Comparative anti-thrombotic activity and haemorrhagic adverse effect of nattokinase and tissue-type plasminogen activator

Abstract

Anti-thrombotic activity and safety of nattokinase, an enzyme produced by Bacillus subtilis during soybean fermentation, were investigated in comparison with tissue-type plasminogen activator (t-PA). Carotid arterial thrombosis was produced with a FeCl₃-soaked paper, followed by intravenous injection of nattokinase or t-PA. Nattokinase and t-PA delayed thrombus formation, near-fully (> 90%) inhibiting at 75 and 8.5 mg/kg, respectively. As adverse effects, t-PA induced petechial haemorrhage at 10 mg/kg in the lungs and thymus, and extensive bleeding at 20 mg/kg. Nattokinase also caused pulmonary haemorrhage from 300 mg/kg. Collectively, the standard safety margins (SSMs) for t-PA and nattokinase were calculated to be 1.2 and 4.0, respectively. Combinational treatment with dexamethasone (2 mg/kg) increased the efficacy and safety of t-PA and nattokinase, widening their SSMs to 2.4 and 8.0, respectively. The results indicate that nattokinase delayed thrombus formation and dissolved thrombi, and that nattokinase could be a good candidate anti-thrombotic agent with relatively-low haemorrhagic risk.

Keywords: Dexamethasone; Haemorrhage; Nattokinase; Thrombosis; Tissue-type plasminogen activator.

Fig. 1

Effects of nattokinase (A) and tissue-type plasminogen activator (t-PA; B) on the carotid artery thrombosis induced by FeCl₃ application outside the arterial wall. Nattokinase or t-PA were intravenously injected 5 min after FeCl₃ exposure. ○: vehicle (phosphate-buffered saline), ▽: 25 mg/kg nattokinase or 5 mg/kg t-PA, □: 50 mg/kg nattokinase or 7.5 mg/kg t-PA; ◇: 75 mg/kg nattokinase or 8.5 mg/kg t-PA, △: 100 mg/kg nattokinase or 10 mg/kg t-PA

Fig. 2

Effects of nattokinase (A, B) and tissue-type plasminogen activator (t-PA; C, D) alone or in combination with dexamethasone on the carotid artery thrombosis induced by FeCl₃ application outside the arterial wall. Nattokinase, t-PA or dexamethasone were intravenously injected 5 min after FeCl₃ exposure. ○: vehicle (phosphate-buffered saline), ●: dexamethasone (2 mg/kg) alone, ▽: nattokinase (25 mg/kg) or t-PA (5 mg/kg) alone, ▼: nattokinase (25 mg/kg) or t-PA (5 mg/kg) plus dexamethasone, □: nattokinase (50 mg/kg) or t-PA (7.5 mg/kg) alone, ■: nattokinase (50 mg/kg) or t-PA (7.5 mg/kg) plus dexamethasone

Fig. 3

Representative findings of carotid arterial thrombi produced by FeCl₃ application outside the arterial wall. Vehicle (phosphate-buffered saline, PBS), dexamethasone (Dexa; 2 mg/kg), nattokinase (Natto; 25 mg/kg) or tissue-type plasminogen activator (t-PA; 5 mg/kg) were intravenously injected 5 min after FeCl₃ exposure

Fig. 4

Representative gross and microscopic findings of haemorrhage (arrows) in the lungs (A) and thymus (B) after intravenous injection of nattokinase (100–300 mg/kg)

Fig. 5

Representative gross and microscopic findings of haemorrhage (arrows) in the lungs (A) and thymus (B) after intravenous injection of tissue-type plasminogen activator (t-PA; 10–30 mg/kg)