The Kinetics of Inhibition of the Action of Thrombin by the Fibrinopeptides Formed during the Clotting of Fibrinogen

 

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Summary

Various investigators have separated the coagulation products formed when fibrinogen is clotted with thrombin and identified fibrinopeptides A and B. Two other peaks are observed in the chromatogram of the products of coagulation, but these have mostly been dismissed by other workers. They have been identified by us as amino acids, smaller peptides and amorphous material (37). We have re-chromatographed these peaks and identified several amino acids. In a closed system of fibrinogen and thrombin, the only reaction products should be fibrin and peptide A and peptide B. This reasoning has come about because thrombin has been reported to be specific for the glycyl-arginyl peptide bond. It is suggested that thrombin also breaks other peptide linkages and the Peptide A and Peptide B are attacked by thrombin to yield proteolytic products. Thrombin is therefore probably not specific for the glycyl-arginyl bond but will react on other linkages as well.

If the aforementioned is correct then the fibrinopeptides A and B would cause an inhibition with the coagulation mechanism itself. We have shown that an inhibition does occur. We suggest that there is an autoinhibition to the clotting mechanism that might be a control mechanism in the human body.

The experiment was designed for coagulation to occur under controlled conditions of temperature and time. Purified reactants were used. We assembled an apparatus to record visually the speed of the initial reaction, the rate of the reaction, and the density of the final clot formed after a specific time.

The figures we derived made available to us data whereby we could calculate and plot the information to show the mechanism and suggest that such an inhibition does exist and also further suggest that it might be competitive.

In order to prove true competitive inhibition it is necessary to fulfill the criteria of the Lineweaver-Burk plot. This has been done. We have also satisfied other criteria of Dixon (29) and Bergman (31) that suggest true competitive inhibition.

^* American Society of Clinical Pathology-Sheard-Sanford prize-winning project (1965) for senior medical students.

^** Please send requests for reprints to Dr. Marvin Murray, M. D., Ph. D., Associate Professor of Pathology.

• References

• 1 Bettelheim F. R, Bailey K. The products of the action of thrombin on fibrinogen. Biochim. biophys. Acta (Amst.) 9: 578 1952;
  CrossrefPubMedGoogle Scholar
• 2 Foranei L. Fibrino-peptide. Biochem. J 52: 200 1952;
  CrossrefPubMedGoogle Scholar
• 3 Bettelheim F. R. The clotting of fibrinogen. II. Fractionation of peptide material liberated. Biochim. biophys. Acta (Amst.) 19: 121 1956;
  CrossrefPubMedGoogle Scholar
• 4 Folk J. E, Gladner J. A, Levin Y. Thrombin-induced formation of co-fibrin. III. Acid degradation studies and summary of sequential evidence on peptide A. J. biol. Chem 234: 2317 1959;
  PubMedGoogle Scholar
• 5 Sjoquist J, Blombäck B, Wallen P. Amino acid sequence of bovine fibrinopeptides. Arkiv for Kemi 16: 425 1960;
  PubMedGoogle Scholar
• 6 Blombäck B, Wallen P, Sjoquist J. On the amino acid sequence of bovine fibrinopeptides. Acta chem. scand 13: 819 1959;
  CrossrefPubMedGoogle Scholar
• 7 Blombäck B, Sjoquist J. Studies on fibrinopeptides from different species. Acta chem. Scand 14: 493 1960;
  CrossrefPubMedGoogle Scholar
• 8 Bailey K, Bettelheim F. R, Lorand L, Middlebrook W. R. Action of thrombin in the clotting of fibrinogen. Nature (Lond.) 167: 233 1951;
  CrossrefPubMedGoogle Scholar
• 9 Blombäck B, Yamashima I. The determination of N-terminal amino acids during the conversion of fibrinogen to fibrin. Acta chem. scand 11: 194 1957;
  CrossrefPubMedGoogle Scholar
• 10 Folk J. E, Gladner J. A, Laki K. The thrombin-induced formation of co-fibrin. II. Preliminary amino acid sequence studies on peptides A and B. J. biol. Chem 234: 67 1959;
  PubMedGoogle Scholar
• 11 Laki K, Mommaerts W. F. Transition of fibrinogen to fibrin as a two-step reaction. Nature (Lond.) 155: 664 1945;
  PubMedGoogle Scholar
• 12 Laki K. The polymerization of proteins: The action of thrombin on fibrinogen. Arch, biochem 32: 317 1951;
  CrossrefPubMedGoogle Scholar
• 13 Seegers W. H. Activation of purified prothrombin. Proc. Soc. exp. Biol. (N. Y.) 72: 677 1949;
  CrossrefPubMedGoogle Scholar
• 14 Seegers W. H, Casillas H. G, Shepard R. S, Thomas W. R, Halick P. Some properties of thrombin preparations. Canad. J. Biochem 37: 775 1959;
  CrossrefPubMedGoogle Scholar
• 15 Scheraga H. A, Laskowski M. The fibrinogen-fibrin conversion. Advanc. Protein. Chem 12: 43 1957;
  PubMedGoogle Scholar
• 16 Latallo Z. S, Fletcher A. P, Alkjaersig N, Sherry S. Influence of pH, ionic strength, neutral ions, and thrombin on fibrin polymerization. Amer. J. Physiol 202: 675 1962;
  PubMedGoogle Scholar
• 17 Landaburu R. H, Seegers W. H. Proteolytic and polymerase activity of thrombin. Amer. J. Physiol 198: 173 1960;
  PubMedGoogle Scholar
• 18 Blombäck B, Vestermark A. Isolation of librino-peptides by chromatography. Arkiv for Kemil2: 173 1958;
  PubMedGoogle Scholar
• 19 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;
  CrossrefPubMedGoogle Scholar
• 20 Triantaphyllopoulos D. C. Nature of the inhibition of thrombin by the anticoagulant fraction of incubated fibrinogen. Fed. Proc 18: 161 1959;
  PubMedGoogle Scholar
• 21 Triantaphyllopoulos D. C. Anticoagulant effect of incubated fibrinogen. Canad. J. Biochem 30: 250 1958;
  PubMedGoogle Scholar
• 22 Lorand L, Yudkin E. P. The effect of arginylpeptides on the clotting of fibrinogen with thrombin. Biochim. biophys. Acta (Amst.) 25: 437 1957;
  PubMedGoogle Scholar
• 23 Blombäck B, Blombäck M. Purification of human and bovine fibrinogen. Arkiv for Kemi 10: 415 1956;
  PubMedGoogle Scholar
• 24 Peterson E. A, Sober H. A. Chromatography of proteins. I. Cellulose ion-exchange adsorbents. J. Amer. chem. Soc 78: 751 1956;
  CrossrefPubMedGoogle Scholar
• 25 Peterson E. A, Sober H. A. Variable gradient device for chromatography. Analyt. Chem 31: 857 1959;
  CrossrefPubMedGoogle Scholar
• 26 Schroeder W. A, Jones R. T, Cormick J, McCalla K. Chromatographic separation of peptides on ion exchange resins. Analyt. Chem 34: 1570 1962;
  CrossrefPubMedGoogle Scholar
• 27 Hirs C. H. W, Moore S, Stein W. H. Peptides obtained by tryptic hydrolysis of perforane acid oxidized ribonuclease. J. biol. Chem 219: 623 1956;
  PubMedGoogle Scholar
• 28 Lowry O. H, Rosebrough N. J, Farr A. L, Randall R. J. Protein measurement with the Folin Phenol reagent. J. biol. Chem 193: 265 1951;
  PubMedGoogle Scholar
• 29 Dixon M. The determination of enzyme inhibitor constants. Biochem. J 55: 170 1953;
  CrossrefPubMedGoogle Scholar
• 30 Lineweaver H, Burk D. The determination of enzyme association constants. J. Amer. chem. Soc 55: 658 1934;
  PubMedGoogle Scholar
• 31 Bergman F, Shemoni A. The enzymatic hydrolysis of alkyl fibroacetates. Biochem. J 55: 50 1953;
  CrossrefPubMedGoogle Scholar
• 32 Piez K. A, Morris L. A modified procedure for the automatic analysis of amino acids. Analyt. Biochem 1: 187 1960;
  CrossrefPubMedGoogle Scholar
• 33 Hamilton P. B. Ion exchange chromatography of amino acids (Effect of resin particle size on column performance). Analyt. Chem 30: 914 1958;
  CrossrefPubMedGoogle Scholar
• 34 Rasmussen P. S. Purification of thrombin by chromatography. Biochim. biophys. Acta (Amst.) 16: 157 1955;
  CrossrefPubMedGoogle Scholar
• 35 Hemker II C, Hemker P. W, Loeliger E. A. Kinetic aspects of the interaction of blood clotting enzymes. I. Derivation of basic formulas. Thrombos. Diathes. haemorrh. (Stuttg.) 12: 155 1965;
  PubMedGoogle Scholar
• 36 Blombäck B. Chemical aspects of fibrinogen and fibrin. In: Fibrinogen and Fibrin Turnover of Clotting Factors. Eds. Hunter R. B, Koller F, Beek E. Thrombos. Diathes. haemorrh., Suppl. 13, p. 29-39 (1963)
  PubMedGoogle Scholar
• 37 Murray M, Gray Jr. L. The formation of fibrin variants. J. exp. molec. Path 3: 287 1964;
  PubMedGoogle Scholar