Abstract
Streptokinase is a virulence factor of streptococci and acts as a plasminogen activator to generate the serine protease plasmin which promotes bacterial metastasis. Streptokinase isolated from group C streptococci has been used therapeutically as a thrombolytic agent for many years and its mechanism of action has been extensively studied. However, group A streptococci are associated with invasive and potentially fatal infections, but less detail is available on the mechanism of action of streptokinase from these bacteria. We have expressed recombinant streptokinase from a group C strain to investigate the therapeutic molecule (here termed rSK-H46A) and a molecule isolated from a cluster 2a strain from group A (rSK-M1GAS) which is known to produce the fibrinogen binding, M1 protein, and is associated with life-threatening disease. Detailed enzyme kinetic models have been prepared which show how fibrinogen-streptokinase-plasminogen complexes regulate plasmin generation, and also the effect of fibrin interactions. As is the case with rSK-H46A our data with rSK-M1GAS support a “trigger and bullet” mechanism requiring the initial formation of SK•plasminogen complexes which are replaced by more active SK•plasmin as plasmin becomes available. This model includes the important fibrinogen interactions that stimulate plasmin generation. In a fibrin matrix rSK-M1GAS has a 24 fold higher specific activity than the fibrin-specific thrombolytic agent, tissue plasminogen activator, and 15 fold higher specific activity than rSK-H46A. However, in vivo fibrin specificity would be undermined by fibrinogen stimulation. Given the observed importance of M1 surface receptors or released M1 protein to virulence of cluster 2a strain streptococci, studies on streptokinase activity regulation by fibrin and fibrinogen may provide additional routes to addressing bacterial invasion and infectious diseases.
Highlights
Streptokinase (SK) is a bacterial plasminogen (Pgn) binding and activating protein widely used as a therapeutic thrombolytic agent [1]
The mechanism underlying this stimulation is different for the two full length Recombinant SK (rSK) variants as can be seen from the individual kcat and KM values. rSK-H46A stimulates Pm activity by increasing the kcat against S-2251, whereas rSK-M1GAS improves the KM for the substrate
It can be seen that there are differences in the way rSK-H46A and rSK-M1GAS interact with Pm and modulate activity, but a significant effect of rSK binding is to protect Pm from inhibition by Fgn through changes in substrate specificity of bound Pm as noted in other studies on peptide substrates for example [32]
Summary
Streptokinase (SK) is a bacterial plasminogen (Pgn) binding and activating protein widely used as a therapeutic thrombolytic agent [1]. The commercial molecule is isolated from Group C streptococci and is able to activate Pgn in solution and is not fibrin specific [2]. Subsequent development of Pgn activator drugs, so-called second and third generation thrombolytics. Streptokinase from Streptococcus Pyogenes highlighted the desirability of fibrin specificity and led to the development of the tissue Pgn activator (tPA) family of drugs [3]. Studying the mechanisms of Pgn activation by microbial activators is helpful in better understanding infection [6] and has been applied to the development of fibrin specific thrombolytics, such as staphylokinase [7]and engineered versions of streptokinase [8, 9]
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