Analysis of five streptokinase formulations using the euglobulin lysis test and the plasminogen activation assay

Abstract

Streptokinase, a 47-kDa protein isolated and secreted by most group A, C and G ß-hemolytic streptococci, interacts with and activates human protein plasminogen to form an active complex capable of converting other plasminogen molecules to plasmin. Our objective was to compare five streptokinase formulations commercially available in Brazil in terms of their activity in the in vitro tests of euglobulin clot formation and of the hydrolysis of the plasmin-specific substrate S-2251™. Euglobulin lysis time was determined using a 96-well microtiter plate. Initially, human thrombin (10 IU/ml) and streptokinase were placed in individual wells, clot formation was initiated by the addition of plasma euglobulin, and turbidity was measured at 340 nm every 30 s. In the second assay, plasminogen activation was measured using the plasmin-specific substrate S-2251™. Streptase™ was used as the reference formulation because it presented the strongest fibrinolytic activity in the euglobulin lysis test. The Unitinase™ and Solustrep™ formulations were the weakest, showing about 50% activity compared to the reference formulation. All streptokinases tested activated plasminogen but significant differences were observed. In terms of total S-2251™ activity per vial, Streptase™ (75.7 ± 5.0 units) and Streptonase™ (94.7 ± 4.6 units) had the highest activity, while Unitinase™ (31.0 ± 2.4 units) and Strek™ (32.9 ± 3.3 units) had the weakest activity. Solustrep™ (53.3 ± 2.7 units) presented intermediate activity. The variations among the different formulations for both euglobulin lysis test and chromogenic substrate hydrolysis correlated with the SDS-PAGE densitometric results for the amount of 47-kDa protein. These data show that the commercially available clinical streptokinase formulations vary significantly in their in vitro activity. Whether these differences have clinical implications needs to be investigated.

Development and characterization of highly selective target-sensitive liposomes for the delivery of streptokinase: in vitro/in vivo studies

“…The SK activity was determined according to the previously reported method (Bode et al, 1991;Couto et al, 2004) with a slight modification. Entrapment efficiency was calculated by the following equation: C/T Â 100 (C: amount of SK incorporated into liposome, T: total amount of SK).…”

Constitutive Expression and Optimization of Nutrients for Streptokinase Production by Pichia pastoris Using Statistical Methods

“…Fibrin Clot Lysis Assay [25] A 1.0-ml mixture of 20 μl plasminogen (120 IU/ml) and 1.0 ml of fibrinogen (2 mg/ml) were added to 0.5 ml of thrombin (33 IU/ml). Subsequently 0.5 ml of streptokinase mixture was added and incubated at 37°C.…”

Optimization of culture media for extracellular expression of streptokinase in Escherichia coli using response surface methodology in combination with Plackett-Burman Design

“…SK is not originally a protease; however on complexation with plasminogen it gives rise to plasmin that can dissolve the fibrin network of a blood clot and solubilize degradation products [2,3]. Heterologous production of SK in Escherichia coli (E. coli), as the most widely used prokaryotic system is useful method for industrial applications [4].…”