In recent years, in order to enhance the efficacy and safety of fibrinolytic therapy, many researches were pursued to discover fibrinolytic enzymes from food-grade microorganisms. Among them, much attention was paid to fibrinolytic enzymes isolated from Douchi.
In this study, many experiments were carried out to test the thrombolytic effect of Douchi fibrinolytic enzyme from Bacillus subtilis LD-8547 in vitro and in vivo. The assay demonstrated that in the groups treated with more than 518 U of DFE, the anticoagulation effect was obvious on animal blood, and the effect was enhanced with increasing dose of DFE. The cascade and waterfall hypotheses of blood coagulation indicate that the coagulation process is divided into three stages: the formation of prothrombin activator, thrombin formation and fibrin formation. In this study, the result indicated that DFE can inhibit the extrinsic coagulation system, which may be the key to antithrombotic and thrombolytic activity. And in the low dose group (259 U), there is no or fewer side effects in bleeding and does not affect the body's normal clotting mechanism. Therefore, low dose DFE is recommended for the long-term use to prevent thrombosis.
Antithrombotic or thrombolytic drugs can block the pathway of thrombus formation. The fundamental task of thrombolytic therapy is the degradation of fibrin by plasmin, which can be activated by the activators from inactive plasminogen. SK and UK depend on this indirect activation pathway. In addition to this indirect working mechanism, thrombolytic enzyme can dissolve fibrin directly, which was proved by our thrombolysis experiments in vitro. Our studies demonstrated that DFE displayed strong thrombolytic ability in mouse. Its effect is better than clinically used UK.
Thrombus formed via the effects of cruor, anticoagulation, fibrinolytic system, haemorheology, vascular endothelial cells, platelets and other factors. Animal thrombosis models are the most effective way to evaluate the function of thrombolytic agents. As an experimental model of peripheral obstructive disease, carrageenan-induced thrombosis in mice was used, because of its advantages of simple induction in small laboratory animals and easy to observe without killing the animals. In this study, the results showed that DFE significantly inhibited tail thrombus formation after the injection of carrageenan. The antithrombotic effect of DFE is even more obvious than some newly reported fibrinolytic enzymes such as Subtilisin QK [16
The oxidative damage of the endarterium induced by FeCl3 is the traditional method to establish the Animal Blood Bolts Model, and it is convenient, fast and the results are reproducible. A moderate concentration (10%) of FeCl3 was used in this study, and the fibrinolytic system could dissolve fibrin, which is the main component of thrombus. The impacts on the fibrinolytic system are the key to evaluate the effect of thrombolytic drugs. In the study, markedly thrombolytic effect of DFE was showed in the in vivo test after the carotid artery thrombosis was made.
Microorganism is an important source of thrombolytic enzymes. With the fast growth and easy control characteristics, microorganism can be manually controlled to obtain the target product. With the development of fermentation industry and extraction of fermentation products, there will be a broad prospect of clinical use of this thrombolytic agent.
Many factors play important roles in thrombosis. Molecular mechanisms on effective prevention and treatment of thrombosis of the DFE from Bacillus subtilis LD-8547 is not clear now. Therefore, a further study is needed in the future.