The Use of DEAE Cellulose in the Preparation of a Factor VIII (AHG)-free Bovine Fibrinogen Reagent

W. F Stapp

doi:10.1055/s-0038-1654562

Thrombosis and Haemostasis, Inhaltsverzeichnis

Thromb Haemost 1961; 6(02): 287-307
DOI: 10.1055/s-0038-1654562

Originalarbeiten — Original Articles — Travaux Originaux

Schattauer GmbH

The Use of DEAE Cellulose in the Preparation of a Factor VIII (AHG)-free Bovine Fibrinogen Reagent^[*]

Authors

W. F Stapp^**

¹Central Coagulation Laboratory (Prof. Dr. E. Deutsch), The First Medical University Clinic (Prof. Dr. E. Lauda), University of Vienna Medical School, Vienna, Austria

Abstract

Summary

Adequate fibrinogen reactivity is suggested as an additional requirement for specific fibrinogen reagents, to be added to the well known requirements of protein concentration, optimum pH, and optimum ionic strength. Reasons and a method for testing are outlined.

A method for the preparation of a specific fibrinogen reagent, deficient in factor VIII, from BaSO₄ adsorbed bovine plasma is described in detail. The method described utilizes either fresh or regenerated DEAE cellulose as an adsorbent in a non-column chromatographic adsorption procedure, associated with a dialysis procedure in which the final fibrinogen product is concentrated and precipitated as a “euglobulin”. The “euglobulin” fibrinogen precipitate was then dissolved/suspended in buffered saline at a specific pH and NaCl concentration. Essentially the method can be briefly outlined into 5 separate steps:

1. Dialysis of BaSO₄ adsorbed bovine plasma against 0.5% NaCl at pH 6.6 in 0.005 M Imidazole-HCl buffer.

2. Washing of DEAE cellulose with the same type of buffered NaCl solution to specific conditions.

3. Mixing of the dialyzed BaSO₄ adsorbed bovine plasma with the washed DEAE cellulose and separation by centrifugation.

4. Concentration of the cellulose adsorbed plasma as a “euglobulin” fibrinogen precipitate by dialysis against running tap water.

5. Dissolving of the “euglobulin” fibrinogen precipitate in 0.9% NaCl buffered at pH 6.8.

Fibrinogen preparations, prepared with the use of regenerated DEAE cellulose were found to be similar to those prepared by fresh DEAE cellulose in fibrinogen reactivity and deficiency in factor VIII activity, but were found to have less fibrinogen concentration than those prepared using fresh DEAE cellulose.

Suggestions were also made for possibly increasing the over-all yield of the fibrinogen reagent through use of a “washing-elution” procedure.

The procedure that was used for regenerating the DEAE cellulose was also described in detail and commented upon.

Volltext

Referenzen

References
(1) Alexander B, De Vries A. Human Prothrombin: Quantitative studies on the labile factor and the restorative effects of normal hypofibrinogenemie and hemophilic plasma on the prothrombin time of stored plasma. J. clin. Invest. 28: 24 1949;
(2) Astrup T. Biochemistry of Blood Coagulation. Thesis Acta physiol, scand.. 7 (Suppl. 21) 47 1944;
(3) Astrup T, Darling S. Investigations on Fibrinogen. Acta physiol. scand. 4: 45 1942;
(4) Bounameaux Y. Dosage du facteur VIII en un temps. Acta haemat. (Basel) 17: 355 1957;
(5) Banfi R. G, Tanturi C. A, Bay R. Prothrombin in preserved blood. J. Lab. clin. Med. 30: 512 1945;
(6) Bidwell E. The purification of bovine antihaemophilic globulin. Brit. J. Haemat. 1: 35 1955;
(7) Biggs R, Macfarlane R. G. Human Blood Coagulation and its Disorders. 342 Blackwell Scientific Publications; Oxford: 1953
(8) Blombäck B, Blombäck M. Purification of human and bovine fibrinogen. Ark. Kemi. 10: 415 1956;
(9) Blombäck M. Purification of antihemophilic globulin. I. Some studies on stability of the antihemophilic globulin activity in Fraction I—0 and a method for its partial separation from fibrinogen. Ark. Kemi. 12: 387 1958;
(10) Cohn E. J, Strong L. E, Huges Jr W. L, Mulford D. J, Ashworth J. N, Melin M, Taylor H. L. Preparation of serum and plasma proteins IV. A system for the separation into fractions of protein and lipoprotein components of biological tissues and fluids. J. Amer. chem. Soc. 68: 459 1946;
(11) van Creveld S, Hoorweg P. G, den Ottolander G. J. H, Veder H. A. Isolation of the antihemophilic factor from human plasma. Acta haemat. (Basel) 15: 1 1956;
(12) van Creveld S, Veder H. A, Pascha C. N. The separation of AHF from Fibrinogen. II. Thromb. Diath. haem. 4: 211 1960;
(13) van Creveld S, Veder H. A, Pascha C. N, Kroeze W. G. The separation of AHF from Fibrinogen. Thromb. Diath. haem. 3: 572 1959;
(14) Didisheim P. An artificial reagent for the diagnosis of classical hemophilia. Science 129: 389 1959;
(15) Fahey J. L, McCoy P. G, Goulian M. Chromatography of serum proteins in normal and pathological sera; the distribution of protein bound carbohydrate and cholesterol, Siderophilin, thyroxin binding protein, B₁₂ binding protein, alkaline and acid phosphatases, radio iodinated albumin and myeloproteins. J. clin. Invest. 37: 272 1958;
(16) Frédéricq L. „De l’existence dans le plasma sanguine d’une substance albummoide se coagulant a + 56° C.” Gand 1877. Extrait des Annales de la Société de Médecine de Gand.
(17) Graham J. B, Penick G. D, Brinkhous K. M. Utilization of Antihemophilic factor during clotting of canine blood and plasma. Amer. J. Physiol. 165: 710 1951;
(18) Hammersten O. Untersuchungen über die sog. Fibringeneratoren, den Faserstoff und die Gerinnung des Fibrinogens. Kap. I. Eiweißkörper und verwandte Stoffe. Malys Jahresber. d. Tierchemie 8: 15 1876;
(19) Jaques L. B. The reducing properties of fibrinogen. Biochem. J. 37: 344 1943;
(20) Jung E. G. A rapid quantitative assay of Factor VIII (AHG) without the use of hemophilia A plasma. Thromb. Diath. haem. 4: 323 1960;
(21) Kaplan S, Friedman I. A. Effects of ultraviolet ray irradiation on clotting mechanism of plasma. J. Amer. med. Ass. 147: 229 1951;
(22) Keckwick R. A, Mackay M. E, Record B. R. Fractionation of human plasma with ether. Nature 157: 629 1946;
(23) Lorand L, Laki K. A simple method for purifying an activator of prothrombin. Biochim. biophys. Acta 13: 448 1954;
(24) 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;
(25) Mertz E. T, Owen C. A. Imidazole buffer: Its use in blood clotting studies. Proc. Soc. exp. Biol. (N. Y.) 43: 204 1940;
(26) Owren P. A. The coagulation of blood. Investigations on a new clotting factor. Thesis. Acta med. scand. 128 (Suppl. 194) 125 1947;
(27) Peterson E. A, Sober H. A. Chromatography of Proteins I. Cellulose Ion Exchange Adsorbents. J. Amer. chem. Soc. 78: 751 1956;
(28) Pool J. G, Robinson J. Observations on plasma banking and transfusion procedures for haemophilia patients using a quantitative assay for AHG. Brit. J. Haemat. 5: 24 1959;
(29) Porath J. Some cellulose ion exchangers of low substitution and their chromatographic application. Ark. Kemi. 11: 97 1956;
(30) Quick A. J. Hemorrhagic Diseases. a) 415 b) p. 34—35, c) p. 432. Lea and Febiger; Philadelphia: 1957
(31) Ratnoff O. D. An accelerating property of plasma for the coagulation of fibrinogen by thrombin. J. clin. Invest. 33: 1175 1954;
(32) Rosenfeld L, Shinowara G. Y. Enzyme studies on human blood. The fibrinogen content of stored plasma. J. Lab. clin. Med. 37: 311 1951;
(33) Seegers W. H, Landaburu R, Fenichel R. L. Isolation of platelet cofactor I from plasma and some of the properties of the preparation. Amer. J. Physiol. 190: 1 1957;
(34) Schulz F. H. Eine einfache volumetrische Fibrinbestimmung. Ärztl. Lab. 1: 107 1955;
(35) Shinowara G. Enzyme studies on human blood. XL The isolation and characterization of thromboplastic cell and plasma components. J. Lab. clin. Med. 38: 11 1951;
(36) Soulier J. P. A new adsorption agent for coagulation factors. J. clin. Path. 12: 303 1959;
(37) Soulier J. P, Gobbiet T, Larrieu J. J. Séparation du fiibrinogène et du facteur antihémophilique A. Rev. Hémat. 12: 481 1957;
(38) Stefanini M. Antithrombin activity of stored plasma. Amer. J. clin. Path. 18: 537 1948;
(39) Stormorken H. Reactivity of stored plasma to thrombin, with reference to the fibrinogen conversion accelerator and heparinoid activity. Brit. J. Haemat. 3: 299 1957;
(40) Surgenor D. M, Steele B. B. Studies on the nature of the antihemophilic activity of normal human plasma. Sixth Annual Symposium on Blood. Thromb. Diath. haem. 1: 563 1957;
(41) Triantaphyllopoulas D. C, Quick A. J, Greenwall T. J. Action of disodium ethylenediamine tetracetate on blood coagulation. Evidence of the development of heparinoid activity during incubation or aeration of plasma. Blood 10: 534 1955;
(42) 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. Thromb. Diath. haem. 1: 1 1957;
(43) Ware A. G, Guest M. M, Seegers W. H. Fibrinogen: with special reference to its preparation and certain properties of the product. Arch. Biochem. 13: 231 1947;
(44) Warner E. D, Brinkhous K, Smith H. P. The titration of prothrombin in certain plasmas. Arch. Path. (Chicago) 18: 587 1934;
(45) Williams T. L. An Introduction to Chromatography. 21 1947—1948 Reprint Edition Blackie and Son Limited; London and Glasgow.:

The Use of DEAE Cellulose in the Preparation of a Factor VIII (AHG)-free Bovine Fibrinogen Reagent[*]

Authors

The Use of DEAE Cellulose in the Preparation of a Factor VIII (AHG)-free Bovine Fibrinogen Reagent^[*]