Download PDF
Thromb Haemost 1986; 55(03): 352-356
DOI: 10.1055/s-0038-1661562
Original Article
Schattauer GmbH Stuttgart

Binding of Various Thrombin Fractions to Fibrin and the Influence of AT-III on Their Adsorption

D W T Nilsen
The Haematological Research Laboratory, Medical Department, Ullevål Hospital, Rikshospitalet, Oslo, Norway
,
F Brosstad
**   The Research Institute for Internal Medicine, Rikshospitalet, Oslo, Norway
,
P Kierulf
*   The Central Laboratory, Ullevål Hospital, Rikshospitalet, Oslo, Norway
,
H C Godal
The Haematological Research Laboratory, Medical Department, Ullevål Hospital, Rikshospitalet, Oslo, Norway
› Author Affiliations
Further Information

Publication History

Received 03 September 1985

Accepted 27 March 1986

Publication Date:
18 July 2018 (online)

  PDF Download  Permissions and Reprints

Summary

Human thrombin with high affinity for fibrin was obtained by subjecting purified thrombin to affinity chromatography on Sepharose insolubilized fibrin monomers, after addition of a radioiodinated subsample of thrombin, molar ratio 1:600. As judged by radioprofiling of the electrophoretic distribution of high-affinity thrombin on 10 per cent polyacrylamide gel containing urea/SDS, the preparation consisted of 70 per cent a-thrombin, 28 per cent β-thrombin and only 2 per cent γ-thrombin. Although a-thrombin was bound more strongly to insolubilized fibrin monomers than the other subfractions, complete separation of the individual components could not be achieved.

High-affinity thrombin was employed for studies on thrombin adsorption to polymerized fibrin, assuming equal behaviour of labelled and unlabelled thrombin.

o avoid passive entrapment of thrombin within the fibrin meshwork at physiological pH, ionic strength and calcium concentration, the optimal fibrinogen concentration was found to be 2.94 umol/1. During such conditions, adsorption of thrombin to polymerized fibrin did not exceed 65 per cent of added thrombin, despite an increasing availability of fibrin-related thrombin binding domains obtained by reducing the thrombin concentration.

Adsorption of thrombin to polymerized fibrin increased by 25 per cent when the ionic strength was reduced to 0.05 mol/1. These findings suggest the presence of thrombin subfractions with different affinities for polymerized fibrin.

Aggregates of high-affinity thrombin formed during its preparation by affinity chromatography, but were prevented by adding polyethylene glycol (m.w. 6,000, final conc. 6.6 g/1). Such aggregates were not inactivated by AT-III, but could still adsorb to polymerized fibrin.

In its monomeric form fibrin-adsorbed thrombin was inactivated by AT-III and could then be extruded from the clot upon syneresis and retraction, but a small fraction remained attached to polymerized fibrin, exhibiting minor amidolytic activity.

Keywords

Thrombin - Fibrin - Antithrombin III - Clot interactions
 

  • References

  • 1 Winter JH, Bennett B, McTaggart F, Douglas AS. Lipoprotein fractions and antithrombin III consumption during clotting. Thromb Haemostas 1982; 47: 236-238
    PubMedGoogle Scholar
  • 2 Nilsen DWT, Jeremic M, Weisert OK, Fϕnstelien E, Godal HC. Functional and antigenic antithrombin III values in plasma and serum from 115 regular blood donors and antithrombin III in prediction of postoperative thrombosis. Thromb Haemostas 1983; 50: 530-533
    PubMedGoogle Scholar
  • 3 Seegers WH. Multiple protein interactions as exhibited by the blood clotting mechanism. J Phys colloid Chem 1947; 51: 198-206
    PubMedGoogle Scholar
  • 4 Liu CY, Nossel HL, Kaplan KL. The binding of thrombin by fibrin. J Biol Chem 1979; 254: 10421-10425
    PubMedGoogle Scholar
  • 5 Abildgaard U, Fagerhol MK, Egeberg O. Comparison of progressive antithrombin activity and the concentrations of three thrombin inhibitors in human plasma. Scand J Clin Invest 1970; 26: 349-354
    CrossrefPubMedGoogle Scholar
  • 6 Hensen A, Loeliger EA. Antithrombin III. Its metabolism and its function in blood coagulation. Thrombos Diathes Haemorrh 1963; 9 (Suppl. 01) 1-79
    PubMedGoogle Scholar
  • 7 Francis CW, Markham RE, Barlow GH, Florach TM, Dobrzynski DM, Marder VJ. Thrombin activity of fibrin thrombi and soluble plasmic derivatives. J Lab Clin Med 1983; 102: 220-230
    PubMedGoogle Scholar
  • 8 Bloom AL. The release of thrombin from fibrin by fibrinolysis. Br J Haematol 1962; 8: 129-133
    CrossrefPubMedGoogle Scholar
  • 9 Owren PA. The coagulation of blood. Investigations on a new clotting factor. Acta Med Scand 1947; 128 (Suppl. 194) 1-327
    PubMedGoogle Scholar
  • 10 Jakobsen E, Kierulf P. A modified beta-alanine precipitation procedure to prepare fibrinogen free of antithrombin III and plasminogen. Thromb Res 1973; 3: 145-159
    CrossrefPubMedGoogle Scholar
  • 11 Nesheim ME, Prendergast FG, Mann KG. Interactions of a fluorescent active-site-directed inhibitor of thrombin: dansylarginine N-(3-Ethyl-1.5-pentanediyl) amide. Biochemistry 1979; 18 (06) 996-1003
    CrossrefPubMedGoogle Scholar
  • 12 Weber K, Osborn M. The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel eletrophoresis. J Biol Chem 1969; 244: 4406-4412
    PubMedGoogle Scholar
  • 13 McConahey PJ, Dixon FJ. A method of trace iodination of proteins for immunologic studies. Int Arch Allergy Appl Immunol 1966; 29: 185-189
    CrossrefPubMedGoogle Scholar
  • 14 Heene DL, Matthias FR. Adsorption of fibrinogen derivatives on insolubilized fibrinogen and fibrin monomer. Thromb Res 1973 2: 137-154
    PubMedGoogle Scholar
  • 15 Kaminski M, McDonagh J. Studies on the mechanism of thrombin. J Biol Chem 1983; 258: 10530-10535
    PubMedGoogle Scholar
  • 16 Nossel HL, Yudelman I, Canfield RE, Butler VP, Spanondes K, Wilner GD, Qureshi GD. Measurement of fibrinopeptide A in human blood. J Clin Invest 1974; 54: 43-53
    CrossrefPubMedGoogle Scholar
  • 17 Jacobsson K. Studies on the determination of fibrinogen in human blood. Scand J Clin Invest 1955; 7 (Suppl. 14) 1-14
    CrossrefPubMedGoogle Scholar
  • 18 Caspary EA, Kekwick RA. Some physicochemical properties of human fibrinogen. Biochem J 1957; 67: 41-48
    CrossrefPubMedGoogle Scholar
  • 19 Fenton JW, Landis BH, Walz DA, Finlayson JS. Human thrombins. In: Chemistry and biology of thrombin Lund blad. et al. (eds.) pp 43-70 Ann Arbor; 1977
  • 20 Ødegaard OR, Lie M, Abildgaard U. Heparin cofactor activity measured with an amidolytic method. Thromb Res 1975; 6: 287-294
    CrossrefPubMedGoogle Scholar
  • 21 Conard J, Brosstad F, Lie Larsen M, Samama M, Abildgaard U. Molar antithrombin concentration in normal human plasma. Haemostasis 1983; 13: 363-368
    PubMedGoogle Scholar
  • 22 Ferry JD, Morrison PR. Preparation and properties of serum and plasma proteins. VIII. The conversion of human fibrinogen to fibrin under various conditions. J Am Chem Soc 1947; 69: 388-400
    CrossrefPubMedGoogle Scholar
  • 23 Blomback B, Okada M. Fibrin gel structure and clotting time. Thromb Res 1982; 25: 51-70
    CrossrefPubMedGoogle Scholar
  • 24 Wilner GD, Danitz MP, Mudd MS, Hsieh K-H, Fenton JW. Selective immobilization of a-thrombin by surface bound fibrin. J Lab Clin Med 1981; 97 (03) 403-411
    PubMedGoogle Scholar
  • 25 Nilsen DW T, Kierulf P, Godal HC. Adsorption of human thrombin to fibrin, and the influence of AT-III on thrombin adsorption. Thromb Haemostas 1985; 54: 88 (Abstr)
    PubMedGoogle Scholar
  • 26 Shapiro SS, Anderson DB. Thrombin inhibition in normal plasma. In: Chemistry and biology of thrombin Lund blad. et al. (eds) pp 361-374 Ann Arbor; 1977
  • 27 Wasiewski W, Fasco MJ, Martin BM, Detwiler TS, Fenton JW. Thrombin adsorption to surfaces and prevention with polyethylene glycol 6.000. Thromb Res 1976; 8: 881-886
    CrossrefPubMedGoogle Scholar