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Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1989 Apr;86(8):2568–2571. doi: 10.1073/pnas.86.8.2568

Plasmin-mediated fibrinolysis by variant recombinant tissue plasminogen activators.

S Urano 1, A R Metzger 1, F J Castellino 1
PMCID: PMC286958  PMID: 2523073

Abstract

A rapid and quantitative fibrinolytic assay has been used to measure the overall activity of a recombinant tissue plasminogen activator (rTPA) preparation for dissolution of a fibrin clot by its ability to activate [Glu1]plasminogen (containing glutamic acid at position 1) to plasmin. A standard curve constructed for wild-type two-chain rTPA that contains, from the amino terminus, the finger (F)-growth factor (E)-kringle 1 (K1)-kringle 2 (K2)-serine protease (P) domains was used to assess the overall fibrin-dissolving abilities of variant recombinant molecules. Two-chain deletion mutants lacking the E domain, the F-E domains, the F-E-K1 domains, and the K1-K2 domains yielded activities ranging from 22% to 35% of the overall activity of wild-type two-chain rTPA, suggesting that both the K2 and F domains are individually responsible for a portion of the function of the molecule. Comparison of variant molecules containing F-K1-K2-P and F-K2-K2-P domains showed that the latter variant possessed a 4-fold higher activity (1.4-fold greater than that of wild-type two-chain rTPA), indicating that, for the activity measured, the presence of K2 leads to a greater effectiveness than that of K1. A plasmin cleavage-resistant mutant (Arg-275----Ser) has been used to assess possible differences in one- and two-chain rTPA in this overall activity, the former displaying 86% of the activity of the latter, suggesting that such differences are indeed small. Finally, the proper covalent attachment of the light and heavy chains of two-chain rTPA are very important to its overall fibrinolytic activity, since replacement of Cys-264 with glycine and concomitant disruption of one of the covalent attachment sites of the two chains provides a variant of rTPA with less than 2% of the activity of the wild-type two-chain molecule. The effector molecule, epsilon-amino hexanoic acid (epsilon Ahx; epsilon-aminocaproic acid), inhibits the overall fibrinolytic effect of rTPA in this system, with an effective Ki of approximately 1.5 mM. Its efficacy, as measured by the Ki, is independent of the presence of the epsilon Ahx binding regions of plasminogen and rTPA and is similar to the efficacy obtained when urokinase was the activator in place of wild-type two-chain rTPA or when activation of plasminogen was bypassed as a result of provision of preformed plasmin to the assay. The results suggest that in the overall clot lysis system, an important epsilon Ahx binding site may exist on fibrin that inhibits its dissolution by plasmin.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

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