We obtained recombinant variants of human antibody FI6 broadly specific to hemagglutinins of the influenza A virus. On the basis of a bi-promoter (CMV, hEF1-HTLV) vector, we developed genetic constructs for the expression of the heavy and light chains of the immunoglobulins of IgA1-, IgA2m1-, and IgG-isotypes. Following transfection and selection, stable Chinese hamster ovary (CHO) cell lines were produced. The antibodies of IgA1-, IgA2m1-, and IgG-isotypes were purified from culture media. We performed an immunochemical characterization and studied their interactions with influenza A strains of the H1N1- and H3N2-subtypes. It was shown that recombinant FI6 variants of the IgA-isotype retain the properties of the parental IgG antibody to demonstrate specificity to all the strains tested. The strongest binding was observed for the H1N1 subtype, which belongs to hemagglutinins of phylogenetic group I.

•Thrombolytic activity of SK-PAMAM conjugates depends on dendrimer generation and modification degree of SK amino groups.•SK-dendrimers conjugates cause less fibrinogenolysis than free SK.•Compared with SK-G1.5 and SK-G2.5 conjugates, SK-G3.5 conjugates have the highest thrombolytic activity.

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 alpha(2)-antiplasmin levels in the surrounding plasma at 37 degrees 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. (C) 2015 Elsevier B.V. All rights reserved.

The comparative in vitro study of the kinetics of various reactions involved in the process of thrombolysis initiated by streptokinase (SK) and staphylokinase (STA) has been carried out. It has been shown that upon the interaction of plasminogen (Pg) with SK or STA in equimolar quantities, the formation rate and the specific esterase activity of the complex plasmin (Pm) with SK (Pm•SK) is higher than those of the complex Pm•STA. The catalytic efficiency (k cat/K m) of hydrolysis of the chromogenic plasmin substrates by Pm•SK complex is 2 times higher than by Pm•STA complex. In the absence of fibrin, the catalytic efficiency (k Pg/K Pg) of activation of Glu-plasminogen and Lys-plasminogen glycoform II by Pm•SK complex is higher than by Pm•STA complex, but the presence of fibrin increases k Pg/K Pg of activation of both plasminogens by Pm•STA complex much more than by Pm•SK complex due to a decrease in K Pg In contrast to STA (15.5 kDa), an SK molecule (47 kDa) creates remarkable steric hindrances for the interaction of plasmin in Pm•SK complex with protein inhibitors. In addition, SK causes higher fibrinogen degradation in plasma than STA. It has been shown that Pm•SK and Pm•STA complexes lyse fibrin clots in buffer with similar rates, while the rate of lysis of plasma clots, immersed in plasma, by Pm•STA complex is remarkably higher than in the case of Pm•SK complex. It has been revealed that the species specificity of STA and SK is determined mainly by the rate of formation and the efficiency of Pm•SK and Pm•STA complexes in the activation of autologous plasminogen. The lysis efficiency of plasma clots of mammals falls in the series: human > dog > rabbit for SK and dog > human > rabbit for STA. The results show that in the purified system SK is a more effective plasminogen activator than STA. In the system containing fibrin and α2-AP, the activator and fibrinolytic activities of STA are higher than those of SK, due to the increased stability in plasma and fibrin specificity of STA, the fast reaction of the complex Pm•STA with α2AP, and the ability of the STA to recycling in the presence of α2AP.