Download PDF
Thromb Haemost 1967; 18(01/02): 211-222
DOI: 10.1055/s-0038-1655030
Originalarbeiten — Original Articles — Travaux Originaux
Schattauer GmbH

A Study of the Reaction Product of Factor VIII and Factor IX by Gel Filtration[*]

C Hougie
1   MRC Coagulation Unit, Oxford, England, and Department of Pathology, University of Washington Seattle, Washington
,
K. W. E Denson
1   MRC Coagulation Unit, Oxford, England, and Department of Pathology, University of Washington Seattle, Washington
,
Rosemary Biggs
1   MRC Coagulation Unit, Oxford, England, and Department of Pathology, University of Washington Seattle, Washington
› Author Affiliations
Further Information

Publication History

Publication Date:
26 June 2018 (online)

Also available at
    Permissions and Reprints

Summary

The products of the reaction of activated factor IX with factor VIII, phospholipid and calcium have been studied using gel filtration on Sephadex G-200. Both factor VIII and phospholipid are completely excluded from the gel (distribution coefficient or KD of zero) while factor IXa behaves like a protein similar in size to albumin (KD approx. 0.4). Factor IXa complexes with phospholipid when calcium ions are added to the eluting buffer. When factor VIII, factor IXa, phospholipid and calcium are applied to the column and eluted with added Ca++ in the eluting buffer, a labile product with a KD of 0 is formed. The product apparently dissociates in the absence of added calcium ions in the eluting buffer. In the presence of calcium ions, the distribution coefficient of factor IX, when applied alone to the column, increased. The above findings are analogous to those reported between activated factor X, thrombin-activated factor V, phospholipid and calcium occurring later in the coagulation process.

* This work was done during the tenure of an Established Investigatorship of the American Heart Association by C. Hougie and was supported in part by Public Health Service Research Grants Nos. HE. 05994-06 and HE. 05995-06 from the National Heart Institute.


 

  • References

  • 1 Macfarlane R. G. An enzyme cascade in the blood clotting mechanism, and its function as a biochemical amplifier. Nature (Lond) 202: 498 1964;
    CrossrefPubMedSearch in Google Scholar
  • 2 Davie E. W, Ratnoff O. D. Waterfall sequence for intrinsic blood clotting. Science 145: 1310 1964;
    CrossrefPubMedSearch in Google Scholar
  • 3 Davie E. W, Ratnoff O. D. The Proteins of Blood Coagulation. The Proteins. 03: 359 1965;
    PubMedSearch in Google Scholar
  • 4 Macfarlane R. G. Observations on the Interaction of Factors VIII, IX and X. Thrombos. Diathes. haemorrh. (Stuttg.) 9 Suppl. 02: 221 1963;
    PubMedSearch in Google Scholar
  • 5 Sjpaet T. H, Cintron J. Pathways to Blood Coagulation Product I Formation. Blood. 21: 745 1963;
    PubMedSearch in Google Scholar
  • 6 Macfarlane R. G, Biggs R, Ash B. J, Denson K. W. E. The activation and consumption of factor X in recalcified plasma. The effect of added Factor VIII and Russell’s viper venom. Brit. J. Haemat. 10: 530 1964;
    PubMedSearch in Google Scholar
  • 7 Hougie G. Sequential Reactions of Factors VIII, IX, and X. In: Hemophilioid Diseases. 3d. ed. 225 Brinkhous K. M. ed. University of North Carolina Press; Chapel Hill: 1964
    Search in Google Scholar
  • 8 Lundblad R. L, Davie E. W. The activation of antihemophilic factor (factor VIII) by activated Christmas factor (activated factor IX). Biochemistry 03: 1720 1964;
    CrossrefPubMedSearch in Google Scholar
  • 9 Lundblad R. L, Davie E. W. The activation of Stuart factor (factor X) by activated antihemophilic factor (activated factor VIII). Biochemistry 04: 113 1965;
    CrossrefPubMedSearch in Google Scholar
  • 10 Lundblad R. L, Thesis Ph. D. The stepwise activation of antihemophilic factor and Stuart factor during normal blood coagulation. University of Washington; 1965.:
  • 11 Biggs R, Macfarlane R. G, Denson K. W. E, Ash B. J. The interaction of factors VIII and IX. Brit. J. Haemat. 11: 276 1965;
    CrossrefPubMedSearch in Google Scholar
  • 12 Macfarlane R. G. The coagulant action of Russell’s viper venom; The use of antivenom in defining its reaction with a serum factor. Brit. J. Haemat. 07: 496 1961;
    CrossrefPubMedSearch in Google Scholar
  • 13 Esnouf M. P, Williams W. J. The isolation and purification of a bovine-plasma protein which is a substrate for the coagulant fraction of Russell’s viper venom. Biochemistry 84: 62 1962;
    CrossrefPubMedSearch in Google Scholar
  • 14 Pechet L, Alexander B. Activation of Prothrombin, Factors VII and X by Proteolytic Pathway. Fed. Proc. 19: 64 1960;
    PubMedSearch in Google Scholar
  • 15 Alexander B, Pechet L, Kliman A. Proteolysis, Fibrinolysis and Coagulation Significance in Thrombolytic Therapy. Circulation 26: 596 1962;
    CrossrefPubMedSearch in Google Scholar
  • 16 Ferguson J. H, Ennis E. G. W. Enzymes and blood clotting. II. Auto prothrombin C, Russell’s viper venom (stypven) and trypsin. Thrombos. Diathes. haemorrh. (Stuttg) 09: 62 1962;
    PubMedSearch in Google Scholar
  • 17 Yin E. T. Kinetics of Human Blood Coagulation Induced by Trypsin. Thrombos. Diathes. haemorrh. (Stuttg) 12: 307 1964;
    PubMedSearch in Google Scholar
  • 18 Papahadjopoulos D, Hougie C, Hanahan D. J. Purification and properties of bovine factor V: A change of molecular size during blood coagulation. Biochemistry 03: 264 1964;
    CrossrefPubMedSearch in Google Scholar
  • 19 Lewis J. H. Effects of epsilon amino caproic acid (EACA) on survival of fibrinogen 1-131 and on fibrinolytic and coagulation factors in dogs. Proc. Soc. exp. Biol. (N. Y) 116: 120 1964;
    CrossrefPubMedSearch in Google Scholar
  • 20 Folch J. Brain cephalin mixture of phosphatides. Separation from it of phosphatidyl serine, phosphatidyl ethanolamine and fraction containing inositol phosphatide. J. biol. Chem. 146: 35 1942;
    PubMedSearch in Google Scholar
  • 21 Biggs R, Macfarlane R. G. Human Blood Coagulation and its Disorders. 3d. ed. Blackwell Scientific Publications Oxford; 1962
    Search in Google Scholar
  • 22 Bell W. M, Alton H. G. Brain extract as substitute for platelet suspensions in thromboplastin generation test. Nature (Lond) 174: 880 1954;
    CrossrefPubMedSearch in Google Scholar
  • 23 Gornall A. G, Bardawill C. J, David M. M. Determination of serum proteins by means of biuretreaction. J. biol. Chem. 177: 751 1949;
    PubMedSearch in Google Scholar
  • 24 Denson K. W. The specific assay of Prower-Stuart factor and factor VII biuret reaction. Acta haemat. (Basel) 25: 105 1961;
    CrossrefPubMedSearch in Google Scholar
  • 25 Langdell R. D, Wagner R. H, Brinkhous K. M. Effect of antihemophilic factor on one-stage clotting tests: Presumptive test for hemophilia and simple one-stage antihemophilic factor assay procedure. J. Lab. clin. Med. 41: 637 1953;
    PubMedSearch in Google Scholar
  • 26 Proctor R. R, Rapaport S. The partial thromboplastin time with kaolin. A simple screening test for first stage plasma clotting factor deficiencies. Amer. J. clin. Path. 36: 212 1961;
    PubMedSearch in Google Scholar
  • 27 Bidwell E. Purification of bovine antihaemophilic globulin. Brit. J. Haemat. 01: 35 1955;
    CrossrefPubMedSearch in Google Scholar
  • 28 Bidwell E, Dike G. W. R, Denson K. W. E. Experiments with Factor VIII Separated from Fibrinogen by Electrophoresis in Free Buffer Film. Brit. J. Haemat. 12: 583-594 1966;
    CrossrefPubMedSearch in Google Scholar
  • 29 Bell W. M, Alton H. G. Brain extract as substitute for platelet suspensions in thromboplastin generation test. Nature (Lond) 174: 880 1954;
    CrossrefPubMedSearch in Google Scholar
  • 30 Gornall A. G, Bardawill G. J, David M. M. Determination of serum proteins by means of biuret reaction. J. biol. Chem. 177: 751 1949;
    PubMedSearch in Google Scholar
  • 31 Denson K. W. The specific assay of Prower-Stuart factor and factor VII biuret reaction. Acta haemat. (Basel) 25: 105 1961;
    CrossrefPubMedSearch in Google Scholar
  • 32 Langdell R. D, Wagner R. H, Brinkhous K. M. Effect of antihemophilic factor on one stage clotting tests: Presumptive test for hemophilia and simple one-stage antihemophilic factor assay procedure. J. Lab. clin. Med. 41: 637 1953;
    PubMedSearch in Google Scholar
  • 33 Proctor R. R, Rapaport S. The partial thromboplastin time with kaolin. A simple screening test for first stage plasma clotting factor deficiencies. Amer. J. clin. Path. 36: 212 1961;
    PubMedSearch in Google Scholar
  • 34 Bidwell E. Purification of bovine antihaemophilic globulin. Brit. J. Haemat. 01: 35 1955;
    CrossrefPubMedSearch in Google Scholar
  • 35 Bidwell E, Dike G. W. R, Denson K. W. E. Experiments with Factor VIII Separated from Fibrinogen by Electrophoresis in Free Buffer Film. Brit. J. Haemat. 12: 583 1966;
    CrossrefPubMedSearch in Google Scholar
  • 36 Denson K. W. D. Phil. Thesis. University of Oxford; 1966
    Search in Google Scholar
  • 37 Esnouf M. P, Williams W. J. The isolation and purification of a bovine-plasma protein which is a substrate for the coagulant fraction of Russell’s viper venom. Biochem. J. 84: 62 1962;
    CrossrefPubMedSearch in Google Scholar
  • 38 Nossel H. L. In: The Contact Phase of Blood Coagulation. 124-128 Blackwell Scientific Publications Oxford; 1964
    Search in Google Scholar
  • 39 Flodin P, Killander J. Fractionation of Human-Serum Proteins by Gel Filtration. Biochim. biophys. Acta (Amst) 63: 403 1962;
    CrossrefPubMedSearch in Google Scholar
  • 40 Flodin P. Methodoligical aspects of gel filtration with special reference to desalting operations. J. Chromatog. 05: 103 1961;
    CrossrefPubMedSearch in Google Scholar
  • 41 Papahadjopoulos D. D. Phil. Thesis: Studies in Blood Coagulation, Interactions between Proteins and Lipids. University of Washington; 1963
    Search in Google Scholar
  • 42 Hanahan D. J, Papahadjopoulos D. Interactions of Phospholipids with Coagulation Factors. Conf. Int. Comm. Blood Clot. Factors, Amsterdam, 71 1964;
    PubMedSearch in Google Scholar
  • 43 Denson K. W. D. Phil. Thesis: University of Oxford; 1966
    Search in Google Scholar
  • 44 Esnouf M. P, Williams W. J. The isolation and purification of a bovine-plasma protein which is a substrate for the coagulant fraction of Russell’s viper venom. Biochem. J. 84: 62 1962;
    CrossrefPubMedSearch in Google Scholar
  • 45 Nossel H. L. In: The Contact Phase of Blood Coagulation. 124-128 Blackwell Scientific Publications Oxford; 1964
    Search in Google Scholar
  • 46 Flodin P, Killander J. Fractionation of Human-Serum Proteins by Gel Filtration. Biochim. biophys. Acta (Amst) 63: 403 1962;
    CrossrefPubMedSearch in Google Scholar
  • 47 Flodin P. Methodological aspects of gel filtration with special reference to desalting operations. J. Chromatog. 05: 103 1961;
    CrossrefPubMedSearch in Google Scholar
  • 48 Papahadjopoulos D. D. Phil. Thesis: Studies in Blood Coagulation, Interactions between Proteins and Lipids. University of Washington. 1963
    Search in Google Scholar
  • 49 Hahan D. J, Papahadjopoulos D. Interactions of Phospholipids with Coagulation Factors. Conf. Int. Comm. Blood Clot. Factors, Amsterdam, 71 1964;
    PubMedSearch in Google Scholar
  • 50 Breckenridge R. T, Ratnojf O. D. The role of proaccelerin in human blood coagulation. Evidence that proaccelerin is converted to a prothrombin-converting principle by activated Stuart factor: With notes on the anticoagulant action of soybean, trypsin inhibitor, protamine sulfate and hexadimethrine bromide. J. clin. Invest. 44: 302 1965;
    CrossrefPubMedSearch in Google Scholar
  • 51 Esnouf M. P, Jobin F. Lipids in Prothrombin Conversion. Conf. Int. Comm. Blood Clot. Factors, Amsterdam, 103 1964;
    PubMedSearch in Google Scholar