Cleavage of blood coagulation factor XIII and fibrinogen by thrombin during in vitro clotting.

Thrombin cleavage of blood coagulation Factor XIII (a2b2) and fibrinogen was studied during in vitro clotting to determine the physiologic sequence of these events. First, the time course of fibrin formation and cleavage of Factor XIII was measured in platelet-rich plasma. Cleavage of fibrinogen was measured by using a radioimmunoassay for fibrinopeptide A. Conversion of trace amounts of radioiodinated a-chains of 125I-Factor XIII to thrombin-modified a-chains was measured in unreduced 10% sodium dodecyl sulfate-polyacrylamide gels. During spontaneous clotting, a similar percentage of 125I-Factor XIII and fibrinogen was cleaved at each time point. Visible gelation of polymerized fibrin monomer occurred when 24 +/- 8% of fibrinogen was cleaved and 21 +/- 6% of Factor XIII was converted to Factor XIII'. Thrombin cleavage of Factor XIII and fibrinogen was also studied in platelet-poor plasma to which thrombin was added. In order to measure Factor XIIIa activity, fibrin polymerization was completely inhibited by the addition of Gly-Pro-Arg-Pro. Factor XIIIa formation was measured by the incorporation of [3H]putrescine into casein. The concentration of added thrombin required to cleave 50% of fibrinogen and Factor XIII was 0.65 U/ml and 0.35 U/ml, respectively. The rate of cleavage of fibrinogen by thrombin was 43-fold greater than cleavage of Factor XIII. Lower Gly-Pro-Arg-Pro concentrations were used to determine the effects of incompletely inhibiting fibrin polymerization on cleavage of Factor XIII and fibrinogen. Thrombin cleavage of Factor XIII but not fibrinogen was dependent on the extent of fibrin polymerization. The more marked the degree of inhibition of fibrin polymerization, the slower the rate of Factor XIIIa formation. Thus, in platelet-rich plasma, thrombin cleavage of Factor XIII and fibrinogen are closely related events during spontaneous clotting. Furthermore, cleavage of Factor XIII during clotting is enhanced by fibrin polymerization in platelet-poor plasma.

[1]  J. Finlayson,et al.  The ɛ-(γ-Glutamyl)Lysine Crosslink and the Catalytic Role of Transglutaminases , 1977 .

[2]  H. Scheraga,et al.  Comparison of structures of various human fibrinogens and a derivative thereof by a study of the kinetics of release of fibrinopeptides. , 1984, Biochemistry.

[3]  U. K. Laemmli,et al.  Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4 , 1970, Nature.

[4]  M. Shuman,et al.  The zymogen forms of blood coagulation factor XIII bind specifically to fibrinogen. , 1982, The Journal of biological chemistry.

[5]  L. Lorand,et al.  A filter paper assay for transamidating enzymes using radioactive amine substrates. , 1972, Analytical biochemistry.

[6]  L. Lorand,et al.  Ca2+-related regulatory function of fibrinogen. , 1978, Proceedings of the National Academy of Sciences of the United States of America.

[7]  C. Skrzynia,et al.  Characterization of the catalytic subunit of factor XIII by radioimmunoassay. , 1982, Blood.

[8]  L. Lorand,et al.  Promotion of thrombin-catalyzed activation of factor XIII by fibrinogen. , 1983, Biochemistry.

[9]  H. Cummins,et al.  Fibrin polymerization and release of fibrinopeptide B by thrombin. , 1982, Thrombosis research.

[10]  L. Lorand,et al.  Calcium-dependent unmasking of active center cysteine during activation of fibrin stabilizing factor. , 1974, Biochemistry.

[11]  L. Lorand,et al.  Diagnostic and genetic studies on fibrin-stabilizing factor with a new assay based on amine incorporation. , 1969, The Journal of clinical investigation.

[12]  A. Loewy,et al.  Factor XIII Assay by an Isotope Method , 1970, British journal of haematology.

[13]  J. Hermans,et al.  Fibrin: Structure and Interactions , 1982, Seminars in thrombosis and hemostasis.

[14]  E. Mihályi.  Physicochemical studies of bovine fibrinogen. IV. Ultraviolet absorption and its relation to the structure of the molecule. , 1968, Biochemistry.

[15]  B. Blombäck,et al.  A two-step fibrinogen–fibrin transition in blood coagulation , 1978, Nature.

[16]  C. Greenberg,et al.  Measurement of blood coagulation Factor XIIIa formation in plasma containing glycyl-L-prolyl-L-arginyl-L-proline. , 1985, Analytical biochemistry.

[17]  R. Doolittle,et al.  Amino acid sequence studies on factor XIII and the peptide released during its activation by thrombin. , 1974, Biochemistry.

[18]  F. Rickles,et al.  Fibrinopeptide A in acute leukemia: relationship of activation of blood coagulation to disease activity , 1981 .

[19]  S. Pizzo,et al.  Human Factor XIII from plasma and platelets. Molecular weights, subunit structures, proteolytic activation, and cross-linking of fibrinogen and fibrin. , 1973, The Journal of biological chemistry.

[20]  L. Lorand,et al.  Fibrin-stabilizing factor (factor XIII). , 1976, Methods in enzymology.