Effects of human fibrinogen and its cleavage products on activation of human plasminogen by streptokinase

Chibber, Bakshy A. K.; Morris, Joseph P.; Castellino, Francis J.

doi:10.1021/bi00335a006

Cited by 68 publications

(50 citation statements)

References 26 publications

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“…SK binding to Pg and conformational expression of the Pg catalytic site in the SK⅐Pg* complex occurred rapidly and without rate-controlling intermediates on the seconds time scale. The slow (minutes) isomerization of the SK⅐Pg* complex reported in previous studies (22,23) with [Glu]Pg at low temperature and/or low chloride concentrations was not observed in our studies. This does not mean that this conformational change does not occur but likely represents differences in the experimental conditions, where this event apparently occurs rapidly under our conditions.…”

Section: Discussioncontrasting

confidence: 73%

“…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.…”

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confidence: 99%

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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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Section: Discussioncontrasting

confidence: 73%

mentioning

confidence: 99%

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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“…Non-proteolytic activation of Pg by SK is closely coupled with enzymatic activation of Pg by Pg-SK and plasmin-SK complexes, by their cleavage of the Arg 561 -Val 562 bond (Bajaj and Castellino, 1977;GonzalezGronow et al, 1978;Davidson et al, 1990). Similar to activation by the physiological proteinases, the rate of SK-initiated plasmin formation is regulated by interactions with fibrin (Strickland et al, 1982;Chibber et al, 1985;Cassels et al, 1987), Pg conformational equilibria (Chibber and Castellino, 1986), and plasmin proteolytic reactions (Strickland et al, 1982). Studies aimed at defining the molecular mechanisms of Pg activation have been complicated by the concurrent plasmin-catalyzed reactions.…”

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confidence: 99%

Analogs of Human Plasminogen That Are Labeled with Fluorescence Probes at the Catalytic Site of the Zymogen

Bock

Day

Verhamme

et al. 1996

Journal of Biological Chemistry

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“…Recent work from our laboratory has shown that at very early reaction times, at least two types of plasminogen activator activities, with differing abilities to be enhanced by fibrinogen, are resolved in SK-HPg complexes (11). During this work, we also discovered that these enzymic activities also displayed differing sensitivity to anions, a consideration that we felt could be exploited to allow us to understand better the mechanism of development of plasminogen activator activity in complexes of SK and HPg and to enable us to isolate effectively the different activator species such that they could be studied independently.…”

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confidence: 99%

Rapid formation of an anion-sensitive active site in stoichiometric complexes of streptokinase and human [Glu1]plasminogen.

Chibber¹,

Radek²,

Morris³

1986

Proc. Natl. Acad. Sci. U.S.A.

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We have examined the time-dependent appearance of amidolytic activity in equimolar complexes of streptokinase (SK) and human [Glul]plasminogen (HPg) under various conditions. When stoichiometric levels of the two proteins are incubated and assayed in hypotonic buffers at 4°C, amidolytic activity toward the chromogenic substrate D-ValLeu-Lys-p-nitroanilide (S-2251), within the resulting complex, appears with an observed first-order rate constant of 1.03 ± 0.06/min. On the other hand, when the assay for amidolytic activity is conducted at a Cl-concentration of 0.15 M, this same activity develops with an observed first-order rate constant of 0.13 ± 0.01/min. Under all conditions of assay of importance to the mechanism proposed, the only molecular components present are SK and H]Pg. The rate of appearance of an enzyme species displaying amidolytic activity is dependent on the anion in its assay; a much slower rate constant is obtained with Cl than with AcO-. These observations are consistent with the formation, within the complex, of an early anion-sensitive active site (SK-HPg*) that is converted to a form (SK-HPg') that is much less sensitive to the presence of anions. During the time period of this process, no conversion of plasminogen to plasmin occurs within the complex. Steadystate kinetic properties of SK-HPg* and SK-HPg' have been measured toward the substrate S-2251. Consistent with the mechanism suggested above, the amidolytic activity of SK-HPg* is inhibited by Cl to a much greater extent than is that of SK-HPg'.Fibrinolytic activity in mammalian plasma is primarily expressed through the action of plasmin, a serine protease generated consequent to activation of its plasma protein precursor, plasminogen. This activation occurs as a result of cleavage of a single peptide bond, ArgSWq-Vall6l, in the zymogen (1) and is mediated by a variety of proteins, such as urokinase (2), streptokinase (SK) (3), and tissue activators (4). Of these, the bacterial protein SK is unique in that it does not appear to be a protease, since a synthetic substrate, or a protein substrate, for SK, other than plasminogen, has not as yet been identified.Current understanding of the mechanism whereby SK activates human plasminogen (HPg) is based on studies that correlate appearance of enzymic activity with the molecular species of proteins present in equimolar complexes of HPg and SK (for a comprehensive review of this area, see ref. 5). From such work, it has been concluded that the major activators of HPg are equimolar complexes of SK and HPg (6), in which an active site has developed in the plasminogen moiety of the complex (7), and a similar complex of SK and human plasmin (HPm), which uses the active site present in HPm for its activator activity (8). This latter complex forms by intramolecular (9, 10) peptide bond cleavage of HPg, within the SK-HPg complex, and/or by stoichiometric interaction of SK with previously formed HPm.Recent work from our laboratory has shown that at very early reaction times, at least two types o...

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Effects of human fibrinogen and its cleavage products on activation of human plasminogen by streptokinase

Cited by 68 publications

References 26 publications

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

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

Analogs of Human Plasminogen That Are Labeled with Fluorescence Probes at the Catalytic Site of the Zymogen

Rapid formation of an anion-sensitive active site in stoichiometric complexes of streptokinase and human [Glu1]plasminogen.

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