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

Abstract

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 αβ1 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 α2-antiplasmin. However, when fibrin-bound, the activities of αβ1 strand mutants were similar to that of wild-type (WT) SK and resistant to α2-antiplasmin. Mutation of Ile1 of SK also prevented nonproteolytic active site induction in bound Pg. However, unlike αβ1 strand mutants, the functional defect of Ile1 mutants was not relieved by fibrin, and complexes of Ile1 mutants and plasmin were resistant to α2-antiplasmin. Plasmin enhanced the activities of αβ1 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 α domain regulates rapid active site induction in bound Pg, contributes to the resistance of the SK-plasmin complex to α2-antiplasmin, and controls fibrin-independent Pg activation. 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 αβ1 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 α2-antiplasmin. However, when fibrin-bound, the activities of αβ1 strand mutants were similar to that of wild-type (WT) SK and resistant to α2-antiplasmin. Mutation of Ile1 of SK also prevented nonproteolytic active site induction in bound Pg. However, unlike αβ1 strand mutants, the functional defect of Ile1 mutants was not relieved by fibrin, and complexes of Ile1 mutants and plasmin were resistant to α2-antiplasmin. Plasmin enhanced the activities of αβ1 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 α domain regulates rapid active site induction in bound Pg, contributes to the resistance of the SK-plasmin complex to α2-antiplasmin, and controls fibrin-independent Pg activation. Streptokinase (SK), 1The abbreviations used are: SK, streptokinase; PA, plasminogen activator; TBS, Tris-buffered saline; WT, wild-type. 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 (1Boyle M.D.P. Lottenberg R. Thromb. Haemostasis. 1997; 77: 1-10Google Scholar). Streptokinase is also used as a clot-dissolving agent to treat thrombotic diseases (2Reddy K.N. Enzyme. 1988; 40: 79-89Google Scholar). 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 (3Bajaj A.P. Castellino F.J. J. Biol. Chem. 1977; 252: 492-498Google Scholar). 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 α2-antiplasmin (4Cederholm-Williams S. De Cock F. Lijnen H. Collen D. Eur. J. Biochem. 1979; 100: 125-132Google Scholar). 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 (2Reddy K.N. Enzyme. 1988; 40: 79-89Google Scholar). Streptokinase contains three domains, denoted α (residues 1–150), β (residues 151–287), and γ (residues 288–414) (5Wang X. Lin X. Loy J. Tang J. Zhang X. Science. 1998; 281: 1662-1665Google Scholar). Each domain binds plasminogen, although none can activate plasminogen independently (5Wang X. Lin X. Loy J. Tang J. Zhang X. Science. 1998; 281: 1662-1665Google Scholar, 6Conejero-Lara F. Parrado J. Azuaga A.I. Dobson C.M. Ponting C.P. Protein Sci. 1998; 7: 2190-2199Google Scholar, 7Rodriguez P. Fuentes P. Barro M. Alvarez J.G. Munoz E. Collen D. Lijnen H.R. Eur. J. Biochem. 1995; 229: 83-90Google Scholar, 8Shi G.-Y. Wu D.-H. Biochem. J. 1994; 304: 235-241Google Scholar). Several lines of evidence suggest that residues 1–59 of the SK α 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 (8Shi G.-Y. Wu D.-H. Biochem. J. 1994; 304: 235-241Google Scholar, 9Nihalani D. Kumar R. Rajagopal K. Sahni G. Protein Sci. 1998; 7: 637-648Google Scholar). 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 (7Rodriguez P. Fuentes P. Barro M. Alvarez J.G. Munoz E. Collen D. Lijnen H.R. Eur. J. Biochem. 1995; 229: 83-90Google Scholar, 10Fay W.P. Bokka L.V. Thromb. Haemostasis. 1998; 79: 985-991Google Scholar, 11Wang S. Reed G. Hedstrom L. Biochemistry. 1999; 38: 5232-5240Google Scholar, 12Young K.-C. Shi G.-Y. Chang Y.-F. Chang L.-C. Lai M.-D. Chuang W.-J. Wu H.-L. J. Biol. Chem. 1995; 270: 29601-29606Google Scholar). Incubation of peptides containing amino acids 1–59 of SK with inactive deletion mutants lacking these residues restores PA activity (8Shi G.-Y. Wu D.-H. Biochem. J. 1994; 304: 235-241Google Scholar, 9Nihalani D. Kumar R. Rajagopal K. Sahni G. Protein Sci. 1998; 7: 637-648Google Scholar, 13Parrado J. Conejero-Lara F. Smith R.A.G. Marshall J.M. Ponting C.P. Dobson C.M. Protein Sci. 1996; 5: 693-704Google Scholar). 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 (11Wang S. Reed G. Hedstrom L. Biochemistry. 1999; 38: 5232-5240Google Scholar, 14Wang S. Reed G. Hedstrom L. Eur. J. Biochem. 2000; 267: 3994-4001Google Scholar). 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 (15Reed G.L. Houng A.K. Liu L. Parhami-Seren B. Matsueda L.H. Wang S. Hedstrom L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 8879-8883Google Scholar). Several questions regarding the function of the amino-terminal fragment (residues 1–59) of the SK α 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 (11Wang S. Reed G. Hedstrom L. Biochemistry. 1999; 38: 5232-5240Google Scholar, 14Wang S. Reed G. Hedstrom L. Eur. J. Biochem. 2000; 267: 3994-4001Google Scholar), less is known about the function of other specific residues (16Lee S.H. Jeong S.T. Kim I.C. Byun S.M. Biochem. Mol. Biol. Int. 1997; 41: 199-207Google Scholar, 17Kim D. Lee S. Kim I. Kim S. Byun S. Thromb Res. 2000; 99: 93-98Google Scholar, 18Lee B.R. Park S.K. Kim J.H. Byun S.M. Biochem. Biophys. Res. Commun. 1989; 165: 1085-1090Google Scholar, 19Liu L. Sazonova I.Y. Turner R.B. Chowdhry S.A. Tsai J. Houng A.K. Reed G.L. J. Biol. Chem. 2000; 275: 37686-37691Google Scholar). 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. Reagents—Glu-plasminogen was purified from human plasma by lysine-Sepharose chromatography (20Deutsch D. Mertz E. Science. 1970; 170: 1095-1096Google Scholar). Purified plasminogen was incubated with d-Phe-Phe-Arg a plasmin and Plasmin was by plasminogen with plasmin substrate was from α2-antiplasmin and fibrin were from human plasminogen resistant to at was and purified as Castellino F.J. Protein 1999; Scholar). was from and of amino acids, N-terminal wild-type SK from was and purified as W.P. Bokka L.V. Thromb. Haemostasis. 1998; 79: 985-991Google Scholar). mutations were the streptokinase by the to the Ile1 and of amino acids within residues 1–59 of SK were to as D. L. J. Biol. Chem. Scholar). SK mutants lacking Ile1 or residues 1–59 were by a as W.P. J. Biol. Chem. Scholar). mutations were by were in as mature and purified to as W.P. Bokka L.V. Thromb. Haemostasis. 1998; 79: 985-991Google Scholar). Protein analysis of Ile1 and deletion mutants the presence of the N-terminal capacity of streptokinase to generate amidolytic activity when in complex with plasminogen was by SK plasminogen and in at and the of at for The amidolytic activity of as of was by the of mutant complex by the of wild-type SK complex and by from concentrations of wild-type SK-plasminogen complex that was a function of complex SK plasminogen activator activity was by SK plasminogen and in at and the The plasminogen activator activity of as of was by the of mutant SK by the of wild-type SK and by of wild-type SK that was a function of SK the of study the role of the SK α domain in the resistance of the SK-plasmin complex to inhibition by α2-antiplasmin, SK and plasmin were incubated for at and α2-antiplasmin was amidolytic activity of complexes was by of of on of study the effects of fibrin on the amidolytic activity of SK-plasminogen SK Pg and were incubated in with in the absence or presence of and the of the was of fibrin on PA activities of complexes of SK and wild-type plasminogen were by SK and and study the amidolytic and PA activities of SK-plasminogen complexes bound to fibrin were by and in of and at were for with containing a and with plasminogen or Pg in of containing was to were and SK was the amidolytic activity of was to in the absence or presence of α2-antiplasmin and the of was the PA activity of were after SK, and of wild-type plasminogen was to at were and to containing to plasmin human plasma containing a of was in the presence of a and by the of and at were with containing and in plasma containing streptokinase and of at the of was by in the plasma with in the to its in concentrations were as (15Reed G.L. Houng A.K. Liu L. Parhami-Seren B. Matsueda L.H. Wang S. Hedstrom L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 8879-8883Google Scholar). concentrations of purified were with the was by the was with the were with R. was as Castellino F.J. Protein 1999; Scholar). were at with a with a and a of Mutations on SK PA an extensive panel of mutations within residues 1–59 of SK. Mutations by a not SK PA activity suggesting that none of the of residues in this was for normal activator activity. a in PA activity was for SK Mutation of Ile1 of SK to an a in PA activity that of wild-type that was by a lag in the of plasmin after which the PA activity of SK was from that of wild-type SK of amino acids (residues which is by a is within the SK amino-terminal and this are associated with a in PA activity D. Kumar R. Rajagopal K. Sahni G. Protein Sci. 1998; 7: 637-648Google Scholar, 10Fay W.P. Bokka L.V. Thromb. Haemostasis. 1998; 79: 985-991Google Scholar, Tang J. Thromb. Res. Scholar). a of mutations between and of SK. Several mutations within this PA activity to that of wild-type SK and the PA activities of SK and SK were reduced to and that of wild-type SK, of SK and that exhibited a of capacity of wild-type SK and SK exhibited of and However, the of mutants SK and as by in the and not from of wild-type SK not These suggested that the mutations between and 24 the of the α domain of and plasminogen activator activities of streptokinase mutants in complex with plasminogen are of activity of SK-plasminogen complex of wild-type SK-plasminogen activator activity of SK-plasminogen complex of wild-type SK-plasminogen in a of capacity of recombinant Each to the of the of the the of which by in a of Mutations on SK mutations at only in the amidolytic activities of SK-plasminogen complexes The amidolytic activity of plasminogen in complex with SK was greater than that of wild-type complex and The amidolytic activity during a incubation of SK with was although of were after a prolonged lag the of mutations between and 24 of SK markedly the amidolytic activity of the activator However, in which of amidolytic activity during incubation of SK and plasminogen was by of and a of plasminogen was to the to PA function the PA activity of SK was from that of wild-type SK, the PA activities of mutants were not or markedly reduced not These suggested that an active site was complexes containing mutations at of SK substrate plasminogen complexes containing mutations at of SK were in substrate plasminogen to plasmin. of the in the of by SK α the capacity of recombinant SKs to induce amidolytic activity in Pg a mutant incapable of to plasmin Castellino F.J. Protein 1999; Scholar). SK of the mutants was to generate amidolytic activity during a incubation at not other mutants amidolytic activity with that of wild-type SK during incubation with Pg for SK which exhibited an activity of that of wild-type SK. These suggested that mutations at or of SK prevented of an active site in plasminogen. The of plasmin to of wild-type plasminogen and SK SK or SK at to a greater than by the amidolytic activity of plasmin but on wild-type SK However, the of plasmin not SK SK or SK to generate amidolytic activity in Pg not suggesting that plasmin active site by a mechanism that of the plasminogen Arg561-Val562 bond. with these analysis of of SK and wild-type plasminogen that the of amidolytic activity with the of plasminogen within the activator complex to the of amidolytic activity of complexes composed of wild-type SK and wild-type plasminogen was and of plasminogen to plasmin analysis and amidolytic activity of SK SK or SK and wild-type plasminogen were incubated at the of the were and to analysis wild-type SK and of amidolytic activity by incubation with are at the of and B. plasmin plasmin SK, and of The was three with similar of SK α SK or SK mutants were incubated with Pg in the presence or absence of fibrin and amidolytic activity was Whereas SKs containing mutations between and 24 not generate amidolytic activity in the absence of fibrin, activities in the presence of fibrin were with that of wild-type SK However, fibrin on the markedly reduced capacity of SK to generate amidolytic activity in Pg the PA activities of complexes containing Pg and SKs with mutations at residues were with containing wild-type SK, the PA activity of complexes containing SK was markedly and the PA activity of complexes containing was not These that mutations within the strand of the α domain 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 in the presence of fibrin, Ile1 was for SK to efficiently induce active site in plasminogen by a nonproteolytic of by and by study the role of the SK α domain in the resistance of the SK-plasmin complex to inhibition by α2-antiplasmin, incubated SK and plasmin for and α2-antiplasmin amidolytic activity of complexes was by of inhibition of wild-type SK-plasmin complex by was inhibition of by was during incubation of plasmin with the SK and of plasmin and other mutants in were not by α2-antiplasmin not with these completely the of amidolytic activity during a incubation of SK or SK at with plasminogen and plasmin and However, complexes by plasminogen and SK or SK in were resistant to inhibition by α2-antiplasmin These suggested that the SK α domain contributes to the resistance of the SK-plasmin complex to inhibition by α2-antiplasmin and that mutations within the strand of the α domain mutants with resistance to of on the of PA activity of SK-(60–414) is it is markedly enhanced by fibrin (15Reed G.L. Houng A.K. Liu L. Parhami-Seren B. Matsueda L.H. Wang S. Hedstrom L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 8879-8883Google Scholar). We a of to mechanisms underlying the of fibrin on SK-(60–414) not generate amidolytic activity during incubation with Pg in the absence or presence of fibrin suggesting that fibrin not for residues 1–59 of SK to necessary to induce an active site in plasminogen by a nonproteolytic However, fibrin 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 The of plasmin the of PA activity during incubation of SK-(60–414) and wild-type plasminogen in the presence of fibrin but not in its absence These suggested that activation of plasminogen by SK-(60–414) not only fibrin but also complex to SK-plasminogen with the plasmin as a in the plasminogen However, the of plasminogen activation by in the presence of fibrin, was markedly than that by wild-type SK. studies suggested that SK-(60–414) a greater fibrinolytic potency than wild-type SK (15Reed G.L. Houng A.K. Liu L. Parhami-Seren B. Matsueda L.H. Wang S. Hedstrom L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 8879-8883Google Scholar). We compared the capacity of wild-type SK and SK-(60–414) to plasma a of wild-type SK exhibited greater fibrinolytic potency than SK-(60–414) a of wild-type SK was to SK-(60–414) at but at SK-(60–414) extensive than wild-type SK during with wild-type SK was at a of but was at a of of plasma was with of These suggested that SK-(60–414) greater fibrinolytic potency than wild-type SK only in which plasma concentrations of wild-type SK are to induce extensive activation of plasminogen in thereby plasma and plasminogen. with this the of purified plasminogen to plasma during the fibrinolytic of wild-type SK but on SK-(60–414) of the mutants by a exhibited a defect in amidolytic or PA activity during incubation with wild-type plasminogen. However, when the panel of mutants was with Pg in which active site can only a nonproteolytic the capacity of SK to induce amidolytic activity was to Residues of SK are in the strand of the α These suggest an important role of the strand in an active site in plasminogen and the hypothesis that a salt bridge between Glu39 of SK and Arg719 of plasminogen an important but not role in the of an active complex (5Wang X. Lin X. Loy J. Tang J. Zhang X. Science. 1998; 281: 1662-1665Google Scholar). We also that of to the in a in SK amidolytic activity. the activator of SK is in to the active site of plasmin (5Wang X. Lin X. Loy J. Tang J. Zhang X. Science. 1998; 281: 1662-1665Google Scholar). is that mutations at or to this or effects that substrate to the active These the of to SK Mutations in the αβ1 strand (residues 17–26) of SK markedly reduced the of an active complex during incubation with plasminogen. Mutations between residues and 24 may the or of the SK terminus and prevented Ile1 from a for it to with Asp740 of plasminogen to induce active site by a nonproteolytic mechanism S. Reed G. Hedstrom L. Eur. J. Biochem. 2000; 267: 3994-4001Google Scholar). However, complexes of plasmin and SKs mutations between were in PA activity, complexes of SK or and plasmin exhibited normal PA activity. These suggested that although Ile1 of SK a key role in an active site in plasminogen (11Wang S. Reed G. Hedstrom L. Biochemistry. 1999; 38: 5232-5240Google Scholar, 14Wang S. Reed G. Hedstrom L. Eur. J. Biochem. 2000; 267: 3994-4001Google Scholar), Ile1 is not for normal of substrate plasminogen by the and the of PA function of mutants was not by effects on residues within the of the α mutations in this may protein and substrate by the activator with this mutations in this were associated with in SK, as by However, the of in the of these mutants and the that fibrin the capacity of mutants (but not to induce amidolytic activity in Pg suggested that in α domain were not and were The SK αβ1 strand has extensive with residues of plasminogen, with Arg719 a with of SK (5Wang X. Lin X. Loy J. Tang J. Zhang X. Science. 1998; 281: 1662-1665Google Scholar). the hypothesis that between residues of plasminogen and the strand of the SK α domain is for rapid of a functional of of by SK Ile1 of SK to for SK to induce an active site in plasminogen (11Wang S. Reed G. Hedstrom L. Biochemistry. 1999; 38: 5232-5240Google Scholar, 14Wang S. Reed G. Hedstrom L. Eur. J. Biochem. 2000; 267: 3994-4001Google Scholar), complexes of plasminogen and SK N-terminal deletion mutants lacking Ile1 after a lag generate nearly normal amidolytic activity (7Rodriguez P. Fuentes P. Barro M. Alvarez J.G. Munoz E. Collen D. Lijnen H.R. Eur. J. Biochem. 1995; 229: 83-90Google Scholar, 10Fay W.P. Bokka L.V. Thromb. Haemostasis. 1998; 79: 985-991Google Scholar, 11Wang S. Reed G. Hedstrom L. Biochemistry. 1999; 38: 5232-5240Google Scholar, 12Young K.-C. Shi G.-Y. Chang Y.-F. Chang L.-C. Lai M.-D. Chuang W.-J. Wu H.-L. J. Biol. Chem. 1995; 270: 29601-29606Google Scholar). studies suggest that active SK complexes can activate inactive SK complexes by hydrolyzing the Arg561-Val562 plasminogen as by Wang S. Reed G. Hedstrom L. Eur. J. Biochem. 2000; 267: 3994-4001Google Scholar). that SK mutants lacking normal function of due to of that or due to effects of other of which binds to SK to generate active complexes that activation of other resulting in an activation mechanism similar to that of H.R. B. F. K. S. Collen D. J. Biol. Chem. Scholar). of Residues 1–59 of SK in Plasminogen of residues 1–59 of SK it by a poorly mechanism (15Reed G.L. Houng A.K. Liu L. Parhami-Seren B. Matsueda L.H. Wang S. Hedstrom L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 8879-8883Google Scholar). We that fibrin not SK-(60–414) to generate amidolytic activity in Pg suggesting that fibrin for the N-terminal fragment of SK to of an activator complex by a However, suggest that plasmin is fibrin can for the N-terminal fragment of SK to a reduced of substrate plasminogen, free complexes are of PA activity. The of the fibrin is to plasmin to fibrin plasminogen activation by tissue-type plasminogen and plasmin is resistant to inhibition by α2-antiplasmin Thromb. Haemostasis. Scholar). suggest that the fibrin of SK-(60–414) is for by its of fibrin and plasmin to generate a complex with PA activity, thereby plasminogen activation to the Reed that SK-(60–414) was a fibrinolytic agent to wild-type SK at concentrations (15Reed G.L. Houng A.K. Liu L. Parhami-Seren B. Matsueda L.H. Wang S. Hedstrom L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 8879-8883Google Scholar). that SK-(60–414) less than wild-type SK at low concentrations but than wild-type SK at higher The fibrinolytic of wild-type SK SK-(60–414) at concentrations is with The of SK-(60–414) wild-type SK at higher concentrations to due to the capacity of wild-type SK, but not to activate plasminogen in thereby to and an of S. J. 1085-1090Google Scholar). with this the of purified plasminogen to plasma by wild-type SK, but not SK N-terminal deletion mutants been as (15Reed G.L. Houng A.K. Liu L. Parhami-Seren B. Matsueda L.H. Wang S. Hedstrom L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 8879-8883Google Scholar), it is important to that fibrinolytic on plasma The of fibrin on the amidolytic and PA activities of SK αβ1 strand mutants was similar to that with although the presence of fibrin, the activities of SK αβ1 strand mutants were greater than that of SK-(60–414) and with that of wild-type SK. However, in the presence of fibrin, Ile1 is to efficiently induce an active site in plasminogen by a nonproteolytic We that the fibrin of αβ1 strand mutants is by by fibrin that the of Ile1 of SK to a salt bridge with Asp740 of plasminogen, which necessary for nonproteolytic active site induction S. Reed G. Hedstrom L. Eur. J. Biochem. 2000; 267: 3994-4001Google Scholar). of SK α in of the to studies suggest an important role of the SK α domain in mediating the resistance of the SK-plasmin complex to inhibition by studies that the of for plasmin is markedly reduced compared with wild-type SK W.P. J. Biol. Chem. 2000; 275: Scholar), suggesting that residues 1–59 of SK play a key role in the of the SK-plasmin is that deletion of residues 1–59 or of residues within the αβ1 strand to SK from the SK-plasmin complex and plasmin. studies key residues within residues 1–59 of SK that its functional and the mechanisms by which the SK α domain the rapid of a functional activator fibrin-independent plasminogen and contributes to the resistance of the activator complex to inhibition by α2-antiplasmin. studies are to the of SK α domain mutants as plasminogen