Plasmin (EC 3.4.21.7) is a key enzyme of the fibrinolytic system, responsible for the degradation of fibrin clot and maintaining blood fluidity. Hence, alterations of the fibrinolytic capacity of blood plasma may contribute to thrombotic or bleeding complications. The aim of this study was to determine effects of a bufadienolide-rich fraction, isolated from roots of Kalanchoe daigremontiana (0.05-50 μg/ml) on enzymatic properties of plasmin. Hydrolysis of a synthetic substrate S-2251 (H-D-Valyl-l-leucyl-l-lysine-p-nitroaniline dihydrochloride) by plasmin revealed that the bufadienolide-rich fraction had a diverse effect on this enzyme, dependently on the concentration range. While the lower concentrations of the examined fraction (0.05-2.5 μg/ml) significantly enhanced the amidolytic activity of plasmin, at 25-50 μg/ml concentrations, the enzyme was evidently inhibited (by about 60%). The Lineweaver-Burk plot indicated on an uncompetitive inhibition of plasmin. Inhibitory effects (up to 80%) were also found in the streptokinase-induced plasminogen activation to plasmin. Docking results suggest that only some of compounds (mostly bersaldegenin 1-acetate (10), bryotoxin (13) and hovetrichoside C (17)) were bound to plasminogen/plasmin, depending on the presence or absence of the substrate in the active site. The obtained findings suggest allosteric regulation of plasminogen activation and plasmin activity by components of the examined fraction.
Keywords: Bufadienolide; Inhibitor; Plasmin; Plasminogen.
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