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Comparative in vitro study of the kinetics of various reactions involved in the process of thrombolysis initiated by streptokinase (SK) and staphylokinase (STA) was carried out. It was shown that at the interaction of an equimolar ratio of plasminogen (Pg) with SK or STA the rate of formation and the specific esterase activity of the complex plasmin (Pm) · SK are higher than those of the complex Pm · STA. The catalytic efficiency (kcat/Km) of hydrolysis of the chromogenic plasmin substrates by Pm · SK complex was 2 times higher than by Pm · STA complex. In the absence of fibrin catalytic efficiency (kPg/K(Pg)) of activation of Glu-plasminogen and Lys-plasminogen glycoform II by Pm · SK complex was higher than by Pm · STA complex, but the pres- ence of fibrin increased kPg/K(Pg)) activation of both plasminogens by Pm · STA complex significantly stronger than by Pm · SK complex due to the decrease in K(Pg)). In contrast to STA (15.5 kDa), SK molecule (47 kDa) creates significant steric hindrances for the interaction of plasmin in Pm · SK complex with protein inhibi- tors. In addition, SK caused greater fibrinogen degradation than STA. It is 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 plas- ma, by Pm · STA complex are significantly higher than those by Pm · SK complex. It was revealed that the species specificity of STA and S K 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 fell in the series: human > dog > rabbit for SK and the dog > human > rabbit for STA. The results show that in the purified system SK is a more effective activator of plasminogen than STA. In the system con- taining 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 recyclization in the presence of α2AP.

By variation of incubation time of streptokinase (SK) with activated polyethylene glycol (M 2 and 5 kDa, PEG2 and PEG5) it was obtained covalent SK-PEG2 and SK-PEG5 conjugates with different modification degrees of amino groups of protein and their properties were studied in vitro as compared with free SK. It was shown, that maximal stable and retaining 80% fibrinolytic activity SK-PEG2 and SK-PEG5 conjugates are formed when the modification degrees of amino groups of protein are 54 and 52%, respectively. At interaction of the given conjugates with equimolar plasminogen concentration it were formed the plasmin (Pm)·SK-PEG2 and Pm·SK-PEG5 activator complexes, the maximal amidase activity of which is equal to activity of unmodified Pm·SK complex. It was found, that the catalytic efficiency of plasminogen activation (kPg/KPg) by Pm·SK-PEG2 complex is some higher (2.84 min(-1) μM(-1)) and by Pm·SK-PEG5 complex is lower (1.17 min(-1) μM(-1)), than that by unmodified Pm·SK complex (2.1 min(-1) μM(-1)). Investigation of lysis kinetics of human plasma clots and depletion of plasminogen and fibrinogen in plasma under the action of free SK and SK-PEG2 and SK-PEG5 conjugates showed, that the latter's have high thrombolytic activity (89 and 72%, respectively) and cause 3.5-4 fold lower side effects, than free SK. Obtained by us SK-PEG2 and SK-PEG5 conjugates with increased stability and decreased side effects may be used in the therapy of thrombotic disorders.

Thrombolytic therapy by high doses of streptokinase (SK) that are stipulated by its rapid clearance is accompanied by side effects. In this work for the purpose of lifetime prolongation in bloodstream and decrease in side effects SK was included in microcapsules from water-soluble polyethyleneglygol (PEG) using the double emulsification method. By variation of the emulsification conditions, molecular weight of PEG (20 or 40 kDa) and PEG/SK ratio (12 or 8 mg PEG/1000 IU SK) it was obtained four preparations of PEG-microcapsules with high percent of SK inclusion (approximately 90-91%), which has completely preserved its fibrinolytic activity and released from microcapsules with different rates. The time of SK full release from obtained PEG-microcapsules was varied from 45 to 90 min (pH 7.4; 37 degrees C). The comparative in vitro study ofthrombolytic and side effects of free SK and SK*PEG-microcapsules was conducted. It was found that at equal doses (500 IU/mL) the lysis rates of human plasma clots under the action of encapsulated preparations of SK (with the exception of a small lag period) were equal to the lysis rate induced by free SK. Besides, SK*PEG-microcapsules caused a less exhaustion of plasminogen and fibrinogen in plasma than free SK.

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/ K) 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/ K) 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/ K of activation of both plasminogens by Pm•STA complex much more than by Pm•SK complex due to a decrease in K 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 α-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 αAP, and the ability of the STA to recycling in the presence of αAP. [ABSTRACT FROM AUTHOR]

Thrombolytic therapy with high doses of streptokinase (SK), which are required due to its rapid clearance from the bloodstream, is accompanied by side effects. In this work, the SK was incorporated into water-soluble polyethylene glycol (PEG) microcapsules with the double emulsification method in order to increase its lifetime in bloodstream and decrease side effects. Four preparations of SK*PEG-microcapsules with a high degree of the SK enclosure (∼90-91%) and total retention of fibrinolytic activity were produced under varying emulsification conditions (PEG molecular mass 20 or 40 kDa and PEG/SK ratio 12 or 8 mg of PEG/1000 IU SK). SK was released from the PEG-microcapsules at different rates: the time of complete release varied from 45 to 90 min (pH 7.4, 37°C). Comparative in vitro study of thrombolytic activity and side effects of the SK in a free and encapsulated state was conducted. It was found that the rate of human plasma clot lysis under the action of encapsulated SK preparations is equal (with exception of a short lag-period) to the rate of lysis induced by the free SK, provided that the doses were equal (500 IU/mL). Furthermore, the SK*PEG-microcapsules caused the reduced exhaustion of plasminogen and fibrinogen in plasma when compared with the free SK. [ABSTRACT FROM AUTHOR]