Activity Regulation by Fibrinogen and Fibrin of Streptokinase from Streptococcus Pyogenes

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.

Measuring fibrinolysis: from research to routine diagnostic assays

“…For example, very high inhibition rate constants (of the order of 10 7 mol L −1 s −1 ) are often quoted for α2AP and PAI‐1, but these are modulated by fibrinogen and fibrin in vivo . Solution studies are definitely easier to perform than studies in a fibrin matrix and are useful for plasminogen activators such as urokinase (uPA) or therapeutic streptokinase (but note, streptokinase variants from other streptococcus strains do interact with fibrinogen and fibrin ). Experience has shown that caution is needed when using tPA with soluble stimulators such as cyanogen bromide fragments of fibrinogen , compared with fibrin , as the kinetics of binding and plasminogen activation are different.…”

Binding of human plasminogen and high-molecular-mass kininogen by cell surface-exposed proteins of Candida parapsilosis

“…Secreted or surface-located microbial proteases often possess an ability to activate or inactivate particular enzymatic components of these proteolytic cascades or to degrade dedicated proteinase inhibitors, thus leading to deregulation of the physiological functionality of such systems (Maeda & Yamamoto, 1996;Potempa et al, 2000;Kozik et al, 2015a;Gogol et al, 2016;Huish et al, 2017). Moreover, a great variety of bacterial or fungal surface-exposed proteins were repeatedly reported to be involved in the interactions with human plasma proteins (Smeesters et al, 2010;Rapala-Kozik et al, 2011;Wollein Waldetoft et al, 2012;Castiblanco-Valencia et al, 2016).…”

Structural Biology and Protein Engineering of Thrombolytics

“…It is naturally produced by the strains of β-hemolytic streptococci which utilize it for overcoming the host’s defensive fibrin barrier and for promoting bacterial metastasis and colonization [183,184]. There are several streptokinases from different streptococci which vary in their structure, but the only variant of streptokinase currently used as a thrombolytic agent originates from the group C streptococci (streptokinase SK-H46A from Streptococcus equisimilis strain H46A) and lacks a significant stimulation by fibrin [185,186]. Despite its moderate half-life in vivo (approximately 10 min) and significantly lower cost, the largest disadvantages are the lack of fibrin specificity and immunogenicity due to its bacterial origin [47,[187], [188], [189]].…”

“…Due to the α domain, streptokinase does not require fibrin and activates plasminogen independently [186,194,200]. Stimulating experiments confirmed that neither fibrin nor fibrinogen had stimulatory effects on activation, preventing targeted thrombolysis on the surface of fibrin clots [185,186]. The activation independent of fibrin can lead to bleeding complications which represents one of the main drawbacks of the currently used streptokinase variant.…”