Structural consideration of the formation of the activation complex between the staphylokinase-like streptococcal plasminogen activator PadA and bovine plasminogen

The characteristics of a streptococcal plasminogen activator (PA) displaying specificity for ruminant plasminogen (Plg) were defined using molecular approaches. The 16-kDa secreted protein PadA was found to be prevalent in Streptococcus dysgalactiae subspecies dysgalactiae isolated from cases of bovine mastitis and septic arthritis in lambs. PadA was able to activate bovine, ovine and caprine Plg, but not human Plg. Amino acid sequence analysis identified a limited level of homology to other streptococcal PAs, including streptokinase; however, PadA was found to align well with and match in size the staphylococcal PA, staphylokinase. Recombinant PadA was used to investigate interaction with bovine Plg, leading to formation of an activator complex that was capable of recruiting and converting further substrate Plg into plasmin. Individual non-overlapping peptides of PadA or bovine microplasminogen were found to block the interaction between PadA and bovine Plg, preventing the formation of the activation complex. Homology modelling based upon structures of staphylokinase complexed with human microplasminogen supported these findings by placing critical residues in close proximity to the plasmin component of the activation complex.     

Staphylococcus aureus: Staphylokinase

Staphylokinase is a 136 aa long bacteriophage encoded protein expressed by lysogenic strains of Staphylococcus aureus. Present understanding of the role of staphylokinase during bacterial infection is based on its interaction with the host proteins, alpha-defensins and plasminogen. alpha-Defensins are bactericidal peptides originating from human neutrophils. Binding of staphylokinase to alpha-defensins abolishes their bactericidal properties, which makes staphylokinase a vital tool for staphylococcal resistance to host innate immunity. Complex binding between staphylokinase and plasminogen results in the formation of active plasmin, a broad-spectrum proteolytic enzyme facilitating bacterial penetration into the surrounding tissues. We have recently shown high levels of staphylokinase expression in clinical isolates of skin and mucosal origin and relative low levels in isolates invading internal organs. These findings are supported by sepsis studies using isogenic S. aureus strains demonstrating increased bacterial load in the absence of staphylokinase production. Our observations indicate that staphylokinase favours symbiosis of staphylococci with the host that makes it an important colonization factor.     

Molecular mechanisms of plasminogen activation: bacterial cofactors provide clues

Plasminogen activation is a key event in the fibrinolytic system that results in the dissolution of blood clots, and also promotes cell migration and tissue remodelling. The recent structure determinations of microplasmin in complex with the bacterial plasminogen activators staphylokinase and streptokinase have provided novel insights into the molecular mechanisms of plasminogen activation and cofactor function. These bacterial proteins are cofactor molecules that contribute to exosite formation and enhance the substrate presentation to the enzyme. At the same time, they modulate the specificity of plasmin towards substrates and inhibitors, making a 'specificity switch' possible.     

A chromogenic assay for the detection of plasmin generated by plasminogen activator immobilized on nitrocellulose using a para-nitroanilide synthetic peptide substrate

A direct solid phase chromogenic assay has been developed for the detection of plasmin (EC 3.4.21.7), generated by the interaction of a nitrocellulose-bound plasminogen activator, using the plasmin specific tripeptide substrate, H-D-valyl-leucyl-lysine - p-nitroaniline. para-Nitroaniline released by the cleavage of the lysine - p-nitroaniline bound by plasmin was derivatized to its diazonium salt and subsequently coupled to N-1-napthylethylenediamine in situ to form a diazoamino of an intense red color at the site of the plasminogen activator. This method was used to assay for the streptococcal plasminogen activator, streptokinase, not only in crude bacterial supernatants, but also to detect streptokinase secreted by individual bacterial colonies. In addition, this solid phase assay was used to identify monoclonal antibodies specific for streptokinase which could inhibit the activation of human plasminogen by streptokinase. This method also permitted simultaneous immunological and biochemical identification of the plasminogen activator, thus permitting unequivocal comparative observations. This assay is quantitative and sensitive to nanogram amounts of activator comparable to those obtained with soluble assays. This method may also be applicable for the detection of other plasminogen activators, such as tissue plasminogen activator, urokinase, and staphylokinase, and also for the detection of immobilized proteases which can cleave other substrates derivatized with p-nitroaniline. The reagents used in this assay are inexpensive and easy to prepare.     

Bacterial plasminogen activators and receptors

Invasive bacterial pathogens intervene at various stages and by various mechanisms with the mammalian plasminogen/plasmin system. A vast number of pathogens express plasmin(ogen) receptors that immobilize plasmin(ogen) on the bacterial surface, an event that enhances activation of plasminogen by mammalian plasminogen activators. Bacteria also influence secretion of plasminogen activators and their inhibitors from mammalian cells. The prokaryotic plasminogen activators streptokinase and staphylokinase form a complex with plasmin(ogen) and thus enhance plasminogen activation. The Pla surface protease of Yersinia pestis resembles mammalian activators in function and converts plasminogen to plasmin by limited proteolysis. In essence, plasminogen receptors and activators turn bacteria into proteolytic organisms using a host-derived system. In Gram-negative bacteria, the filamentous surface appendages fimbriae and flagella form a major group of plasminogen receptors. In Gram-positive bacteria, surface-bound enzyme molecules as well as M-protein-related structures have been identified as plasminogen receptors, the former receptor type also occurs on mammalian cells. Plasmin is a broad-spectrum serine protease that degrades fibrin and noncollagenous proteins of extracellular matrices and activates latent procollagenases. Consequently, plasmin generated on or activated by Haemophilus influenzae, Salmonella typhimurium, Streptococcus pneumoniae, Y. pestis, and Borrelia burgdorferi has been shown to degrade mammalian extracellular matrices. In a few instances plasminogen activation has been shown to enhance bacterial metastasis in vitro through reconstituted basement membrane or epithelial cell monolayers. In vivo evidence for a role of plasminogen activation in pathogenesis is limited to Y. pestis, Borrelia, and group A streptococci. Bacterial proteases may also directly activate latent procollagenases or inactivate protease inhibitors of human plasma, and thus contribute to tissue damage and bacterial spread across tissue barriers.     

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.     

Specificity of thrombin: I. Esterolytic properties of thrombin,plasma, trypsin,and chymotrypsin with Nβ-substituted guanidino derivatives of p-nitrophenyl-p′-guanidinobenzoate

Thrombin exhibits a restricted specificity, relative to plasmin, trypsin, and chymotrypsin, for a series of derivatives of the titrant substrate p-nitrophenyl-p′-guanidinobenzoate (NPGB). Substitution on the beta guanidino nitrogen of NPGB with an n-butyl, n-hexyl, cyclo-hexyl, or benzyl residue does not prevent the esterolytic cleavage of these derivatives but does markedly alter their substrate properties with the four enzymes investigated.All four enzymes cleave NPGB at equivalent concentrations by releasing p-nitrophenol as pre-steady-state burst reactions followed by its steady-state production. Both chymotrypsin and trypsin similarly display burst reactions with the derivatives at corresponding concentrations. The acyl-enzyme intermediates formed with chymotrypsin, however, are more stable for the derivatives than for NPGB, and those formed with trypsin are less stable. In contrast, plasmin and thrombin exhibit incomplete burst reactions with the derivatives at these concentrations. Except for the cyclo-hexyl derivative, with which plasmin does not react, the derivatives relative to NPGB were cleaved faster by plasmin than by thrombin. These cleavages with thrombin, moreover, were competitively inhibited by benzamidine. Kinetic data obtained for thrombin further indicated that the substituent groups of derivatives hindered the initial formation of enzyme-substrate complexes. These results suggest that thrombin and, most likely, plasmin have restricted primary binding-site regions for small molecule substrates which do not readily accommodate bulky substituent groups.In addition, increasing concentrations of glycerol were found to greatly alter the esterolytic properties of thrombin for the compounds studied. This effect was demonstrated by increased deacylation rates with NPGB and by decreased cleavage rates with the n-butyl derivative.     

alpha Domain deletion converts streptokinase into a fibrin-dependent plasminogen activator through mechanisms akin to staphylokinase and tissue plasminogen activator

The mechanism of action of plasminogen (Pg) activators may affect their therapeutic properties in humans. Streptokinase (SK) is a robust Pg activator in physiologic fluids in the absence of fibrin. Deletion of a "catalytic switch" (SK residues 1-59), alters the conformation of the SK alpha domain and converts SKDelta59 into a fibrin-dependent Pg activator through unknown mechanisms. We show that the SK alpha domain binds avidly to the Pg kringle domains that maintain Glu-Pg in a tightly folded conformation. By virtue of deletion of SK residues 1-59, SKDelta59 loses the ability to unfold Glu-Pg during complex formation and becomes incapable of nonproteolytic active site formation. In this manner, SKDelta59 behaves more like staphylokinase than like SK; it requires plasmin to form a functional activator complex, and in this complex SKDelta59 does not protect plasmin from inhibition by alpha(2)-antiplasmin. At the same time, SKDelta59 is unlike staphylokinase or SK and is more like tissue Pg activator, because it is a poor activator of the tightly folded form of Glu-Pg in physiologic solutions. SKDelta59 can only activate Glu-Pg when it was unfolded by fibrin interactions or by Cl(-)-deficient buffers. Taken together, these studies indicate that an intact alpha domain confers on SK the ability to nonproteolytically activate Glu-Pg, to unfold and process Glu-Pg substrate in physiologic solutions, and to alter the substrate-inhibitor interactions of plasmin in the activator complex. The loss of an intact alpha domain makes SKDelta59 activate Pg through classical "fibrin-dependent mechanisms" (akin to both staphylokinase and tissue Pg activator) that include: 1) a marked preference for a fibrin-bound or unfolded Glu-Pg substrate, 2) a requirement for plasmin in the activator complex, and 3) the creation of an activator complex with plasmin that is readily inhibited by alpha(2)-antiplasmin.     

Kinetic and structural characterization of a two-domain streptokinase: dissection of domain functionality

The mammalian protease plasminogen can be activated by bacterial activators, the three-domain (alpha, beta, gamma) streptokinases and the one-domain (alpha) staphylokinases. These activators act as plasmin(ogen) cofactors, and the resulting complexes initiate proteolytic activity of host plasminogen which facilitates bacterial colonization of the host organism. We have investigated the kinetic mechanism of the plasminogen activation mediated by a novel two-domain (alpha, beta) streptokinase isolated from Streptococcus uberis (Sk(U)) with specificity toward bovine plasminogen. The interaction between Sk(U) and plasminogen occurred in two steps: (1) rapid association of the proteins and (2) slow transition to the active complex Sk(U)-PgA. The complex Sk(U)-PgA converted plasminogen to plasmin with the following parameters: K(m) < or = 1.5 microM and k(cat) = 0.55 s(-)(1). The ability of proteolytic fragments of Sk(U) to activate plasminogen was investigated. Only two C-terminal segments (97-261 and 123-261), which both contain the beta-domain (126-261), were shown to be active. They initiated plasminogen activation in complex with plasmin, but not with plasminogen, and thereby exhibited functional similarity to the staphylokinase. The fusion protein His(6)-Sk(U) (i.e., Sk(U) with a small N-terminal tag) acted exclusively in complex with plasmin as well. These observations demonstrate that (1) the N-terminal alpha-domain, including a native N-terminus, was necessary for "virgin" activation of the associated plasminogen in the Sk(U)-PgA complex and (2) the C-terminal beta-domain of Sk(U) is important for recognition of the substrate in the Sk(U)-PgA complex.     

Activation of plasminogen by pro-urokinase. II. Kinetics

The kinetics of the activation of plasminogen by recombinant pro-urokinase obtained by expression of human urokinase cDNA in Escherichia coli was studied. The conversion of pro-urokinase (U) and plasminogen (P) to urokinase (u) and plasmin (p) is represented by a sequence of three reactions which each obey Michaelis-Menten kinetics, i.e. (Formula: see text). In this model, pro-urokinase formally behaves as an enzyme in Reaction I and as a substrate in reaction II. The experimentally measured overall rates of formation of urokinase and plasmin are in good agreement with those calculated from the kinetic parameters and the initial concentrations of pro-urokinase and plasminogen, confirming the validity of the model. It appears that recombinant pro-urokinase is an equally potent activator of plasminogen (k2/Km = 0.05 microM-1 s-1), as in urokinase (k"2/K"m = 0.02 microM-1 s-1). This is due to the fact that the proenzyme, which is virtually inactive toward low Mr substrates for urokinase, forms an intermediate of the Michaelis-Menten type with plasminogen, with a much higher affinity than that of the active enzyme with its substrate. This is an exceptional phenomenon among the serine proteases.     

Mechanism of action of θ-amino acids on plasminogen activation and fibrinolysis induced by staphylokinase

Stimulation of Lys-plasminogen (Lys-Pg) and Glu-plasminogen (Glu-Pg) activation under the action of staphylokinase and Glu-Pg activation under the action of preformed plasmin-staphylokinase activator complex (Pm-STA) by low concentrations and inhibition by high concentrations of omega-amino acids (>90-140 mM) were found. Maximal stimulation of the activation was observed at concentrations of L-lysine, 6-aminohexanoic acid (6-AHA), and trans-(4-aminomethyl)cyclohexanecarboxylic acid 8.0, 2.0, and 0.8 mM, respectively. In contrast, the Lys-Pg activation rate by Pm-STA complex sharply decreased when concentrations of omega-amino acids exceeded the above-mentioned values. It was found that formation of Pm-STA complex from a mixture of equimolar concentrations of staphylokinase and Glu-Pg or Lys-Pg is stimulated by low concentrations (maximal at 10 mM) of 6-AHA. Negligible increase in the specific activities of plasmin and Pm-STA complex was detected at higher concentrations of 6-AHA (to maximal at 70 and 50 mM, respectively). Inhibitory effects of omega-amino acids on the rate of fibrinolysis induced by staphylokinase, Pm-STA complex, and plasmin were compared. It was found that inhibition of staphylokinase-induced fibrinolysis by omega-amino acids includes blocking of the reactions of Pm-STA complex formation, plasminogen activation by this complex, and lysis of fibrin by forming plasmin as a result of displacement of plasminogen and plasmin from the fibrin surface. Thus, the slow stage of Pm-STA complex formation plays an important role in the mechanism of action of omega-amino acids on Glu-Pg activation and fibrinolysis induced by staphylokinase. In addition to alpha-->beta change of Glu-Pg conformation, stimulation of Pm-STA complex formation leads to increase in Glu-Pg activation rate in the presence of low concentrations of omega-amino acids. Inhibition of Pm-STA complex formation on fibrin surface by omega-amino acids is responsible for appearance of long lag phases on curves of fibrinolysis induced by staphylokinase.     

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.     

Binding of plasminogen to extracellular matrix

We have previously demonstrated that plasminogen immobilized on various surfaces forms a substrate for efficient conversion to plasmin by tissue plasminogen activator (t-PA) (Silverstein, R. L., Nachman, R. L., Leung, L. L. K., and Harpel, R. C. (1985) J. Biol. Chem. 260, 10346-10352). We now report the binding of human plasminogen to the extracellular matrix synthesized in vitro by cultured endothelial cell monolayers. The binding was specific, saturable at plasma plasminogen concentrations, reversible, and lysine-binding site-dependent. Functional studies demonstrated that matrix immobilized plasminogen was a much better substrate for t-PA than was fluid phase plasminogen as shown by a 100-fold decrease in Km. Activation of plasminogen by t-PA and urokinase on the matrix was equally efficient. The plasmin generated on the matrix, in marked contrast to fluid phase, was protected from its fast-acting inhibitor, alpha 2-plasmin inhibitor. Matrix-associated plasmin converted bound Glu- into Lys-plasminogen, which in turn is more rapidly activated to plasmin by t-PA. The extracellular matrix not only binds and localizes plasminogen but also improves plasminogen activation kinetics and prolongs plasmin activity in the subendothelial microenvironment.     

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.     

Photoaffinity labeling of functionally different lysine-binding sites in human plasminogen and plasmin

Photoaffinity labeling of human plasmin using 4-azidobenzoylglycyl-L-lysine inhibits clot lysis activity, while the activity toward the active-site titrant, p-nitrophenyl-p'-guanidinobenzoate, or alpha-casein are maintained. Photoaffinity labeling of native Glu-plasminogen with the same reagent causes incorporation of approximately 1.5 mol label per mol plasminogen. This labeled plasminogen can be activated to plasmin by either urokinase or streptokinase. The resulting plasmin has full clot lysis activity and can be subsequently photoaffinity labeled with a loss of clot lysis activity. The rate of activation of labeled plasminogen by urokinase is increased relative to that of native plasminogen. epsilon-Aminocaproic acid blocks incorporation of photoaffinity label into both plasminogen and plasmin, indicating that the labeling is specific to the lysine-binding sites. The labels are located in the kringle 1+2+3 fragment in either photoaffinity-labeled plasminogen or plasmin. These results indicate that the specific lysine-binding site blocked in plasmin acts in concert with the active-site in binding and using fibrin as a substrate. This clot lysis regulating site is not available for labeling in plasminogen, but is exposed or changed upon activation to plasmin. The different lysine-binding sites labeled in plasminogen may regulate the conformation of the molecule as evidence by an enhanced rate of activation to plasmin.     

Homocysteine and its thiolactone impair plasmin activity induced by urokinase or streptokinase in vitro

Mechanisms of homocysteine (Hcy) contribution to thrombosis are complex and only partly recognized. The available data suggest that the prothrombotic activity of homocysteine may be not only a result of the changes in coagulation process and endothelial dysfunction, but also the dysfunction of fibrinolysis. The aim of the present work was to assess the effects of homocysteine (10-100 μM mM) and its thiolactone (HTL, 0.1-1 μM) on plasminogen and plasmin functions in vitro. The amidolytic activity of generated plasmin in Hcy or HTL-treated plasminogen and plasma samples was measured by the hydrolysis of chromogenic substrate. Effects of Hcy and HTL on proteolytic activity of plasmin were monitored electrophoretically, by using of fibrinogen as a substrate. The exposure of human plasma and purified plasminogen to Hcy or HTL resulted in the decrease of urokinase-induced plasmin activity. In plasminogen samples treated with the highest concentration of homocysteine (100 μM) or thiolactone (1 μM), the activity of plasmin was inhibited by about 50%. In plasma samples, a reduction of amidolytic activity by about 30% (for 100 μM Hcy) and 40% (for 1 μM HTL), was observed. Both Hcy and HTL were also able to diminish the streptokinase-induced proteolytic activity of plasmin. In conclusion, the results obtained in this study demonstrate that Hcy and HTL may affect fibrinolytic properties of plasminogen and plasma, leading to the decrease of plasmin activity.     

Interference of active site specific reagents in plasminogen-streptokinase active site formation

We have recently observed slow, non-Michaelis-Menten kinetics of activation of native cat plasminogen by catalytic concentrations of streptokinase. In order to understand the reasons for this phenomenon, we undertook to study the formation of the plasminogen-streptokinase activator complex under the same plasminogen activation conditions. The results obtained in this study show that the potential active site in both cat and human plasminogen is capable of binding strongly the specific substrates (S) p-nitrophenyl p-guanidinobenzoate (NPGB) and H-D-valyl-L-leucyl-L-lysyl-p-nitroanilide, through the active site is incapable of hydrolyzing these substrates. Binding studies support these and the following conclusions. Streptokinase binds to this zymogen-substrate complex to create the ternary plasminogen-S-streptokinase complex, which then slowly converts to an acylated plasminogen-streptokinase form. This acylation reaction is 550 times slower than acylation of the preformed plasminogen-streptokinase complex by NPGB. The same reaction also occurs with human plasminogen, though the acylation reaction is 10 times faster than when the cat zymogen is used. NPGB binds specifically to plasminogen but not to streptokinase. These studies proved that inhibition of cat plasminogen activation by streptokinase occurs at the level of activator complex formation. We conclude from our studies that streptokinase binding to both cat and human plasminogen occurs at the potential active site of the zymogen. Consequently, it is probable that plasminogen activation in vivo is inhibited by binding of active site specific inhibitors to plasminogen.     

Activation of pro-urokinase and plasminogen on human sarcoma cells: a proteolytic system with surface-bound reactants

Human HT-1080 fibrosarcoma cells produce urokinase-type plasminogen activator (u-PA) and type 1 plasminogen activator inhibitor (PAI-1). We found that after incubation of monolayer cultures with purified native human plasminogen in serum-containing medium, bound plasmin activity could be eluted from the cells with tranexamic acid, an analogue of lysine. The bound plasmin was the result of plasminogen activation on the cell surface; plasmin activity was not taken up onto cells after deliberate addition of plasmin to the serum-containing medium. The cell surface plasmin formation was inhibited by an anticatalytic monoclonal antibody to u-PA, indicating that this enzyme was responsible for the activation. Preincubation of the cells with diisopropyl fluorophosphate-inhibited u-PA led to a decrease in surface-bound plasmin, indicating that a large part, if not all, of the cell surface plasminogen activation was catalyzed by surface-bound u-PA. In the absence of plasminogen, most of the cell surface u-PA was present in its single-chain proenzyme form, while addition of plasminogen led to formation of cell-bound two-chain u-PA. The latter reaction was catalyzed by cell-bound plasmin. Cell-bound u-PA was accessible to inhibition by endogenous PAI-1 and by added PAI-2, while the cell-bound plasmin was inaccessible to serum inhibitors, but accessible to added aprotinin and an anticatalytic monoclonal antibody. A model for cell surface plasminogen activation is proposed in which plasminogen binding to cells from serum medium is followed by plasminogen activation by trace amounts of bound active u-PA, to form bound plasmin, which in turn serves to produce more active u-PA from bound pro-u-PA. This exponential process is subject to regulation by endogenous PAI-1 and limited to the pericellular space.     

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.     

Plasminogen activator-anti-human fibrinogen conjugate

A covalent conjugate between the plasminogen activator urokinase and polyclonal rabbit anti-human fibrinogen has been formed using the heterobifunctional coupling reagent N-succinimidyl 3-(2-pyridyldithio) propionate. The resultant urokinase-anti-human fibrinogen conjugate was separated from unreacted material by gel filtration. The conjugate exhibited amidase activity against the small chromogenic substrate pyroglutamyl-glycyl-arginine-p-nitroanilide as well as plasminogen activator activity in an assay employing plasminogen and the plasmin substrate D-valyl-leucyl-lysine-p-nitroanilide. Retention of antibody specificity for fibrinogen was demonstrated using an enzyme linked immunoassay procedure. The conjugate was found to have greater stability in human plasma than unconjugated urokinase.