Activation of human plasminogen by streptokinase: new aspects of fibrinolysis regulation

It is established fact, that SK-initiated fibrinolysis is decelerated, when oxygen is removed from solutions; that SK possesses superoxide dismutase-like activity and that its activating function is sharply inhibited by superoxide radical scavengers. The point in discussion is the possibility of oxygen-dependent Pg activation, which is not related to functions of serine proteinase-like activators. The realization of oxygen-radical mechanisms is supposed to depend on activation of zymogens of other serine proteinases and proteinase reactions effect.     

Kinetic mechanism of the activation of human plasminogen by streptokinase.

A method of determining the initial rate of plasminogen activation has been developed. The method has been used to investigate the mechanism of activation of human plasminogen by streptokinase. Plasmin formation follows saturation kinetics. Inhibition of plasmin formation by epsilon-aminocaproic acid is uncompetitive with a Ki of 0.6 mM. A model consistent with the data is that streptokinase induces a conformational change in the plasminogen molecule, producing an active center which cleaves an internal peptide bond to produce plasmin. Thus, streptokinase functions as a catalytic allosteric effector.     

Domain interactions between streptokinase and human plasminogen.

Plasmin (Pm), the main fibrinolytic protease in the plasma, is derived from its zymogen plasminogen (Plg) by cleavage of a peptide bond at Arg(561)-Val(562). Streptokinase (SK), a widely used thrombolytic agent, is an efficient activator of human Plg. Both are multiple-domain proteins that form a tight 1:1 complex. The Plg moiety gains catalytic activity, without peptide bond cleavage, allowing the complex to activate other Plg molecules to Pm by conventional proteolysis. We report here studies on the interactions between individual domains of the two proteins and their roles in Plg activation. Individually, all three SK domains activated native Plg. While the SK alpha domain was the most active, its activity was uniquely dependent on the presence of Pm. The SK gamma domain also induced the formation of an active site in Plg(R561A), a mutant that resists proteolytic activation. The alpha and gamma domains together yielded synergistic activity, both in Plg activation and in Plg(R561A) active site formation. However, the synergistic activity of the latter was dependent on the correct N-terminal isoleucine in the alpha domain. Binding studies using surface plasmon resonance indicated that all three domains of SK interact with the Plg catalytic domain and that the beta domain additionally interacts with Plg kringle 5. These results suggest mechanistic steps in SK-mediated Plg activation. In the case of free Plg, complex formation is initiated by the rapid and obligatory interaction between the SK beta domain and Plg kringle 5. After binding of all SK domains to the catalytic domain of Plg, the SK alpha and gamma domains cooperatively induce the formation of an active site within the Plg moiety of the activator complex. Substrate Plg is then recognized by the activator complex through interactions predominately mediated by the SK alpha domain.     

Purification of streptokinase by affinity chromatography on immobilized acylated human plasminogen.

Streptokinase is an extracellular protein produced by several strains of streptococci. It functions in the species-specific conversion of plasminogen to plasmin. In this paper we describe the purification of streptokinase by affinity chromatography on human plasminogen acylated with p'-nitrophenyl p-guanidinobenzoate. The acylated and non-acylated plasminogen and plasmin were coupled to cyanogen bromide-activated Sepharose 4B and evaluated for streptokinase purification. These results show that a homogeneous preparation of streptokinase with high specific activity and high yield can be obtained using acylated plasminogen. This method permits the binding of one milligram of streptokinase per milliliter of swollen gel.     

Effects of plasminogen, streptokinase and their equimolar complexes with pyruvate kinase on the human neuroblastoma IMR-32 cells

The system of extracellular proteolysing, consists of plasminogen (PGn), its active protease (plasmin), PGn activation and PGn activators inhibitors, influences the nervous tissue functions, their growth, differentiation and proliferation in both, normal and pathological conditions. The purpose of the investigation was to study the effects of exogenous PGn, its activator streptokinase (SK), PK and their equimolar complex on the morpho-functional state neuroblastoma IMR-32 cells. PGn, SK, PK and their complexes stimulated cells proliferation during 1-3 days of incubation, shown by cell quantity increase. We also observed DNA, RNA and protein increase. The low lactate dehydrogenase efflux was evidence of that an addition of the proteins under investigation in the culture medium prevented the development of degenerative alterations connected with serum deprivation. The levels of extracellular PGn-activator activity, as measured by the biochemical fibrinolytic assay, increased over SK. This SK effect vanished on the 3rd day when SK formed complexes with PK. New original facts obtained testify the probability of initiation of neoplastic transformation and tumor growth potentiation.     

Effects of plasminogen, streptokinase, and their equimolar complexes with pyruvate kinase on the human neuroblastoma IMR-32 cells

The system of extracellular proteolysis consisted of plasminogen (PGn), its active protease, plasmin, and PGn activators and their inhibitors affect the growth, differentiation, and proliferation of nervous cells both under normal and pathological conditions. The purpose of our investigation was to study the effects of exogenous PGn, its activator, streptokinase (SK), pyruvate kinase (PK), and their equimolar complexes on morphological and functional properties of IMR-32 neuroblastoma cells. It has been found that PGn, SK, PK, and their complexes stimulate cell proliferation during 1–3 days of incubation. We also observed increased DNA, RNA, and protein content. The low-lactate dehydrogenase (LDH) efflux indicated that the addition of the proteins we assayed to the culture medium prevented the development of degenerative processes caused by serum deprivation. The levels of extracellular PGn-activator activity, as measured by the fibrinolytic method, increased in the presence of SK. The SK effect vanished if SK was in the complex with PK on the 3rd day of cultivation. New original facts were obtained to testify the probability of initiation of neoplastic transformation and tumor growth potentiation.     

Analysis of the interactions between streptokinase domains and human plasminogen.

The contrasting roles of streptokinase (SK) domains in binding human Glu1-plasminogen (Plg) have been studied using a set of proteolytic fragments, each of which encompasses one or more of SK's three structural domains (A, B, C). Direct binding experiments have been performed using gel filtration chromatography and surface plasmon resonance. The latter technique has allowed estimation of association and dissociation rate constants for interactions between Plg and intact SK or SK fragments. Each of the SK fragments that contains domain B (fragments A2-B-C, A2-B, B-C, and B) binds Plg with similar affinity, at a level approximately 100- to 1,000-fold lower than intact SK. Experiments using 10 mM 6-aminohexanoic acid or 50 mM benzamidine demonstrate that either of these two lysine analogues abolishes interaction of domain B with Plg. Isolated domain C does not show detectable binding to Plg. Moreover, the additional presence of domain C within other SK fragments (B-C and A2-B-C) does not alter significantly their affinities for Plg. In addition, Plg-binding by a noncovalent complex of two SK fragments that contains domains A and B is similar to that of domain B. By contrast, species containing domain B and both domains A and C (intact SK and the two-chain complex A1 x A2-B-C) show a significantly higher affinity for Plg, which could not be completely inhibited by saturating amounts of 6-AHA. These results show that SK domain B interacts with Plg in a lysine-dependent manner and that although domains A and C do not appear independently to possess affinity for Plg, they function cooperatively to establish the additional interactions with Plg to form an efficient native-like Plg activator complex.     

Effects of human fibrinogen and its cleavage products on activation of human plasminogen by streptokinase

The influence of human fibrinogen (Fg) and its terminal plasminolytic digestion products, fragment D and fragment E, on the kinetics of activation of human plasminogen (Pg) by catalytic levels of streptokinase (SK) has been investigated. Both Fg and fragment D enhanced the rates of activation of human Glu1-Pg, Lys77-Pg, and Val442-Pg. Fragment E was refractive in this regard. In the case of Glu1-Pg, the Km for activation by SK, 0.4 microM, was not affected by the presence of Fg or fragment D. The kcat for this same reaction, 0.12 s-1, was elevated to 0.3 s-1 at saturating levels of these effector molecules. On the other hand, the Km for activation of Lys77-Pg, 0.5 microM, was decreased to 0.09 microM, whereas the kcat, 0.33 s-1, was not altered in the presence of saturating concentrations of Fg or fragment D. In the case of Val442-Pg, the Km for this same activation, 2.0 microM, was lowered to 0.4 microM and 0.25 microM in the presence of Fg and fragment D, respectively. The kcat for this process, 1.0 s-1, was unchanged in the presence of these agents. The concentrations of Fg (KFg) and fragment D (KFD) that led to half-maximal stimulation of the activation rates were determined. For Fg with Glu1-Pg, Lys77-Pg, and Val442-Pg, the KFg values were 0.08 microM, 0.14 microM, and 0.17 microM, respectively. The KFD values for these same plasminogens were 0.25 microM, 2.0 microM, and 1.7 microM, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)