Thrombolytic Therapy: Current Status

Marder, Victor J.; Sherry, Sol

doi:10.1056/nejm198806093182306

Cited by 485 publications

(117 citation statements)

References 94 publications

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“…4A) showing a 4-to 6-fold increase in potency: plasma clearance does not play a role in the plasma clot assay. Marder and Sherry (27) In summary, r-scuPA(32kDa)-59D8 demonstrates that it is possible to design a plasminogen activator chimera in which the activities of the components, though comparable to those of the native proteins, manifest increased selectivity and potency when combined in a single molecule.…”

Section: Resultsmentioning

confidence: 96%

A recombinant chimeric plasminogen activator with high affinity for fibrin has increased thrombolytic potency in vitro and in vivo.

Runge

Quertermous

Zavodny

et al. 1991

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

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A recombinant plasminogen activator with high fibrin affinity and specificity was expressed by transfecting hybridoma cells with a plasmid that combines sequence coding for low molecular mass (32 kDa) single-chain urokinasetype plasminogen activator [scuPA(32kDa)J and anti-fibrin monoclonal antibody 59D8. The expression of the recombinant molecule [r-scuPA(32kDa)-59D8] was optimized by replacing the 3' untranslated region (initially that of high molecular mass scuPA) in the plasmid with the 3' untranslated region of either 13-globin or mouse immunoglobulin. This modification resulted in a >100-fold improvement in the level of protein expression.The 103-kDa r-scuPA(32kDa)-59D8 protein displayed catalytic activity indistinguishable from that of high molecular mass scuPA and fibrin binding comparable to that of native antibody 59D8. r-scuPA(32kDa)-59D8 was 6 times more potent than high molecular mass scuPA in lysing a human plasma clot in vitro and was 20 times more potent than high molecular mass scuPA in the rabbit jugular vein model of thrombolysis.Molecules of this type may serve as prototypes for highly specific, antibody-targeted enzymes suitable for human use.Acute thrombotic occlusion of a major epicardial coronary artery results in myocardial infarction, the single most common cause of death in industrialized societies. The use of thrombolytic therapy in patients with acute myocardial infarction has resulted in a significant reduction in mortality (1-3). At its present stage of development, however, thrombolytic therapy is limited by (i) significant bleeding at high doses (intracranial hemorrhage causes stroke or death in 0.1-0.5% of patients who receive plasminogen activators)(1-4), (ii) the failure to restore blood flow in 20%o of patients or the fact that thrombotic reocclusion after cessation of therapy occurs in 15-25% of patients (5), and (iii) the lag between initiation of therapy and clot lysis, which averages about 60 min (5).These limitations have prompted generation (by recombinant DNA technology) of hundreds of plasminogen activator mutants, which have produced, at best, only modest improvements in thrombolytic efficacy. Most investigators have either rearranged (or duplicated or deleted) various plasminogen activator functional domains or altered their posttranslational modification (6-10). We and others (11-16) have pursued an alternative strategy, the generation ofchemical conjugates or recombinant molecules with domains for both plasminogen activator activity and high-affinity fibrin binding [conferred by a monoclonal antibody (17) that binds to fibrin, the principal component of a thrombus, but not fibrinogen, its circulating precursor]. Here we report the generation and characterization (in vitro and in vivo) of a recombinant molecule that is fibrin selective by two different mechanisms. It combines a high-affinity anti-fibrin antibody, 59D8, with a low molecular mass (32 kDa) single-chain urokinase-type plasminogen activator [scuPA(32kDa)], a fibrin-selective plasminogen activato...

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Section: Resultsmentioning

confidence: 96%

A recombinant chimeric plasminogen activator with high affinity for fibrin has increased thrombolytic potency in vitro and in vivo.

Runge

Quertermous

Zavodny

et al. 1991

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

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“…If tissue plasminogen activator is employed in the zymogen activation process, a fibrin surface is required, and plasmin is produced in the vicinity of its own substrate (47). By contrast, if streptokinase is employed, the fibrin surface is no longer required (48,49). Plasmin can be generated at a site distant from fibrin, and the levels of plasmin may become prematurely depleted before this plasmin has an opportunity to reach its target.…”

Section: Discussionmentioning

confidence: 99%

Examining Thrombin Hydrolysis of the Factor XIII Activation Peptide Segment Leads to a Proposal for Explaining the Cardioprotective Effects Observed with the Factor XIII V34L Mutation

Trumbo

Maurer

2000

Journal of Biological Chemistry

130

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In the blood coagulation cascade, thrombin cleaves fibrinopeptides A and B from fibrinogen revealing sites for fibrin polymerization that lead to insoluble clot formation. Factor XIII stabilizes this clot by catalyzing the formation of intermolecular cross-links in the fibrin network. Thrombin activates the Factor XIII a 2 dimer by cleaving the Factor XIII activation peptide segment at the Arg 37 -Gly 38 peptide bond. Using a high performance liquid chromatography assay, the kinetic constants K m , k cat , and k cat /K m were determined for thrombin hydrolysis of fibrinogen A␣-(7-20), Factor XIII activation peptide-(28 -41), and Factor XIII activation peptide-(28 -41) with a Val 34 to Leu substitution. This Val to Leu mutation has been correlated with protection from myocardial infarction. In the absence of fibrin, the Factor XIII activation peptide-(28 -41) exhibits a 10-fold lower k cat /K m value than fibrinogen A␣-(7-20). With the Factor XIII V34L mutation, decreases in K m and increases in k cat produce a 6-fold increase in k cat /K m relative to the wild-type Factor XIII sequence. A review of the x-ray crystal structures of known substrates and inhibitors of thrombin leads to a hypothesis that the new Leu generates a peptide with more extensive interactions with the surface of thrombin. As a result, the Factor XIII V34L is proposed to be susceptible to wasteful conversion of zymogen to activated enzyme. Premature depletion may provide cardioprotective effects.Fibrinogen is composed of three chains A␣, B␤, and ␥ arranged into the dimer (A␣B␤␥) 2 . In blood coagulation, the serine protease thrombin cleaves the N-terminal portions of the A␣ and B␤ chains. For the A␣ chain, cleavage occurs at the Arg 16 -Gly 17 peptide bond and fibrinopeptide A (FpA) 1 is released; whereas, for the B␤ chain, cleavage occurs at the Arg 14 -Gly 15 peptide bond and fibrinopeptide B (FpB) is released. Removal of the fibrinopeptides leads to exposure of fibrin polymerization sites that react to form an insoluble blood clot (reviewed in Ref. 1).Activated Factor XIII helps stabilize this clot structure by catalyzing the formation of intermolecular ␥-glutamyl-⑀-lysine cross-links in the fibrin network and in fibrin-enzyme complexes. Factor XIII is a member of a family of enzymes known as transglutaminases that have a catalytic triad, similar to cysteine proteases, composed of amino acids Cys 314 , His 373, and Asp 396 . In plasma, Factor XIII is expressed as a zymogen of the form a 2 b 2 . In the presence of thrombin and calcium, the a 2 unit is released and activated. By contrast, platelet Factor XIII is expressed as the zymogen a 2 unit (reviewed in Ref. 2).The Factor XIII a 2 dimer contains in the N-terminal portion of each monomer a sequence known as the activation peptide (3, 4). Each activation peptide segment crosses the dimer interface and extends over the catalytic site of the opposing Factor XIII a subunit. Cleavage of the activation peptide segments by thrombin at the Arg 37 -Gly 38 peptide bond aids in exposure of the Fact...

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“…The two molecular precursors of S-NO-t-PA (NO and t-PA) are among the products specifically secreted by the endothelium to counteract thrombogenic processes such as platelet activation, augmented local vasoconstriction, and activation of the coagulation system (29). t-PA converts plasminogen to plasmin on fibrin and platelet thrombi, which in turn induces fibrinolysis and platelet disaggregation (1,3).…”

Section: Discussionmentioning

confidence: 99%

S-nitrosylation of tissue-type plasminogen activator confers vasodilatory and antiplatelet properties on the enzyme.

Stamler

Simon

Jaraki

et al. 1992

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

170

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Tissue-type plasminogen activator (t-PA) reacts upon exposure to endothelium-derived relaxing factor (EDRF) by way of the enzyme's single free sulfhydryl to form a stable S-nitrosothiol protein adduct. S-nitrosylation endows t-PA with potent vasodilatory and antiplatelet properties that are accompanied by elevations in intracellular cyclic GMP analogous to those induced by low molecular weight (e.g., S-nitroso amino acid) S-nitrosothiols. Moreover, this chemical modification does not adversely affect the catalytic efficiency of t-PA, the fibrin stimulation of this activity, the binding of t-PA to fibrinogen, or the interaction of the enzyme with its physiologic serine protease inhibitor, plasminogen-activator inhibitor type I. The coupling of vasodilatory, antiplatelet, and fibrinolytic properties in one molecule makes the S-nitrosylated t-PA a unique molecular species and may provide insight into the mechanisms by which the endothelium maintains vessel patency. These data also suggest a pharmacologic approach to treatment of thromboocclusive disorders.

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Thrombolytic Therapy: Current Status

Cited by 485 publications

References 94 publications

A recombinant chimeric plasminogen activator with high affinity for fibrin has increased thrombolytic potency in vitro and in vivo.

A recombinant chimeric plasminogen activator with high affinity for fibrin has increased thrombolytic potency in vitro and in vivo.

Examining Thrombin Hydrolysis of the Factor XIII Activation Peptide Segment Leads to a Proposal for Explaining the Cardioprotective Effects Observed with the Factor XIII V34L Mutation

S-nitrosylation of tissue-type plasminogen activator confers vasodilatory and antiplatelet properties on the enzyme.

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