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J. Biol. Chem., Vol. 277, Issue 30, 26846-26851, July 26, 2002

This Article Abstract Full Text (PDF) All Versions of this Article: 277/30/26846    most recent M202999200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Gladysheva, I. P. Articles by Reed, G. L. Search for Related Content PubMed PubMed Citation Articles by Gladysheva, I. P. Articles by Reed, G. L. Social Bookmarking                      What's this?

Chimerism Reveals a Role for the Streptokinase -Domain in Nonproteolytic Active Site Formation, Substrate, and Inhibitor Interactions^*

Inna P. Gladysheva, Irina Y. Sazonova, Shakeel A. Chowdhry, Lin Liu, Ryan B. Turner, and Guy L. Reed

From the Cardiovascular Biology Laboratory, Harvard School of Public Health and the Massachusetts General Hospital, Boston, Massachusetts 02114

Received for publication, March 28, 2002, and in revised form, May 16, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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 -domain. To examine this hypothesis, a chimeric streptokinase, SKswap, was created by swapping the SK -domain with the homologous -domain of Streptococcus uberis Pg activator (SUPA or PauA, SK uberis), a streptokinase that cannot activate human plasminogen. SKswap formed a tight complex with microplasminogen with an affinity comparable with streptokinase. The SKswap-plasmin complex also activated human plasminogen with catalytic efficiencies (k_cat/K_m = 16.8 versus 15.2 µM¹ min¹) comparable with streptokinase. However, SKswap was incapable of nonproteolytic active site generation and activated plasminogen by a staphylokinase mechanism. When compared with streptokinase complexes, SKswap-plasmin and SKswap-microplasmin complexes had altered affinities for low molecular weight substrates. The SKswap-plasmin complex also was less resistant than the streptokinase-plasmin complex to inhibition by ₂-antiplasmin and was readily inhibited by soybean trypsin inhibitor. Thus, in addition to mediating high affinity binding to plasmin(ogen), the streptokinase -domain is required for nonproteolytic active site generation and specifically modulates the interactions of the complex with substrates and inhibitors.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The plasmin system dissolves blood clots and remains a fruitful target for biomedical research aimed at saving lives and preventing disability in patients with cardiovascular diseases. Plasmin is a serine protease that cleaves fibrin, the protein matrix of blood clots (1, 2). Plasminogen activators convert the zymogen plasminogen (Pg)¹ to plasmin by direct enzymatic cleavage of a single Arg⁵⁶¹-Val⁵⁶² peptide bond yielding a heavy chain, which contains five kringle domains, and a light chain, which contains a protease domain (microplasmin). Streptokinase (SK) and staphylokinase (SAK) are two classes of microbial Pg activators that have evolved to regulate and target the activity of the Pg-plasmin system (3-6). Although SK has been used to treat heart attacks for >30 years (7), the structural bases for its unique molecular properties are just now being elucidated (8).

Both SK and SAK form a cofactor-enzyme complex with plasmin that cleaves Pg substrate (9-12). However, SK contains three domains (, , and ) (8), whereas SAK only contains one domain (13, 14). Consequently, the interactions of SK with plasmin and Pg (P(g)) are more extensive and complex. For example, SK has 4100 Å² (-domain = 1650 Å², -domain = 950 Å², and -domain = 1500 Å²) of intermolecular contact with microplasmin (8), whereas SAK has only 1750 Å² of contact (13). Thus, SK forms a tighter and more stable complex with P(g) (15, 16) than SAK (6, 17). The stability of SK·P(g) complex may contribute to the major mechanistic differences between SK and SAK in Pg activation. In this stable complex, SK profoundly alters the interactions of plasmin with substrates (fibrin and Pg) and inhibitors (₂-antiplasmin), whereas SAK does not (18). In this complex, SK also has the unique property of nonproteolytically generating or rearranging the latent active site of Pg to make it catalytically active (Pg*), although SAK does not (3, 19).

The ability of SK to form a tight stable complex with Pg arises in large part from high affinity binding interactions between the -domains of SK and Pg (20-23). However, catalytically, the SK-microplasmin complex is a more efficient Pg activator than the complex of SK with the entire P(g) molecule despite the fact that SK binds to microplasmin with lower affinity than it does to plasmin (24, 25). In the SK-microplasmin complex, the - and -domains have extensive intermolecular contacts with microplasmin, whereas the -domain has relatively few interactions with microplasmin (8). Still, a number of studies have shown that the -domain is required for plasminogen activation (25-27). However, the paucity of interactions of the -domain with microplasmin suggests that the -domain does not contribute substantially to the stability or the catalytic function of this activator complex. If this is true, the -domain may serve merely as a linker or spacer unit that optimally positions the - and -domains for functional interactions with microplasmin and substrate (21). Alternatively, the function of the -domain may be to modulate the interactions of substrates with the activator complex.

The goal of these studies was to examine the role of the SK -domain in the function of the SK·P(g) and SK-microplasmin(ogen) activator complex. These studies took advantage of the exquisitely restricted ability of SK molecules to activate mammalian Pgs from other species. For example, SK activates human Pg but not bovine or horse Pg, whereas Streptococcus uberis Pg activator (SUPA or PauA, SK uberis) activates bovine but not human Pg (28-30). The molecular swapping of these two closely related -domains, SK -domain and -domain of SUPA, to create SK·SUPA·SK chimera (SKswap) allowed us to elucidate the role of the -domain in 1) active site generation, 2) Pg activation and, 3) interactions with plasmin inhibitor ₂-antiplasmin.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Cloning, Expression, and Purification of Recombinant Proteins-- Recombinant SUPA, SK, micro-Pg, and micro-PgR561A mutant were cloned, expressed in bacteria, purified, and characterized as described previously (20, 30-32). The SUPA sequence was modeled on the SK structural coordinates using a Swiss model program (www.expasy.ch). Residues 126-261 of the SUPA -domain were found to closely recapitulate residues 146-281 of the SK -domain. Consequently, the cDNA for the SKswap chimera was generated in such a way that the codons for residues 146-281 of SK were replaced by the codons for residues 126-281 of SUPA by PCR with an overlap extension as we have described previously (33). The SKswap chimera cDNA was ligated into the pMALc vector (New England Biolabs, Beverly, MA) for expression as a maltose-binding fusion protein in bacteria (20). The maltose-binding protein-SKswap fusion protein was absorbed on DEAE Affi-Gel Blue-agarose, and purified protein was eluted between 75 and 160 nM NaCl by a linear gradient of 0-250 mM NaCl in 10 mM phosphate buffer, pH 7.2. The chimeric structure was cleaved from maltose-binding protein by treatment with Factor Xa (New England Biolabs) for 5 h at room temperature in 200 mM Tris-HCl buffer, pH 8.0, 100 mM NaCl, 2 mM CaCl₂. The separated SKswap was analyzed by SDS-PAGE and Western blot analysis using monoclonal antibody 9D10 directed to residues 1-13 of SK -domain to confirm that the NH₂ terminus remained intact after cleavage (34).

Protein Labeling-- Human Pg (95% Glu-Pg, Chromogenix, Milano, Italy) was radioiodinated by the Iodogen method (35), and the specific radioactivity was determined as described previously (36).

Active Site Titration-- The molar quantity of active sites generated nonproteolytically by the SK in human Pg or by SUPA in bovine Pg was determined at 37 °C in a Hitachi 2500 fluorescence spectrophotometer by active site titration with the fluorogenic substrate 4-methylumbelliferyl p-guanidinobenzoate (MUGB, Sigma) as described previously (20, 37). The activity of SKswap (35-39% active) was determined by an indirect titration method. Human Pg (98 nM) was preincubated with different amounts of SKswap (0-200 nM) and then with a stoichiometric amount of SK (98 nM) for 8 min on ice. Subsequently, the hydrolysis of MUGB was monitored, and the active concentration of SKswap was calculated from a graph that showed an inverse linear dependence of SK·Pg active concentration on the total SKswap concentration.

Active Site Generation-- All of the experiments subsequently described were performed in an assay buffer (50 mM Tris-HCl, 100 mM NaCl, pH 7.4) with 0.5 mM S2251 (H-D-valyl-L-leucyl-L-lysine-p-nitroanilide dihydrochloride, Chromogenix). The ability of SKswap to generate active sites in the Pg molecule was examined using a recombinant noncleavable micro-PgR561A mutant that can only be activated through nonproteolytic mechanisms (38). Equimolar complexes of micro-PgR561A·SKswap or micro-PgR561A·SK (final concentration 50 nM) were prepared by mixing micro-PgR561A and SK or SKswap for 10 min to 24 h at 37 °C. The complexes were transferred to a thermostatically regulated (37 °C) quartz cuvette containing assay buffer and S2251 in a total volume of 300 µl, and the change of absorbance was monitored at 405 nm for 10 min in a Cary 100-Bio spectrophotometer.

Amidolysis Kinetic Assays of the SK, SUPA, or SKswap Activator Complexes-- The amidase kinetic parameters of the activator complexes were measured with a p-nitroanilide substrate S2251 as described previously (31, 39). The equimolar complexes (final concentration 5 nM) were prepared by mixing human plasmin (Sigma) or human microplasmin and SK, SUPA, or SKswap for 10 min on ice. Microplasmin was generated from micro-Pg by urokinase (1:30 ratio) at 37 °C for 1 h. The reaction was initiated by the addition of enzyme complexes to assay buffer containing various concentrations of S2251 (final concentration 50-1500 µM) in a total volume of 100 µl in microtiter plates at 37 °C. The generation of amidolytic activity at 405 nm was monitored at 37 °C for 10 min in a Thermomax microplate reader (Molecular Devices Corp., Menlo Park, CA). Less than 10% H-D-Val-L-Leu-L-Lys-nitroanilide was consumed during the course of the reaction. The data were plotted as velocity/substrate and analyzed by hyperbolic curve fitting with the Sigma Plot program. A ^1 M at 405 nm of 10,000 was used for p-nitroanilide.

Steady-state Pg Activation Kinetic Parameters-- The kinetics of Pg activation by activator complexes were studied as described previously (39). Stoichiometric activator complexes (final concentration 5-25 nM) were formed by mixing human plasmin or microplasmin and SK or SKswap on ice for 10 min. The activators were added to microtiter plates containing assay buffer, 0.5 mM S2251, and human Glu-Pg (95% Glu-Pg) (final concentration 500-1600 nM) or microplasminogen (100-900 nM) in a total volume of 100 µl. The reactions were carried out at 37 °C in a microplate reader. Initial reaction rates were obtained from the first 300 s by plotting A₄₀₅/min², and the apparent Michaelis-Menten and catalytic rate constants were calculated by fitting the data to a hyperbolic curve as described previously (39) using the Sigma Plot program.

Complex Formation between SKswap and Micro-PgR561A-- Unlike SK, SKswap cannot generate an active site in micro-PgR561A mutant to convert it to the enzyme, micro-PgR561A*. Hence, the ability of SKswap to form an inactive complex with micro-PgR561A mutant was examined indirectly using SKswap as an inhibitor of active site generation and consequent amidolysis by the SK-micro-PgR561A*. The micro-PgR561A mutant (final concentration 50 nM) was preincubated for 25 min at 37 °C with different amounts of SKswap (final concentration 0-200 nM) and then with a stoichiometric amount of SK (final concentration 50 nM) for 15 min at 37 °C. The preincubated complex was added to a cuvette containing assay buffer and S2251 (final concentration 0.5 mM). The hydrolysis of S2251 was monitored at 405 nm in a Cary 100-Bio spectrophotometer. Because only the complex with SK has amidolytic activity, the fraction of Pg bound to SK can be determined by the rate of S2251 hydrolysis. The apparent inhibition constant was calculated as described previously (40) using Equation 1

(Eq. 1)

where a is the fraction of total enzyme, SK-micro-PgR561A*, which is not bound to the inhibitor, [I]_o is a total inhibitor SKswap concentration, and K_i(app) is the dissociation constant of microplasmin-R561A·SKswap complex without accounting for the influence of the substrate.

Inhibition of Amidolysis of Human Plasmin, SK-Plasmin, and SKswap-Plasmin-- Human plasmin (final concentration 5 or 50 nM) or stoichiometric complexes of SK-plasmin or SKswap-plasmin (final concentration 5 or 50 nM) were mixed together for 3 min at room temperature and then incubated for 10 min at room temperature with increasing concentrations of ₂-antiplasmin (final concentrations 0-15 nM) or soybean Kunitz-type trypsin inhibitor (SBTI) (final concentration 0-800 nM). The reaction was initiated by the addition of assay buffer containing S2251 (0.5 mM) in a total volume of 100 µl in microtiter plates at 37 °C. The change of absorbance at 405 nm was monitored in a microplate reader for 10 min.

Binding of SK and SKswap to Human Pg-- The wells of microtiter plates were coated with SK (5 µg/ml) for 1 h at room temperature. The wells were washed, and nonspecific protein binding sites were blocked with 1% bovine serum albumin. Radioiodinated human Glu-Pg (50,000 cpm/25 µl) was added to the wells for 1 h in the presence of increasing concentrations of SK (0.5-500 µg/ml) or SKswap (0.5-500 µg/ml). Afterward, the wells were washed and -counted.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The Role of the -domain in Human Glu-Pg Activation-- Molecular modeling studies indicated that residues 126-261 of the SUPA -domain closely recapitulated the structure of the SK -domain (residues 146-281) (30). These sequences were swapped to create a chimeric SK molecule, which consisted of SK -domain, SUPA -domain, and SK -domain (SKswap). The ability of SK, SUPA, and SKswap chimera to activate human Glu-Pg was compared. As expected, SK but not SUPA activated human Glu-Pg. The chimeric SKswap retained the ability of SK to activate human Glu-Pg (Fig. 1).

View larger version (18K): [in this window] [in a new window]   Fig. 1.   Activation of Glu-Pg by SKswap, SKswap-plasmin activator complex, SK, and SUPA. The activation of human Pg (final concentration 300 nM) by SKswap, SK, SUPA, or stoichiometric complexes of SKswap-plasmin (final concentration 20 nM) was measured at 405 nm and 37 °C using S2251 substrate (final concentration 0.5 mM) in 50 mM Tris-HCl, 100 mM NaCl, pH 7.4 buffer.

Role of the -Domain in Nonproteolytic Active Site Generation-- A fundamental distinction between a SK and SAK mechanism is the ability of SK to generate an active site in Pg under conditions in which proteolysis is blocked (19, 41). We examined nonproteolytic active site generation by SKswap to determine whether the -domain played a role in this process. These studies used a recombinant micro-Pg (micro-PgR561A) in which the activation bond sequence Arg⁵⁶¹-Val⁵⁶² had been mutated to prevent proteolytic cleavage to the plasmin. SK generated an active site within micro-PgR561A mutant as we have previously shown (32, 38), but the SKswap did not generate an active site even after 24-h incubation at 37 °C. Similarly, when active site titration experiments were performed in the presence of excess acylating agent, MUGB, SK was capable of nonproteolytic active site generation, but SKswap was not (data not shown).

Stable Complex Formation Between SKswap and P(g)-- The SK -domain has been shown to be required for high affinity binding interactions between SK and Pg (20, 22). Thus, a potential explanation for the failure of the SKswap to generate an active site was that it was incapable of forming a complex with the micro-PgR561A mutant. However, in binding experiments, radioiodinated human Pg bound to SKswap at levels comparable with SK (Fig. 2). In addition, SKswap was an efficient inhibitor of active site generation, and amidolysis by SK-micro-Pg*R561A (Fig. 3, inhibition constant of 15 nM). Thus, SKswap formed a complex with micro-PgR561A but was incapable of nonproteolytic active site formation, suggesting that like SAK, SKswap activated Glu-Pg by forming an activator complex with plasmin.

View larger version (16K): [in this window] [in a new window]   Fig. 2.   Competition between SK and SKswap for binding to 125I-Glu-Pg. The wells of microtiter plates were coated with SK (5 µg/ml) for 1 h at room temperature. Nonspecific binding was blocked by 1% bovine serum albumin. After washing, 125I-Glu-Pg (50,000 cpm/25 µl) was added to wells for 1 h in the presence of SK () or SKswap () (0.5-500 µg/µl). After 1 h, the wells were washed and -counted.

View larger version (13K): [in this window] [in a new window]   Fig. 3.   SKswap inhibits active site generation in micro-PgR561A by SK. The micro-PgR561A mutant (final concentration 50 nM) was preincubated with different amounts of SKswap (final concentration 0-200 nM) for 25 min at 37 °C and then with a stoichiometric amount of SK for 15 min at 37 °C. Preincubated complex was added to a cuvette containing assay buffer (50 mM Tris-HCl, 100 mM NaCl, pH7.4) and S2251 (final concentration 0.5 mM). The hydrolysis of S2251 was monitored for 10 min at 405 nm.

Amidase Parameters of Various Enzymes and Activator Species-- Kinetic studies were performed to determine whether the -domain contributes to substrate processing by SK activator complex. The kinetic parameters of cleavage of the tripeptide substrate S2251 at pH 7.4 and 37 °C by human plasmin, microplasmin, and activator complexes are shown in Table I. The apparent Michaelis-Menten constants, K_m, were ~4-fold higher for microplasmin than plasmin. When SK formed a complex with plasmin or microplasmin, it decreased the K_m by ~2-4-fold. When SKswap formed a complex with plasmin, it also decreased the K_m by ~4-fold, but it had little effect on the K_m for microplasmin. In contrast, when SUPA formed a complex with plasmin, it decreased the K_m by ~7-fold. Thus, SKswap had distinctive effects on amidolysis kinetics by various plasmins, which were intermediate between those of SK and SUPA.

Kinetics of Pg Activation by Various Activator Complexes-- Because previous studies noted above had indicated that SKswap activated Pg through forming a complex with plasmin, we examined Pg activation by SKswap-plasmin or SKswap-microplasmin complexes. Although the K_m and k_cat were lower, the overall catalytic efficiencies of the SKswap-plasmin complex for Glu-Pg activation were comparable with that of the SK-plasmin complex and similar to the catalytic efficiency of SK for Glu-Pg (Table II).

The kinetic constants for Glu-Pg and micro-Pg activation by SKswap or SK activator complexes are also summarized in Table II. The SKswap-microplasmin and SK-microplasmin complexes showed similar catalytic efficiencies (k_cat/K_m) as activators of micro-Pg. In contrast, the SK-microplasmin complex was a more catalytically efficient (52-fold) activator of Glu-Pg than the SKswap-microplasmin complex, largely because of a higher catalytic rate constant (18-fold). The affinity of SKswap-microplasmin complex for Glu-Pg substrate was 14-fold less than SKswap-plasmin. Consequently, the SKswap-plasmin activator complex was more catalytically efficient.

Inhibition of Plasmin, SK-Plasmin, and SKswap-Plasmin Complexes by ₂-Antiplasmin and SBTI-- The fact that SK alters the resistance of plasmin to inhibitors is a hallmark of the affinity and stability of the SK-plasmin complex versus the SAK-plasmin complex (18). We examined whether the -domain played a role in the interactions of the SK-plasmin activator complex with ₂-antiplasmin or the reversible inhibitor SBTI. When compared with human plasmin alone, preformed complexes of SK or SKswap with plasmin resisted inhibition by increasing the doses of ₂-antiplasmin (Fig. 4A). However, in these studies, the SKswap-plasmin complex was less resistant to ₂-antiplasmin inhibition than the SK-plasmin activator complex. The preformed SK-plasmin complex was completely resistant to the inhibitory effect of the SBTI, whereas plasmin and the SKswap-plasmin complex were completely inhibited (Fig. 4B).

View larger version (17K): [in this window] [in a new window]   Fig. 4.   Inhibition of human plasmin, SK-plasmin, and SKswap-plasmin complexes by 2-antiplasmin and SBTI. A, human plasmin (5 nM, ) or stoichiometric complexes (5 nM) of SK-plasmin () or SKswap-plasmin () were incubated for 10 min at room temperature with 2-antiplasmin (final concentration 0-15 nM). B, human plasmin (50 nM, ) or stoichiometric complexes (50 nM) of SK-plasmin () or SKswap-plasmin () were incubated for 10 min at room temperature with SBTI (final concentration 0-800 nM). The reactions were initiated by the addition of the enzyme-inhibitor complex to assay buffer (50 mM Tris-HCl, 100 mM NaCl, pH 7.4) containing S2251 (0.5 mM), and the residual activities of the enzyme complexes were determined by measuring the absorption at 405 nm.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The sequence and structural homology between SK -domain and -domain of SUPA (27.4%) (28, 30) provided an opportunity to determine the role of the -domain in the function of the SK-Pg activator complex. A SKswap chimera was examined for its function in 1) active site generation, 2) Pg activation, and 3) interactions with the plasmin inhibitors ₂-antiplasmin and SBTI. The SKswap retained the ability to activate human Pg similar to the parent SK molecule (Fig. 1). However, despite being an efficient activator, the SKswap lost its ability to activate Glu-Pg nonproteolytically, which is a hallmark of SK and SUPA. Instead, SKswap activated Pg by a staphylokinase-like mechanism through forming an activator complex with plasmin.

The conclusion that SKswap activated human Pg through staphylokinase-like and not streptokinase-like mechanism was supported by the following results. 1) SKswap was unable to generate an active site in the uncleavable micro-PgR561A mutant (Fig. 2). 2) SKswap did not form an active site in Pg, which was titratable with MUGB. 3) The activation of Glu-Pg by SKswap-Pgs complexes preincubated on ice or at 37 °C gave artifactually lower kinetics constants than SKswap-plasmin complexes (data not shown). The switch from a streptokinase-like to staphylokinase-like Pg activation mechanism was unexpected, because both SK and SUPA activate Pg (human or bovine) through streptokinase-like mechanisms (3, 4, 30). Moreover, recent studies have emphasized the roles of the SK - and -domains but not the SK -domains in this process (32, 38, 42). In addition, the SK -domain alone cannot independently induce active site formation in Pg,² and the isolated -domain of SUPA (residues 97-261) was also incapable of virgin activation of Pg (29). An important distinction between SK and SAK is the ability of SK to form a very tight stable complex with Pg (6, 15-17), which is mediated in large part through the SK -domain (20, 22, 25, 43). The fact that SKswap activated Glu-Pg by a staphylokinase mechanism suggested that it might not form a tight stable complex with Glu-Pg like SK. However, the affinity of Glu-Pg for SKswap was comparable with or better than the affinity of Glu-Pg for SK (Fig. 2). This finding suggests that high affinity binding of SK perhaps is not sufficient for active site generation in Pg and that interactions among specific residues of the -domain with the protease domain may be required for this process. Molecular modeling predicts that the SK -domain has few significant intermolecular contacts (e.g. SK Glu²¹⁸, Asp²²⁰, Asp²³⁸) with microplasmin (e.g. Arg⁵⁸², Ser⁶⁰⁸, Arg⁶¹⁰, and Ser⁶¹²) in the activator complex. The primary sequence and structural alignments indicate that these residues and those mediating weaker interactions are not conserved between SK and SUPA.

Molecular modeling studies have supported earlier biochemical studies indicating that the SK -domain may be important for interactions of the activator complex with substrates and inhibitors (14, 44). Despite their strong overall structural similarities, the replacement of the SK -domain with the SUPA -domain alters amidolytic and Pg activation kinetics. The affinity (1/K_m) of the SKswap-plasmin complex for S2251 is 2-fold higher than that of SK-plasmin activator complex. The affinity of the SKswap-plasmin complex for human Pg is ~6-fold higher than that of SK-plasmin activator complex, although the overall catalytic efficiency was unchanged. In addition, the -domain also appears to modulate the interaction of the activator complex with covalent and noncovalent inhibitors. The SKswap-plasmin activator complex was more readily inhibited by ₂-antiplasmin, the physiologically important regulator of plasmin, than the SK-plasmin activator complex (Fig. 4A). The effects were most pronounced with SBTI, a reversible protease inhibitor. SBTI had no effect on the hydrolysis of SK-human plasmin activator complex (45). However, SKswap-plasmin activator complex and plasmin were completely inhibited by SBTI under these experimental conditions (Fig 4B). Taken together, these data suggest that the -domain structure determines the interactions of the activator complex with small molecular weight and large physiologic substrates as well as with protein inhibitors.

Previous studies have indicated that the -domain of SK plays a critical role in forming a high affinity stable complex with P(g) (20-23), although these interactions are not appreciated in the recent crystal structure with microplasmin (8). Recent studies have provided strong evidence for functional interactions of SK with the kringle domains in the activator complex and substrate. Indeed, the activator complex formation with full-length Pg occurs through a kringle-dependent mechanism (25, 42). Our studies indicate that the minimal activator complex of SK-microplasmin preferentially activates the kringle containing Glu-Pg substrate with a catalytic efficiency that is 24-fold greater than the kringleless micro-Pg substrate. In contrast, the SKswap-microplasmin complex activates Glu-Pg and micro-Pg with efficiencies that are comparable (within 2-fold) to each other (Table II) and to the activation of micro-Pg substrate by the SK-microplasmin complex (Table II). However, the SK-microplasmin complex has a significantly higher (8-fold) activator activity than the SK-plasmin complex toward Glu-Pg (Table II), whereas the SKswap-microplasmin complex activates Glu-Pg substrate 7-fold less efficiently than the SKswap-plasmin complex. Taken together, these kinetic results indicate that kringles not only in the Pg substrate structure but also in the structure of Pg activator complex may also influence the efficiency of Pg activation in part through a -domain-dependent mechanism.

	FOOTNOTES

^* This work was supported in part by National Institutes of Health Grant HL-57314 (to G. L. R.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Cardiovascular Biology Laboratory, HSPH II-127, 677 Huntington Ave., Boston, MA 02115. Tel.: 617-432-4992; Fax: 617-432-0033; E-mail: guyreed@hsph.harvard.edu.

Published, JBC Papers in Press, May 16, 2002, DOI 10.1074/jbc.M202999200

² I. Y. Sazonova and G. L. Reed, unpublished data.

	ABBREVIATIONS

The abbreviations used are: Pg, plasminogen; micro-PgR561A, microplasmin-Pg with residue 561 arginine mutated to alanine; SK, streptokinase; SAK, staphylokinase; SUPA, Streptococcus uberis plasminogen activator; SKswap, SK·SUPA·SK chimera; SBTI, soybean Kunitz-type trypsin inhibitor; S2251, H-D-valyl-L-leucyl-L-lysine-p-nitroanilide dihydrochloride; MUGB, 4-methylumbelliferyl p-guanidinobenzoate.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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