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Thromb Haemost 1961; 05(02): 218-249
DOI: 10.1055/s-0038-1654921
Originalarbeiten – Original Article – Travaux Originaux
Schattauer GmbH

Activation of Purified Prothrombin to Autoprothrombin I or Autoprothrombin II (Platelet Cofactor II or Autoprothrombin II-A)[*]

Eberhard F. Mammen
1   Department of Physiology and Pharmacology, Wayne State University, College of Medicine, Detroit, Michigan, USA
,
William R. Thomas
1   Department of Physiology and Pharmacology, Wayne State University, College of Medicine, Detroit, Michigan, USA
,
Walter H. Seegers
1   Department of Physiology and Pharmacology, Wayne State University, College of Medicine, Detroit, Michigan, USA
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Publikationsdatum:
21. Juni 2018 (online)

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Summary

Autoprothrombin I, autoprothrombin II, and autoprothrombin II-A have been derived from bovine prothrombin and concentrated.

Autoprothrombin I is formed when thrombin is used to activate prothrombin which has been chromatographed on IRC-50 or diethylaminoethyl cellulose. The activation is inhibited by calcium ions, and goes more rapidly as the alkalinity is increased up to pH 9.7. During the activation the liberation of acidic groups predominates over that of the basic groups. In activation mixtures proline, alanine, threonine and valine were found as N-terminal amino acids. The activity was identified with a fraction that contained N-terminal valine, but an alteration can be made with IRC-50 so that the activity is all retained with a change in N-terminal amino acid. The activity is tentatively ascribed to a molecule that has a sedimentation constant of 3.8 in an ultracentrifuge. The autoprothrombin I was found to be soluble in ammonium sulfate solution at 50% of saturation and precipitated when the salt concentration is at 70% of saturation. There was very little loss of activity associated with drying from the frozen state at pH 8.0. Acetone precipitation destroyed the activity, and acidification predisposed to a loss in activity.

All prothrombin preparations we have worked with develop autoprothrombin I activity in the presence of platelet factor 3 and calcium ions, but not, if the calcium ions are not there. The solubility of the protein is the same as when obtained by thrombin activation or by separation from serum.

Autoprothrombin II is formed when purified thrombin is used to activate non-chromatographed prothrombin. The activation is inhibited with calcium ions, and there is a pH optimum between 7 and 8. During activation nonbuffered solutions become acidic. The activity is identified with a fraction in which the N-terminal amino acid is proline and the C-terminal amino acid is tyrosine. The activity was concentrated in a fraction that was practically homogenous upon ultracentrifuge analysis. The sedimentation constant is near S = 3.7 (tentative). The activity is easily precipitated at 50% of saturation with ammonium sulfate. No activity was lost upon dialysis or drying from the frozen state around pH 7 to 8. The concentrates with autoprothrombin I activity are precipitinogenic against antiprothrombin and anti-bovine serum. The autoprothrombin II of serum is also precipitated at 50% of saturation with ammonium sulfate.

Autoprothrombin II-A is an anticoagulant that was found to be closely related to autoprothrombin II in physical chemical characteristics. The anticoagulant had arginine as N-terminal amino acid and tyrosine as C-terminal amino acid. After IRC-50 resin chromatography the autoprothrombin II-A protein was found to be changed and had autoprothrombin II activity, N-terminal proline and C-terminal tyrosine. The autoprothrombin II-A activity was retained during dialysis and during drying from the frozen state. The sedimentation constant found with the ultracentrifuge was 3.8. The activity was also found in concentrates from serum.

Autoprothrombin II corrects the recalcified clotting time of PTC deficient plasma. Hemophilia A blood clots rapidly with autoprothrombin II. The recalcified clotting time of hemophilia A plasma is also brought to the normal range with concentrates of autoprothrombin II. In general the prothrombin utilization does not seem to be increased when the procoagulant is added to hemophilic blood or plasma.

* This investigation was supported by a research grant H-2026 from the National Heart Institute, National Institutes of Health, U. S. Public Health Service.


 

  • References

  • 1 Seegers W. H, Smith H. P, Warner E. D, Brinkhous K. M. The purification of prothrombin. J. biol. Chem. 123: 751 1938;
    PubMedGoogle Scholar
  • 2 Seegers W. H. The purification of prothrombin. Rec. chem. Progress. 13: 143 1952;
    PubMedGoogle Scholar
  • 3 Seegers W. H, Loomis E. C, Vandenbelt J. M. Preparation of prothrombin products: Isolation of prothrombin and its properties. Arch. Biochem. 06: 85 1945;
    PubMedGoogle Scholar
  • 4 Miller K. D. Chromatographic isolation of plasma prothrombin and trans-a-glucosylase. J. biol. Chem. 231: 987 1958;
    PubMedGoogle Scholar
  • 5 Lamy F, Waugh D. F. Certain properties of bovine prothrombin. J. biol. Chem. 203: 489 1953;
    PubMedGoogle Scholar
  • 6 Laki K, Kominz D. R, Symonds P, Lorand L, Seegers W. H. The amino acid composition of bovine prothrombin. Arch. Biochem. Biophys. 49: 276 1954;
    CrossrefPubMedGoogle Scholar
  • 7 Seegers W. H, Andrews E. B, McClaughry R. I. Formation of prothrombin derivatives from purified prothrombin. Amer. J. Physiol. 164: 722 1951;
    PubMedGoogle Scholar
  • 8 Seegers W. H, Thomas W. R, Landaburu R. H, Mammen E. F. Protbrombin: Living enzyme of my blood. Rec. chem. Progress. in press, 1960
    PubMedGoogle Scholar
  • 9 Landaburu R. H, Seegers W. H. Further studies of prothrombin derivatives. Proc. Soc. exp. Biol. (N. Y) 94: 708 1957;
    CrossrefPubMedGoogle Scholar
  • 10 Seegers W. H, Landaburu R. H, Johnson J. F. Thrombin-E as a fibrinolytic enzyme. Science 131: 726 1960;
    CrossrefPubMedGoogle Scholar
  • 11 McClaughry R. I, Seegers W. H. Nature of an accelerator of prothrombin activation arising during storage of prothrombin. Proc. Soc. exp. Biol. (N. Y) 80: 372 1952;
    CrossrefPubMedGoogle Scholar
  • 12 Alkjaersig N, Abe T, Johnson S. A, Seegers W. H. An accelerator of prothrombin activation derived from prothrombin. Amer. J. Physiol. 182: 443 1955;
    PubMedGoogle Scholar
  • 13 Seegers W. H, Alkjaersig N, Johnson S. A. Formation of autoprothrombin in solutions containing purified prothrombin and purified platelet factor 3. Amer. J. Physiol. 181: 589 1955;
    PubMedGoogle Scholar
  • 14 Mertz E. T, Seegers W. H, Smith H. P. Inactivation of prothrombin by purified thrombin solutions. Proc. Soc. exp. Biol. (N. Y) 41: 657 1939;
    CrossrefPubMedGoogle Scholar
  • 15 Seegers W. H, Johnson S. A. Conversion of prothrombin to autoprothrombin II (platelet cofactor II) and its relation to the blood clotting mechanism. Amer. J. Physiol. 184: 259 1956;
    PubMedGoogle Scholar
  • 16 Penner J. A, Duckert F, Johnson S. A, Seegers W. H. Conversion of prothrombin to autoprothrombin II. Canad. J. Biochem. 34: 1199 1956;
    PubMedGoogle Scholar
  • 17 Johnson S. A, Seegers W. H. The reduction of autoprothrombin II activity with Dicumarol. Circulation Res. 04: 182 1956;
    CrossrefPubMedGoogle Scholar
  • 18 Aggeler P. M, White S. G, Glendening M. B, Page E. W, Leake T. B, Bates G. Plasma thromboplastin component (PTC) deficiency: A new disease resembling hemophilia. Proc. Soc. exp. Biol. (N. Y) 79: 692 1952;
    CrossrefPubMedGoogle Scholar
  • 19 Biggs R, Douglas A. S, Macfarlane R. G, Dacie J. V, Pitney W. R, Merskey C, O’Brien J. R. Christmas disease. A condition previously mistaken for haemophilia. Brit. med. J. 02: 1378 1952;
    CrossrefPubMedGoogle Scholar
  • 20 Seegers W. H, Levine W. G, Shepard R. S. Further studies on the purification of thrombin. Canad. J. Biochem. 36: 603 1958;
    PubMedGoogle Scholar
  • 21 Ware A. G, Seegers W. H. Two-stage procedure for the quantitative determination of prothrombin concentration. Amer. J. clin. Path. 19: 471 1949;
    PubMedGoogle Scholar
  • 22 Seegers W. H, Smith H. P. Factors which influence the activity of purified thrombin. Amer. J. Physiol. 137: 348 1942;
    PubMedGoogle Scholar
  • 23 Carter J. R, Warner E. D. Quantitative estimation of plasma acceleratorglobulin. Proc. Soc. exp. Biol. (N. Y) 75: 223 1950;
    CrossrefPubMedGoogle Scholar
  • 24 Johnson S. A. Activation of purified prothrombin with hemophilic plasma. Amer. J. clin. Path. 23: 875 1953;
    PubMedGoogle Scholar
  • 25 Johnson S. A, Seegers W. H, Koppel J. L, Olwin J. H. The reduction of platelet cofactor II (autoprothrombin II) activity with anticoagulants. Thromb. Diath. haem. 01: 158 1957;
    PubMedGoogle Scholar
  • 26 Edman P. Preparation of phenyl thiohydantoins from some natural amino acids. Acta chem. scand. 04: 277 1950;
    CrossrefPubMedGoogle Scholar
  • 27 Sjöquist J. Paper strip identification of phenyl thiohydantoins. Acta chem. scand. 07: 447 1953;
    CrossrefPubMedGoogle Scholar
  • 28 Edman P, Sjöquist J. Identification and semiquantitative determination of phenyl thiohydantoins. Acta chem. scand. 10: 1507 1956;
    CrossrefPubMedGoogle Scholar
  • 29 Blombäck B, Yamashina I. On the N-terminal amino acid in fibrinogen and fibrin. Ark. Kemie. 12: 299 1958;
    PubMedGoogle Scholar
  • 30 Seegers W. H, Casillas G, Shepard R. S, Thomas W. R, Halick P. Some properties of thrombin preparations. Canad. J. Biochem. 37: 776 1959;
    PubMedGoogle Scholar
  • 31 Tibbs J. Identification of the free carboxo-groups in peptides and proteins. Nature (London) 168: 910 1951;
    PubMedGoogle Scholar
  • 32 Halick P, Seegers W. H. Antigenic properties of purified bovine prothrombin. Amer. J. Physiol. 187: 103 1956;
    PubMedGoogle Scholar
  • 33 Ware A. G, Seegers W. H. Studies on prothrombin: Purification, inactivation with thrombin, and activation with thromboplastin and calcium. J. biol. Chem. 174: 565 1948;
    PubMedGoogle Scholar
  • 34 Penner J. A, Seegers W. H. Activation of prothrombin. Amer. J. Physiol. 186: 343 1956;
    PubMedGoogle Scholar
  • 35 Seegers W. H, Alkjaersig N. Comparative properties of purified human and bovine prothrombin. Amer. J. Physiol. 172: 731 1953;
    PubMedGoogle Scholar
  • 36 Yphantis D. A, Waugh D. F. Ultracentrifugal characterization by direct measurement of activity. II. Experimental. J. physical Chem. 60: 630 1956;
    CrossrefPubMedGoogle Scholar
  • 37 McClaughry R. I. Concentration of a prothrombin conversion accelerator and thrombin precursor from serum. Amer. J. Physiol. 186: 335 1956;
    PubMedGoogle Scholar
  • 38 Ouchterlony O. Antigen-antibody reactions in gels. IV. Types of reactions in coordinated systems of diffusion. Acta path, microbiol. scand. 32: 231 1953;
    PubMedGoogle Scholar
  • 39 Seegers W. H, Landaburu R. H, Holburn R. R, Tocantins L. M. Clotting of hemophilic blood with purified platelet cofactor I, platelet factor 3 and threone. Proc. Soc. exp. Biol. (N. Y) 95: 583 1957;
    CrossrefPubMedGoogle Scholar
  • 40 Johnson J. F, Mammen E. F, Seegers W. H. Prothrombin utilization following addition of platelet cofactor I concentrates to hemophilic plasma. Thromb. Diath. haem. 03: 588 1959;
    PubMedGoogle Scholar
  • 41 Johnson S. A, Rutzky J, Schneider C. L, Seegers W. H. Activation of purified prothrombin with hemophilia plasma. Proc. 4th Internat. Congr. Internat. Soc. Hemat., Buenos Aires. 1952
    PubMedGoogle Scholar
  • 42 Schulman I, Smith C. H. Hemorrhagic disease in an infant due to deficiency of a previously undescribed clotting factor. Blood 07: 794 1952;
    PubMedGoogle Scholar
  • 43 Johnson S. A, McClaughry R. I, Seegers W. H. Nature of the blood clotting mechanisms in hemophilia. J. Mich. State med. Soc. 54: 797 1955;
    PubMedGoogle Scholar
  • 44 Johnson S. A, Seegers W. H. Platelet cofactor I activity in the hemophilias. In: Brinkhous K. M. (Edit.): Hemophilia and Hemophiloid Diseases. 27 The University of North Carolina Press; Chapel Hill: 1957
    Google Scholar
  • 45 Owen C. A, Hoffmann G. R, Ziffren S. E, Smith H. P. Blood coagulation during infancy. Proc. Soc. exp. Biol. (N. Y) 41: 81 1939;
    PubMedGoogle Scholar
  • 46 Johnson J. F, Seegers W. H. Laboratory observations on parahemophilia and proconvertin deficient plasma. Mich. State, med. J. 52: 537 1953;
    PubMedGoogle Scholar
  • 47 Seegers W. H, Alkjaersig N. Nature of the blood coagulation mechanisms in SPCA plasma. Circulation Res. 03: 514 1955;
    CrossrefPubMedGoogle Scholar
  • 48 Seegers W. H, Alkjaersig N, Johnson S. A. On the nature of the blood coagulation mechanisms in certain clinical states. Amer. J. clin. Path. 25: 983 1955;
    PubMedGoogle Scholar
  • 49 Seegers W. H. Activation of purified prothrombin. Proc. Soc. exp. Biol. (N. Y) 72: 677 1949;
    CrossrefPubMedGoogle Scholar
  • 50 Seegers W. H, Mammen E, Lee J. M, Landaburu R. H, Cho M. H, Baker W. J, Shepard R. S. Further studies on the purification of platelet cofactor I. In: Brinkhous K. M. (Edit.): Hemophilia and other Hemorrhagic states. 38 The University of North Carolina Press; Chapel Hill: 1959
    Google Scholar
  • 51 Shulman S, Landaburu R. H, Seegers W. H. Biophysical studies on platelet cofactor I preparations. Thromb. Diath. haem.. in press, 1960
    PubMedGoogle Scholar