Download PDF
Thromb Haemost 1966; 15(03/04): 327-337
DOI: 10.1055/s-0038-1649434
Originalarbeiten — Original Articles — Travaux Originaux
Schattauer GmbH

Human Antihemophilic Factor (AHF) Purification A Comparison of Two Procedures[*]

J. P Hurt
1   Departments of Pathology and Biochemistry, University of North Carolina, Chapel Hill, North Carolina
,
R. H Wagner   **)
1   Departments of Pathology and Biochemistry, University of North Carolina, Chapel Hill, North Carolina
,
K. M Brinkhous
1   Departments of Pathology and Biochemistry, University of North Carolina, Chapel Hill, North Carolina
› Author Affiliations
Further Information

Publication History

Publication Date:
24 July 2018 (online)

    Permissions and Reprints

Summary

A method is described for the precipitation from plasma of a human AHF fraction (Fraction AA) with beta alanine. The Fraction AA is further purified by reprecipitation with beta alanine or by adsorption with Florigel or bentonite. Reprecipitation with beta alanine is a convenient step as no additional equipment or reagents are required for the second precipitation. All reagents can be rendered sterile and free of pyrogens. When the precipitates are quantitatively dissolved, the yields are greater than 70% and purification ranges from 300-600 times. The adsorption procedure also has advantages. Adsorption adds only 45 minutes to a 6 hour fractionation. AHF recovery is as high as 70% with a purification of 3,000 to 5,000 times.

*) This investigation was supported by ITSPHS grants HE-01648 and HE-06350 from the National Institutes of Health.


**) This investigator was supported by Public Health Service Research Career Program Award K3-HE-1147.


 

  • References

  • 1 Addis T. The pathogenesis of hereditary haemophilia. J. Path. Bact 15: 427 1911;
    CrossrefPubMedGoogle Scholar
  • 2 Pool J. G, Hershgold E. J, Pappenhagen A. P. High-potency antihaemophilic factor concentrate prepared from cryoglobulin precipitate. Nature (Lond) 203: 312 1964;
    PubMedGoogle Scholar
  • 3 Wagner R. H, Brinkhous K. M, Penick G. D. The antihemophilic factor: Some problems in its study. Hemophilia and Other Hemorrhagic States. Ed. Brinkhous K. M. The University of North Carolina Press; Chapel Hill, North Carolina: 7 1959
    Google Scholar
  • 4 Wagner R. H, Pate D, Brinkhous K. M. Further purification of antihemophilic factor (AHF) from dog plasma. Fed. Proc 13: 445 1954;
    PubMedGoogle Scholar
  • 5 Cohn E. J, Strong L. E, Hughes Jr. W. L, Mulford D. J, Ashworth J. N, Melin M, Taylor H. L. Preparation and properties of serum and plasma proteins. IV. A system for the separation into fractions of the protein and lipoprotein components of biological tissues and fluids. J. Amer. chem. Soc 68: 459 1946;
    CrossrefPubMedGoogle Scholar
  • 6 Blombäck B, Blombäck M. Purification of human and bovine fibrinogen. Arkiv Kemi 10: 415 1956;
    PubMedGoogle Scholar
  • 7 Kekwick R. A, Mackay M. E, Record B. R. Fractionation of human plasma with ether. Nature (Lond) 157: 629 1946;
    PubMedGoogle Scholar
  • 8 Bidwell E. The purification of bovine antihaemophilic globulin. Brit. J. Haemat 01: 35 1955;
    CrossrefPubMedGoogle Scholar
  • 9 Simonetti G, Casillas G, Pavlovsky A. Purification du facteur VIII antihémophilique (FAH). Hémostase 01: 57 1961;
    PubMedGoogle Scholar
  • 10 Creveld S, van Veder H. A, Pascha C. N, Kroeze W. F. The separation of AHF from fibrinogen. Thrombos. Diathes. haemorrh. (Stuttg) 03: 572 1959;
    PubMedGoogle Scholar
  • 11 Lozner E. L, Kark R, Taylor F. H. L. The coagulation defect in hemophilia: The clot promoting activity in hemophilia of berkefelded normal human plasma free from fibrinogen and prothrombin. J. clin. Invest 18: 603 1939;
    CrossrefPubMedGoogle Scholar
  • 12 Creveld S, van Hoorweg P. G, den Ottolander G. J. H, Veder M. A. Isolation of the antihaemophilic factor from human plasma. Acta haemat. (Basel) 15: 1 1956;
    CrossrefPubMedGoogle Scholar
  • 13 Surgenor D. M, Steele B. B. Studies on the nature of the antihemophilic activity of normal plasma. Thrombos. Diathes. haemorrh. (Stuttg) 01: 563 1957;
    PubMedGoogle Scholar
  • 14 Cohn E. J, Gnrd F. R. N, Surgenor D. M, Barnes B. A, Brown R. K, Derouaux G, Gillespie J. M, Kahnt F. W, Lever W. F, Liu C. H, Mittelman D, Mouton R. F, Schmid K, Uroma E. A system for the separation of the components of human blood: Quantitative procedures for the separation of the protein components of human plasma. J. Amer. chem. Soc 72 4: 65 1950;
    PubMedGoogle Scholar
  • 15 Blombäck B, Blombäck M, Struwe I. Studies on blood clotting Factor VIII. Arkiv Kemi 18: 223 1961;
    PubMedGoogle Scholar
  • 16 Creveld S, van Veder H. A, Pascha C. N. The separation of AHF from fibrinogen. II. Thrombos. Diathes. haemorrh. (Stuttg) 04: 211 1960;
    PubMedGoogle Scholar
  • 17 Simonetti C, Casillas G, Pavlovsky A. Tannic acid purification of Factor VIII. The Hemophilias. Ed. Brinkhous K. M. The University of North Carolina Press; Chapel Hill, North Carolina: 100 1964
    Google Scholar
  • 18 Wagner R. H. Purification of antihemophilic factor (AHF) by precipitation with aliphatic amino acids. Proc. of 9th Intern. Hemat. Cong; Mexico City, Mexico: 1962
    Google Scholar
  • 19 Wagner R. H, McLester W. D, Smith M, Brinkhous K. M. Purification of antihemophilic factor (factor VIII) by amino acid precipitation. Thrombos. Diathes. haemorrh. (Stuttg) 11: 64 1964;
    PubMedGoogle Scholar
  • 20 Roberts H. R, Graham J. B, Webster W. P, Penick G. D. Plasma transfusion therapy in hemophilia. The Hemophilias. Ed. Brinkhous K. M. The University of North Carolina Press; Chapel Hill, North Carolina: 326 1964
    Google Scholar
  • 21 Webster W. P, Roberts H. R, Thelin G. M, Wagner R. H, Brinkhous K. M. Clinical use of a glycine precipitated antihemophilic fraction. Amer. J. med. Sci 250: 643 1965;
    CrossrefPubMedGoogle Scholar
  • 22 Wagner R. H, Richardson B. A, Brinkhous K. M. A study of the separation of fibrinogen and antihemophilic factor (AHF) in canine, porcine, and human plasmas. Thrombos. Diathes. haemorrh. (Stuttg) 01: 1 1957;
    PubMedGoogle Scholar
  • 23 Soulier J. P. Séparation du fibrinogène et du facteur antihémophilique A. II. A l’aide de la bentonite. Path, et Biol 07: 2451 1959;
    PubMedGoogle Scholar
  • 24 Ménaché D, Fauvert R, Soulier J. P. Utilisation en hépatologie d’une fraction contenant la prothrombine, le complexe proconvertine-facteur Stuart et le facteur anti-hémophilique B (P.P.B.). Path et Biol 07: 2515 1959;
    PubMedGoogle Scholar
  • 25 Quick A. J. The clinical application of the hippuric acicl and the prothrombin tests. Amer. J. clin. Path 10: 222 1940;
    PubMedGoogle Scholar
  • 26 Mertz E. T, Owens G. Imidazole buffer: Its use in blood clotting studies. Proc. Soc. exp. Biol. (N.Y) 43: 204 1940;
    CrossrefPubMedGoogle Scholar
  • 27 Thelin G. M, Wagner R. H. Sedimentation of plasma antihemophilic factor. Arch. Biochem 95: 70 1961;
    CrossrefPubMedGoogle Scholar
  • 28 Langdell R. D, Wagner R. H, Brinkhous K. M. Effect of antihemophilic factor on one- stage clotting tests. J. Lab. clin. Med 41: 637 1953;
    PubMedGoogle Scholar
  • 29 McLendon W. W, Barrow E. M, Hurt J. P, Bryan F. T, Wagner R. H. Modified one- stage antihemophilic factor assay and its use in testing human AHF fractions. Fed. Proc 20: 57 1961;
    PubMedGoogle Scholar
  • 30 Nye S. W, Graham J. B, Brinkhous K. M. The partial thromboplastin time as a screening test for the detection of latent bleeders. Amer. J. med. Sei 243: 279 1962;
    CrossrefPubMedGoogle Scholar
  • 31 Rodman Jr. N. F, Barrow E. M, Graham J. B. Diagnosis and control of the hemophilioid states with the partial thromboplastin time (PTT) test. Amer. J. clin. Path 29: 525 1958;
    PubMedGoogle Scholar
  • 32 Graham J. B, Buckwalter J. A, Hartley L. J, Brinkhous K. M. Canine hemophilia: Observations on the course, the clotting anomaly, and the effect of blood transfusions. J. exp. Med 90: 97 1949;
    CrossrefPubMedGoogle Scholar
  • 33 Clay Mineral Standards, Preliminary Reports. A.P. I. Research Project, Pub. Columbia University; New York, New York: (May 1951).
    Google Scholar
  • 34 Brinkhous K. M, Langdell R. D, Penick G. D, Graham J. B, Wagner R. H. Newer approaches to the study of hemophilia and hemophilioid states. J.A.M.A 154: 481 1954;
    CrossrefPubMedGoogle Scholar
  • 35 Blombäck B, Blombäck M, Nilsson I. M. Note on the purification of human antihemophilic globulin. Acta chem. scand 12: 1878 1958;
    CrossrefPubMedGoogle Scholar
  • 36 Her Ralph K. The Colloid Chemistry of Silica and Silicates. Cornell Press; Ithaca, New York: 223 1955
    Google Scholar