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.     

An elastase-dependent pathway of plasminogen activation

In reaction mixtures containing Glu-plasminogen, alpha 2-antiplasmin, and tissue plasminogen activator or urokinase, either pancreatic or leukocyte elastase enhances the rate of plasminogen activation by 2 or more orders of magnitude. This effect is the consequence of several reactions. (a) In concentrations on the order of 100 nM, elastase degrades plasminogen within 10 min to yield des-kringle1-4-plasminogen (mini-plasminogen), which is 10-fold more efficient than Glu-plasminogen as a substrate for plasminogen activators. Des-kringle1-4-plasminogen is insensitive to cofactor activities of fibrin(ogen) fragments or an endothelial cell cofactor. (b) Des-kringle1-4-plasmin is one-tenth as sensitive as plasmin to inhibition by alpha 2-antiplasmin: k" = 10(6) M-1 s-1 versus 10(7) M-1 s-1. (c) alpha 2-Antiplasmin is disabled efficiently by elastase, with a k" of 20,000 M-1 s-1. The elastase-dependent reactions are not influenced by 6-aminohexanoate. In diluted (10-fold) blood plasma, the capacity of endogenous inhibitors to block plasmin expression is suppressed by 30 microM elastase. It is proposed that elastases provide an alternative pathway for Glu-plasminogen activation and a mechanism for controlling initiation of fibrinolysis by urokinase-type plasminogen activators.     

Streptococcus uberis plasminogen activator (SUPA) activates human plasminogen through novel species-specific and fibrin-targeted mechanisms

Bacterial plasminogen (Pg) activators generate plasmin to degrade fibrin blood clots and other proteins that modulate the pathogenesis of infection, yet despite strong homology between mammalian Pgs, the activity of bacterial Pg activators is thought to be restricted to the Pg of their host mammalian species. Thus, we found that Streptococcus uberis Pg activator (SUPA), isolated from a Streptococcus species that infects cows but not humans, robustly activated bovine but not human Pg in purified systems and in plasma. Consistent with this, SUPA formed a higher avidity complex (118-fold) with bovine Pg than with human Pg and non-proteolytically activated bovine but not human Pg. Surprisingly, however, the presence of human fibrin overrides the species-restricted action of SUPA. First, human fibrin enhanced the binding avidity of SUPA for human Pg by 4-8-fold in the presence and absence of chloride ion (a negative regulator). Second, although SUPA did not protect plasmin from inactivation by α(2)-antiplasmin, fibrin did protect human plasmin, which formed a 31-fold higher avidity complex with SUPA than Pg. Third, fibrin significantly enhanced Pg activation by reducing the K(m) (4-fold) and improving the catalytic efficiency of the SUPA complex (6-fold). Taken together, these data suggest that indirect molecular interactions may override the species-restricted activity of bacterial Pg activators; this may affect the pathogenesis of infections or may be exploited to facilitate the design of new blood clot-dissolving drugs.     

On the mechanism of fibrin-specific plasminogen activation by staphylokinase

The mechanism of plasminogen activation by recombinant staphylokinase was studied both in the absence and in the presence of fibrin, in purified systems, and in human plasma. Staphylokinase, like streptokinase, forms a stoichiometric complex with plasminogen that activates plasminogen following Michaelis-Menten kinetics with Km = 7.0 microM and k2 = 1.5 s-1. In purified systems, alpha 2-antiplasmin inhibits the plasminogen-staphylokinase complex with k1(app) = 2.7 +/- 0.30 x 10(6) M-1 s-1 (mean +/- S.D., n = 12), but not the plasminogen-streptokinase complex. Addition of 6-aminohexanoic acid induces a concentration-dependent reduction of k1(app) to 2.0 +/- 0.17 x 10(4) M-1 s-1 (mean +/- S.D., n = 5) at concentrations greater than or equal to 30 mM, with a 50% reduction at a 6-aminohexanoic acid concentration of 60 microM. Staphylokinase does not bind to fibrin, and fibrin stimulates the initial rate of plasminogen activation by staphylokinase only 4-fold. Staphylokinase induces a dose-dependent lysis of a 0.12-ml 125I-fibrin-labeled human plasma clot submersed in 0.5 ml of citrated human plasma; 50% lysis in 2 h is obtained with 17 nM staphylokinase and is associated with only 5% plasma fibrinogen degradation. Corresponding values for streptokinase are 68 nM and more than 90% fibrinogen degradation. In the absence of a fibrin clot, 50% fibrinogen degradation in human plasma in 2 h requires 790 nM staphylokinase, but only 4.4 nM streptokinase. These results suggest the following mechanism for relatively fibrin-specific clot lysis with staphylokinase in a plasma milieu. In plasma in the absence of fibrin, the plasminogen-staphylokinase complex is rapidly neutralized by alpha 2-antiplasmin, thus preventing systemic plasminogen activation. In the presence of fibrin, the lysine-binding sites of the plasminogen-staphylokinase complex are occupied and inhibition by alpha 2-antiplasmin is retarded, thus allowing preferential plasminogen activation at the fibrin surface.     

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.     

Kinetics of the reactions between streptokinase, plasmin and alpha 2-antiplasmin.

Streptokinase reacts very rapidly with human plasmin (rate constant 5.4 S 10(7) M-1 s-1) forming a 1:1 stoichiometric complex which has a dissociation constant of 5 X 10(-11) M. This plasmin-streptokinase complex is 10(5) times less reactive towards alpha 2-antiplasmin than plasmin, the inhibition rate constant being 1.4 X 10(2) M-1 s-1. The loss of reactivity of the streptokinase-plasmin complex towards alpha 2-antiplasmin is independent of the lysine binding sites in plasmin since low-Mr plasmin, which lacks these sites, and plasmin in which the sites have been blocked by 6-aminohexanoic acid, are both equally unreactive towards alpha 2-antiplasmin on reaction with streptokinase. The plasmin-streptokinase complex binds to Sepharose-lysine and Sepharose-fibrin monomer in the same fashion as free plasmin, showing that the lysine binding sites are fully exposed in the complex. Bovine plasmin is rapidly inhibited by human alpha 2-antiplasmin (k1 = 1.6 X 10(6) M-1 s-1) and similarly loses reactivity towards the inhibitor on complex formation with streptokinase (50% binding at 0.4 microM streptokinase).     

On the specific interaction between the lysine-binding sites in plasmin and complementary sites in alpha2-antiplasmin and in fibrinogen.

Plasminogen and plasminogen derivatives which contain lysine-binding sites were found to decrease the reaction rate between plasmin and alpha2-antiplasmin by competing with plasmin for the complementary site(s) in alpha2-antiplasmin. The dissocwation constant Kd for the interaction between intact plasminogen (Glu-plasminogen) and alpha2-antiplasmin is 4.0 microM but those for Lys-plasminogen or TLCK-plasmin are about 10-fold lower indicating a stronger interaction. The lysine-binding site(s) which is situated in triple-loops 1--3 in the plasmin A-chain is mainly responsible for the interaction with alpha2-antiplasmin. The interaction between Glu-plasminogen and alpha2-antiplasmin furthermore enhances the activation of Glu-plasminogen by urokinase to a comparable extent as 6-aminohexanoic acid, suggesting that similar conformational changes occur in the proenzyme after complex formation. Fibrinogen, fibrinogen digested with plasmin, purified fragment E and purified fragment D interfere with the reaction between plasmin and alpha2-antiplasmin by competing with alpha2-antiplasmin for the lysine-binding site(s) in the plasmin A-chain. The Kd obtained for these interactions varied between 0.2 microM and 1.4 microM; fragment E being the most effective. Thus the fibrinogen molecule contains several complementary sites to the lysine-binding sites located both in its NH2-terminal and COOH-terminal regions; these sites are to a large extent.     

On the molecular interactions between plasminogen-staphylokinase, alpha 2-antiplasmin and fibrin.

The molecular interactions between the plasminogen-staphylokinase complex, alpha 2-antiplasmin and fibrin were studied by measuring the effect of CNBr-digested fibrinogen on the inhibition rate of the plasminogen-staphylokinase complex by alpha 2-antiplasmin. The second-order rate constant for the inhibition of plasminogen-staphylokinase by alpha 2-antiplasmin was 2.7 +/- 0.3.10(6) M-1 s-1 (mean +/- S.D.; n = 7). Addition of CNBr-digested fibrinogen, but not of fibrinogen, resulted in a concentration-dependent reduction of the apparent inhibition rate constant, with a 50 percent reduction at a concentration of 5 nM CNBr-digested fibrinogen. The second-order rate constant for the inhibition of the low-Mr plasminogen-staphylokinase complex (plasminogen lacking the kringle structures comprising the lysine-binding sites) by alpha 2-antiplasmin was about 30-fold lower (9.3 +/- 0.7.10(4) M-1 s-1, mean +/- S.D.; n = 4) than that of plasminogen-staphylokinase and was not affected by addition of CNBr-digested fibrinogen. Inhibition of the plasminogen-staphylokinase complex by the chloromethylketone D-Val-Phe-Lys-Ch2Cl is 9-fold less efficient than that of plasmin (k2/Ki of 700 M-1 s-1 versus 6300 M-1 s-1). Our results confirm and establish that rapid inhibition of plasminogen-staphylokinase by alpha 2-antiplasmin requires the availability of the lysine-binding sites in the plasminogen moiety of the complex. Fibrin, but not fibrinogen, reduces the inhibition rate by alpha 2-antiplasmin by competition for interaction with the lysine-binding site. Protection of the plasminogen-staphylokinase complex bound to fibrin from rapid inhibition by alpha 2-antiplasmin thus appears to contribute to the fibrin-specificity of clot lysis with staphylokinase in a plasma milieu, by allowing preferential plasminogen activation at the fibrin surface, while the free complex is rapidly inhibited in plasma.     

Kinetics of in vitro fibrinolysis by plasmin and a plasmin-streptokinase equimolar complex]

Kinetics of fibrinolysis by plasmin and plasmin streptokinase complex have been studied using fibrin gels formed from purified fibrin and human blood plasma. The gels were placed into buffer or blood plasma. The contributions of plasminogen and alpha 2-antiplasmin present or absent in both phases to the kinetics of fibrinolysis were quantitatively estimated. In the complex catalyzed fibrinolysis, plasminogen activation reaction dominated whereas in plasmin-catalyzed fibrinolysis, the inhibitor involved reaction, suppressing the process, prevailed.     

Plasminogen activation with single-chain urokinase-type plasminogen activator (scu-PA). Studies with active site mutagenized plasminogen (Ser740----Ala) and plasmin-resistant scu-PA (Lys158----Glu)

The mechanism of the activation of plasminogen by single-chain urokinase-type plasminogen activator (single-chain u-PA, scu-PA) was studied using rscu-PA-Glu158, a recombinant plasmin-resistant mutant of human scu-PA obtained by site-specific mutagenesis of Lys158 to Glu, and rPlg-Ala740, a recombinant human plasminogen in which the catalytic site is destroyed by mutagenesis of the active-site Ser740 to Ala. Conversion of 125I-labeled single-chain plasminogen to two-chain plasmin was quantitated on reduced sodium dodecyl sulfate-gel electrophoresis combined with autoradiography and radioisotope counting of gels bands. The efficiencies of both rscu-PA-Glu158 and rscu-PA for the activation of rPlg-Ala740 and of natural plasminogen were comparable and were 250-500-fold lower than that of recombinant two-chain u-PA (rtcu-PA) for rscu-PA-Glu158 and 100-200-fold lower for rscu-PA. Pretreatment of rscu-PA-Glu158 or rscu-PA with excess alpha 2-antiplasmin, which efficiently neutralizes all contaminating rtcu-PA, did not significantly reduce the catalytic efficiency of these single-chain moieties, indicating that they have a low but significant intrinsic plasminogen activating potential. The low intrinsic catalytic efficiency of rscu-PA for the conversion of plasminogen to plasmin may be sufficient to generate trace amounts of plasmin, which may regulate plasminogen activation by converting poorly active rscu-PA to very active rtcu-PA.     

Effect of streptokinase on the interaction of alpha-2-antiplasmin with different sites of plasmin molecule

Interaction of streptokinase and alpha-2-antiplasmin with plasmin and plasminogen fragments was compared. Binding sites on the enzyme become half-saturated, streptokinase and alpha-2-antiplasmin concentration being 8.5 and 30 nM, respectively. 6-Aminohexanoic acid in concentration of 20 mM reduces the adsorption of streptokinase and and alpha-2-antiplasmin by 20 and 60%, respectively. From all the investigated fragments, streptokinase shows the greatest affinity for mini-plasminogen and alpha-2-antiplasmin for kringles 1-3. Both proteins in the presence of 20 mM 6-aminohexanoic acid do not bind with kringle domains. Arginine dose 0.1 M does not influence streptokinase adsorption on mini-plasminogen and decreases the value of alpha-2-antiplasmin binding with mini-plasminogen by 50%. The data obtained indicate that plasminogen molecule has the sites of the highest affinity for streptokinase on the serine-proteinase domain, however for alpha-2-antiplasmin it is in the kringles 1-3. Streptokinase with equimolar quantity in respect of alpha-2-antiplasmin inhibits the adsorption of alpha-2-antiplasmin on the plasmin by 70% and in the presence of 6-aminohexanoic acid it is inhibited completely. Addition of streptokinase also increases the influence of increasing concentration of the acid. Inhibiting influence of streptokinase decreases, and that of 6-aminohexanoic acid increases, when plasmin is modified with diisopropylfluorophosphate in its active centre. At the same time maximum inhibition of streptokinase adsorption on the plasmin at different concentrations of alpha-2-antiplasmin and 6-aminohexanoic acid accounts for only 20%. We suppose that in the process of complex formation streptokinase competes with alpha-2-antiplasmin for the binding sites on the catalytic domain of the plasmin. Partial or complete blocking of the plasmin active centre contact zone by streptokinase effectively protects it from inhibition by alpha-2-antiplasmin.     

Zymogen-activation kinetics. Modulatory effects of trans-4-(aminomethyl)cyclohexane-1-carboxylic acid and poly-D-lysine on plasminogen activation.

The kinetics of plasminogen activation catalysed by urokinase and tissue-type plasminogen activator were investigated. Kinetic measurements are performed by means of a specific chromogenic peptide substrate for plasmin, D-valyl-L-leucyl-L-lysine 4-nitroanilide. Two methods are proposed for the analysis of the resulting progress curve of nitroaniline formation in terms of zymogen-activation kinetics: a graphical transformation of the parabolic curve and transformation of the curve for nitroaniline production into a linear progress curve by the addition of a specific inhibitor of plasmin, bovine pancreatic trypsin inhibitor. The two methods give similar results, suggesting that the reaction between activator and plasminogen is a simple second-order reaction at least at plasminogen concentrations up to about 10 microM. The kinetics of both Glu1-plasminogen (residues 1-790) and Lys77-plasminogen (residues 77-790) activation were investigated. The results confirm previous observations showing that trans-4-(aminomethyl)cyclohexane-1-carboxylic acid at relatively low concentrations enhances the activation rate of Glu1-plasminogen but not that of Lys77-plasminogen. At higher concentrations both Glu1- and Lys77-plasminogen activation are inhibited. The concentration interval for the inhibition of urokinase-catalysed reactions is shown to be very different from that of the tissue-plasminogen activator system. Evidence is presented indicating that binding to the active site of urokinase (KD = 2.0 mM) is responsible for the inhibition of the urokinase system, binding to the active site of tissue-plasminogen activator is approx. 100-fold weaker, and inhibition of the tissue-plasminogen activator system, when monitored by plasmin activity, is mainly due to plasmin inhibition. Poly-D-lysine (Mr 160 000) causes a marked enhancement of plasminogen activation catalysed by tissue-plasminogen activator but not by urokinase. Bell-shaped curves of enhancement as a function of the logarithm of poly-D-lysine concentration are obtained for both Glu1- and Lys77-plasminogen activation, with a maximal effect at about 10 mg/litre. The enhancement of Glu1-plasminogen activation exerted by trans-4-(aminomethyl)cyclohexane-1-carboxylic acid is additive to that of poly-D-lysine, whereas poly-D-lysine-induced enhancement of Lys77-plasminogen activation is abolished by trans-4-(aminomethyl)cyclohexane-1-carboxylic acid. Analogies are drawn up between the effector functions of poly-D-lysine and fibrin on the catalytic activity of tissue-plasminogen activator.     

Structural elements that govern the substrate specificity of the clot-dissolving enzyme plasmin

There is remarkable homology between the core structures of plasmin, a fibrin clot-degrading enzyme, and factor D, a complement-activating enzyme, despite markedly different biological functions. We postulated that sequence divergence in the loop structures between these two enzymes mediated the unique substrate and inhibitor interactions of plasmin. Recombinant microplasminogens chimerized with factor D sequences at loops 3, 5, and 7 were cleaved by the plasminogen activator urokinase and developed titratable active sites. Chimerization abolished functional interactions with the plasminogen activator streptokinase but did not block complex formation. The microplasmin chimeras showed enhanced resistance (k(i) decreased up to two to three times) to inactivation of microplasmin by alpha(2)-antiplasmin. Microplasmin chimerization had minimal ( approximately 2 fold) effects on the catalytic efficiency for cleavage of small substrates and did not alter the cleavage of fibrin. However, microplasmin and the microplasmin chimeras showed enhanced abilities to degrade fibrin in plasma clots suspended in human plasma. These studies indicate that loop regions of the protease domain of plasmin are important for interactions with substrates, regulatory molecules, and inhibitors. Because modification of these regions affected substrate and inhibitor interactions, loop chimerization may hold promise for improving the clot dissolving properties of this enzyme.     

Kinetics of activated thrombin-activatable fibrinolysis inhibitor (TAFIa)-catalyzed cleavage of C-terminal lysine residues of fibrin degradation products and removal of plasminogen-binding sites

Partial digestion of fibrin by plasmin exposes C-terminal lysine residues, which comprise new binding sites for both plasminogen and tissue-type plasminogen activator (tPA). This binding increases the catalytic efficiency of plasminogen activation by 3000-fold compared with tPA alone. The activated thrombin-activatable fibrinolysis inhibitor (TAFIa) attenuates fibrinolysis by removing these residues, which causes a 97% reduction in tPA catalytic efficiency. The aim of this study was to determine the kinetics of TAFIa-catalyzed lysine cleavage from fibrin degradation products and the kinetics of loss of plasminogen-binding sites. We show that the k(cat) and K(m) of Glu(1)-plasminogen (Glu-Pg)-binding site removal are 2.34 s(-1) and 142.6 nm, respectively, implying a catalytic efficiency of 16.21 μm(-1) s(-1). The corresponding values of Lys(77)/Lys(78)-plasminogen (Lys-Pg)-binding site removal are 0.89 s(-1) and 96 nm implying a catalytic efficiency of 9.23 μm(-1) s(-1). These catalytic efficiencies of plasminogen-binding site removal by TAFIa are the highest of any TAFIa-catalyzed reaction with a biological substrate reported to date and suggest that plasmin-modified fibrin is a primary physiological substrate for TAFIa. We also show that the catalytic efficiency of cleavage of all C-terminal lysine residues, whether they are involved in plasminogen binding or not, is 1.10 μm(-1) s(-1). Interestingly, this value increases to 3.85 μm(-1) s(-1) in the presence of Glu-Pg. These changes are due to a decrease in K(m). This suggests that an interaction between TAFIa and plasminogen comprises a component of the reaction mechanism, the plausibility of which was established by showing that TAFIa binds both Glu-Pg and Lys-Pg.     

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.     

Engineering of Plasmin-Resistant Forms of Streptokinase and Their Production in Bacillus subtilis: Streptokinase with Longer Functional Half-Life

The short in vivo half-life of streptokinase limits its efficacy as an efficient blood clot-dissolving agent. During the clot-dissolving process, streptokinase is processed to smaller intermediates by plasmin. Two of the major processing sites are Lys59 and Lys386. We engineered two versions of streptokinase with either one of the lysine residues changed to glutamine and a third version with both mutations. These mutant streptokinase proteins (muteins) were produced by secretion with the protease-deficient Bacillus subtilis WB600 as the host. The purified muteins retained comparable kinetics parameters in plasminogen activation and showed different degrees of resistance to plasmin depending on the nature of the mutation. Muteins with double mutations had half-lives that were extended 21-fold when assayed in a 1:1 molar ratio with plasminogen in vitro and showed better plasminogen activation activity with time in the radial caseinolysis assay. This study indicates that plasmin-mediated processing leads to the inactivation of streptokinase and is not required to convert streptokinase to its active form. Plasmin-resistant forms of streptokinase can be engineered without affecting their activity, and blockage of the N-terminal cleavage site is essential to generate engineered streptokinase with a longer in vitro functional half-life.     

Characterization of plasmin-mediated activation of plasma procarboxypeptidase B. Modulation by glycosaminoglycans

Plasma carboxypeptidase B (PCB) is an exopeptidase that exerts an antifibrinolytic effect by releasing C-terminal Lys and Arg residues from partially degraded fibrin. PCB is produced in plasma via limited proteolysis of the zymogen, pro-PCB. In this report, we show that the K(m) (55 nM) for plasmin-catalyzed activation of pro-PCB is similar to the plasma concentration of pro-PCB (50-70 nM), whereas the K(m) for the thrombin- or thrombin:thrombomodulin-catalyzed reaction is 10-40-fold higher than the pro-PCB level in plasma. Additionally, tissue-type plasminogen activator triggers activation of pro-PCB in blood plasma in a reaction that is stimulated by a neutralizing antibody versus alpha(2)-antiplasmin. Together, these results show that plasmin-mediated activation of pro-PCB can occur in blood plasma. Heparin (UH) and other anionic glycosaminoglycans stimulate pro-PCB activation by plasmin but not by thrombin or thrombin:thrombomodulin. Pro-PCB is a more favorable substrate for plasmin in the presence of UH (16-fold increase in k(cat)/K(m)). UH also stabilizes PCB against spontaneous inactivation. The presence of UH in clots prepared with prothrombin-deficient plasma delays tissue-type plasminogen activator-triggered lysis; this effect of UH on clot lysis is blocked by a PCB inhibitor from potato tubers. These results show that UH accelerates plasmin-catalyzed activation of pro-PCB in plasma and PCB, in turn, stabilizes fibrin against fibrinolysis. We propose that glycosaminoglycans in the subendothelial extracellular matrix serve to augment the levels of PCB activity thereby stabilizing blood clots at sites where there is a breach in the integrity of the vasculature.     

Regulation of nonproteolytic active site formation in plasminogen

Streptokinase may be less effective at saving lives in patients with heart attacks because it explosively generates plasmin in the bloodstream at sites distant from fibrin clots. We hypothesized that this rapid plasmin generation is due to SK's singular capacity to nonproteolytically generate the active protease SK x Pg*, and we examined whether the kringle domains regulate this process. An SK mutant lacking Ile-1 (deltaIle1-SK) does not form SK x Pg*, although it will form complexes with plasmin that can activate plasminogen. When compared to SK, deltaIle1-SK diminished the generation of plasmin in plasma by more than 30-fold, demonstrating that the formation of SK x Pg* plays an important role in SK activity in the blood. The rate of SK x Pg* formation (measured by an active site titrant) was much slower in Glu-Pg, which contains five kringle domains, than in Pg forms containing one kringle (mini-Pg) or no kringles (micro-Pg). In a similar manner, Streptococcus uberis Pg activator (SUPA), an SK-like molecule, generated SUPA x Pg* much slower with bovine Pg than bovine micro-Pg. The velocity of SK x Pg* formation was regulated by agents that influence the conformation of Pg through interactions with the kringle domains. Chloride ions, which maintain the compact Pg conformation, hindered SK x Pg* formation. In contrast, epsilon-aminocaproic acid, fibrin, and fibrinogen, which induce an extended Pg conformation, accelerated the formation of SK x Pg*. In summary, the explosive generation of plasmin in blood or plasma, which diminishes SK's therapeutic effects, is attributable to the formation of SK x Pg*, and this process is governed by kringle domains.     

Structure-function analysis of the streptokinase amino terminus (residues 1-59)

Streptokinase (SK) binds to plasminogen (Pg) to form a complex that converts substrate Pg to plasmin. Residues 1-59 of SK regulate its capacity to induce an active site in bound Pg by a nonproteolytic mechanism and to activate substrate Pg in a fibrin-independent manner. We analyzed 24 SK mutants to better define the functional properties of SK-(1-59). Mutations within the alphabeta1 strand (residues 17-26) of SK completely prevented nonproteolytic active site induction in bound Pg and rendered SK incapable of protecting plasmin from inhibition by alpha2-antiplasmin. However, when fibrin-bound, the activities of alphabeta1 strand mutants were similar to that of wild-type (WT) SK and resistant to alpha2-antiplasmin. Mutation of Ile1 of SK also prevented nonproteolytic active site induction in bound Pg. However, unlike alphabeta1 strand mutants, the functional defect of Ile1 mutants was not relieved by fibrin, and complexes of Ile1 mutants and plasmin were resistant to alpha2-antiplasmin. Plasmin enhanced the activities of alphabeta1 strand and Ile1 mutants, suggesting that SK-plasmin complexes activated mutant SK.Pg complexes by hydrolyzing the Pg Arg561-Val562 bond. Mutational analysis of Glu39 of SK suggested that a salt bridge between Glu39 and Arg719 of Pg is important, but not essential, for nonproteolytic active site induction in Pg. Deleting residues 1-59 rendered SK dependent on plasmin and fibrin to generate plasminogen activator (PA) activity. However, the PA activity of SK-(60-414) in the presence of fibrin was markedly reduced compared with WT SK. Despite its reduced PA activity, the fibrinolytic potency of SK-(60-414) was greater than that of WT SK at higher (but not lower) SK concentrations due to its capacity to deplete plasma Pg. These studies define mechanisms by which the SK alpha domain regulates rapid active site induction in bound Pg, contributes to the resistance of the SK-plasmin complex to alpha2-antiplasmin, and controls fibrin-independent Pg activation.     

A monoclonal antibody directed against the high-affinity lysine-binding site (LBS) of human plasminogen. Role of LBS in the regulation of fibrinolysis

One of thirty murine monoclonal antibodies, raised by immunization with human plasmin-alpha 2-antiplasmin complex, was found to be directed against the high-affinity lysine-binding site in plasminogen. Indeed, this antibody (MA-HAL) reacted with plasminogen and with a fragment of plasminogen composed of the first three triple-loop structures (LBS I) and was displaced by 6-aminohexanoic acid (50% displacement at 25 microM). In competitive radioimmunoassays the binding of radiolabeled plasminogen to MA-HAL was reduced to 50% with 2.3 microM alpha 2-antiplasmin or 1.3 microM histidine-rich glycoprotein, which corresponds to the known dissociation constants between these ligands and the high-affinity lysine-binding site of plasminogen. MA-HAL did not influence the activation of plasminogen by tissue-type plasminogen activator in the absence of CNBr-digested fibrinogen, but abolished the effect of CNBr-digested fibrinogen on the Michaelis constant of the reaction. MA-HAL reduced the reaction rate between plasmin and alpha 2-antiplasmin by a factor 20 and abolished the binding of plasminogen to fibrin. These results indicate that MA-HAL specifically binds to and masks the high-affinity lysine-binding site of plasminogen. It therefore is a useful tool for the investigation of the role of this structure in the regulation of fibrinolysis, both at the level of fibrin-stimulated activation of plasminogen and of the inhibition of generated plasmin.