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

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Abstract

(J.A. Kline). 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; K<inf>M</inf> = 268.78 ± 19.12, 324.90 ± 8.43 μM and K<inf>cat</inf> = 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 (K<inf>i</inf>) for both plasmin and δ-plasmin ranging from K<inf>i</inf> < 4 μM for pentamidine to K<inf>i</inf> > 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.

Original languageEnglish
Pages (from-to)358-362
Number of pages5
JournalBiochemical and Biophysical Research Communications
Volume457
Issue number3
DOIs
StatePublished - Feb 13 2015

Fingerprint

Fibrinolysin
Derivatives
Kringles
benzamidine
Thrombosis
Pentamidine
Molecules
Serine Proteases

Keywords

  • Benzamidine
  • Delta-plasmin
  • Drug delivery
  • Fibrinolytic
  • Inhibitors
  • Nanoparticle
  • Plasmin
  • Tunable

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Cell Biology
  • Molecular Biology
  • Medicine(all)

Cite this

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title = "Comparative study on the inhibition of plasmin and delta-plasmin via benzamidine derivatives",
abstract = "(J.A. Kline). 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.",
keywords = "Benzamidine, Delta-plasmin, Drug delivery, Fibrinolytic, Inhibitors, Nanoparticle, Plasmin, Tunable",
author = "Nathan Alves and Jeffrey Kline",
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language = "English",
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AU - Kline, Jeffrey

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N2 - (J.A. Kline). 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.

AB - (J.A. Kline). 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.

KW - Benzamidine

KW - Delta-plasmin

KW - Drug delivery

KW - Fibrinolytic

KW - Inhibitors

KW - Nanoparticle

KW - Plasmin

KW - Tunable

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