Making tissue-type plasminogen activator more fibrin specific

Paoni, Nicholas F.; Chow, Alice M.; Peña, Luisa; Keyt, Bruce A.; Zoller, Mark J.; Bennett, William F.

doi:10.1093/protein/6.5.529

Cited by 32 publications

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“…Molecular details of the stimulation of t-PA by fibrin, a complex process that almost certainly involves multiple points of contact between the two proteins, remain obscure (22)(23)(24)(25)(26)(27). Fibrin stimulation of two-chain t-PA may occur through a single mechanism; stimulation of single-chain t-PA by fibrin co-factors, however, appears to utilize at least two distinct mechanisms.…”

Section: Zymogen-like Variant Of T-pa 29mentioning

confidence: 99%

Converting Tissue Type Plasminogen Activator into a Zymogen

Tachias

Madison

1997

Journal of Biological Chemistry

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In stark contrast to most other members of the chymotrypsin family of serine proteases, tissue type plasminogen activator (t-PA) is not synthesized and secreted as a true zymogen. Instead, single-chain t-PA exhibits very significant catalytic activity. Consequently, the zymogenicity, or ratio of the catalytic efficiencies of the mature, two-chain enzyme and the single-chain precursor, is only 3-9 for t-PA. Both we and others have previously proposed that Lys 156 may contribute directly to this exceptional property of t-PA by forming interactions that selectively stabilize the active conformation of the single-chain enzyme. To test this hypothesis we created variants of t-PA in which Lys 156 was replaced by a tyrosine residue. As predicted, the K156Y mutation selectively suppressed the activity of the single-chain enzyme and thereby substantially enhanced the enzyme's zymogenicity. In addition, however, this mutation produced a very dramatic increase in the ability of single-chain t-PA to discriminate among distinct fibrin co-factors. Compared with wild type t-PA, one of the variants characterized in this study, t-PA/R15E,K156Y, possessed substantially enhanced response to and selectivity among fibrin co-factors, resistance to inhibition by plasminogen activator inhibitor type 1, and significantly increased zymogenicity. The combination of these properties, and the maintenance of full activity in the presence of fibrin, suggest that the R15E,K156Y mutations may extend the therapeutic range of t-PA.Proteases are normally synthesized as inactive precursors or zymogens that must either be proteolytically processed or bind to a specific co-factor to develop substantial catalytic activity. The increase in catalytic efficiency after zymogen activation, or zymogenicity, varies widely among individual members of the (chymo)trypsin family but, in almost all cases, is dramatic. For example, strong zymogens such as trypsinogen, chymotrypsinogen, or plasminogen are almost completely inactive with measured zymogenicities of 10 4 to 10 6 (1, 2). Other serine proteases exhibit intermediate zymogenicity. The enzymatic activity of Factor XIIa is 4000-fold greater than that of Factor XII (3), and the catalytic efficiency of urokinase is 250-fold greater than that of prourokinase (4). By contrast, the catalytic activities of single-and two-chain t-PA 1 vary by a factor of only 3-9 (5-9).We have suggested previously that the unusually high catalytic activity of single-chain t-PA results both from the absence of interactions, present in typical zymogens, that stabilize (an) inactive conformation(s) of the zymogen and the presence of interactions, absent in typical zymogens, that stabilize an active conformation of the single-chain enzyme (8 -10). Recent studies have provided substantial support for this hypothesis. We demonstrated that the absence of the zymogen triad contributes to the enzymatic activity of single-chain t-PA (8, 9), and two groups have suggested that Lys 156 2 stabilizes an active conformation of single-chain t-PA (...

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Section: Zymogen-like Variant Of T-pa 29mentioning

confidence: 99%

Converting Tissue Type Plasminogen Activator into a Zymogen

Tachias

Madison

1997

Journal of Biological Chemistry

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“…To accomplish this, fibrin acts as a template to which both t-PA and Pg bind (4). The fibrin-binding properties of t-PA have been ascribed to its finger and second kringle (K 2 ) domains (5,6), although recent studies suggest that the protease domain also influences the interaction of t-PA with fibrin (4,7,8). The binding of both Glu-and Lys-plasminogen (Glu-Pg and Lys-Pg, respectively) to fibrin is entirely kringle-mediated, with Lys-Pg having higher affinity for fibrin than Glu-Pg (9).…”

mentioning

confidence: 99%

Characterization of the Interactions of Plasminogen and Tissue and Vampire Bat Plasminogen Activators with Fibrinogen, Fibrin, and the Complex of d-Dimer Noncovalently Linked to Fragment E

Stewart¹,

Fredenburgh²,

Weitz

1998

Journal of Biological Chemistry

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Vampire bat plasminogen activator (b-PA) causes less fibrinogen (Fg) consumption than tissue-type plasminogen activator (t-PA). Herein, we demonstrate that this occurs because the complex of D-dimer noncovalently linked to fragment E ((DD)E), the most abundant degradation product of cross-linked fibrin, as well as Fg, stimulate plasminogen (Pg) activation by t-PA more than b-PA. To explain these findings, we characterized the interactions of t-PA, b-PA, Lys-Pg, and Glu-Pg with Fg and (DD)E using right angle light scattering spectroscopy. In addition, interactions with fibrin were determined by clotting Fg in the presence of various amounts of t-PA, b-PA, Lys-Pg, or Glu-Pg and quantifying unbound material in the supernatant after centrifugation. Glu-Pg and Tissue-type plasminogen activator (t-PA)1 is a naturally occurring serine protease that initiates fibrinolysis by converting plasminogen (Pg) to plasmin (1). Not only is fibrin the target for plasmin attack, but fibrin also stimulates t-PA-mediated Pg activation (2, 3). To accomplish this, fibrin acts as a template to which both t-PA and Pg bind (4). The fibrin-binding properties of t-PA have been ascribed to its finger and second kringle (K 2 ) domains (5, 6), although recent studies suggest that the protease domain also influences the interaction of t-PA with fibrin (4, 7, 8). The binding of both Glu-and Lys-plasminogen (Glu-Pg and Lys-Pg, respectively) to fibrin is entirely kringle-mediated, with Lys-Pg having higher affinity for fibrin than Glu-Pg (9). As a functional consequence of t-PA interaction with fibrin, the catalytic efficiency of t-PA-mediated Pg activation is 2-3 orders in magnitude higher in the presence of fibrin than in its absence (3, 10). In contrast to fibrin, fibrinogen (Fg) stimulates Pg activation by t-PA only 25-fold (3, 10). Based on these considerations, t-PA is designated a fibrin-specific plasminogen activator (11). Despite this designation, t-PA causes systemic plasminemia and fibrinogenolysis when given to patients (12). In recent studies, we have demonstrated that t-PA causes systemic plasminemia, because, like intact fibrin, soluble fibrin degradation products stimulate t-PA-mediated Pg activation (13). Furthermore, we have identified the (DD)E complex as the fibrin derivative primarily responsible for this effect (14) and have shown that the stimulatory activity of (DD)E is similar to that of fibrin.2 (DD)E, a complex of D-dimer noncovalently bound to fragment E, is the major degradation product of cross-linked fibrin (15). As a potent stimulator of t-PA-mediated activation of Pg, (DD)E generated during thrombus dissolution has the potential to induce systemic plasminemia (12,15).The limited fibrin specificity of t-PA has prompted the development of plasminogen activators with greater selectivity for fibrin (16). One such agent is the plasminogen activator isolated from the saliva of vampire bats (Desmodus rotundus) (17). Full-length vampire bat salivary plasminogen activator (designated DSPA␣ 1 ) has over 72% amino acid seque...

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“…In the past few years, domain-domain interaction has been proposed: Horrevoets et 44) These results indicate that the sugar chain at Asn448, which is in the serine protease domain, may also affect the fibrin binding, although we did not evaluate the influence of this sugar chain on the fibrin binding. Moreover, each domain is thought to be necessary for t-PA to exhibit physiological activity.…”

Section: Discussionmentioning

confidence: 87%

Influence of Sugar Chain on Fibrin Affinity of Recombinant t-PA.

Aoki¹,

Shimizu

Shimonishi

et al. 2001

Biological & Pharmaceutical Bulletin

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Tissue plasminogen activator (t-PA) is a serine protease that cleaves plasminogen into its active form, plasmin. The plasmin then subsequently degrades fibrin.1,2) The activity of t-PA is markedly stimulated in the presence of fibrin. 2)Human t-PA produced by recombinant DNA technology is presently available for clinical use in patients with acute myocardial infarction. [3][4][5] Although t-PA is thought to be clinically superior to streptokinase and urokinase because of its specific affinity for fibrin, the rapid clearance of t-PA from circulating blood sometimes requires the administration of high doses to obtain therapeutic blood levels, which can lead to bleeding incidents due to a decrease in plasma fibrinogen levels.6,7) To solve this problem, several types of mutant type t-PAs (mt-PAs) have been developed. [8][9][10][11][12][13][14] Although most of them are superior to wild type t-PA (WT t-PA) in terms of plasma half life, they are inferior to WT t-PA in terms of activity. They are generally produced by modification of the peptide chain, therefore, these modifications cause the loss of activity and fibrin affinity. [15][16][17] t-PA is composed of 5 domains: finger, epidermal growth factor (EGF)-like, kringle 1 and 2, and serine protease domain. All of them are assumed to be necessary for t-PA to exhibit physiological activity, especially the finger (F) and kringle 2 (K2) domains are indispensable for fibrin affinity. 18,19) In contrast, modification of the sugar chain is not expected to affect the function of each domain. t-PA is glycosylated at three sites, Asn117, Asn184, and Asn448. The sugar moiety at Asn117 carries a high mannose structure, whereas those at Asn184 and Asn448 carry complex type oligosaccharides.20) The Asn184 glycosylation site has been shown to be variably glycosylated by steric hindrance between the kringle 1 and 2 domains.21) This partial glycosylation results in t-PA being expressed as two major glycoforms, termed Type I and Type II, which are defined by the presence and absence of the complex type oligosaccharide at Asn184, respectively. Although most studies have been made on the role of the sugar chain in the plasma clearance of t-PA, [22][23][24][25] little is known about the influence of the sugar chains on the fibrin affinity. In this study, we determined the influence of the sugar chain on the fibrin affinity using 4 kinds of t-PAs, which have differing sugar chain structures.It has been reported that the binding of t-PA to fibrin is mediated by two discrete domains, the F and K2 domains. 26,27) Binding mediated through these domains can be distinguished on the basis of competition for fibrin binding by lysine analogues such as e-amino caproic acid (EACA), which eliminates K2 dependent binding, but does not eliminate F dependent binding. We also demonstrated the role of sugar chains in the fibrin affinity mediated via these domains one by one, using EACA in this study. MATERIALS AND METHODSChemicals Recombinant WT t-PA (lot R8803, 1.3 mg/ ml) and Gln117 t-PA (lot GYL013, ...

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Making tissue-type plasminogen activator more fibrin specific

Cited by 32 publications

References 0 publications

Converting Tissue Type Plasminogen Activator into a Zymogen

Converting Tissue Type Plasminogen Activator into a Zymogen

Characterization of the Interactions of Plasminogen and Tissue and Vampire Bat Plasminogen Activators with Fibrinogen, Fibrin, and the Complex of d-Dimer Noncovalently Linked to Fragment E

Influence of Sugar Chain on Fibrin Affinity of Recombinant t-PA.

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