Mapping of the antigenic regions of streptokinase in humans after streptokinase therapy.

Streptokinase (SK) is efficaciously used as a thrombolytic drug for the treatment of myocardial infarction. Being a bacterial protein, SK is immunogenic in humans. Therefore, resulting from SK therapy, patients become immunized and anti-SK antibody (Ab) titers rise post-treatment. High Ab titers might provoke severe immune reactions during SK therapy and neutralize SK activity, preventing effective thrombolysis. Spot synthesis combined with peptide library techniques is a useful tool for studying protein-peptide interactions on continuous cellulose membranes. Here, we report on the mapping of antigenic regions of SK using a spot-synthesized peptide library and human total sera from patients receiving SK therapy. All tested samples have high anti-SK Ab titers and most of them show significant SK neutralizing capacity. Individual variations in peptide recognition were detected. However, patients treated with SK tend, in general, to show a common regional binding pattern, including residues 1-20, 130-149, 170-189, and 390-399. This is the first study reporting the probing of a cellulose-bound set of peptides with total human sera.     

On the plasminogen-activating function of streptokinase.

1. Several hypotheses have been advanced to explain the activating function of streptokinase. The predominant hypothesis suggests a stable equimolar streptokinase-plasmin(ogen) complex, activating free plasminogen by an active centre, which is located in the plasmin(ogen) part of the complex. 2. This hypothesis cannot explain a number of phenomena and certain accumulated experimental data, for example: rabbit and bovine plasminogen activation by streptokinase, not forming stable complexes with these plasminogens; possible activation with pH less than or equal to 2, in the presence of urea, during modification of streptokinase tyrosine residues, i.e. when these two proteins cannot form a stable complex. 3. On the basis of acquired experimental data the following concept is suggested: the activating function of streptokinase is oxygen-dependent and is realised with the help of superoxide radical due to the O(2-.)-generating ability of plasminogen and the O(2-.)-converting ability of streptokinase.     

Characterization of highly purified native streptokinase and altered streptokinase after alkaline treatment.

Physical and chemical data are reported for highly purified native streptokinase (staphylokinase, EC 3.4.99.22) (Kabikinase) and streptokinase treated with an alkaline agent (altered streptokinase). The mol. wts. were similar and were determined to be 50 200 by sedimentation equilibrium methods, polyacrylamide gradient gel electrophoresis and sodium dodecylsulphate-polyacrylamide gel electrophoresis. The sedimentation coefficient so20,w of native and altered streptokinase was found to be 3.37 S. The frictional ratio and the absorptivity (A1%1cm) at 280 nm of native streptokinase was found to be 1.29 and 7.5, respectively. Native streptokinase showed essentially a single band in the isoelectro-focusing pattern (pI 5.2), while altered streptokinase showed at least two separate bands. Polyacrylamide gel electrophoresis in the presence of Triton X-100 exhibited one band for native streptokinase but altered streptokinase showd two bands. At pH 12 the biological and immunological activity of streptokinase was markedly decreased in a time-dependent reaction. The amino-terminal amino acid of the two streptokinase forms was isoleucine and the carboxyl-terminal amino acid of native streptokinase was tyrosine. Peptide analysis showed that some peptides in altered streptokinase exhibited higher mobility compared to native streptokinase. The data suggest that streptokinase undergoes a conformational change when incubated in alkaline media, but no simultaneous loss of peptides was observed.     

Role of the streptokinase alpha-domain in the interactions of streptokinase with plasminogen and plasmin

The role of the streptokinase (SK) alpha-domain in plasminogen (Pg) and plasmin (Pm) interactions was investigated in quantitative binding studies employing active site fluorescein-labeled [Glu]Pg, [Lys]Pg, and [Lys]Pm, and the SK truncation mutants, SK-(55-414), SK-(70-414), and SK-(152-414). Lysine binding site (LBS)-dependent and -independent binding were resolved from the effects of the lysine analog, 6-aminohexanoic acid. The mutants bound indistinguishably, consistent with unfolding of the alpha-domain on deletion of SK-(1-54). The affinity of SK for [Glu]Pg was LBS-independent, and although [Lys]Pg affinity was enhanced 13-fold by LBS interactions, the LBS-independent free energy contributions were indistinguishable. alpha-Domain truncation reduced the affinity of SK for [Glu]Pg 2-7-fold and [Lys]Pg 相似文献   

Unfolding and aggregation during the thermal denaturation of streptokinase.

The thermal denaturation of streptokinase from Streptococcus equisimilis (SK) together with that of a set of fragments encompassing each of its three domains has been investigated using differential scanning calorimetry (DSC). Analysis of the effects of pH, sample concentration and heating rates on the DSC thermograms has allowed us to find conditions where thermal unfolding occurs unequivocally under equilibrium. Under these conditions, pH 7.0 and a sample concentration of less than approximately 1.5 mg x mL(-1), or pH 8.0, the heat capacity curves of intact SK can be quantitatively described by three independent two-state transitions, each of which compares well with the two-state transition observed for the corresponding isolated SK domain. The results indicate that each structural domain of SK behaves as a single cooperative unfolding unit under equilibrium conditions. At pH 7.0 and high sample concentration, or at pH 6.0 at any concentration investigated, the thermal unfolding of domain A was accompanied by the time-dependent formation of aggregates of SK. This produces a severe deformation of the DSC curves, which become concentration dependent and kinetically controlled, and thus precludes their proper analysis by standard deconvolution methods. A simple model involving time-dependent, high-order aggregation may account for the observed effects. Limited-proteolysis experiments suggest that in the aggregates the N-terminal segment 1-63 and the whole of SK domain C are at least partially structured, while domain B is highly unstructured. Unfolding of domain A, under conditions where the N-terminal segment 1-63 has a high propensity for beta sheet structure and a partially formed hydrophobic core, gives rise to rapid aggregation. It is likely that this region is able to act as a nucleus for the aggregation of the full-length protein.     

Treatment of deep vein thrombosis with streptokinase.

From September 1962 to May 1972 145 patients with acute or subacute deep vein thrombosis confirmed by phlebography were treated with streptokinase. During the same period 42 patients considered unfit for thrombolytic therapy were treated with herapin and oral anticoagulants. The results, assessed by repeat phlebography, in 93 of the patients treated with streptokinase were compared with those in 42 patients treated with heparin. The age, sex, and severity of occlusion were roughly similar in both groups. Streptokinase treatment was successful in 42 per cent, partially successful in 25 per cent, and unsuccessful in 32 per cent of the 93 patients compared with none, 10 per cent, and 88 percent respectively in the 42 patients treated with heparin. Streptokinase was more effective when the thrombus was in proximal rather than calf veins. Thrombi of more than six days old were readily lysed. Plasma fibrinogen levels were below 0-8 g/1 (80 mg/100 ml) in nearly all patients successfully treated. The incidence of pulmonary embolism was no greater with streptokinase than with heparin treatment. Only prolonged follow-up would show whether thrombolytic treatment would be effective in preventing late complications of deep vein thrombosis such as chronic venous insufficiency.     

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.     

Specificity role of the streptokinase C-terminal domain in plasminogen activation.

Several pathogenic bacteria secrete plasminogen activator proteins. Streptokinase (SKe) produced by Streptococcus equisimilis and staphylokinase secreted from Staphylococcus aureus are human plasminogen activators and streptokinase (SKu), produced by Streptococcus uberis, is a bovine plasminogen activator. Thus, the fusion proteins among these activators can explain the function of each domain of SKe. Replacement of the SKalpha domain with staphylokinase donated the staphylokinase-like activation activity to SKe, and the SKbetagamma domain played a role of nonproteolytic activation of plasminogen. Recombinant SKu also activated human plasminogen by staphylokinase-like activation mode. Because SKu has homology with SKe, the bovine plasminogen activation activities of SKe fragments were checked. SKebetagamma among them had activation activity with bovine plasminogen. This means that the C-terminal domain (gamma-domain) of streptokinase determines plasminogen species necessary for activation and converses the ability of substrate recognition to human species.     

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.     

The streptokinase gene

The subject of this paper is the molecular cloning, nucleotide sequencing, and expression in heterologous hosts of the streptokinase gene (skc) from the group C streptococcal strain H46A. The skc gene shows no extended regions homologous to the staphylokinase gene.     

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.     

Limited proteolysis of streptokinase and properties of some fragments.

Limited proteolysis of streptokinase (Sk) by trypsin and thermolysin was performed under various incubation conditions and analysed by polyacrylamide gel electrophoresis. Several fragments (Sk1, Tr27, Tr17, Th26, and Th16) were isolated and characterized further. The N-terminal sequences of Tr27, Tr17, Th26, Th16 and the C-terminal sequences of Tr27 and Th26 were determined by partial sequencing. The evidence available allows the positioning of these fragments within the Sk sequence. Fragment Sk1 is obtained by carefully standardized tryptic digestion of Sk and gel chromatography under non-denaturing conditions. Sk1 is formed by a large polypeptide Ser60-Lys293 and non-covalently bonded smaller polypeptides composed of amino acids from the N-terminal region Ile1-Lys59 of Sk. Fragment Tr27 consists of the large polypeptide Ser60-Lys293 of Sk1, and can be obtained from Sk1 by removal of the smaller N-terminal polypeptides under denaturing conditions. Fragment Th26 is composed of amino acids Phe63-His291. The N-termini of fragments Tr17 and Th16 start with Glu148 and Ile151. From their electrophoretically-determined sizes it can be concluded that they most probably have the same C-terminal amino acids, Lys293 and His291, as fragments Tr27 and Th26, respectively. Secondary structure elements of similar composition were found in all the fragments studied using circular dichroism (c.d.) and infrared (i.r.) measurements. Differential scanning calorimetric (d.s.c.) measurements were performed in order to correlate the sequence regions of Sk to energetic folding units of the protein. Fragments Sk1, Tr27, Th26, Tr17, and Th16 show one melting peak in the temperature range from 42.8 to 46.1 degrees C (thermal unfolding stage). For fragment Sk1, this melting peak can be separated by deconvolution into two transitions at T1 = 46.1 degree C and T2 = 47.3 degrees C with delta H1 = 450 kJ/mol and delta H2 = 219 kJ/mol, respectively. Fragments Tr17 and Th16 show one two-state transition at T = 42.8 degrees C with delta H = 326 kJ/mol.     

Conformational properties of streptokinase

The conformational properties of streptokinase (SK) have been assessed by the techniques of differential scanning calorimetry, circular dichroism (CD), and through a combinational approach employing several algorithms which are predictive of secondary structural characteristics. In low ionic strength buffers, SK undergoes a reversible two-state thermal transition with a temperature of maximum heat capacity (Tm) of 46.1 +/- 0.9, a delta Hcal of 98 +/- 11 kcal/mol and a delta Hcal/delta HvH of approximately 1. In high ionic strength buffers, similar calorimetric properties were obtained with the exception that the delta Hcal/delta HvH values were considerably less than 1, indicating the existence of an additional irreversible thermally induced alteration in the molecule, most likely resulting in its aggregation. The effect of pH on the thermal unfolding properties of SK was determined. The results demonstrated that single two-state thermal transitions were obtained, with progressively decreasing Tm values, as the pH was reduced from 6.4 to 3.4, indicating a destabilization of the entire molecule at reduced pH. In the alkaline region, between pH 8.4 and 9.4, stabilization of a separate region of the molecule was obtained, as evidenced by an increase in the delta Hcal/delta HvH to values approximating 2. CD analysis was performed in order to estimate secondary structural characteristics of SK. The best fit of secondary structural parameters to the experimental CD spectrum provided estimates of 17% helices, 28% beta-sheet, 21% beta-turns, and 34% disordered structures. Both the intensity of the spectral band at 208 nm and the level of antiparallel beta-sheet strongly suggest that SK is an alpha + beta protein.