Thrombolytic activity of cheonggukjang kinase in recovery from brain damage in a rat cerebral embolic stroke model

Cheonggukjang is a soybean paste made by fermenting whole cooked soybeans with Bacillus subtilis. Cheonggukjang contains a fibrinolytic enzyme that could provide clinical applications for removing blood clots. In the present study, the term ＂cheonggukjang kinase＂ （CGK） was used to refer to this fibrinolytic enzyme. The thrombolytic effects of CGK were analyzed in a rat model of cerebral embolic stroke produced by middle cerebral artery occlusion （MCAO）. Results from fibrin and platelet-rich clot lysis assays demonstrated that thrombolytic activity was greatest in CGKs, which were cultured for 40 hours. In addition, T50, the time needed to decompose 50% of the clot, did not change with plasminogen treatment, indicating that CGK was not a plasminogen activator, but was rather presumed to act as a plasmin-like protein. An intravenous infusion of CGK （1 U plasmin-like activity/100 μg CGK/kg） at 1 hour after MCAO resulted in removal of clots in a rat model of cerebral embolic stroke. CGK-treated groups exhibited a significant dose-dependent reduction in infarct volume. CGK treatment also improved functional recovery, as assessed by neurological deficit scores. Decreased infarct volume and improved functional recovery following CGK treatment was greater compared with recombinant tissue plasminogen activator （10 mg/kg）. These results suggested that CGK effectively reduced infarct volume and improved functional recovery following ischemic brain injury. CGK exhibits a number of potential clinical applications ir the treatment of cerebral embolic stroke.

A comparative study of the antioxidant,antimicrobial,cytotoxic and thrombolytic potential of the fruits and leaves of Spondias dulcis

Objective:To investigate the antioxidant,antimicrobial,cytotoxic and thrombolytic property of the fruits and leaves of Spondias dulcis（S.dulcis）.Methods:Methanolic extracts of fruits and leaves of S.dulcis were partitioned with chloroform and dichloromethane.The antioxidant potential of the crude extract and partitioned fractions were evaluated in terms of total phenolic content,total flavonoid content,DPPH radical scavenging potential,reducing potential and total antioxidant capacity by specific standard procedures.The antimicrobial activity was evaluated using disc diffusion method.The cytotoxicity was evaluated by using brine shrimp lethality bioassay and compared with vincristine sulfate.The thrombolytic activity was compared with streptokinase.Results:The methanolic fruit extract exhibited the highest phenolic content,flavonoid content and antioxidant capacity,among the other extracts,with the highest DPPH radical scavenging activity at a concentration of 10μg/mL(IC50:1.91μg/mL)and maximum reducing power at a concentration of 100μg/ml.(EC50:3.58μg/mL).Though all extract showed moderate antimicrobial activity against the bacterial strains,weak or no activity against fungus.The range of LC50value of all extracts was 1.335-14.057μg/mL which was far lower than the cut off index for cytotoxicity.All extracts exhibited statistically significant（P【0.001）thrombolytic activity.Conclusions:Our study suggested that 5.dulcis exhibits antimicrobial activities against a wide variety of strains while it possesses significant antioxidant,cytotoxic and thrombolytic aclivily.

Thrombolytic effect of rAcAP5 and its mechanism

Ancylostoma caninum anticoagulant peptide 5 （AcAP5） has been reported as an FXa inhibitor. We found that this peptide showed potent inhibitory activity on activated thrombin-activatable fibrinolysis inhibitor （TAFIa）, which protects the fibrin clot against lysis. This study investigated the effects of recombinant AcAP5 （rAcAP5） on fibrinolytic activity in vitro and on thrombolytic activity in vitro and in vivo. In addition, euglobulin lysis time （ELT）, fibrinogen content and fibrin degradation product （FDP） in normal rat plasma were all determined to evaluate the influence ofrAcAP5 on fibrinolytic activity in circulation blood. TAFIa activity was detected by colorimetry; fibrinolysis in vitro was determined with turbidimetry. Thrombolysis in vitro was measured by thrombus weight reduction; thrombolysis in vivo was conducted by arteriovenous shunt method with thrombus weight reduction. ELT, fibrinogen content and FDP were detected using routine method, rAcAP5 concentration-dependently inhibited TAFIa activity in vitro with an IC50 of 63.7 nmol/L, rAcAP5 （5-40 nmol/L） significantly accelerated urokinase-induced clot lysis of rabbit plasma and shortened fibrinolysis time （P〈0.01）. rAcAP5 at 1.5 ktmol/L enhanced the reduced weight of thrombus induced by urokinase （P〈0.05）, and it also reduced thrombus weight （P〈0.05） in vitro when used alone. In an arteriovenous shunt thrombolytic model, rAcAP5 （50-200 μg/kg, i.v.） dose-dependently enhanced the reduced weight of the thrombus, rAcAP5 at effective doses failed to influence ELT, fibrinogen contents and FDP in normal rat circulation plasma. In conclusion, rAcAP5 is a thrombolytic peptide, which may be attributed to its TAFIa inhibition, rAcAP5 may only exert thrombolytic activity on the thrombus site but not influence the fibrinolysis activity and fibrinogen in circulation plasma. These findings suggest that rAcAP5 is a potential thrombolytic candidate agent.