Structure-Function Analysis of the Streptokinase Amino Terminus (Residues 1–59)*

Lakshmi V. Mundada {ddagger}, Mary Prorok §, Melanie E. DeFord §, Mariana Figuera §, Francis J. Castellino § and William P. Fay {ddagger} 

From the {ddagger}Research Service, Ann Arbor Veterans Affairs Hospital and the Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, Michigan 48109 and the §Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana 46556

Received for publication, February 20, 2003 , and in revised form, April 18, 2003.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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 {alpha}{beta}1 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 {alpha}2-antiplasmin. However, when fibrin-bound, the activities of {alpha}{beta}1 strand mutants were similar to that of wild-type (WT) SK and resistant to {alpha}2-antiplasmin. Mutation of Ile1 of SK also prevented nonproteolytic active site induction in bound Pg. However, unlike {alpha}{beta}1 strand mutants, the functional defect of Ile1 mutants was not relieved by fibrin, and complexes of Ile1 mutants and plasmin were resistant to {alpha}2-antiplasmin. Plasmin enhanced the activities of {alpha}{beta}1 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 {alpha}2-antiplasmin, and controls fibrin-independent Pg activation.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Streptokinase (SK),1 a protein secreted by several streptococcal species, binds and activates human plasminogen. Streptokinase may play an important role in streptococcal virulence by generating proteolytic activity at the bacterial cell surface, thereby facilitating invasion of host tissues (1). Streptokinase is also used as a clot-dissolving agent to treat thrombotic diseases (2). Unlike mammalian plasminogen activators, which activate plasminogen by limited proteolysis, streptokinase lacks intrinsic enzymatic activity. Rather, SK binds to plasminogen and induces within it an active site by a nonproteolytic mechanism (3). The SK-plasminogen complex converts free plasminogen molecules to plasmin. Plasminogen within the activator complex also is converted to plasmin by a poorly understood proteolytic mechanism, and the SK-plasmin complex is resistant to inactivation by {alpha}2-antiplasmin (4).

Streptokinase consists of 440 amino acids, including a 26-amino acid N-terminal signal peptide that is cleaved during secretion to yield the mature 414-amino acid protein of Mr 47,000 (2). Streptokinase contains three domains, denoted {alpha} (residues 1–150), {beta} (residues 151–287), and {gamma} (residues 288–414) (5). Each domain binds plasminogen, although none can activate plasminogen independently (58). Several lines of evidence suggest that residues 1–59 of the SK {alpha} domain play a key role in plasminogen activation. The Lys59-Ser60 peptide bond of SK is cleaved very rapidly upon incubation of SK with plasminogen, and the resulting N-terminal peptide remains associated with the SK-plasminogen complex (8, 9). SK deletion mutants lacking residues 1–59 form complexes with plasminogen that exhibit markedly reduced plasminogen activator (PA) activity, despite the fact that these mutant SK-plasminogen complexes can generate near normal amidolytic activity after prolonged incubation (7, 1012). Incubation of peptides containing amino acids 1–59 of SK with inactive deletion mutants lacking these residues restores PA activity (8, 9, 13). Deletion of Ile1 of SK markedly inhibits its capacity to induce an active site in plasminogen, which supports the hypothesis that establishment of a salt bridge between Ile1 of SK and Asp740 of plasminogen is necessary for SK to induce an active site in plasminogen by a nonproteolytic mechanism (11, 14). Residues 1–59 also appear to play an important role in determining the capacity of SK to efficiently activate plasminogen in the absence of fibrin, a property that distinguishes SK from tissue-type plasminogen activator. Whereas full-length SK efficiently activates plasminogen in the absence or presence of fibrin, the very low PA activity of SK-(60–414) is greatly enhanced by fibrin (15).

Several questions regarding the function of the amino-terminal fragment (residues 1–59) of the SK {alpha} domain remain unanswered. First, what residues play key roles in mediating the functional effects of the amino-terminal fragment on plasminogen activation? Although the role of Ile1 of SK in plasminogen activation has been examined in some detail (11, 14), less is known about the function of other specific residues (1619). Second, what is the mechanism by which complexes composed of plasminogen and SK mutants lacking portions of the amino terminus attain normal or nearly normal amidolytic activity, but only after a prolonged lag phase? And third, what are the mechanisms underlying the fibrin specificity exhibited by SK mutants lacking residues 1–59? The purpose of this study was to address these issues by generating and characterizing an extensive panel of recombinant SKs carrying mutations within residues 1–59.


    EXPERIMENTAL PROCEDURES
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Reagents—Glu-plasminogen was purified from human plasma by lysine-Sepharose chromatography (20). Purified plasminogen was incubated with D-Phe-Phe-Arg chloromethylketone dihydrochloride (Calbiochem-Novabiochem), a plasmin inhibitor, and dialyzed extensively before use. Plasmin was prepared by incubating plasminogen (2 mg/ml) with urokinase (0.5 units/ml; American Diagnostica). Chromogenic plasmin substrate (S-2251) was from Chromogenix. Human {alpha}2-antiplasmin and soluble fibrin fragments (Desafib-X) were from American Diagnostica. Recombinant human plasminogen resistant to hydrolysis at Arg561 (Pg R561A) was generated and purified as described (21). Human {alpha}-thrombin was from Calbiochem.

Generation and Purification of Recombinant SK—Mature (i.e. 414 amino acids, N-terminal isoleucine), wild-type SK (derived from Streptococcus equisimilis H46A) was generated and purified as described previously (10). Point mutations were introduced into the streptokinase coding sequence by using the pSELECT mutagenesis vector (Promega) according to the manufacturer's instructions. Ile1 and all clusters of charged amino acids within residues 1–59 of SK were mutated to alanine residues, as described previously (22). SK mutants lacking Ile1 (i.e. SK-(2–414)) or residues 1–59 (i.e. SK-(60–414)) were generated by a PCR-based strategy, as described (23). All mutations were confirmed by DNA sequencing. Mutant streptokinases were subcloned into pET-3a, expressed in Escherichia coli strain BL21(DE3)pLysS as mature length proteins, and purified to homogeneity as described (10). Protein sequence analysis of Ile1 point and deletion mutants confirmed the presence of the intended N-terminal residue.

Functional Assays—The capacity of streptokinase to generate amidolytic activity when in complex with plasminogen was determined by incubating SK (180 nM), plasminogen (60 nM), and S-2251 (300 µM) in 0.01 M Tris-HCl, 0.14 M NaCl, pH 7.5 (TBS), at room temperature and monitoring the absorbance of reaction mixtures at 405 nM (A405) for 15 min. The amidolytic activity of each complex, expressed as percentage of wild type, was calculated by dividing the A405/min of mutant complex by the A405/min of wild-type SK complex and then multiplying by 100. Standard curves constructed from different concentrations of wild-type SK-plasminogen complex revealed that A405/min was a linear function of complex concentration. SK plasminogen activator activity was studied by incubating SK (3.5 nM), plasminogen (700 nM), and S-2251 (300 µM) in TBS at room temperature and measuring the A405 15 min later. The plasminogen activator activity of each mutant, expressed as percentage of wild type, was calculated by dividing the A405 of mutant SK by the A405 of wild-type SK and then multiplying by 100. Standard curves of wild-type SK revealed that A405 was a linear function of SK concentration over the range of 0–3.5 nM. To study the role of the SK {alpha} domain in the resistance of the SK-plasmin complex to inhibition by {alpha}2-antiplasmin, SK (50 nM) and plasmin (10 nM) were incubated for 5 min at room temperature, and then {alpha}2-antiplasmin (50 nM) was added. Five minutes later, residual amidolytic activity of complexes was determined by hydrolysis of S-2251.

Effects of Fibrin on Activity of SK-Plasminogen Complex—To study the effects of fibrin on the amidolytic activity of SK-plasminogen complexes, SK (240 nM), Pg R561A (120 nM), and S-2251 (500 µM) were incubated in TBS with 1 mg/ml bovine serum albumin in the absence or presence of Desafib X (desAA fibrinogen X; 1.2 µM), and the A405 of the reaction mixture was monitored. Effects of fibrin on PA activities of complexes of SK and wild-type plasminogen were determined by incubating SK (3.5–16.5 nM), Glu-plasminogen (700 nM), Desafib X (1 µM), and S-2251 (300 µM) and monitoring A405. To study the amidolytic and PA activities of SK-plasminogen complexes bound to insoluble fibrin matrices, fibrin-coated microtiter plate wells (96 wells/plate) were prepared by incubating fibrinogen (1.4 µM) and thrombin (0.5 units/ml) in 100 µl of phosphate-buffered saline (0.05 M sodium phosphate, 0.15 M NaCl, pH 7.5) and air-drying plates overnight at 37 °C. Wells were blocked for 1 h with 3% bovine serum albumin containing D-Phe-Pro-Arg chloromethylketone dihydrochloride, a thrombin inhibitor (10 µM; Calbiochem), and washed five times with rinsing buffer (0.9% NaCl, 0.05% Triton X-100). Wild-type plasminogen or Pg R561A (50–100 nM in 100 µl of TBS containing 1 mg/ml bovine serum albumin) was added to wells. One hour later, wells were washed extensively, and then SK (0–150 nM) was added. To measure the amidolytic activity of complexes, S-2251 (300 µM) was added to wells in the absence or presence of {alpha}2-antiplasmin (240 nM), and the A405 of wells was monitored. To measure the PA activity of fibrin-bound complexes, wells were washed extensively 30 min after adding SK, and then 100 µl of wild-type plasminogen (1 µM) was added to wells. After 20 min at room temperature, 25 µl were removed and transferred to wells containing S-2251 (final concentration 300 µM) to determine plasmin formation.

Plasma Clot Lysis—Pooled, citrated human plasma (50 µl), containing a trace amount of 125I-human fibrinogen (Amersham Biosciences), was clotted in the presence of a plastic rod (to facilitate clot washing and transfer) by the addition of thrombin (1 unit/ml) and CaCl2 (5 mM). After 1 h at 37 °C, clots were washed five times with TBS containing hirudin (1 µg/ml) and then suspended in citrated plasma (450 µl) containing streptokinase (10–50 nM) and hirudin (1 µg/ml). After 0–120 min of gentle rocking at 37 °C, the percentage of clot lysis was calculated by comparing radioactive counts in the plasma with those present in the washed clot prior to its placement in plasma. Plasma fibrinogen concentrations were measured as described (15).

Other Methods—Protein concentrations of purified proteins were determined with the BCA Reagent (Pierce). DNA sequencing was performed by the Sanger method. SDS-PAGE was performed with the PhastSystem (Amersham Biosciences). Gels were stained with Coomassie Brilliant Blue R. Differential scanning calorimetry was performed as described (21). CD spectra analyses were performed at 25 °C with a JASCO J-600 spectropolarimeter with a 1.0-nm bandwidth and a 0.5-nm step resolution.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Effects of Point Mutations on SK PA Activity—We generated an extensive panel of point mutations within residues 1–59 of SK. Mutations targeted by a charged-to-alanine strategy did not significantly decrease SK PA activity (Table I), suggesting that none of the clusters of charged residues in this region was essential for normal activator activity. Of note, a 3-fold increase in PA activity was observed for SK K36A. Mutation of Ile1 of SK to an alanine caused a moderate decrease in PA activity (to 23 ± 7% that of wild-type SK) that was characterized by a lag phase in the appearance of plasmin formation, after which the PA activity of SK I1A was indistinguishable from that of wild-type SK (Fig. 1). A relatively long series of hydrophobic amino acids (residues 11–26), which is missed by a charged-to-alanine mutagenesis strategy, is located within the SK amino-terminal fragment, and deletions into this region are associated with a marked reduction in PA activity (9, 10, 24). Therefore, we generated a series of neutral-to-charged mutations between positions 11 and 26 of SK. Several mutations within this region decreased PA activity to ~1% that of wild-type SK (i.e. L18D, V20D, V20K, and V22D), whereas the PA activities of SK V19D and SK G24D were reduced to 13.6 ± 5% and 2.5 ± 2% that of wild-type SK, respectively. Differential scanning calorimetry of SK S16D, L18D, and G24D revealed that they exhibited a single temperature of maximum heat capacity (Tm) of ~45 °C, whereas wild-type SK and SK K36A exhibited two Tm of ~45 °C and 55–60 °C (Table II). However, the secondary structural characteristics of mutants exhibiting abnormal calorimetry profiles (i.e. SK S16D, L18D, and G24D), as evaluated by CD spectroscopy in the near-UV (240–320 nm) and far-UV (190–260 nm) regions, did not differ from those of wild-type SK (data not shown). These results suggested that the mutations introduced between positions 18 and 24 altered the tertiary structure of the {alpha} domain of SK.


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TABLE I
Amidolytic and plasminogen activator activities of streptokinase mutants in complex with plasminogen Data are mean ± 1 S.D. of triplicate experiments.

 


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FIG. 1.
Plasminogen activator activity of SK I1A. Plasminogen (700 nM) was incubated alone or with wild-type SK or SK I1A (3.5 nM), and plasmin formation was monitored by hydrolysis of S-2251 (300 µM). The experiment was repeated in triplicate with indistinguishable results.

 

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TABLE II
Temperature(s) of maximum heat capacity (Tm) of recombinant streptokinases Each Tm corresponds to the midpoint of the peak of the raw thermogram. Values shown represent the mean of two separate determinations, which varied by <2%.

 

Effects of Point Mutations on SK Amidolytic Activity— Charged-to-alanine mutations induced at most only minor reductions in the amidolytic activities of SK-plasminogen complexes (Table I). The amidolytic activity of plasminogen in complex with SK K36A was ~2-fold greater than that of wild-type complex (Table I and Fig. 2). The amidolytic activity observed during a 15-min incubation of SK I1A with Glu-plasminogen was severely reduced, although gradually increasing rates of S-2251 hydrolysis were observed after a prolonged lag phase (Fig. 2). Similarly, the cluster of mutations between positions 18 and 24 of SK markedly inhibited the amidolytic activity of the activator complex. However, in two-stage assays (in which delayed generation of amidolytic activity during incubation of equimolar SK and plasminogen was confirmed by hydrolysis of S-2251 (stage 1) and then a 200-fold molar excess of plasminogen was added to the reaction mixture to assess PA function (stage 2)), the PA activity of SK I1A was indistinguishable from that of wild-type SK, whereas the PA activities of position 18–24 mutants were not detectable or markedly reduced (data not shown). These results suggested that once an active site was formed, complexes containing mutations at position 1 of SK processed substrate plasminogen normally, whereas complexes containing mutations at positions 18–24 of SK were severely defective in converting substrate plasminogen to plasmin.



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FIG. 2.
Amidolytic activities of SK-plasminogen complexes. Plasminogen (80 nM) was incubated alone or with SK (240 nM), and generation of amidolytic activity was monitored by hydrolysis of S-2251 (300 µM). The experiment was performed at least three times with similar results.

 

Analysis of Mechanisms Underlying the Lag in the Generation of Amidolytic Activity by SK {alpha} Domain Mutants—We studied the capacity of recombinant SKs (240 nM) to induce amidolytic activity in Pg R561A (120 nM), a mutant zymogen incapable of conversion to plasmin (21). Neither SK I1A nor any of the position 18–24 mutants was able to generate amidolytic activity during a 1-h incubation at 37 °C (data not shown). All other point mutants induced amidolytic activity comparable with that of wild-type SK during incubation with Pg R561A, except for SK E39A,D41A, which exhibited an activity of 18.1 ± 5.5% that of wild-type SK. These results suggested that mutations at position 1 or positions 18–24 of SK prevented rapid, proteolysis-independent formation of an active site in complexed plasminogen. The addition of plasmin (1.6 nM) to reaction mixtures of wild-type plasminogen (80 nM) and SK I1A, SK L18D, or SK G24D (each at 240 nM) accelerated S-2251 hydrolysis to a much greater extent than could be explained simply by the amidolytic activity of added plasmin (Fig. 3, B–D) but had no significant effect on wild-type SK (Fig. 3A). However, the addition of trace plasmin did not enable SK I1A, SK L18D, or SK G24D to generate amidolytic activity in Pg R561A (data not shown), suggesting that plasmin catalyzed active site formation by a mechanism that involved hydrolysis of the plasminogen Arg561-Val562 bond. Consistent with these data, SDS-PAGE analysis of equimolar reaction mixtures of SK I1A and wild-type plasminogen revealed that the appearance of amidolytic activity correlated with the delayed conversion of plasminogen within the activator complex to plasmin, whereas the appearance of amidolytic activity of complexes composed of wild-type SK and wild-type plasminogen was immediate and preceded conversion of plasminogen to plasmin (Fig. 4).



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FIG. 3.
Effects of plasmin and {alpha}2-antiplasmin on the amidolytic activities of activator complexes. Streptokinase (240 nM) was incubated with plasminogen (80 nM), and generation of amidolytic activity was monitored by hydrolysis of S-2251 (300 µM). Plasmin (PL; 1.6 nM) with or without {alpha}2-antiplasmin (AP; 240 nM) was added to some reaction mixtures, as indicated. A, wild-type SK. The dashed line represents a control reaction consisting of plasmin (1.6 nM) and S-2251. The open circles represent incubation of plasminogen (80 nM) and S-2251 in the absence of SK. B, SK I1A; C, SK L18D; D, SK G24D.

 


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FIG. 4.
SDS-PAGE analysis and amidolytic activity of SK I1A-plasminogen complexes. Wild-type SK or SK I1A (7.8 µM) and wild-type plasminogen (7.8 µM) were incubated at 15 °C. At the indicated time points, samples of the reaction mixture were removed and subjected to SDS-PAGE analysis under reducing conditions (A, wild-type SK; B, SK I1A) and measurement of amidolytic activity (after 100-fold dilution) by incubation with 300 µM S-2251 (C). Molecular masses (in kDa) are shown at the right of A and B. Plg, plasminogen; Pl-HC, plasmin heavy chain; PL-LC, plasmin light chain; SK, native and proteolytically modified forms of streptokinase. The experiment was performed three times with similar results.

 

Fibrin Dependence of SK {alpha} Domain Point Mutants—Wild-type SK or SK point mutants were incubated with Pg R561A in the presence or absence of soluble fibrin fragments, and amidolytic activity was monitored. Whereas SKs containing mutations between positions 18 and 24 did not generate significant amidolytic activity in the absence of fibrin, their activities in the presence of fibrin were comparable with that of wild-type SK (Fig. 5). However, fibrin had no significant effect on the markedly reduced capacity of SK I1A to generate amidolytic activity in Pg R561A. Similarly, the PA activities of fibrin-bound complexes containing Pg R561A and SKs with mutations at residues 18–24 were comparable with those containing wild-type SK, whereas the PA activity of fibrin-bound complexes containing SK I1A was markedly reduced, and the PA activity of fibrin-bound complexes containing SK-(2–414) was undetectable (data not shown). These results indicated that 1) point mutations within the {beta}1 strand of the {alpha} domain generated SKs that exhibited markedly reduced amidolytic and PA activities in the absence of fibrin but normal or nearly normal activities in the presence of fibrin, and 2) even in the presence of fibrin, Ile1 was required for SK to efficiently induce active site formation in plasminogen by a nonproteolytic mechanism.



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FIG. 5.
Effect of fibrin on the amidolytic activities of complexes of Pg R561A and SK. Pg R561A (120 nM) was incubated with wild-type SK (240 nM), the indicated SK mutants (240 nM), or buffer (No SK) in the absence (white bars) or presence (black bars) of soluble fibrin fragments (1.2 µM). Amidolytic activity was measured by hydrolysis of S-2251. Data represent mean ± 1 S.D. of three or four experiments.

 

Inhibition of Activator Complexes by {alpha}2-Antiplasmin and Modulation by Fibrin—To study the role of the SK {alpha} domain in the resistance of the SK-plasmin complex to inhibition by {alpha}2-antiplasmin, we incubated SK (50 nM) and plasmin (10 nM) for 5 min and then added {alpha}2-antiplasmin (50 nM). Five minutes later, residual amidolytic activity of complexes was determined by hydrolysis of S-2251. No significant inhibition of wild-type SK-plasmin complex by antiplasmin was observed. In contrast, marked inhibition of S-2251 hydrolysis by antiplasmin was observed during incubation of plasmin with the following SK mutants: S16K, L18D, V19D, V20K, V20D, V22D, G24D, and SK-(60–414). Complexes of plasmin and all other point mutants listed in Table I were not inhibited by {alpha}2-antiplasmin (data not shown). Consistent with these results, antiplasmin (240 nM) essentially completely inhibited the generation of amidolytic activity during a 1-h incubation of SK L18D or SK G24D (each at 240 nM) with plasminogen (80 nM) and trace plasmin (1.6 nM; Fig. 3, C and D). However, complexes formed by incubating plasminogen and SK L18D or SK G24D in fibrin-coated microtiter plate wells were resistant to inhibition by {alpha}2-antiplasmin (Fig. 6). These results suggested that the SK {alpha} domain contributes to the resistance of the SK-plasmin complex to inhibition by {alpha}2-antiplasmin and that mutations within the {beta}1 strand of the {alpha} domain generated mutants with fibrin-dependent resistance to antiplasmin.



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FIG. 6.
Resistance of fibrin-bound activator complexes to inhibition by {alpha}2-antiplasmin. Plasminogen (50 nM) was incubated in fibrin-coated wells for 1 h, and then wells were washed, and SK (150 nM) and S-2251 (300 µM) were added in the absence or presence of {alpha}2-antiplasmin (240 nM). Control fibrin-coated wells contained Pg R561A but no SK. Amidolytic activity of fibrin-bound complexes was determined by measuring the rate of S-2251 hydrolysis. Data represent mean ± 1 S.D. of three experiments.

 

Effects of Fibrin on the Activity of SK-(60–414)—The PA activity of SK-(60–414) is severely reduced, yet it is markedly enhanced by fibrin (15). We performed a series of experiments to identify potential mechanisms underlying the effect of fibrin on SK-(60–414). SK-(60–414) (240 nM) did not generate amidolytic activity during incubation with Pg R561A (120 nM), either in the absence or presence of soluble fibrin fragments (1.2 µM; Fig. 5), suggesting that fibrin could not substitute for residues 1–59 of SK to provide structural elements necessary to induce an active site in plasminogen by a nonproteolytic mechanism. However, fibrin significantly enhanced the capacity of complexes of SK-(60–414) and wild-type plasminogen to generate PA activity, although the specific activity of SK-(60–414) in the presence of fibrin was markedly less than that of wild-type SK (Fig. 7A). The addition of trace plasmin potently accelerated the generation of PA activity during incubation of SK-(60–414) and wild-type plasminogen in the presence of fibrin but not in its absence (Fig. 7B). These results suggested that activation of plasminogen by SK-(60–414) required not only fibrin but also SK-(60–414)-plasmin complex (as opposed to SK-plasminogen complex), with the plasmin provided as a trace contaminant in the plasminogen preparation. However, the rate of plasminogen activation by SK-(60–414)-plasmin, even in the presence of fibrin, was markedly slower than that induced by wild-type SK.



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FIG. 7.
Effect of fibrin and plasmin on the plasminogen activator activity of SK-(60–414). A, effect of fibrin. Wild-type SK or SK-(60–414) (10 nM) was incubated with plasminogen (Plg; 300 nM) and S-2251 (300 µM) in the absence or presence of soluble fibrin fragments (Fn; 1 µM), and plasmin formation was determined by measuring substrate hydrolysis. Control reactions lacking SK are shown. B, effect of plasmin. SK-(60–414) (10 nM) was incubated with plasminogen (Plg; 300 nM) and S-2251 (300 µM). Soluble fibrin fragments (Fn;1 µM) and/or plasmin (PL; 6 nM) were added to some reactions, as indicated, and a control reaction containing plasminogen, plasmin, and fibrin, but lacking SK-(60–414), was included. Plasmin formation was determined by measuring S-2251 hydrolysis.

 

Previous studies suggested that SK-(60–414) exhibits a greater fibrinolytic potency than wild-type SK (15). We compared the capacity of wild-type SK and SK-(60–414) to lyse plasma clots. At a concentration of 10 nM, wild-type SK exhibited much greater fibrinolytic potency than SK-(60–414) (Fig. 8A). At a concentration of 50 nM, wild-type SK was superior to SK-(60–414) at early time points (15–30 min) but at later time points SK-(60–414) induced more extensive clot lysis than did wild-type SK (Fig. 8B). Plasma fibrinogen depletion during clot lysis with wild-type SK was relatively mild at a concentration of 10 nM but was severe at a concentration of 50 nM (Fig. 8C). In contrast, minimal depletion of plasma fibrinogen was observed with either concentration of SK-(60–414). These results suggested that SK-(60–414) exhibits greater fibrinolytic potency than wild-type SK only under conditions in which plasma concentrations of wild-type SK are high enough to induce extensive activation of plasminogen in plasma, thereby depleting plasma fibrinogen and plasminogen. Consistent with this hypothesis, the addition of purified plasminogen to plasma during clot lysis potentiated the fibrinolytic efficacy of wild-type SK but had no detectable effect on SK-(60–414) (Fig. 8B).



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FIG. 8.
Induction of plasma clot lysis by wild-type SK and SK-(60–414). Radiolabeled fibrin clots were suspended in hirudin-anticoagulated plasma. Wild-type SK or SK-(60–414) was added, and clot lysis was monitored by measuring radioactive counts in the plasma at the indicated time points. A, concentration of SK was 10 nM. A control reaction lacking SK is also shown. B, concentration of SK was 50 nM.As indicated, additional plasminogen (Pg; 0.43 nmol) was added to some reaction mixtures at each time point at which clot lysis was measured. C, capacity of wild-type SK and SK-(60–414) to deplete plasma fibrinogen. Wild-type SK or SK-(60–414) (each at a concentration of 10 or 50 nM) was added to plasma, and 2 h later, plasma fibrinogen concentration was measured. Results are standardized to control reactions, which lacked SK. Data represent the mean of triplicate experiments.

 


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
None of the mutants generated by a charged-to-alanine mutagenesis strategy exhibited a significant defect in amidolytic or PA activity during incubation with wild-type plasminogen. However, when the panel of charged-to-alanine mutants was screened with Pg R561A (i.e. under conditions in which active site formation can occur only via a nonproteolytic mechanism), the capacity of SK E39A,D41A to induce amidolytic activity was moderately to severely reduced. Residues 39–41 of SK are located in the {beta}2 strand of the {alpha} domain. These results suggest an important role of the {alpha}{beta}2 strand in establishing an active site in plasminogen and support the hypothesis that a salt bridge between Glu39 of SK and Arg719 of plasminogen plays an important but not essential role in the formation of an active complex (5). We also observed that mutation of Lys36, located immediately adjacent to the {beta}2 strand, resulted in a significant increase in SK amidolytic activity. Within the activator complex, Lys36 of SK is in close proximity to the active site of plasmin (5). It is possible that mutations at or close to this residue produce steric or charge effects that facilitate substrate access to the active site. These results support the feasibility of using mutagenesis to increase SK function.

Mutations in the {alpha}{beta}1 strand (residues 17–26) of SK markedly reduced the formation of an active complex during incubation with plasminogen. Mutations between residues 18 and 24 may have altered the position or mobility of the SK N terminus and prevented Ile1 from assuming a conformation required for it to interact with Asp740 of plasminogen to induce active site formation by a nonproteolytic mechanism (14). However, complexes of plasmin and SKs harboring mutations between positions 18–24 were severely defective in PA activity, whereas complexes of SK I1A or SK-(2–414) and plasmin exhibited normal PA activity. These results suggested that 1) although Ile1 of SK plays a key role in inducing an active site in complexed plasminogen (11, 14), Ile1 is not required for normal processing of substrate plasminogen by the complex, and 2) the loss of PA function of position 18–24 mutants was not mediated simply by steric effects on Ile1. Since residues 18–24 reside within the hydrophobic core of the {alpha} domain, mutations in this region may have disrupted protein folding and substrate turnover by the activator complex. Consistent with this hypothesis, mutations in this region were associated with apparent structural changes in SK, as assessed by differential scanning calorimetry. However, the lack of significant changes in the CD spectra of these mutants and the observation that fibrin restored the capacity of position 18–24 mutants (but not SK-(60–414)) to induce amidolytic activity in Pg R561A suggested that their potential alterations in {alpha} domain structure were not irreversible and were probably minor. The SK {alpha}{beta}1 strand has extensive contact with residues 713–721 of plasminogen, with Arg719 forming a van der Waals contact with Val19 of SK (5). Our data support the hypothesis that contact between residues 713–721 of plasminogen and the {beta}1 strand of the SK {alpha} domain is essential for rapid formation of a fully functional complex.

Mechanism of Delayed Generation of Amidolytic Activity by SK Mutants Lacking Ile1Although Ile1 of SK appears to be required for SK to induce an active site in complexed plasminogen (11, 14), complexes of plasminogen and SK N-terminal deletion mutants lacking Ile1 can, after a lag phase, generate nearly normal amidolytic activity (7, 1012). Our studies suggest that active (i.e. SK I1A-plasmin) complexes can activate inactive (i.e. SK I1A-plasminogen) complexes by hydrolyzing the latter's Arg561-Val562 plasminogen bond, as hypothesized by Wang et al. (14). It appears that SK mutants lacking normal function of Ile1, due to mutation of that residue or due to steric effects of other mutations, require trace amounts of plasmin, which binds to SK to generate active complexes that trigger progressive activation of other complexes, resulting in an activation mechanism similar to that of staphylokinase (25).

Role of Residues 1–59 of SK in Fibrin-independent Plasminogen Activation—Deletion of residues 1–59 of SK renders it fibrin-dependent by a poorly defined mechanism (15). We found that fibrin did not enable SK-(60–414) to generate amidolytic activity in Pg R561A, suggesting that fibrin cannot substitute for the N-terminal fragment of SK to enable formation of an activator complex by a proteolysis-independent mechanism. However, our results suggest that once plasmin is generated, fibrin can partially substitute for the N-terminal fragment of SK to enable a reduced turnover rate of substrate plasminogen, whereas free SK-(60–414)-plasmin complexes are essentially devoid of PA activity. The environment of the fibrin clot is ideally suited to provide plasmin to "prime" SK-(60–414), since fibrin enhances plasminogen activation by tissue-type plasminogen activator, and fibrin-bound plasmin is resistant to inhibition by {alpha}2-antiplasmin (26). Our data suggest that the fibrin selectivity of SK-(60–414) is accounted for by its requirement of both fibrin and plasmin to generate a complex with PA activity, thereby targeting plasminogen activation to the clot surface. Reed et al. found that SK-(60–414) was a superior fibrinolytic agent to wild-type SK at all concentrations studied (15). In contrast, we found that SK-(60–414) induced less clot lysis than wild-type SK at low concentrations but more lysis than wild-type SK at higher concentrations. The fibrinolytic superiority of wild-type SK over SK-(60–414) at lower concentrations is consistent with our kinetic data. The superiority of SK-(60–414) over wild-type SK at higher concentrations appears to be due to the capacity of wild-type SK, but not SK-(60–414), to activate plasminogen in plasma, thereby leading to "plasminogen steal" and an arrest of clot lysis (27). Consistent with this hypothesis, the addition of purified plasminogen to plasma accelerated clot lysis by wild-type SK, but not SK-(60–414). Since SK N-terminal deletion mutants have been proposed as potential therapeutic agents (15), it is important to realize that their fibrinolytic efficacy depends on their plasma concentration.

The effect of fibrin on the amidolytic and PA activities of SK {alpha}{beta}1 strand point mutants was similar to that observed with SK-(60–414), although more potent. In the presence of fibrin, the activities of SK {alpha}{beta}1 strand point mutants were significantly greater than that of SK-(60–414) and comparable with that of wild-type SK. However, even in the presence of fibrin, Ile1 is required to efficiently induce an active site in plasminogen by a nonproteolytic mechanism. We hypothesize that the fibrin dependence of {alpha}{beta}1 strand mutants is explained by conformational changes induced by fibrin that restore the ability of Ile1 of SK to establish a salt bridge with Asp740 of plasminogen, which appears necessary for nonproteolytic active site induction (14).

Role of SK {alpha} Domain in Resistance of the Activator Complex to {alpha}2-Antiplasmin—Our studies suggest an important role of the SK {alpha} domain in mediating the resistance of the SK-plasmin complex to inhibition by antiplasmin. Previous studies demonstrated that the affinity of SK-(55–414) for plasmin is markedly reduced compared with wild-type SK (28), suggesting that residues 1–59 of SK play a key role in maintaining the cohesiveness of the SK-plasmin complex. It is possible that deletion of residues 1–59 or mutation of residues within the {alpha}{beta}1 strand enable antiplasmin to displace SK from the SK-plasmin complex and inhibit plasmin.

In summary, our studies identify key residues within residues 1–59 of SK that mediate its functional properties, and they provide insights into the mechanisms by which the SK {alpha} domain coordinates the rapid assembly of a functional activator complex, facilitates fibrin-independent plasminogen activation, and contributes to the resistance of the activator complex to inhibition by {alpha}2-antiplasmin. Further studies are indicated to examine the potential use of SK {alpha} domain mutants as fibrin-specific plasminogen activators.


    FOOTNOTES
 
* This work was supported by a Merit Review Award from the Department of Veterans Affairs (to W. P. F.) and by National Institutes Grants HL 57346 (to W. P. F.) and HL13423 (to F. J. C.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

To whom correspondence should be addressed: University of Michigan Medical Center, 1150 W. Medical Center Dr., Ann Arbor, MI 48109-0644. Tel.: 734-763-7838; Fax: 734-936-2641; E-mail: wfay{at}umich.edu.

1 The abbreviations used are: SK, streptokinase; PA, plasminogen activator; TBS, Tris-buffered saline; WT, wild-type. Back



    REFERENCES
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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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