Studies on the Degradation of Human Fibrinogen by Plasmin and Trypsin^[*]

Summary

The effects of trypsin and plasmin on the functional and physicochemical properties of purified human fibrinogen were observed at various stages of proteolysis. Concentrations of plasmin and trypsin that produced fibrinogenolysis at comparable rates as measured in a pH stat produced, at similar rates, loss of precipitability of fibrinogen by heat and ammonium sulphate and alterations in electrophoretic mobility on starch gel. Trypsin produced a more rapid loss of clottability of fibrinogen and a more rapid appearance of inhibitors of the thrombin-fibrinogen clotting system than did plasmin. Consistent differences were noted between the effects of trypsin and plasmin on the immunoelectrophoretic properties of fibrinogen during the early stages of proteolysis.

These results are consistent with the hypothesis that trypsin initially reacts with the same peptide bonds of fibrinogen that are split by thrombin, but these same bonds do not appear to be split initially by plasmin. Measurement of the various functional and physico-chemical changes produced by the action of trypsin and plasmin on fibrinogen can be used to recognize various stages of proteolysis.

^* These investigations were carried out under contract AT(30-1)1208 between the U. S. Atomic Energy Commission and the Johns Hopkins University and were supported in part by research grant HE-01601 from The National Heart Institute, research career program award AM-K3-3779 and graduate training grant Tl AM-5260 from The National Institute of Arthritis and Metabolic Diseases, and U. S. Public Health Service International Postdoctoral Fellowship F 05-485.

• References

• 1 Virchow R. Über den Faserstoff (1845/46). In:Gesammelte Abhandlungen zur wissenschaftlichen Medizin. Meidinger Frankfurt a. M., 1856
• 2 Hammarsten O. Über das Fibrinogen. Pflüger’s Arch. ges. Physiol 22: 431 1880;
• 3 Garner R. L, Tillett W. S. Biochemical studies on the fibrinolytic activity of hemolytic streptococci. II. Nature of the reaction. J. exp. Med 60: 255 1934;
• 4 Seegers W. H, Nieft M. L, Vandenbelt J. M. Decomposition products of fibrinogen and fibrin. Arch. Biochem 7: 15 1945;
• 5 Nussenzweig V, Seligmann M, Pelmont J, Grabar P. Les produits de dégradation du fibrinogène humain par la plasmine. I. Séparation et propriétés physico-chimiques. Ann. Inst. Pasteur 100: 377 1961;
• 6 Niewiarowski S, Kowalski E, Stachurska J. Influence of fibrinogen derived antithrombin (antithrombin VI) on the blood coagulation system. Acta biochim. pol 6: 43 1959;
• 7 Triantaphyllopoidos D. C. Anticoagulant effect of incubated fibrinogen. Canad. J. Biochem 35: 250 1958;
• 8 Fletcher A. P, Alkjaersig N, Sherry S. Pathogenesis of the coagulation defect developing during pathological plasma proteolytic (“fibrinolytic”) states. I. The significance of fibrinogen proteolysis and circulating fibrinogen breakdown products. J. clin. Invest 41: 896 1962;
• 9 Latallo Z. S, Budzynski A. Z, Lipinski B, Kowalski E. Inhibition of thrombin and of fibrin polymerization, two activities derived from plasmin-digested fibrinogen. Nature (Lond.) 203: 1184 1964;
• 10 Bang N. U, Fletcher A. P, Alkjaersig N, Sherry S. Pathogenesis of the coagulation defect developing during pathological plasma proteolytic (“fibrinolytic”) states. III. Demonstration of abnormal clot structure by electron microscopy. J. clin. Invest 41: 935 1962;
• 11 Alkjaersig N, Fletcher A. P, Sherry S. Pathogenesis of the coagulation defect developing during pathological plasma proteolytic (“fibrinolytic”) states. II. The significance, mechanism and consequences of defective fibrin polymerization. J. clin. Invest 41: 917 1962;
• 12 Pechet L, Alexander B. The effect of certain proteolytic enzymes on the thrombin-fibrinogen interaction. Biochemistry 1: 875 1962;
• 13 Mihalyi E, Godfrey J. E. Digestion of fibrinogen by trypsin: I. Kinetic studies of the reaction. Biochim. biophys. Acta (Amst.) 67: 73 1963;
• 14 Alexander B, Rimon A, Katchalski E. Effects of water-insoluble trypsin derivatives on fibrinogen. Fed. Proc 24: 804 1965;
• 15 Wallen P, Bergström K. Action of thrombin on plasmin digested fibrinogen. Acta chem. scand 22: 574 1958;
• 16 Michaelis L. Der Acetat-Veronal-Puffer. Biochem. Z 234: 139 1931;
• 17 Laki K. The polymerization of proteins: the action of thrombin on fibrinogen. Arch. Biochem 32: 317 1951;
• 18 Morse E. E, Jackson D. P, Gonley G. L. Bole of platelet fibrinogen in the reactions of platelets to thrombin. J. clin. Invest 44: 809 1965;
• 19 Ratnoff G. D, Menzie G. A new method for the determination of fibrinogen in small samples of plasma. J. Lab. clin. Med 37: 316 1951;
• 20 Feinberg J. G. A new quantitative method for antigen-antibody titrations in gels. Nature (Lond.) 177: 530 1956;
• 21 Scheidegger J. J. Une micro-méthode de l’immunoélectrophorèse. Int. Arch. Allergy 7: 103 1955;
• 22 Smithies G. An improved procedure for starch-gel electrophoresis: Further variations in the serum proteins of normal individuals. Biochem. J 71: 585 1959;
• 23 Boyer S. H, Hiner R. Modified apparatus for starch gel electrophoresis. J. Lab. clin. Med 61: 879 1963;
• 24 Parfentjev I. A, Johnson M. L, Cliffton E. E. The determination of plasma fibrinogen by turbidity with ammonium sulphate. Arch. Biochem. Biophys 46: 470 1953;
• 25 Sutherland E. W, Cori C. F, Haynes R, Olsen N. S. Purification of the hyperglycemic-glycogenolytic factor from insulin and from gastric mucosa. J. biol. Chem 180: 825 1949;
• 26 Triantaphyllopoulos D. C. AFIF inhibitor from fibrinogen. Thrombos. Diathes. haemorrh. (Stuttg.) 7 (Suppl. 01) 79 1961;
• 27 Lewis J. H, Wilson J. H. Fibrinogen breakdown products. Amer. J. Physiol 207: 1053 1964;
• 28 Mills D. A, Goyne R, Pollara B, von Korff R. W. The action of plasmin on fibrinogen and fibrin. I. Changes in the N-terminal residues. Biochim. biophys. Acta (Amst.) 86: 527 1964;