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Background: Polyamidoamine (PAMAM) dendrimers are a new class of monodisperse polymers that are used for drug delivery in systemic administrations. The influence of PAMAM dendrimers on components of the blood coagulation system has been extensively studied, but their effect on the activity of the fibrinolysis system has not been studied to date., Methods: The effect of cationic (G1-G3) and anionic (G1.5-G3.5) PAMAM dendrimers on the conformation and function of the main components of the coagulation and fibrinolysis systems was comparatively studied. Changes in overall plasma hemostatic potential, thrombin generation, prothrombin time, thrombin and tPA activities, the fluorescence of fibrinogen and plasminogen, zeta potential, polymerization of fibrinogen, and activation of plasminogen were analyzed to assess coagulofibrinolytic mechanisms of influence of the charge of the dendrimers., Results: Cationic dendrimers increased prothrombin time, suppressed thrombin generation in plasma, and changed the conformation and coagulability of fibrinogen, while anionic dendrimers did not have such effects. Anionic dendrimers slightly reduced tPA activity and altered plasminogen conformation much more strongly than the cationic dendrimers. Plasminogen activation by tPA was strongly inhibited by anionic dendrimers and weakly stimulated by cationic dendrimers. All these effects were enhanced with increasing generation and concentration of the dendrimers., Conclusions: PAMAM-NH 2 dendrimers inhibit the extrinsic activation pathway of the coagulation system and alter the conformation and function of fibrinogen. PAMAM-COOH dendrimers change the conformation of plasminogen and inhibit its activation by tPA. This study gives new insight into the effect of anionic PAMAM dendrimers on the activity of the fibrinolytic system. For intravenous applications, the antifibrinolytic effect of anionic PAMAM dendrimers of generation ≥G2.5 should be considered., Competing Interests: Declaration of competing interest The authors report no conflict of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Background: Mechanisms of fibrin-specificity of tissue plasminogen activator (tPA) and recombinant staphylokinase (STA) are different, therefore we studied in vitro the possibility of the synergy of their combined thrombolytic action., Methods: Thrombolytic effects of tPA, STA and their combinations were measured by lysis rate of human plasma clot and side effects were evaluated by decreasing in fibrinogen, plasminogen and α2-antiplasmin levels in the surrounding plasma at 37°C in vitro., Results: STA and tPA induced dose- and time-dependent clot lysis: 50% lysis in 2 h was obtained with 30 nM tPA and 75 nM STA, respectively. At these concentrations, tPA produced greater degradation of plasma fibrinogen than STA. According to a mathematical analysis of dose-response curves by the isobole method, combinations of tPA and STA caused a considerable synergistic thrombolytic effect. The simultaneous and sequential combinations of tPA (<4 nM) and STA (<35 nM) induced a significant fibrin-specific synergistic thrombolysis, which was more pronounced in 2 h at simultaneous combinations than at sequential addition of STA after 30 min of tPA action. Simultaneous combination of 2.5 nM tPA and 15 nM STA showed a maximal 3-fold increase in thrombolytic effect compared to the expected total effect of the individual agents. Sequential combinations caused a lower depletion of plasma proteins compared to simultaneous combinations., Conclusions: The simultaneous and sequential combinations of tPA and STA possessed synergistic fibrin-specific thrombolytic action on clot lysis in vitro., General Significance: The results show that combined thrombolysis may be more effective and safer than thrombolysis with each activator alone., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Introduction: The binding of plasminogen (Pg) to cell receptors and extracellular ligands facilitates its activation to plasmin, which stimulates the extracellular matrix degradation, neoangiogenesis and tumor invasion. Plasmin can also degrade IgG thereby exposing C-terminal lysine residues. Previously, we have found IgG specifically bounded to Pg in the plasma of patients with malignant tumors., Aim: To identify IgG degraded by plasmin in the plasma of cancer patients., Materials and Methods: Methods of ELISA were used for comparative research of levels of IgG bound to Pg in plasma of patients with the prostate cancer (PC, n=25) and lung cancer (LC, n=17). Plasma of healthy donors (n=29) was used as control. All patients signed informed consent for participation in this study. Affinity chromatography on Pg-sepharose was used for the quantification of IgG. Carboxypeptidase was used for remove of C-terminal lysine residues of the IgG. The program ATTESTAT was used for nonparametric analysis., Results: The frequency of occurence of elevated levels of IgG to Pg in plasma was detected in 68% of patients with PC, 59% of patients with LC and only 12% of healthy women and 10% of healthy men. The quantification of antibodies in plasma samples showed that the quantity of IgG to Pg in patients with PC was 27% from the total amount of IgG and in healthy men - 9%. Treatment of diluted plasma samples with carboxypeptidase B abolished the elevated levels of IgG to Pg, as well as the specific activity of the purified IgG to Pg-sepharose., Conclusions: C-terminal lysine residues which are formed as a result of degradation of native IgG with plasmin can bind to lysine binding sites on the kringle domains of Pg. Increased levels of these degraded IgG can be marker at cancer., (© 2016 Elsevier Ltd. All rights reserved.)

Kinetic parameters (k(Pg) and K(Pg)) were determined for activation of Glu-plasminogen (Glu-Pg) and Lys-plasminogen (Lys-Pg) type I (with N-linked carbohydrate chain at Asn-289) and type II (with unsubstituted Asn-289) by plasmin-staphylokinase (Pm-STA) complex. The K(Pg) values for Glu-Pg I and Lys-Pg I (17.1 and 11.2 microM, respectively) were higher than those for Glu-Pg II and Lys-Pg II (14.9 and 5.4 microM, respectively), while only minor differences in the k(Pg) values were observed between plasminogens type I and type II. Soluble fibrin significantly increased the k(Pg)/K(Pg) values for activation of all four plasminogens due to a decrease in the K(Pg) values but did not alter the k(Pg) values. However, the activation of plasminogens type I was stimulated by fibrin lesser degree than that of plasminogens type II. These findings indicate that N-glycosylation of kringle 3 of plasminogen decreases the stability of Pm-STA-Pg ternary enzyme-substrate complex in solution as well as interferes with its formation and rearrangement on the fibrin surface.

Comparative kinetic and electrophoretic study of the interaction of plasminogen (PG) with equimolar concentrations of staphylokinase (SPK) and streptokinase (SK) at 4 and 37 degreesC showed that the PG--SK complex has fibrinolytic and esterase activities, whereas the PG--SPK complex was inactive. Both esterase and fibrinolytic activities were enhanced during the conversion of the PG--SPK complex to the complex of plasmin (PL) with SPK (PL--SPK) at 37 and 4 degreesC, while the PG--SK complex was rapidly converted to the PL--SK complex with higher esterase activity only at 37 degreesC. The catalytic efficiency of Z-Lys-pNP hydrolysis (kcat/Km) by the preformed PL--SPK complex was twofold lower than that in the case of the PL--SK complex. Incubation of the PL--SPK and PG--SK(PL--SK) complexes at 37 degreesC for 24 h was associated with the degradation of the proteins and with different kinetics of lowering of esterase, plasminogen activator, and fibrinolytic activities. The PL--SPK complex was considerably more stable than the PG--SK(PL--SK) complex; streptokinase degraded more rapidly than staphylokinase. Kinetics of lysis of fibrin clots by the two complexes were similar, but the efficiency of lysis of plasma clots by the PL--SPK complex was significantly higher than that in the case of the PG--SK(PL--SK) complex (at 0.03-1 microM). Probably, unlike streptokinase, staphylokinase which is less susceptible to degradation in the PL--SPK complex and is released from the triple complex alpha2-antiplasmin--PL--SPK, forms a potentially highly active new complex with free molecules of plasminogen in the plasma.

Polyacrylamide beads (microgranules) containing covalently bound alpha-chymotrypsin were obtained. An average diameter of the microgranules is 100--150 micrometers. Prior to the bead polymerization alpha-chymotrypsin was modified by acryloyl chloride. The efficiency of the microgranulated alpha-chymotrypsin action depends on the kinetic properties of the substrates used. The pH optimum for the microgranulated enzyme activity towards N-acetyl-L-tyrosine ethyl ester is shifted by 1,5 units to the alkaline zone. The immobilized alpha-chymotrypsin is completely stable for 100 hrs at 50 degrees.