Proteolytic fragmentation of fibrinogen. II. kinetic modeling of the digestion of human and bovine fibrinogen plasmin or trypsin.

The kinetic data presented in the previous paper (Mihalyi, E., et al. (1976), Biochemistry 15, preceding paper in this issue), with respect to the fragmentation of human the bovine fibrinogen by either plasmin or trypsin, were compared with several chemical kinetic models. The models were derived mathematically on the basis of the three-nodular structure of fibrinogen (Hall, C.E., and Sayter, H.S. (1959), J. BiophysBiochem. Ctyol. 5, 11) and the asymmetrical cleavage sequence first proposed by Marder, V.J., et. al. ((1969) J. Biol. Chem. 244, 2111). The parameters were determined by nonlinear curve fitting. The whole process could be described accurately by only two rate constants. Several variant models were tested and, although a clear cut choice cannot be made, one of these, the protected three-bonds model, appears to give the best fit in most cases. This model assumes that the chain segment that distinguishes F from X protects certain other chains (the bonds) from proteolytic cleavage.     

Bacteroides intermedius binds fibrinogen.

The binding of Bacteroides intermedius VPI 8944 to human fibrinogen has been characterized. The binding is time dependent, at least partially reversible, saturable, and specific. On an average, a maximum of 3,500 fibrinogen molecules bind per bacterial cell, with a dissociation constant of 1.7 X 10(-11) M. These bacteria also exhibit a fibrinogenolytic activity which can be partially inhibited by protease inhibitors. Bacteria release fibrinogenolytic activity into the surrounding medium without loss of binding activity, but more pronounced fibrinogen breakdown occurs when 125I-labeled fibrinogen is associated with the bacteria, suggesting that fibrinogen is degraded at the cell surface. Fibrinogen binding by B. intermedius might represent a mechanism of bacterial tissue adherence.     

Inhibition of plasmin by fibrinogen.

The kinetics of inhibition of the amidolytic activity of plasmin on D-Val-L-Leu-L-Lys p-nitroanilide hydrochloride (S-2251) by fibrinogen and fibrin were determined. Reciprocal (1/v versus 1/[S]) plots of plasmin inhibition by 0.50 microM-fibrinogen showed a non-linear downward curve. The Hill coefficient (h) was 0.68, suggesting negative co-operativity. By contrast, fibrin produced a simple competitive inhibition of plasmin (Ki = 12 micrograms/ml). Addition of 0.1 mM-6-aminohexanoic acid shifted the non-linear curve obtained in the presence of fibrinogen to a straight line as for controls, indicating that 6-aminohexanoic acid abolishes the fibrinogen-induced inhibition. Transient exposure of the enzyme to pH 1.0 abrogates the ability of fibrinogen to inhibit plasmin activity. Acidification had no effect on the Vmax but increased the Km of plasmin. The present evidence for modulation of plasmin reveals a novel mechanism for control of fibrinolysis by fibrinogen, a component of the coagulation system and the precursor of the physiological substrate of plasmin.     

Interaction of fibrinogen with detergent.

Both cationic and anionic detergents were found to precipitate fibrinogen by forming fibrinogen-detergent complexes. These complexes were soluble in distilled water, but the aqueous solutions were very unstable and the complexes precipitated in the presence of salt. In the interaction of fibrinogen with the cationic detergent, stearyltrimethyl-ammonium chloride, approximately 160 molecules of detergent were found to bind to one molecule of fibrinogen. In distilled water, the fibrinogen-stearyltrimethylammonium complex (FG-STA(Cl)) remained soluble in the presence of thrombin [ED 3.4.21.5] although the same peptides were released as those released from fibrinogen. Precipitation of FG-STA(Cl) by salt was found to be closely related to adsorption of the anion of the salt by the complex. Futher addition of salt resulted in solubilization of the precipitate, and the solubilization was also due to further adsorption of the anion onto the precipitate.     

Bleomycin-specific fragmentation of double-stranded DNA.

Brief exposure of covalently closed circular duplex PM2 DNA to low concentrations of the clinical bleomycin mixture (Blenoxane) resulted in specific fragmentation of the genome that does not depend on the presence of superhelical turns. The double-strand breaks are in fact produced at several discrete sites on the PM2 genome but frequently occurring near the HpaII restriction endonuclease cleavage site. Initial rates of formation of nicked circular and linear duplex PM2 DNAs are reduced to different extents as the ionic strength of the reaction is increased. Increasing ionic strength is most effective in reducing the initial rate and overall yield of apparent double-strand scissions compared with single-strand scissions in the bleomycin-treated PM2 DNA.     

Electron microscopic studies of plasmic degradation products of fibrinogen. Implications for the disulfide structure of fibrinogen.

Fibrinogen, coagulable plasmic derivatives (Fragments X) and Fragments Y, D and E were studied by negative staining electron microscopy. Fragment X obtained from Stage 1 digests and fibrinogen were both globular, while Fragment X of Stage 2 digests appeared as a nodular filament. The Stage 1 and Stage 2 Fragment X preparations had approximately the same molecular weight, but could be differentiated by several subtle differences in polypeptide chain structure. Fragments Y and D were also filamentous, although shorter than Fragment X (Stage 2), and Fragment E appeared as a small, compact or folded filament. These results agree with the concept that fibrinogen consists of a strand of nodules connected by thin strands, folded into a compact, spherical shape. The molecule opens up when stabilizing bonds are disrupted or liberated by plasmin. The data are compatible with a fibrinogen molecule in which the two halves are linked by a single locus of disulfide bonds at the amino terminus and with the asymmetric hypothesis of plasmic degradation to Fragments X, Y, D and E.     

A solid-phase radioimmunoassay for fibrinogen.

A solid-phase radioimmunoassay for fibrinogen has been developed utilizing [¹⁴C]-methylated fibrinogen as standard antigen and fibrinogen-specific antibodies covalently linked to Sepharose. Fibrinogen was [¹⁴C]-methylated by reductive alkylation using [¹⁴C] formaldehyde and sodium borohydride. The methylated fibrinogen was unaltered in clotting ability and antigenicity.The assay, an isotope dilution assay, is quantitative for picomole amounts of fibrinogen. It is specific for fibrinogen in homologous plasma and in the presence of a variety of other proteins.