Download PDF
Thromb Haemost 1976; 35(01): 186-190
DOI: 10.1055/s-0038-1647943
Original Article
Schattauer GmbH

Importance of Protease Inhibition in Studies on Purified Factor VIII (Antihaemophilic Factor)

Eugen A. Beck
1   Central Haematology Laboratory, Inselspital, CH-3010 Berne, Switzerland
,
Peter Bachmann
1   Central Haematology Laboratory, Inselspital, CH-3010 Berne, Switzerland
,
Peter Barbier
1   Central Haematology Laboratory, Inselspital, CH-3010 Berne, Switzerland
,
Miha Furlan
1   Central Haematology Laboratory, Inselspital, CH-3010 Berne, Switzerland
› Author Affiliations
Further Information

Publication History

Received 04 November 1975

Accepted 23 November 1975

Publication Date:
02 July 2018 (online)

    Permissions and Reprints

Summary

According to some authors factor VIII procoagulant activity may be dissociable from carrier protein (MW~ 2 × 106) by agarose gel filtration, e.g. at high ionic strength. We were able to reproduce this phenomenon. However, addition of protease inhibitor (Trasylol) prevented the appearance of low molecular weight peak of factor VIII procoagulant activity both at high ionic strength and elevated temperature (37°C). We conclude from our results that procoagulant activity and carrier protein (von Willebrand factor, factor VIII antigen) are closely associated functional sites of native factor VIII macro molecule. Consequently, proteolytic degradation should be avoided in functional and structural studies on factor VIII and especially in preparing factor VIII concentrate for therapeutic use.

 

  • References

  • 12 Beck E. A. 1975. One-stage factor VIII assay improved by continuous stirring. In: Ulutin O.N. , Peake I. R. Haemophilia. Excerpta Medica; Amsterdam, American Elsevier Publishing Company, Inc., New York: 187.
    Google Scholar
  • 13 Cohen R. I, Epstein S. E, Cohen L. S, Dennis L. H. 1968; Alterations of fibrinolysis and blood coagulation induced by exercise, and the role of beta-adrenergic-receptor stimulation. Lancet II: 1264.
    PubMedGoogle Scholar
  • 14 Cooper H. A, Griggs T. R, Wagner R. H. 1973; Factor VIII recombination after dissociation by CaCl2 . Proceedings of the National Academy of Sciences (Wash.) 70: 2326.
    CrossrefPubMedGoogle Scholar
  • 15 Ekert H, Firkin B. G. 1975; Recent advances in haemophilia and von Willebrand disease. Vox Sanguinis 28: 409.
    PubMedGoogle Scholar
  • 16 Lowry O. H, Roseborough N. J, Farr A. L, Randall R. J. 1951; Protein measurement with the Folin phenol reagent. Journal of Biological Chemistry 193: 265.
    PubMedGoogle Scholar
  • 17 McKee P. A, Andersen J. C, Switzer M. E. 1975; Molecular structural studies of human factor VIII. Annals of the New York Academy of Sciences 240: 8.
    CrossrefPubMedGoogle Scholar
  • 18 Owen W. G, Wagner R. H. 1972; a Antihemophilic factor: separation of an active fragment following dissociation by salts or detergents. Thrombosis et Diathesis Haemorrhagica (Stuttg.) 27: 502.
    PubMedGoogle Scholar
  • 19 Owen W. G, Wagner R. H. 1972; b Antihemophilic factor. A new method for purification. Thrombosis Research 1: 71.
    PubMedGoogle Scholar
  • 20 Patek A. J, Taylor F. H. L. 1937; Hemophilia. II. Some properties of a substance obtained from normal human plasma effective in accelerating the coagulation of hemophilic blood. Journal of Clinical Investigation 16: 113.
    PubMedGoogle Scholar
  • 21 Plow E. F, Edgington T. S. 1973; Disciminating neoantigenic differences between fibrinogen and fibrin derivatives. Proceedings of the National Academy of Sciences (Wash.) 70: 1169.
    CrossrefPubMedGoogle Scholar
  • 22 Proctor R. B, Rapaport S. I. 1961; The partial thromboplastin time with kaolin. A simple screening test for first stage plasma clotting factor deficiencies. American Journal of Clinical Pathology 36: 212.
    PubMedGoogle Scholar
  • 23 Ratnoff O. D, Bennet B. 1973; The genetics of hereditary disorders of blood coagulation. Science 779: 1291.
    PubMedGoogle Scholar
  • 24 Shapiro G. A, Andersen J. C, Pizzo V, McKee P. A. 1973; The subunit structure of normal and hemophilic factor VIII. Journal of Clinical Investigation 52: 2198.
    CrossrefPubMedGoogle Scholar
  • 25 Weiss H. J, Kochwa S. 1970; Molecular forms of antihaemophilic globulin in plasma, cryo-precipitate and after thrombin activation. British Journal of Haematology 18: 89.
    CrossrefPubMedGoogle Scholar
  • 26 Weiss H. J, Hoyer L. W, Rickles F. R, Varma A, Rogers J. 1973; Quantitative assay of a plasma factor deficient in von Willebrand’s disease that is necessary for platelet aggregation. Journal of Clinical Investigation 52: 2708.
    CrossrefPubMedGoogle Scholar
  • 27 Zimmerman T. S, Ratnoff O. D, Powell A. E. 1971; Immunologic differentiation of classic hemophilia (factor VIII deficiency) and von Willebrand’s disease, with observations on combined deficiencies of antihemophilic factor and proaccelerin (factor V) and on acquired circulating anticoagulants against antihemophilic factor. Journal of Clinical Investigation 50: 244.
    CrossrefPubMedGoogle Scholar
  • 28 Zimmerman T. S, Roberts J, Edgington T. S. 1975; Factor-VHI-related antigen: multiple molecular forms in human plasma. Proceedings of the National Academy of Sciences (Wash.) 72: 5121.
    CrossrefPubMedGoogle Scholar