kawamura Y, lino M, Takeya H, Ogawa H, Suzuki K. Identification of the oligosaccharide structures of human coagulation factor X activation peptide at each glycosylation site. GlycoconjJ 1995; 12: 173-181
    PubMedGoogle Scholar
  • 33 Bouhours J, Bouhours D. Hydroxylation of CMP-NeuAc controls the expression of N-glycolylneuraminic acid in GM3 ganglioside of the small intestine of inbred rat s. J Biol Chem 1989; 264: 16992-16999
    PubMedGoogle Scholar
  • 34 Mizuochi T, Yamashita K, Fujikawa K, Titani K, Kobata A. The structures of the carbohydrate moieties of bovine blood coagulation factor X. J Biol Chem 1980; 255: 3526-3531
    PubMedGoogle Scholar
  • 35 James HL, Girolami A, Fair DS. Molecular defect in coagulation factor XFriuli results from a substitution of serine for proline at position 343. Blood 1991; 77: 317-323
    PubMedGoogle Scholar
  • 36 Kim DJ, Girolami A, James HL. Characterization of recombinant human coagulation factor XFriuli . Thromb Haemost 1996; 75: 313-317
    Article in Thieme ConnectPubMedGoogle Scholar
  • 37 Sugahara Y, Catalfamo J, Brooks, Hitomi E, Bajaj SP, Kurachi K. Isolation and characterization of canine factor IX. Thromb Haemost 1996; 75: 450-455
    Article in Thieme ConnectPubMedGoogle Scholar