The mechanism of activation of human plasminogen by streptokinase

McClintock, David K.; Bell, Paul H.

doi:10.1016/0006-291x(71)90670-x

Cited by 190 publications

(132 citation statements)

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“…In this paper the term "activation" carries two connotations: it means proteolytic cleavage of mflg when mediated by urokinase; with streptokinase it signifies the appearance of an active enzymatic site, without chain cleavage, in the zymogen- SK complex. in the zymogen a typical serine protease active site that expresses characteristic amidolytic and PA activity (McClintock & Bell, 1971;Reddy & Markus, 1972). Active site formation is achieved without covalent modification of either P/g or SK and, hence, in the absence of any of the structural changes, such as the appearance of a new N-terminus and formation of a salt bridge, that ordinarily accompany proenzyme activation in the chymotrypsinogen family of proteases.…”

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confidence: 99%

Structure and function of microplasminogen: II. Determinants of activation by urokinase and by the bacterial activator streptokinase

Wang

Reich

1995

Protein Sci.

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We have used a group of human microplasminogens (mflg), modified by residue substitutions, insertions, deletions, and chain breaks ( I ) to study the determinants of productive interactions with two plasminogen activators, urokinase (uPA), and streptokinase (SK); ( 2 ) to explore the basis of species specificity in the zymogen-SK complex activity; and (3) to compare active SK complex formation in mPIg and microplasmin (mPlm). Modifications within the disulfide-bonded loop containing the activation site and the adjacent hexadecapeptide upstream sequence showed that uPA recognition elements encompassed R29 at the activation site and multiple elements extending upstream to perhaps 13 residues, all maintained in specific conformational register by the surrounding pairs of disulfide bonds. A generally parallel pattern of structural requirements was observed for active zymogen-SK complex formation. Changes within the loop downstream of the activation site were tolerated well by uPA and poorly by SK. The introduction of selected short bovine (Plg) sequences in human mPlg reduced the activity of the resulting SK complexes. The requirements for active SK complex formation are different for mPlg and mPlm.Keywords: microplasminogen; microplasmin; plasminogen activator; streptokinase; urokinase Plasminogen is the principal serine protease zymogen in the extracellular fluids of vertebrates, and its active form -plasmin -is implicated in pericellular proteolysis associated with a wide range of physiological and pathological processes. Plasminogen is normally recruited for extracellular proteolysis by regulated cellular secretion of plasminogen activators. Two PAS are known in vertebrate organisms, urokinase, and tissue plasminogen activator, the latter, on present knowledge, restricted to placental mammals. Both uPA and tPA are serine proteases belonging to the chymotrypsinogen family of enzymes; both activate Plg by proteolysis at a single site within the polypeptide chain, and both are among the most specific and limited proteases known. Abbreviations: CNBr/HFo, cyanogen bromide in 73.5% formic acid; PA, plasminogen activator; P A , tissue plasminogen activator; u f A , urokinase; Plg, plasminogen; Plm, plasmin; m f l g , microplasminogen; mPlm, microplasmin; mPlgM-, methionineless microplasminogen; mflmM; methionineless microplasmin; mPlg-SK mPlm-SK, the streptokinase complexes of microplasminogen and microplasmin, respectively; SK, streptokinase; mPlg-E,, mPlg-B2, mPlg-E,, human-bovine hybrid mflgs; mPlg-M, human-murine hybrid mPlg; PBS, phosphatebuffered saline; SBTI, soybean trypsin inhibitor. Amino acids are represented by the single-letter code.

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confidence: 99%

Structure and function of microplasminogen: II. Determinants of activation by urokinase and by the bacterial activator streptokinase

Wang

Reich

1995

Protein Sci.

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“…SK possesses no intrinsic catalytic activity but interacts with Pg and Pm, converting both the zymogen and active proteinase into specific proteolytic Pg activators (14 -19). SK binding to Pg results in conformational expression of an active catalytic site on the zymogen without the usual strict requirement for peptide bond cleavage (14,16,17). Pm is generated subsequently by proteolytic cleavage of Arg 561 -Val 562 , and the SK⅐Pm complex propagates Pg activation through expression of a substrate recognition exosite (20,21).…”

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“…Early studies (14,16,17) demonstrated that interaction of SK with Pg produced the activated Pg catalytic site in the SK⅐Pg* complex. Subsequent kinetic studies indicated that Pg activation involved an initially formed SK⅐Pg* activation complex and an isomerized form of the complex (22,23).…”

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Resolution of Conformational Activation in the Kinetic Mechanism of Plasminogen Activation by Streptokinase

Boxrud¹,

Verhamme

Bock

2004

Journal of Biological Chemistry

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Streptokinase (SK) 1 is used clinically as a thrombolytic drug to activate plasminogen (Pg) to plasmin (Pm), the serine proteinase responsible for dissolution of fibrin clots (1). Native [Glu]Pg is a multidomain zymogen consisting of a 77-residue N-terminal peptide, five kringle domains, and a serine proteinase catalytic domain that is activated by cleavage of Arg 561 -Val 562 (2, 3).[Glu]Pg is in a compact conformation, maintained by intramolecular interactions between the N-terminal peptide and lysine-binding sites in kringles 4 and 5 (4 -6). Release of the N-terminal peptide by Pm cleavage generates [Lys]Pg, which adopts an extended conformation and is cleaved by plasminogen activators at a faster rate (5, 7-13). SK possesses no intrinsic catalytic activity but interacts with Pg and Pm, converting both the zymogen and active proteinase into specific proteolytic Pg activators (14 -19). SK binding to Pg results in conformational expression of an active catalytic site on the zymogen without the usual strict requirement for peptide bond cleavage (14,16,17). Pm is generated subsequently by proteolytic cleavage of Arg 561 -Val 562 , and the SK⅐Pm complex propagates Pg activation through expression of a substrate recognition exosite (20,21).It is well established that both conformational and proteolytic activation contribute to SK-induced Pg activation, but there are a number of unresolved questions concerning the mechanism of conformational activation and its coupling to subsequent proteolytic Pm formation. Early studies (14,16,17) demonstrated that interaction of SK with Pg produced the activated Pg catalytic site in the SK⅐Pg* complex. Subsequent kinetic studies indicated that Pg activation involved an initially formed SK⅐Pg* activation complex and an isomerized form of the complex (22, 23). The isomerization was time-, chloride ion-, and fibrinogen-dependent, and the active complexes interconverted slowly under certain experimental conditions. In other studies, a species of Pg isolated from a mixture of SK and Pg was reported to be an active "virgin enzyme" form of free Pg*, suggesting irreversible conformational activation (24). The relationship between SK-Pg binding and conformational activation and its reversibility have not been clearly established.Previous studies of the mechanism of Pg activation by SK have not taken into account the binding affinities of SK for Pg and Pm species, primarily because consistent and reliable equilibrium binding constants have not been available. A number of studies have examined the binding interactions by various experimental approaches but have resulted in a very broad range of dissociation constants, varying from 28 pM to 220 nM for [Glu]Pg (25-32), for example, where the higher affinities may result from Pm generation in SK/Pg mixtures. Our equilibrium binding studies have employed active site-labeled fluorescent Pg and Pm analogs to quantitate SK binding in the absence of proteolytic reactions (21,28,33,34). The results for the active site-labeled proteins support a rela...

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“…The SK and human plasminogen (HPlg) complex can activate Plg to plasmin (Plm) from different mammalian species (3)(4)(5)(6)(7)(8)(9)(10). Plm thus produced in the blood circulation in turn catalyzes the hydrolysis of fibrin and dissolution of blood clots.…”

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“…However, HPlg in interacting with SK becomes a virgin enzyme, which has amidolytic activity, although the activating peptide Arg 560 -Val 561 remains intact (5,6,8,11). Shortly after complex formation, HPlg⅐SK is converted to HPlm⅐SK (12), both of which can act as a Plg activator to catalyze the hydrolysis of the activating peptide bond of other Plgs (7,13,14).…”

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