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.