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Investigation on plasmin-proteolysis of abnormal fibrinogen (Fibrinogen Giessen)
THROMBOSIS RESEARCH Printed in the United States
INVESTIGATION
ON PLASMIN-PROTEOLYSIS
~01.2, pp. 361-364, 1973 Pergamon Press, Inc.
OF ABNORMhL FIBRINOGEN
(FIBRINOGEN ~mssm) W.H.KRAUSE and H.BLEYL Department
of Internal Medicine and Department Chemistry, University
of Clinical
of Giessen,
63 Giessen, Klinikstrasse
32 b, West-Germany
(Received 8.2.1973; in revised form Accepted by Editor L. Rbka)
27.3.1973.
ABST!
In the cases of congenital dysfibrinogenemias so Par known the limited proteolysis by thrombin and reptilase was applied to characterize the abnormal fibrinogen (1). In a number of dysfibrinogenemias a decrease in the rate of fibrinopeptide release by thrombin was shown (2,3,4). In Fibrinogen Detroit (5,6) fibrinopeptide B was ;lot released to any appreciable extent. In fibrinogen Giesse:l the release of fibrinopeptide A did not succeed after incubation with thrombin which was demonstrated by twodimensional paper electrophoresis (7). A lack of cleavage of fibrinopeptide A by reptilase on Fibrinogen Metz was shown by Soria et al. CR). Plasnih digestion could give further information on the structure of abnormal fibrinogen. Therefore plasmin proteolysis was performed on fibrinogen Giessen.
361
362
FIBRINOGEN
GIESSEN
PROTEOLYSIS
METHODS -------Fibrinogen Giessen was isolated according to the method of Kazal et al. (9), huma n fibrinogen Kabi (Deutsche Kabi, Munchen) was used as normal fibrinogen. Both had a clottability of 91%. Fibrinogens were lysed by plasmin (Kabi). 3 ml fibrinogen solution (normal fibrinogen 300 mg%, abnormal fibrinogen 220 mg%) were lysed by 0,l U plasmin/ml at 37OC. After an incubation period of 2,5, 10, 20, 30, 60 and 120 min the lysis was blocked by aprotinin (500 U/ml, Trasylol, Bayer, Leverkusen). The thrombin time was then measured as follows: 0.1 ml plasma, 0.1 ml fibrinogen solution or FDP, 0.1 ml thrombin (Behringwerke, Marburg) 6 U/ml with normal fibrinogen, 60 U/ml with abnormal fibrinogen. A vertical flat bed gel electrophoresis according to Bleyl (10) was used for separation of the lysis products. A polyacrylamide gel from 4% to 12% was prepared in a TRIS-citrate buffer 0.375 M, pH 9. In electrophoresis TRIS-borate buffer 0.067 M, pH 9 was used. In both buffers the SDS concentration was 0.1 g/100 ml.
RESULTS ------The effect of proteolysis by plasmin in fibrinogen Giessen are shown in Table 1. With normal fibrinogen thrombin time was prolonged up to 30 min, indicating maximal accumulation of split product Y. Further plasmin degradation up to 120 min reduces the thrombin time by splitting Y to D and E. With abnormal fibrinogen there was also an increase in the thrombin clotting time which reached a maximum after 5 min and then remained at more than 300 sec. PAA gel electrophoresis of FDP form normal fibrinogen showed the split products X,Y,D and E, where as in fibrinogen Giessen only high molecular split products could be found. (Fig.1) De gration products similar to D and E could be obtained from the abnormal fibrinogen only by increasing the plasmin concentration up to 1 U/ml.
TABLE -------I Effect of fibrinogen degration products (FDP) from normal fibrinogen and fibrinogen Giessen on the thrombin time ___________________-~~-~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ Plasmin lysis 0 2 5 10 30 60 120 time (min) Normal clottingfg/FDP time (s) 25.4
38.6
fg Giessen/FDP clotting time (s) 22.0
55.8 300
47.0 115.2 300 300
300
80.4 300
37.4 300
vo1.2,~0.4
FIBRINOGEN
GIESSEN
363
PROTEOLYSIS
FIGURE 1
i
f
gel electrophoresis of FDP Giessen (Pos. l-7) and normal FDP (Pos. 8-14). Pos. l-7 and 8-14 show split products of incubation with plasmin after 0,2, 10, 20, 30, 60 and 120 min. Fibrinogen Giessen Pos. l-7 showes contamination with albumin at the anodic side.
PAA
DISCUSSION ---------With normal thrombin concentration fibrinogen Giessen reveals a slow coagulation, a lack of cleavage of fibrinopeptide A as well as a delayed aggregation of fibrinmonomers. With plasmin only high molecular thrombin clottable and nonclottable fragments (X and Y) are found, indicating that in the initial stage bonds at the carboxyl-terminal end of thea (A) chain and the NH -terminal end of the 8 (B) chain were split like in normal fibsinogen (11,12,13). It is likely that structure changes in other parts of fibrinogen Giessen impairs the further digestion. REFERENCES -----__---1. MENACHE,D. and GUILLIN,M.C.: Abnormal Fibrinogens. Abstract III. Congress, International Society on Thrombosis and Haemostasis, 1972 Wiesbaden. 2. GRALNICK,H.R., GIVELBER,H.M., SHAINOFF,J.R. and FINLAYSON,J.S.: Fibrinogen Bethesda: a congenital dysfibrinogenemia delayed fibrinopeptide release. J. Clin. Invet.: 50, 1819 (1971).
with
364
FIBRINOGEN
GIESSEN
PROTEOLYSIS
Vo1.2,No.4
3. BECK, E.A., SHAINOFF,J.R., VOGEL,A. and JACKSON, D.P.: Functional evaluation of an inherited abnormal fibrinogen. J. Clin. Invest .: 50, 1874 (1971). 4. SHERMAN,L.A.,GASTON,L.W., KAPLAN,M. and SPIVACK, A.R.: Fibrinogen St. Louis: A new inherited fibrinogen variant, coincidentally associated with hemophilia A. J. Clin. Invest: 51, 590 (1972). 5. BLOMBACK,M., BLOMBACK,B., MAMMEN,E.F. and PRASAD,A.S.: Fibrinogen Detroit a molecular defect in the N-terminal disulphide knot of human fibrinogen. Nature: 218, 134, (1968). 6. BLOMBACK, B. and BLOMBACK,M.: Molecular defects and variants of fibrinogen. Nouv. Rev. franc. d. Hematolog. l0, 671, (1970). 7. KRAUSE,W.H., HEENE,D.L., ZIMMERMANN,P. and LASCH,H.G.: Congenital dysfibrinogenemia (Fibrinogen Giessen). Abstract, III. Congress Internation Society on Thrombosis and Haemostasis, 1972, Wiesbaden. 8. SORIA,J,, SORIA, C., SAMAMA,M., POIROT,E. and KLING,C.: Fibrinogen Troyes-Fibrinogen Metz. Two new cases of congenital dysfibrinogenemia. Thromb. Diath. haemorrh., 27: 619, (1972). 9. KAZAL, L.A., AMSEL,S., MILLER, O.P. and TOKANTINS, L.M.: The preparation and some properties of fibrinogen precipitated from human plasma by glycine. Proc. Sot. exp. Biol. (N.Y.):113: 989, (1963). 10. BLEYL, H. : A new method in polyacrylamide phoresis. To be published,
gel electro-
11. MILLS,D.A.. * A molecular model for the proteolysis of human fibrinogen by plasmin, Biochim. Biophys. Acta: 263, 619, (1972). 12. MILLS,D.A. and KARPATKIN,S.: The initial macromolecular derivatives of human fibrinogen produced by plasmin. Biochim. Biophys. Acta: 271, 163 (1972). 13. FURLAN,M. and BECK,E.A.: Plasmatic degradation of human fibrinogen. I Structural characterization of degradation products. Biochim. Biophys, Acta: 262 , 631 (1972).
INVESTIGATION
ON PLASMIN-PROTEOLYSIS
~01.2, pp. 361-364, 1973 Pergamon Press, Inc.
OF ABNORMhL FIBRINOGEN
(FIBRINOGEN ~mssm) W.H.KRAUSE and H.BLEYL Department
of Internal Medicine and Department Chemistry, University
of Clinical
of Giessen,
63 Giessen, Klinikstrasse
32 b, West-Germany
(Received 8.2.1973; in revised form Accepted by Editor L. Rbka)
27.3.1973.
ABST!
In the cases of congenital dysfibrinogenemias so Par known the limited proteolysis by thrombin and reptilase was applied to characterize the abnormal fibrinogen (1). In a number of dysfibrinogenemias a decrease in the rate of fibrinopeptide release by thrombin was shown (2,3,4). In Fibrinogen Detroit (5,6) fibrinopeptide B was ;lot released to any appreciable extent. In fibrinogen Giesse:l the release of fibrinopeptide A did not succeed after incubation with thrombin which was demonstrated by twodimensional paper electrophoresis (7). A lack of cleavage of fibrinopeptide A by reptilase on Fibrinogen Metz was shown by Soria et al. CR). Plasnih digestion could give further information on the structure of abnormal fibrinogen. Therefore plasmin proteolysis was performed on fibrinogen Giessen.
361
362
FIBRINOGEN
GIESSEN
PROTEOLYSIS
METHODS -------Fibrinogen Giessen was isolated according to the method of Kazal et al. (9), huma n fibrinogen Kabi (Deutsche Kabi, Munchen) was used as normal fibrinogen. Both had a clottability of 91%. Fibrinogens were lysed by plasmin (Kabi). 3 ml fibrinogen solution (normal fibrinogen 300 mg%, abnormal fibrinogen 220 mg%) were lysed by 0,l U plasmin/ml at 37OC. After an incubation period of 2,5, 10, 20, 30, 60 and 120 min the lysis was blocked by aprotinin (500 U/ml, Trasylol, Bayer, Leverkusen). The thrombin time was then measured as follows: 0.1 ml plasma, 0.1 ml fibrinogen solution or FDP, 0.1 ml thrombin (Behringwerke, Marburg) 6 U/ml with normal fibrinogen, 60 U/ml with abnormal fibrinogen. A vertical flat bed gel electrophoresis according to Bleyl (10) was used for separation of the lysis products. A polyacrylamide gel from 4% to 12% was prepared in a TRIS-citrate buffer 0.375 M, pH 9. In electrophoresis TRIS-borate buffer 0.067 M, pH 9 was used. In both buffers the SDS concentration was 0.1 g/100 ml.
RESULTS ------The effect of proteolysis by plasmin in fibrinogen Giessen are shown in Table 1. With normal fibrinogen thrombin time was prolonged up to 30 min, indicating maximal accumulation of split product Y. Further plasmin degradation up to 120 min reduces the thrombin time by splitting Y to D and E. With abnormal fibrinogen there was also an increase in the thrombin clotting time which reached a maximum after 5 min and then remained at more than 300 sec. PAA gel electrophoresis of FDP form normal fibrinogen showed the split products X,Y,D and E, where as in fibrinogen Giessen only high molecular split products could be found. (Fig.1) De gration products similar to D and E could be obtained from the abnormal fibrinogen only by increasing the plasmin concentration up to 1 U/ml.
TABLE -------I Effect of fibrinogen degration products (FDP) from normal fibrinogen and fibrinogen Giessen on the thrombin time ___________________-~~-~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ Plasmin lysis 0 2 5 10 30 60 120 time (min) Normal clottingfg/FDP time (s) 25.4
38.6
fg Giessen/FDP clotting time (s) 22.0
55.8 300
47.0 115.2 300 300
300
80.4 300
37.4 300
vo1.2,~0.4
FIBRINOGEN
GIESSEN
363
PROTEOLYSIS
FIGURE 1
i
f
gel electrophoresis of FDP Giessen (Pos. l-7) and normal FDP (Pos. 8-14). Pos. l-7 and 8-14 show split products of incubation with plasmin after 0,2, 10, 20, 30, 60 and 120 min. Fibrinogen Giessen Pos. l-7 showes contamination with albumin at the anodic side.
PAA
DISCUSSION ---------With normal thrombin concentration fibrinogen Giessen reveals a slow coagulation, a lack of cleavage of fibrinopeptide A as well as a delayed aggregation of fibrinmonomers. With plasmin only high molecular thrombin clottable and nonclottable fragments (X and Y) are found, indicating that in the initial stage bonds at the carboxyl-terminal end of thea (A) chain and the NH -terminal end of the 8 (B) chain were split like in normal fibsinogen (11,12,13). It is likely that structure changes in other parts of fibrinogen Giessen impairs the further digestion. REFERENCES -----__---1. MENACHE,D. and GUILLIN,M.C.: Abnormal Fibrinogens. Abstract III. Congress, International Society on Thrombosis and Haemostasis, 1972 Wiesbaden. 2. GRALNICK,H.R., GIVELBER,H.M., SHAINOFF,J.R. and FINLAYSON,J.S.: Fibrinogen Bethesda: a congenital dysfibrinogenemia delayed fibrinopeptide release. J. Clin. Invet.: 50, 1819 (1971).
with
364
FIBRINOGEN
GIESSEN
PROTEOLYSIS
Vo1.2,No.4
3. BECK, E.A., SHAINOFF,J.R., VOGEL,A. and JACKSON, D.P.: Functional evaluation of an inherited abnormal fibrinogen. J. Clin. Invest .: 50, 1874 (1971). 4. SHERMAN,L.A.,GASTON,L.W., KAPLAN,M. and SPIVACK, A.R.: Fibrinogen St. Louis: A new inherited fibrinogen variant, coincidentally associated with hemophilia A. J. Clin. Invest: 51, 590 (1972). 5. BLOMBACK,M., BLOMBACK,B., MAMMEN,E.F. and PRASAD,A.S.: Fibrinogen Detroit a molecular defect in the N-terminal disulphide knot of human fibrinogen. Nature: 218, 134, (1968). 6. BLOMBACK, B. and BLOMBACK,M.: Molecular defects and variants of fibrinogen. Nouv. Rev. franc. d. Hematolog. l0, 671, (1970). 7. KRAUSE,W.H., HEENE,D.L., ZIMMERMANN,P. and LASCH,H.G.: Congenital dysfibrinogenemia (Fibrinogen Giessen). Abstract, III. Congress Internation Society on Thrombosis and Haemostasis, 1972, Wiesbaden. 8. SORIA,J,, SORIA, C., SAMAMA,M., POIROT,E. and KLING,C.: Fibrinogen Troyes-Fibrinogen Metz. Two new cases of congenital dysfibrinogenemia. Thromb. Diath. haemorrh., 27: 619, (1972). 9. KAZAL, L.A., AMSEL,S., MILLER, O.P. and TOKANTINS, L.M.: The preparation and some properties of fibrinogen precipitated from human plasma by glycine. Proc. Sot. exp. Biol. (N.Y.):113: 989, (1963). 10. BLEYL, H. : A new method in polyacrylamide phoresis. To be published,
gel electro-
11. MILLS,D.A.. * A molecular model for the proteolysis of human fibrinogen by plasmin, Biochim. Biophys. Acta: 263, 619, (1972). 12. MILLS,D.A. and KARPATKIN,S.: The initial macromolecular derivatives of human fibrinogen produced by plasmin. Biochim. Biophys. Acta: 271, 163 (1972). 13. FURLAN,M. and BECK,E.A.: Plasmatic degradation of human fibrinogen. I Structural characterization of degradation products. Biochim. Biophys, Acta: 262 , 631 (1972).