Biochemical and Biophysical Research Communications 2015-02-13

Comparative study on the inhibition of plasmin and delta-plasmin via benzamidine derivatives.

Nathan J Alves, Jeffrey A Kline

Index: Biochem. Biophys. Res. Commun. 457(3) , 358-62, (2015)

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Abstract

The potent fibrinolytic enzyme, plasmin has numerous clinical applications for recannulizing vessels obstructed by thrombus. Despite its diminutive size, 91 kDa, success in the recombinant expression of this serine protease has been limited. For this reason, a truncated non-glycosylated plasmin variant was developed capable of being expressed and purified from E. coli. This mutated plasmin, known as δ-plasmin, eliminates four of the five kringle domains present on native plasmin, retaining only kringle 1 fused directly to the unmodified catalytic domain of plasmin. This study demonstrates that δ-plasmin exhibits similar kinetic characteristics to full length plasmin despite its heavily mutated form; KM = 268.78 ± 19.12, 324.90 ± 8.43 μM and Kcat = 770.48 ± 41.73, 778.21 ± 1.51 1/min for plasmin and δ-plasmin, respectively. A comparative analysis was also carried out to investigate the inhibitory effects of a range of benzamidine based small molecule inhibitors: benzamidine, p-aminobenzamidine, 4-carboxybenzamidine, 4-aminomethyl benzamidine, and pentamidine. All of the small molecule inhibitors, with the exception of unmodified benzamidine, demonstrated comparable competitive inhibition constants (Ki) for both plasmin and δ-plasmin ranging from Ki < 4 μM for pentamidine to Ki > 1000 μM in the case of aminomethyl benzamidine. This result further supports that δ-plasmin retains much of the same functionality as native plasmin despite its greatly reduced size and complexity. This study serves the purpose of demonstrating the tunable inhibition of plasmin and δ-plasmin with potential applications for the improved clinical delivery of δ-plasmin to treat various thrombi.Copyright © 2015 Elsevier Inc. All rights reserved.