An immunogenic region within residues Val1670-Glu1684 of the factor VIII light chain induces antibodies which inhibit binding of factor VIII to von Willebrand factor

We have identified a monoclonal anti-factor VIII (FVIII) antibody, C4, which inhibits the binding of purified human FVIII to purified human von Willebrand factor (vWF). Both whole immunoglobulin C4 and its Fab fragment demonstrated dose-dependent inhibition of FVIII binding to vWF immobilized on the surface of polystyrene beads. Synthetic peptides based on the amino acid sequence of FVIII were tested for the ability to block the binding of C4 to FVIII in an enzyme-linked immunosorbent assay system. A single synthetic FVIII pentadecapeptide, consisting of residues Val1670-Glu1684, was able to inhibit C4 binding to FVIII. Under the conditions used, the Val1670-Glu1684 peptide demonstrated total inhibition of C4 binding at a concentration of 1 microM. Synthetic FVIII peptides flanking and overlapping the Val1670-Glu1684 peptide had no significant inhibitory activity on C4 binding in concentrations up to 100 microM. A polyclonal antibody made to the Val1670-Glu1684 peptide also demonstrated inhibition of FVIII binding to vWF. Polyclonal antibodies made to synthetic FVIII peptides flanking and partially overlapping the Val1670-Glu1684 sequence did not demonstrate such inhibition. Localization of the binding region of the monoclonal anti-FVIII antibody C4 to residues Val1670-Glu1684 suggests that this site is at, or near, a major vWF binding domain of FVIII.     

Animal experimental studies on fibrinolysis with streptokinase]

In contrast to rabbit blood plasma, in guinea pig and rat blood plasma activation of fibrinolysis by streptokinase is achieved after addition of human plasminogen or human plasma only. A simple experimental procedure for testing application forms of streptokinase in rats is described. Fibrinolysis in vivo is more effective after subsequent administration of human plasma and streptokinase in rats than after administration of a mixture of human plasma and streptokinase (activator).     

Molecular processes in the streptokinase thrombolytic therapy

The aim of this research is to evaluate the current streptokinase thrombolytic treatment and to identify or improve new techniques that will base new approaches with a higher efficiency in this area of expertise. In order to be as realistic as possible a new method was set up using magnetic vectorized nanoparticles streptokinase and human blood thrombus. The experimental data confirm the maximum 83% thrombus lyses whenever increase streptokinase concentration. It is very probable to happen because of the presence of high concentration of antiplasmin in the blood that neutralizes around half of the thrombolytic potential of the sanguine plasminogen. The experiment shows also that only free serum plasminogen are available for streptokinase action in order to generate plasmin.     

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.     

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.     

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.     

Plasminogen binding with decapeptide and polypeptide fragments of streptokinase

The plasminogen binding with streptokinase decapeptides, modeling the primary structure of molecule, and chymotryptic fragments of streptokinase have been investigated. The immunoenzymatic assay has shown that plasminogen binds to all streptokinase fragments with the decreasing affinity in the set of fragments: 36 > 30 > 17 > 7 > 11 kDa. Location of the binding sites in streptokinase primary structure was performed using the immobilized decapeptides on plastic pins adopted to IEA. In the presence of 10 mM 6-aminohexanoic acid 11 sites for human Glu- and mini-plasminogens, pig and bovine plasminogens binding have been found. They were of the same location for human, bovine and pig plasminogens. 3 sites were located in plasminogen alpha-domain--T43-A72, N113-T126, Q133-V158, 5 sites in beta-domain--T163-L188, A203-S222, Q239-I264, Y275-L294, T315-L340, and 3 sites in gamma-domain--T361-R362, N377-E392, T397-N410. Participation of linear part of streptokinase polypeptide chain in plasminogen--streptokinase complex formation is suggested.     

Plasminogen activation by antiplasminogen monoclonal antibody IV-1C. Properties and mechanism of reaction

In review the results of investigation of plasminogen(Pg) activation by antiplasminogen monoclonal antibody IV-1c have been presented. Antigenic determinant of IV-1c was localized in Val709-Gly718 site of Pg protease domain. IV-1c completely inhibited the Pg activation by streptokinase, but increased the rate of Pg activation by t-PA and urokinase. Catalytic properties of plasmin in complex with IV-1c were studied. It was found that IV-1c induced catalytic activity in Pg-IV-1c complex. It was shown that Pg and IV-1c interacts in complex by two-centre mechanism: IV-1c binds with Pg by paratope and by N-terminal lysine of gamma-chain and Pg binds to IV-1c by one of the lysine binding sites and by V709-G718 site of protease domain. The influence of pH, temperature, 1.5 mM Ca2+, Mg2+, Sr2+, Ba2+, Co2+, Ni2+ cations and 10 mM Cl-, F-, Ac-, SO4(2-), HPO4(2-) anions on lag and fast phases of Pg activation by VI-1c was investigated. It was revealed that Val709-Gly718 site was determining in Pg activation by IV-1c and streptokinase.     

Species specificity of streptokinase

Streptokinase, a bacterial protein, forms a complex with human plasminogen which results in a conformational change in the plasminogen molecule and the exposure of an active center. The plasminogen-streptokinase complex is an activator of plasminogen and is rapidly converted to a plasmin-streptokinase complex which, in the human, is also an activator of plasminogen. Species differences have been found in the reaction of streptokinase with plasminogen varying from no active complex formation at one extreme to the rapid formation of an active activator complex at the other, with resultant differences in rates of complex formation and the yield of plasmin. Explanation of these species differences at a molecular level are discussed as well as the possible application of complex formation in a variety of biological systems as a mechanism to produce variation in enzyme activities in proportion to the concentration of substrate available.     

Chimerism reveals a role for the streptokinase Beta -domain in nonproteolytic active site formation,substrate, and inhibitor interactions

Streptokinase (SK) and staphylokinase form cofactor-enzyme complexes that promote the degradation of fibrin thrombi by activating human plasminogen. The unique abilities of streptokinase to nonproteolytically activate plasminogen or to alter the interactions of plasmin with substrates and inhibitors may be the result of high affinity binding mediated by the streptokinase beta-domain. To examine this hypothesis, a chimeric streptokinase, SKbetaswap, was created by swapping the SK beta-domain with the homologous beta-domain of Streptococcus uberis Pg activator (SUPA or PauA, SK uberis), a streptokinase that cannot activate human plasminogen. SKbetaswap formed a tight complex with microplasminogen with an affinity comparable with streptokinase. The SKbetaswap-plasmin complex also activated human plasminogen with catalytic efficiencies (k(cat)/K(m) = 16.8 versus 15.2 microm(-1) min(-1)) comparable with streptokinase. However, SKbetaswap was incapable of nonproteolytic active site generation and activated plasminogen by a staphylokinase mechanism. When compared with streptokinase complexes, SKbetaswap-plasmin and SKbetaswap-microplasmin complexes had altered affinities for low molecular weight substrates. The SKbetaswap-plasmin complex also was less resistant than the streptokinase-plasmin complex to inhibition by alpha(2)-antiplasmin and was readily inhibited by soybean trypsin inhibitor. Thus, in addition to mediating high affinity binding to plasmin(ogen), the streptokinase beta-domain is required for nonproteolytic active site generation and specifically modulates the interactions of the complex with substrates and inhibitors.     

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.     

Site-specific alteration of Gly-24 in streptokinase: its effect on plasminogen activation

Oligonucleotide-directed mutagenesis was carried out to replace glycine-24 of streptokinase with histidine, glutamic acid, or alanine. Substitutions with either histidine or glutamic acid resulted in almost complete loss of streptokinase activity but streptokinase replaced with alanine retained its activity. Although streptokinases with histidine-24 or glutamic acid-24 bound normally to human plasminogen, they were not able to generate active plasmin, whereas those with alanine-24 or glycine-24 (wild-type) could generate active plasmin. The results indicate that the small, uncharged alkyl group side-chain on the 24th amino acid residue of streptokinase is indispensable for the activity of the human plasminogen-streptokinase complex.     

Process of platelets activation by streptokinase

This study concerns the influence of streptokinase and antistreptokinase antibodies on rabbits platelets in blood plasma depleted of plasminogen. The immune complex streptokinase-antibody causes platelets activation, whereas other investigated immune complexes didnot express such activity. Platelets aggregation wasnot detected in any case. It was determined that streptokinase induces platelets activation in the rabbit plasma with high titre of antistreptokinase antibodies in absence of plasminogen.     

Integration of human plasminogen or streptokinase into stable complexes with oxidoreductases and pyruvate kinase

Summary The formation of stable equimolar complexes of streptokinase or plasminogen with muscle lactate dehydrogenase or pyruvate kinase, heart mitochondrial malate dehydrogenase and hepatic catalase at pH 7.4, 3.0 and 10.0 was first detected by differential spectroscopy methods. All complexes, except those of plasminogen with dehydrogenases, were resistant to 6 M urea. Judging from circular dichroism spectra, tertiary and secondary structures were considerably changed in the complexes. These changes were significantly dependent upon the nature of interacting proteins; in some cases their structures were more ordered. NAD (but not NADH) hampered the formation of streptokinase complexes with dehydrogenases. The plasminogen-activating function of streptokinase and the ability of plasminogen to be activated by streptokinase in the complexes with oxidoreductases were essentially unchanged. Pyruvate kinase induced a moderate (by 35%) increase in the streptokinase activating function. It is assumed that the formation of complexes of streptokinase or plasminogen with enzymes may serve as a link in metabolic regulation and/or intercellular interactions.     

The mechanism of a bacterial plasminogen activator intermediate between streptokinase and staphylokinase

The therapeutic properties of plasminogen activators are dictated by their mechanism of action. Unlike staphylokinase, a single domain protein, streptokinase, a 3-domain (alpha, beta, and gamma) molecule, nonproteolytically activates human (h)-plasminogen and protects plasmin from inactivation by alpha(2)-antiplasmin. Because a streptokinase-like mechanism was hypothesized to require the streptokinase gamma-domain, we examined the mechanism of action of a novel two-domain (alpha,beta) Streptococcus uberis plasminogen activator (SUPA). Under conditions that quench trace plasmin, SUPA nonproteolytically generated an active site in bovine (b)-plasminogen. SUPA also competitively inhibited the inactivation of plasmin by alpha(2)-antiplasmin. Still, the lag phase in active site generation and plasminogen activation by SUPA was at least 5-fold longer than that of streptokinase. Recombinant streptokinase gamma-domain bound to the b-plasminogen.SUPA complex and significantly reduced these lag phases. The SUPA-b.plasmin complex activated b-plasminogen with kinetic parameters comparable to those of streptokinase for h-plasminogen. The SUPA-b.plasmin complex also activated h-plasminogen but with a lower k(cat) (25-fold) and k(cat)/K(m) (7.9-fold) than SK. We conclude that a gamma-domain is not required for a streptokinase-like activation of b-plasminogen. However, the streptokinase gamma-domain enhances the rates of active site formation in b-plasminogen and this enhancing effect may be required for efficient activation of plasminogen from other species.     

Modification of functional groups of the streptokinase molecule during photooxidation

Photochemical oxidation with methylene blue as photosensitizer results in the destruction of one histidine residue in the streptokinase molecule. This process is characterized by the rate constant corresponding to the modification of free L-histidine and results in partial inactivation of the protein. The rate of protein photo oxidation and photoinactivation is pH-dependent. As can be judged from the results of CD spectroscopy and gel chromatography, in weakly acidic (but not in weakly alkaline) media the reaction results in conformation changes of the streptokinase globule which affect the state of the protein tryptophanyl residue. It was found that the imidazole group destroyed during the photooxidation reaction is not essential either for the specific activity of streptokinase or for the formation of is stable complex with human plasminogen. The specificity of modification of the streptokinase histidine residue during the photooxidation reaction is discussed.     

Identification through combinatorial random and rational mutagenesis of a substrate-interacting exosite in the gamma domain of streptokinase

To identify new structure-function correlations in the γ domain of streptokinase, mutants were generated by error-prone random mutagenesis of the γ domain and its adjoining region in the β domain followed by functional screening specifically for substrate plasminogen activation. Single-site mutants derived from various multipoint mutation clusters identified the importance of discrete residues in the γ domain that are important for substrate processing. Among the various residues, aspartate at position 328 was identified as critical for substrate human plasminogen activation through extensive mutagenesis of its side chain, namely D328R, D328H, D328N, and D328A. Other mutants found to be important in substrate plasminogen activation were, namely, R319H, N339S, K334A, K334E, and L335Q. When examined for their 1:1 interaction with human plasmin, these mutants were found to retain the native-like high affinity for plasmin and also to generate amidolytic activity with partner plasminogen in a manner similar to wild type streptokinase. Moreover, cofactor activities of the mutants precomplexed with plasmin against microplasminogen as the substrate as well as in silico modeling studies suggested that the region 315-340 of the γ domain interacts with the serine protease domain of the macromolecular substrate. Overall, our results identify the presence of a substrate specific exosite in the γ domain of streptokinase.     

The interaction of streptokinase.plasminogen activator complex, tissue-type plasminogen activator, urokinase and their acylated derivatives with fibrin and cyanogen bromide digest of fibrinogen. Relationship to fibrinolytic potency in vitro.

The effects of purified soluble fibrin and of fibrinogen fragments (fibrin mimic) on the activation of Lys-plasminogen (i.e. plasminogen residues 77-790) to plasmin by streptokinase.plasminogen activator complex and by tissue-type plasminogen activator were studied. Dissociation constants of both activators were estimated to lie in the range 90-160 nM (fibrin) and 16-60 nM (CNBr-cleavage fragments of fibrinogen). The kinetic mechanism for both types of activator comprised non-essential enzyme activation via a Rapid Equilibrium Ordered Bireactant sequence. In order to relate the fibrin affinity of plasminogen activators to their fibrinolytic potency, the rate of lysis of supported human plasma clots formed in the presence of unmodified or active-centre-acylated precursors of plasminogen activators was studied as a function of the concentration of enzyme derivative. The concentrations of unmodified enzyme giving 50% lysis/h in this assay were 0.9, 2.0 and 11.0 nM for tissue-type plasminogen activator, streptokinase.plasmin(ogen) and urokinase respectively. However, the potencies of active-centre-acylated derivatives of these enzymes suggested that acylated-tissue plasminogen activator and streptokinase.plasminogen complexes of comparable hydrolytic stability were of comparable potency. Both types of acyl-enzyme were significantly more potent than acyl-urokinases.     

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)     

Integration of human plasminogen or streptokinase into stable complexes with oxidoreductases and pyruvate kinase

The formation of stable equimolar complexes ofstreptokinase or plasminogen with muscle lactatedehydrogenase or pyruvate kinase, heart mitochondrialmalate dehydrogenase and hepatic catalase at pH 7.4,3.0 and 10.0 was first detected by differentialspectroscopy methods. All complexes, except those ofplasminogen with dehydrogenases, were resistant to 6 Murea. Judging from circulardichroism spectra, tertiary and secondary structureswere considerably changed in the complexes. Thesechanges were significantly dependent upon the natureof interacting proteins; in some cases theirstructures were more ordered. NAD (but not NADH)hampered the formation of streptokinase complexes withdehydrogenases. The plasminogen–activating function ofstreptokinase and the ability of plasminogen to beactivated by streptokinase in the complexes withoxidoreductases were essentially unchanged.Pyruvate kinase induced a moderate (by 35%) increasein the streptokinase activating function. It isassumed that the formation of complexes ofstreptokinase or plasminogen with enzymes may serve asa link in metabolic regulation and/or intercellular interactions.