Characterization of the terminal degradation products of canine fibrinogen by plasmin.

Fibrinogen, isolated from canine plasma by the successive procedures of (1) freezing and thawing, (2) fractional precipitation with 25% saturated (HN4)2SO4 and (3) Sepharose 6B gel-filtration, had a molecular weight of 282 000 by the rapid sedimentation equilibrium method. However, a molecular weight for canine fibrinogen of 332 000, which is closer to that reported for human and bovine fibrinogens (340 000 plus or minus 20 000), was obtained from the sum of the molecular weights of the Aalpha, Bbeta and gamma chains, determined from dodecylsulfate gel electrophoretic patterns of reduced fibrinogen. Canine fibrinogen, subjected to proteolysis by urokinase-activated plasminogen for 24 h, contained degradation fragments D and E which were isolated by starch block electrophoresis and Sephadex G-200 gel-filtration. The purified D and E fragments with sedimentation coefficients of 5.0 S and 2.5 S had weight average molecular weights of 89 000 and 42 000, respectively by the rapid sedimentation equilibrium method. The ratio of D to E was 2:1 per parent fibrinogen molecule. Antigenic analysis according to anti-fibrinogen antiserum showed that both D and E fragments were antigenically deficient to native fibrinogen and revealed a reaction of non-identity with each other. Upon immunoelectrophoresis at pH 8.2, D and E had different electrophoretic mobilities. Preliminary studies indicate that based on thrombin time alone, D has anticoagulant activity while E appears to be a coagulation potentiator. Canine fibrinogen apparently consist of two core fragments with dissimilar chemical characteristics in common with the fundamental structures of human and bovine fibrinogens.     

Control of enzymatic degradation of hyaluronan by divalent cations.

Enzymatic degradation of hyaluronan (HA) by testicular hyaluronidase (HAase, hyaluronate 4-glucanohydrolase) requires inclusion of mono- or divalent cations in the reaction mixture. Most divalent cations activated HAase with equal potency; however, Cu2+ suppressed degradation, and Ca2+ showed a concentration-dependent regulation of size of the oligosaccharide products. Careful selection of HAase assay parameters is critical for discovery of novel HAase inhibitors and for preparation of controlled-size oligosaccharide fragments.     

Terminal plasmin degradation products D and E of duck fibrinogen and their effect on polymerization of fibrin

Duck fibrinogen (Mr 320 000) treated with streptokinase-activated human plasminogen in the presence of calcium ions was hydrolysed to terminal core fragments D and E. They were isolated from the digest by: (1) ion-exchange chromatography on DEAE-cellulose, (2) gel filtration on Sephadex G-100, and (3) affinity chromatography with the use of fibrin monomers coupled to CNBr-activated Sepharose. When the native D fragment, D1 was additionally digested by plasmin in the presence of EDTA, more degraded forms D2 and D3 appeared. Molecular weight of D1, D2, D3 and E estimated on SDS-polyacrylamide gel electrophoresis is 100 000, 89 000, 80 000 and 50 000, respectively. It was found that after reduction with 2-mercaptoethanol the fragments D1 and D3 consisted each of three polypeptide chains: alpha, beta, gamma: the gamma-chain of D3 remnant was more degraded (Mr 24 000) as compared with the gamma-chain of D1 remnant (Mr 42 000). Polymerization of both duck and pig fibrin monomers was inhibited by fragments D1 but not by D3.     

Factors influencing the structure of terminal plasmin degradation products of human fibrinogen and fibrin

Experiments have been carried out with fibrinogen and with purified degradation products of fibrinogen and fibrin which demonstrate that the structure of D fragments obtained after prolonged plasmin digestion is influenced by several factors in the media. The previously described protective effect of calcium ions on the gamma-chain carboxy-terminals of fibrinogen against attack has been confirmed by working at high plasmin concentrations and/or in the presence of 2 M urea. Several compounds such as EDTA, EGTA, citrate and iminodiacetic acid appear to have a separate effect. In the absence of calcium ions these compounds appear to make the gamma-chain carboxy-terminal ends of the D and D-dimer fragments more susceptible to plasmin digestion. Finally, as demonstrated by experiments with purified D-E complexes from fibrinogen and with whole fibrinogen digests, the E moiety of the D-E complexes appears to be capable of protecting the D moiety against low plasmin concentrations also in the absence of calcium ions.     

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.     

Fusogenic capacities of divalent cations and effect of liposome size

The initial kinetics of divalent cation (Ca2+, Ba2+, Sr2+) induced fusion of phosphatidylserine (PS) liposomes, LUV, is examined to obtain the fusion rate constant, f11, for two apposed liposomes as a function of bound divalent cation. The aggregation of dimers is rendered very rapid by having Mg2+ in the electrolyte, so that their subsequent fusion is rate limiting to the overall reaction. In this way the fusion kinetics are observed directly. The bound Mg2+, which by itself is unable to induce the PS LUV to fuse, is shown to affect only the aggregation kinetics when the other divalent cations are present. There is a threshold amount of bound divalent cation below which the fusion rate constant f11 is small and above which it rapidly increases with bound divalent cation. These threshold amounts increase in the sequence Ca2+ less than Ba2+ less than Sr2+, which is the same as found previously for sonicated PS liposomes, SUV. While Mg2+ cannot induce fusion of the LUV and much more bound Sr2+ is required to reach the fusion threshold, for Ca2+ and Ba2+ the threshold is the same for PS SUV and LUV. The fusion rate constant for PS liposomes clearly depends upon the amount and identity of bound divalent cation and the size of the liposomes. However, for Ca2+ and Ba2+, this size dependence manifests itself only in the rate of increase of f11 with bound divalent cation, rather than in any greater intrinsic instability of the PS SUV. The destabilization of PS LUV by Mn2+ and Ni2+ is shown to be qualitatively distinct from that induced by the alkaline earth metals.     

Platelet fibrinogen. Identity and initial observations on the mode of its degradation by plasmin.

Fibrinogen has been purified from human platelets. Platelet fibrinogen exhibits a characteristic pattern in agar gel immunoelectrophoresis different from that of plasma fibrinogen. Stepwise plasmin degradation has been used in further elucidation of the molecular properties of the platelet protein. Examination of comparative digests by immunologic and gel electrophoretic methods has revealed that (1) the platelet protein is more resistant to plasmin degradation, (2) the plasmin-produced fragments of platelet fibrinogen differ consistently from those of its plasma counterpart, and (3) platelet fibrinogen is different from fragment X of plasma fibrinogen. It is suggested that platelet fibrinogen may contribute to the stability of the thrombus.     

The effect of divalent cations on the conformation and function of human plasminogen

The activation of native human plasminogen (Glu1-Pg) by tissue plasminogen activator, urinary plasminogen activator (u-PA), and streptokinase is inhibited by the divalent cations Ca2+, Mg2+, and Mn2+. This inhibition is accompanied by a conformational change in the molecule as evidenced by a decrease in Stokes' radius and intrinsic fluorescence. Kinetic analysis indicates that Mn2+ acts as an uncompetitive inhibitor of u-PA-catalyzed Glu1-Pg activation. In contrast to the inhibitory effects of divalent cations on Glu1-Pg, Ca2+ and Mg2+ stimulate the activation of proteolytically modified Lys77-Pg. These observations provide further evidence that Glu1-Pg and Lys77-Pg exhibit differential responses to ligands in the microenvironment.     

The effect of divalent cations on aggregation of Dictyostelium discoideum.

Following the initiation of development, amoebae of Dictyostelium discoideum aggregate chemotactically toward cyclic AMP (cAMP). Adenyl cyclase, cAMP phosphodiesterase, and cAMP binding sites all increase 20--40 fold during the first few hours of development. It has been shown that addition of 1 mM EDTA and 5 mM MgCl2 accelerates the aggregation process. Likewise, the calcium ionophore, A23187, leads to precocious aggregation while 4 X 10(-5) M progesterone considerably delays it. These treatments have now been shown to result in increased accumulation of adenyl cyclase in the case of EDTA and Mg2+ or the ionophore and greatly decreased accumulation in the case of the steroid. Treatment with EDTA and Mg2+ or the ionophore has been shown not only to accelerate aggregation in wild-type amoebae but to overcome complete blocks to aggregation in certain mutant strains. We have found that addition of Mn2+ will also permit aggregation of mutant cells otherwise unable to aggregate. This divalent ion, unlike EDTA and Mg2+ or the ionophore, was shown to directly stimulate adenyl cyclase. Calcium ions were also found to affect the enzyme such that at Ca2+ concentrations found within the cells the great majority of the activity is inhibited. Manganese ions can overcome the inhibition by Ca2+. These findings show that conditions which stimulate aggregation result in increased activity of adenyl cyclase either by increased accumulation of the enzyme or by increased activity of the available enzyme, and support the proposed central role of adenyl cyclase in aggregation.     

The effect of arginine on hydrolysis of fibrinogen by plasmin and mini-plasmin]

The rate of plasmin or Val442-plasmin catalyzed hydrolysis of fibrinogen decreases several times as affected by arginine in high concentrations. The enzyme is shown to be not inhibited by arginine. The observed effect is supposed to depend on saturation of the protein-proteins interaction sites located between 442 and 790 amino acid residues.     

The role of DL and DH degradation products in the reactions of the plasmin hydrolysis of fibrinogen and fibrin]

The mechanism of effect of plasmin hydrolysis degradation products, fragments DL and DH, on fibrinolysis and fibrinogenolysis processes was investigated on the basis of their various structural and functional characteristics. Using electrophoresis of unreduced samples and degradation products concentrations changing kinetics, DH was shown to be the only fragment which possessed an antifibrinolytic effect. Rauleigh's light scattering analysis indicated the ability of fragments DL and DH to co-form with plasminogen reversible equimolar complexes.     

Role of divalent cations in EOG generation

Underwater EOGs in response to 10^–4 M isoamyl acetatewere recorded in vivo in frogs. The olfactory mucosa was superfusedby sodium-free sucrose solutions, and the effect of the additionof millimolar concentrations of sodium, calcium, cobalt, bariumor cadmium was studied. All voltage responses were standardizedaccording to the solution resistivities. Barium and calciumefficiently restored the response; sodium and cobalt were lessefficient. Cobalt did not alter the restoring effect of calcium.Cadmium induced an irreversible blockage irrespective of thepresence of calcium. These results are discussed in terms ofpossible ionic mechanisms mediating olfactory responses.     

Corn leaf phosphoenolpyruvate carboxylases: The effect of divalent cations on activity

Differences in the kinetic properties of corn leaf phosphoenolpyruvate (PEP) carboxylase isoenzymes were found, depending on whether Mg²⁺ or Mn²⁺ was used as the metal cofactor of the reaction. Also, differences in kinetic constants with respect to Mg²⁺ and Mn²⁺ were noticed between the two isoenzymes which further differentiates the two proteins. The catalytic activity of the enzyme in the Mg²⁺-activated system was dependent on a PEP-Mg²⁺ complex and not on the concentration of free Mg²⁺ or free PEP. Kinetics in the presence of total Mg²⁺ and those of PEP-Mg²⁺ suggest a negative cooperative effect with respect to ligand binding with concurrent progressive substrate activation. Magnesium ions, thus, have a special regulatory role in the corn leaf PEP carboxylase reaction.     

Stabilization effect of protons and divalent cations on membrane structures of lipids

Ordered-fluid phase transitions of methylphosphatidic and phosphatidic acid bilayers are discussed theoretically to explain comprehensively various observed data in the presence of Ca2+ or protons. It is shown that the observed data can be explained reasonably by taking account of the interaction among neighboring head-groups with the aid of Ca2+ or protons. As a result, a quantitative explanation is given for the dependence of the phase-transition temperature on pH as well as Ca2+ concentration with the coexistence of monovalent cations. The dependence of the cooperativity of the transition on pH and monovalent cation concentration is also well explained. It is also pointed out that in the ordered-fluid phase transitions, a hysteresis metastable state and phase separation can be expected to appear under some conditions.