fibrin and valyl-leucyl-lysine-4-nitroanilide

A study was carried out to establish an appropriate method for streptokinase (SK) potency determination (biological assay) in order to fulfil the main function of the Instituto Nacional de Medicamentos respecting products marketed in Argentina. The potency of different commercial samples of SK was determined against the International Standard, and three internationally accepted methods were used for this purpose: fibrin plate, clot lysis and chromogenic method. The analysis of results suggests that the fibrin plate method is the least precise and reproducible. The clot lysis and chromogenic methods demonstrated great precision and reproducibility, giving a correlation coefficient of 0.99. It is concluded that both of these methods are best suited to determine potency of SK commercial products.

Topics: Animals; Cattle; Chromogenic Compounds; Evaluation Studies as Topic; Fibrin; Fibrinolysis; Fibrinolytic Agents; Oligopeptides; Reference Standards; Reproducibility of Results; Sensitivity and Specificity; Streptokinase

Poloxamer 407 has shown clinical promise in suppressing surgically related adhesion formation. The mechanisms by which this occurs are not well understood. Since poloxamer 188 has rather dramatic fibrin altering properties, the present study was performed to evaluate the effects of poloxamer 407 on fibrin assembly, structure and dissolution. Studies were performed in platelet-rich plasma (PRP), platelet-poor plasma (PPP) and a purified protein system. Poloxamer 407 enhanced the rate of fibrin assembly, and increased final gel turbidity. As poloxamer 407 concentration rose from 0 to 20 mg/ml in the purified protein system, the final gel optical density (OD) increased from 0.30 to 0.95, and fiber size (mass/length ratio [mu]) increased from 2.4 to 13.4 x 10(13) daltons/cm. Precipitation was noted in the purified system at poloxamer 407 concentrations > or = 20 mg/ml. Over a poloxamer 407 range of 0-20 mg/ml, mu increased from 2.64 to 13.2 x 10(13) daltons/cm in PRP. In PPP, mu increased from 2.95 to 9.25 x 10(13) daltons/cm. In contrast to results with poloxamer 188, clot lysis with tPA (43 IU/ml) was prolonged in the presence of poloxamer 407. At 20 mg of poloxamer 407 per ml, clot lysis was less than 18% complete after 3000 s. For the control, lysis was 50% complete after 1350 s. Poloxamer 407 inhibition of fibrinolysis was due to inhibition of plasminogen activation or plasmin activity. The fibrin altering properties of poloxamer 407 may partially explain some of this agent's interesting clinical properties.

Topics: Blood Coagulation; Fibrin; Humans; Kinetics; Oligopeptides; Poloxalene; Thrombin; Time Factors; Tissue Plasminogen Activator

Development of monoclonal antibodies capable of inhibiting the specific binding of t-PA to fibrin.. After immunization of Balb/c mice with recombinant t-PA (rt-PA) we selected the monoclonal antibody MA3B5 by its ability to inhibit the binding of t-PA to fibrin, and the MA2C1 devoid of this property. The influence of such antibodies was evaluated on fibrin plates, amidolytic assays and clot lysis assays. Furthermore, their interference with the activator bound to fibrin was assayed with a spectrophotometric solid phase assay (SOFIA).. The results showed that MA3B5 totally inhibited the t-PA induced fibrinolytic activity on fibrin plates, reduced amidolytic activity by 86.5% and inhibited the clot lysis induced by t-PA in a dynamic system. In contrast, the MA2C1 showed no inhibition. By assessing the binding of t-PA to fibrin with the SOFIA assay we could demonstrate that only the MA3B5 reduced significantly (up to 90% with 100 micrograms/mL of antibody) the amount of t-PA bound to fibrin surface.. We have purified and characterized a monoclonal antibody which specifically blocks the fibrin binding site of t-PA.

Topics: Animals; Antibodies, Monoclonal; Female; Fibrin; Fibrinolysis; Mice; Mice, Inbred BALB C; Oligopeptides; Protein Binding; Recombinant Proteins; Tissue Plasminogen Activator

This study was conducted to examine the effects of mastitis and stage of lactation on plasminogen activator (PA) activity in milk somatic cells. An assay system, which measures the plasmin-mediated hydrolysis of the chromogenic substrate D-valyl-leucyl-lysine p-nitroanilide, was used to assess PA activity present within milk somatic cells. Milk cell associated PA activity was increased (P < 0.05) by 50% in the presence of fibrin fragments. This suggests that milk somatic cells contain tissue PA which, unlike urokinase PA, is preferentially activated in the presence of fibrin fragments. An increase of the milk somatic cell count from < 5 x 10(4) to > 10(6) cells/ml resulted in an 8-fold increase in PA activity per cell. Elevated levels of PA activity were associated with milk somatic cells isolated from mastitic quarters obtained from cows in early (< 4 months in lactation) or late lactation (> 8 months in lactation). We conclude that PA activity is increased during severe mastitic inflammation. Although the physiological function of this enzyme is as yet unclear, we propose that it may be involved in the conversion of plasminogen to plasmin, contributing to the higher levels of plasmin occurring in milk isolated from mastitic quarters.

Topics: Animals; Cattle; Chromogenic Compounds; Female; Fibrin; Fibrinolysin; Hydrolysis; Lactation; Mastitis, Bovine; Milk; Oligopeptides; Plasminogen Activators; Tissue Plasminogen Activator

The kinetics of plasmin inhibition by alpha 2-antiplasmin (alpha 2AP), alpha 2-macroglobulin (alpha 2M) and leupeptin were studied in the presence of fibrin monomer (Fn) and CNBr fragments of fibrinogen (Fg-CNBr). Active plasmin was detected in continuous and discontinuous assays using the chromogenic substrate D-Val-L-Leu-L-Lys p-nitroanilide hydrochloride (S-2251). The two 'fibrin-like' preparations functioned as hyperbolic mixed-type inhibitors of S-2251 hydrolysis. The dissociation constants (KF) for the binding of plasmin to Fn and Fg-CNBr were 22 nM and 17 nM respectively. Fn and Fg-CNBr inhibited the reaction of plasmin with alpha 2AP: the extent of inhibition depended on the fibrin concentration. In the presence of 800 nM-Fn or 800 nM-Fg-CNBr, the experimental second-order rate constant (K"app.) was decreased from 2.4 x 10(7) M-1.s-1 to 1.2 x 10(6) and 5.3 x 10(5) M-1.s-1 respectively. The effect of Fn and Fg-CNBr on the rate of plasmin inhibition by alpha 2M was even greater. The k"app. value was decreased from 4.0 x 10(5) M-1.s-1 to 8.0 x 10(2) and 1.3 x 10(3) M-1.s-1 in the presence of 800 nM-Fn and -Fg-CNBr respectively. By contrast, the fibrin preparations caused only a small change in the rate of plasmin inhibition by leupeptin. The maximum change in k"app. was 3-fold. All plasmin inhibition curves were linear, suggesting that free and fibrin-bound forms of plasmin remained in equilibrium during the course of reaction with proteinase inhibitors. Fn and Fg-CNBr had no effect on the reaction of miniplasmin with S-2251, alpha 2AP or alpha 2M. When 125I-plasmin was incubated with Fg-CNBr and then allowed to react with a premixed solution of alpha 2AP and alpha 2M, the Fg-CNBr did not significantly change the percentage of plasmin bound to alpha 2AP. These experiments demonstrate that the reaction of plasmin with alpha 2M is inhibited by the non-covalent binding of plasmin to fibrin. We propose that plasmin bound to the surface of a clot is protected from inhibition by alpha 2M as well as by alpha 2AP.

Topics: alpha-Macroglobulins; Chromogenic Compounds; Cyanogen Bromide; Fibrin; Fibrin Fibrinogen Degradation Products; Fibrinogen; Fibrinolysin; Humans; Kinetics; Leupeptins; Oligopeptides

Fibrolase, a fibrinolytic enzyme isolated from Agkistrodon c. contortrix (southern copperhead) venom, solubilizes fibrin primarily by rapid hydrolysis of the alpha and beta chains. Fibrolase is also an A alpha, B beta fibrinogenase. The breakdown products of fibrin and fibrinogen following incubation with fibrolase were different from those observed with plasmin. This enzyme is a metalloprotease that was inhibited by ethylenediaminetetraacetic acid. Fibrolase was inhibited by dithiothreitol, suggesting that disulfide bonds are important for catalytic activity. It was also inhibited by alpha 2-macroglobulin, but not by the soybean or lima bean trypsin inhibitors, diisopropylfluorophosphate, or p-hydroxymercuribenzoate. Unlike thrombolytic agents such as streptokinase, fibrolase does not activate plasminogen as evidenced by the use of plasmin-specific chromogenic substrate S-2251 and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Topics: alpha-Macroglobulins; Animals; Caseins; Crotalid Venoms; Dithiothreitol; Edetic Acid; Fibrin; Fibrin Fibrinogen Degradation Products; Fibrinogen; Fibrinolysis; Fibrinolytic Agents; Humans; Metalloendopeptidases; Oligopeptides; Plasminogen; Thrombin

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.

Topics: Amino Acid Sequence; Aminocaproic Acid; Binding, Competitive; Chromogenic Compounds; Fibrin; Fibrinogen; Fibrinolysin; Hydrogen-Ion Concentration; Kinetics; Molecular Sequence Data; Oligopeptides

Topics: Antibodies, Monoclonal; Chromogenic Compounds; Fibrin; Fibrin Fibrinogen Degradation Products; Humans; Oligopeptides; Plasminogen; Protein Conformation; Tissue Plasminogen Activator

When equimolar amounts of Glu-plasminogen (Glu-plg) and streptokinase (SK) were mixed in the presence of S-2251, SK-plg complex was formed and only gradually converted to SK-plasmin complex. When equimolar amounts of Glu-plg and SK were mixed with fibrinogen or fibrin, Glu-plg was converted faster to plasmin suggesting that Glu-plg molecule in the complex was converted to plasmin. It is thus concluded that SK-plg complex is converted to SK-plasmin complex slowly in the absence of fibrin or fibrinogen. When SK-plg-fibrin(ogen) complex was formed, plasminogen moiety was converted to plasmin faster inside a trimolecular complex of SK, plasminogen and fibrin(ogen).

Topics: Autoradiography; Electrophoresis, Polyacrylamide Gel; Fibrin; Fibrinogen; Fibrinolysin; Humans; Oligopeptides; Plasminogen; Streptokinase

Topics: Electrophoresis, Polyacrylamide Gel; Fibrin; Fibrinogen; Humans; Hydrolysis; Immunoblotting; Kinetics; Macromolecular Substances; Oligopeptides; Plasminogen; Streptokinase

Equimolar concentrations of Lys-plasminogen (Lys-PLG) and streptokinase (SK) were mixed with various concentrations of fibrin, fibrinogen, fragment D or E (potentiating agents). The activity of the mixture was measured by the hydrolysis of S-2251. Kinetic analyses indicated that catalytic rate constant (kcat) of the hydrolysis of S-2251 increased in the presence of increasing amounts of potentiating agents. Km did not change in their presence. Effectiveness of the enhancement of the hydrolysis of S-2251 was in the order of fibrin greater than fibrinogen greater than E greater than D. It could be concluded that a complex of SK, Lys-PLG and potentiating agent is a better enzyme, thus activator, than a complex of SK and Lys-PLG. Since Lys-PLG does not change its conformation and its activation rate upon interaction with fibrin, the enhanced activator activity of complex of SK, Lys-PLG and potentiating agent may not be due to the conformational change of Lys-PLG.

Topics: Chromogenic Compounds; Drug Synergism; Fibrin; Fibrin Fibrinogen Degradation Products; Humans; Hydrolysis; Kinetics; Oligopeptides; Peptide Fragments; Plasminogen; Plasminogen Activators; Protein Conformation; Spectrophotometry; Streptokinase; Thrombin

Topics: Antibodies, Monoclonal; Epitopes; Fibrin; Humans; Oligopeptides; Tissue Plasminogen Activator

When Glu-plasminogen (Glu-plg) was activated by various amounts of streptokinase (SK), Km of the hydrolysis of S-2251 by the mixture of Glu-plg and SK did not change, but Vmax increased with an increase in the amount of SK. Since low concentrations of SK-plg complex do not result in its conversion to the SK-plasmin complex by mutual activation, these results seem to suggest that the SK-plg complex may be a better activator when S-2251 is used as a substrate. When Glu-plg was activated by SK in the presence of fibrin(ogen) or its degradation products (potentiating agents), Km did not change with an increase in the concentration of each potentiating agent, but Vmax increased. The effects of potentiating agents on the kcat values of these activators were in the order of fibrin greater than fibrinogen greater than D greater than E.

Topics: Drug Synergism; Fibrin; Fibrin Fibrinogen Degradation Products; Humans; Hydrolysis; Kinetics; Oligopeptides; Osmolar Concentration; Plasminogen; Plasminogen Activators; Streptokinase; Time Factors

When human plasma was activated by urokinase (UK) in the presence of thrombin, thrombin plus Ca++, Ca++ or in their absence and the plasmin activity was measured by the hydrolysis of S-2251, plasmin activity was higher in the presence of cross-linked or non cross-linked plasma clot. The results of similar experiments utilizing plasma after severe exercise indicated that the hydrolysis of S-2251 by plasma containing tissue plasminogen activator (t-PA) was also higher in cross-linked or non cross-linked plasma clot. Fibrinolysis was faster in thrombin-induced plasma clot, but was later shown significantly in plasma clot induced by thrombin and Ca++, whereas practically no fibrinogenolysis was shown in plasma. When Glu-plasminogen (Glu-plg) was activated by UK in the presence of cross-linked or non cross-linked fibrin and alpha 2 antiplasmin (alpha 2AP), fibrinolysis was faster in cross-linked fibrin than non cross-linked fibrin in the presence of alpha 2AP. No fibrinogenolysis was shown either. Plasmin activity measured by the hydrolysis of S-2251 was also higher in cross-linked or non cross-linked fibrin than in fibrinogen in the presence of alpha 2AP. These results indicate that enhanced activation of Glu-plg by UK or t-PA in the presence of fibrin was a more significant event than the inactivation of plasmin in the plasma clot or purified clot by alpha 2AP cross-linked to fibrin.

Topics: Calcium; Fibrin; Fibrinogen; Fibrinolysis; Humans; Hydrolysis; Oligopeptides; Plasminogen; Tissue Plasminogen Activator; Urokinase-Type Plasminogen Activator

Studies were performed on the activation of a native form of human plasminogen (Glu-plg) or its degraded form (Lys-plg) by streptokinase (SK) in the presence of fibrin, fibrinogen, SK-potentiator, fragment D or E. When Glu-plg (0.1 microM) was activated by 0.5 u/ml of SK in the presence of 100 micrograms of S-2251 and 0.1 microM of fibrin, fibrinogen or their degradation products (potentiating agents), fibrin enhanced the rate of the hydrolysis of S-2251 to the largest extent. Fragments D and E only slightly enhanced it. The order of effectiveness of enhancement was fibrin greater than SK-potentiator greater than fibrinogen greater than D greater than E. When Lys-plg (0.1 microM) was activated by 0.5 u/ml of SK in the presence of potentiating agents, SK-potentiator enhanced the hydrolysis of S-2251 to the largest extent. The enhancement was far less in comparison to the enhancement of the hydrolysis by Glu-plg and SK. The measurement of delta OD405/min at the time of 50% hydrolysis of the substrate was performed in order to compare the effects of concentrations of potentiating agents. The maximum enhancement was obtained at almost an equimolar ratio of plasminogen and fibrin. Fifty percent enhancement was obtained at 0.05 microM for SK-potentiator, 0.072 microM for fibrinogen, 0.21 microM for D and 0.35 microM for E. Fibrin caused the largest extent of enhancement among other potentiating agents. These results may indicate that a trimolecular complex between SK, plasminogen and potentiating agents hydrolyzes S-2251 more effectively than SK-plasminogen complex, thus a trimolecular complex being a better activator than SK-plasminogen complex. Although D and E enhanced only slightly the rate of hydrolysis of S-2251 at equimolar ratio to plasminogen, increase in their concentration resulted in the same extent of enhancement as shown in the presence of fibrinogen or SK-potentiator.

Topics: Fibrin; Fibrin Fibrinogen Degradation Products; Fibrinogen; Humans; Hydrolysis; Kinetics; Oligopeptides; Peptide Fragments; Plasminogen; Spectrophotometry; Streptokinase

An indirect spectrophotometric assay for extrinsic plasminogen activator has been devised, which is based on the parabolic assay of Drapier et al. (5). The system contains activator, plasminogen, the synthetic plasmin substrate H-D-Val-Leu-Lys-pNA (S-2251, Kabi) and a mixture of soluble fibrinogen fragments prepared by treatment of fibrinogen with cyanogen bromide. The addition of these fibrinogen fragments considerably enhances the sensitivity and specificity of the method owing to specific stimulation of the plasminogen activation by extrinsic plasminogen activator. The assay conditions were optimized and the application for extrinsic plasminogen activator measurements in plasma euglobulin fractions is demonstrated.

Topics: Fibrin; Fibrinogen; Humans; Kinetics; Oligopeptides; Plasminogen Activators; Spectrophotometry

A urokinase-activated plasmin (UK-plasmin) preparation was assayed against the International Reference Preparation for Plasmin (IRP-plasmin) using caseinolytic, fibrinolytic, fibrinogenolytic and chromogenic assay methods. The relative potency (using multi-dose bioassays) was estimated by the fibrinolytic method to be about twice that obtained by the caseinolytic, fibrinolytic and chromogenic assay methods. It was found that the UK-plasmin binds to fibrin to a greater extent than does the IRP-plasmin and this is advanced as an explanation for the discrepancy between assay methods. This difference in the binding of the two plasmins to fibrin may mean that it will be difficult to compare the fibrinolytic activities of various plasmin preparations. It is also shown that, during thermal degradation, the IRP-plasmin loses fibrinolytic activity more rapidly than amidolytic activity.

Topics: Binding Sites; Caseins; Chromogenic Compounds; Fibrin; Fibrinogen; Fibrinolysin; Fibrinolysis; Hot Temperature; Humans; Oligopeptides; Preservation, Biological; Urokinase-Type Plasminogen Activator