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.     

The peptide Glu-His-Ile-Pro-Ala binds fibrinogen and inhibits platelet aggregation and adhesion to fibrinogen and vitronectin.

Antiplatelet agents are clinically useful as antithrombotic entities. The importance of antiplatelet agents led us to design, synthesize, and characterize a new antiplatelet peptide. This peptide is a presumptive mimic of a ligand binding site on the platelet fibrinogen receptor. Unlike peptides related to Arg-Gly-Asp-Ser and His-His-Leu-Gly-Gly-Ala-Lys-Gln-Ala-Gly-Asp-Val that bind to the fibrinogen receptor, this peptide binds to fibrinogen. The anticomplementarity hypothesis was used to design this presumptive peptide mimic of the vitronectin binding site on the fibrinogen receptor, glycoprotein IIb/IIIa complexes. The resulting peptide (Glu-His-Ile-Pro-Ala) has the characteristics of a fibrinogen binding site mimic: It binds fibrinogen and inhibits both the adhesion of platelets to fibrinogen and platelet aggregation. The peptide also inhibits the adhesion of platelets to vitronectin. The antiplatelet activity of this mimic peptide was dependent on its amino acid sequence, since closely related analogues were either inactive or less active inhibitors of platelet function than the original peptide. These results demonstrate that the peptide Glu-His-Ile-Pro-Ala has the characteristics expected of a mimic of a glycoprotein IIb/IIIa ligand binding site.     

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.     

Inhibited thrombins. Interactions with fibrinogen and fibrin.

Fibrin-monomer-Sepharose was used to study thrombin binding to fibrin and the role of the enzyme active centre in this interaction. Binding properties of preformed enzyme-inhibitor complexes, as well as inhibition of thrombin already adsorbed to fibrin monomer, were investigated. No apparent difference was found in binding properties of phenylmethanesulphonyl fluoride-, D-Phe-Pro-Arg-CH2Cl- and dansylarginine NN-(3-ethylpentane-1,5-diyl)amide-inhibited thrombins. Also, the elution profile of phenylmethane-sulphonyl fluoride-inhibited thrombin from fibrinogen-Sepharose was identical with that of active thrombin from fibrin-monomer-Sepharose. Thus far the only low-Mr inhibitor that prevents thrombin from binding to fibrin monomer is pyridoxal 5'-phosphate. Preformed hirudin-thrombin complexes do not interact with fibrin. The extent to which the active centre of thrombin associated with fibrin is still accessible to substrates and inhibitors was also studied. Thrombin bound to fibrin hydrolyses a synthetic substrate at the same rate as the free enzyme. Water-soluble low-Mr inhibitors such as D-Phe-Pro-Arg-CH2Cl and dansylarginine NN-(3-ethylpentane-1,5-diyl)amide can readily modify the active centre of the fibrin-associated enzyme, and the active centre is exposed to the degree that displacement of dansylarginine NN-(3-ethylpentane-1,5-diyl)amide by D-Phe-Pro-Arg-CH2Cl is possible without disturbing the binding. Hirudin disrupts the affinity between thrombin and fibrin. These data indicate that the active centre of thrombin associated with fibrin through extended binding is fully exposed and freely accessible. It is possible that extended binding may play a regulatory role in the activation of Factor XIII by thrombin, as well as inactivation of this enzyme by antithrombin III.     

Relations between fibrinogen degradation products and heparinocytes.

In the emergency reaction there is a short-time increase of the values of basophilic leukocytes in connection with normal values of fibrinogen degradation products (FDP). After diminuation of basophils FDP are to be detected. In the chronical ill and under different hormonal contraceptives these two processes are overlapped disturbing a unique negative or positive correlation of heparinocytes and FDP. Concerning the situation under standardized conditions or in single cases worthful conclusions are possible about compensation or decompensation of a latent disseminated intravascular coagulation.     

Assembly and secretion of fibrinogen. Degradation of individual chains.

Hep G2 cells produce surplus A alpha and gamma fibrinogen chains. These excess chains, which are not secreted, exist primarily as free gamma chains and as an A alpha-gamma complex. We have determined the intracellular location and the degradative fate of these polypeptides by treatment with endoglycosidase-H and by inhibiting lysosomal enzyme activity, using NH4Cl, chloroquine, and leupeptin. Free gamma chain and the gamma component of A alpha-gamma are both cleaved by endoglycosidase-H, indicating that the gamma chains accumulate in a pre-Golgi compartment. Lysosomal enzyme inhibitors did not affect the disappearance of free gamma chains but inhibited A alpha-gamma by 50%, suggesting that A alpha-gamma is degraded in lysosomes. The degradative fate of individual chains was determined in transfected COS cells which express but do not secrete single chains. Leupeptin did not affect B beta chain degradation, had very little affect on gamma chain, but markedly inhibited A alpha chain degradation. Antibody to immunoglobulin heavy chain-binding protein (GRP 78) co-immunoprecipitated B beta but not A alpha or gamma chains. Preferential binding of heavy chain-binding protein to B beta was also noted in Hep G2 cells and in chicken hepatocytes. Taken together these studies indicate that B beta and gamma chains are degraded in the endoplasmic reticulum, but only B beta is bound to BiP. By contrast A alpha chains and the A alpha-gamma complex undergo lysosomal degradation.