252301 Biomedical Potentials of Bacterial Fibrinolytic Enzymes

Wednesday, October 31, 2012
Hall B (Convention Center )
Sudhir Rai, LGRD, NICHD, NIH, BETHESDA, MD

Many common disorders are related to thrombosis , of which myocardial infarction and stroke are the most common. Thrombosis is also a clinical problem in various cancers and after surgery, especially orthopedic. Blood clots, mostly comprising fibrin, are formed from fibrinogen by thrombin (EC 3.4.21.5); its cross-linking is catalyzed by factor XIIIa. Fibrin is hydrolyzed by plasmin (EC 3.4.21.7), which is activated from plasminogen by plasminogen activators. Fibrin formation and fibrinolysis are kept in balance by homeostasis. However, when fibrin is not hydrolyzed because of some disorder, thrombosis occurs.

Fibrinolytic agents that are available for clinical use are mostly plasminogen activators, such as a tissue-type plasminogen activator (t-PA), urokinase, and the bacterial plasminogen activator streptokinase. Despite their widespread use, these fibrinolytic agents present important shortcomings, including short half-lives, high cost, and the risk of allergic reactions and bleeding complications. Therefore, research for fibrinolytic agents from other sources continues. To date, fibrinolytic enzymes have been discovered in snake venom, insects, marine creatures, and fermented foods such as Bacillus subtilis natto, Chungkook-jang, and Tempeh. In particular, oral administration of natto or its enzyme (nattokinase) can enhance the release of an endogenous plasminogen activator in both animal models and human subjects.

Recently, serine proteases have attracted interest for their hydrolytic activity toward recalcitrant animal proteins such as collagen, keratin, blood clot and amyloid prion proteins from the perspective of beneficial use of industrial waste and medical applications. We recently found a serine protease from Bacillus sp., through screening of 130 environmental samples. Among them, few isolates showed high hydrolytic activity toward recalcitrant proteins.

In the present study, we demonstrated the remarkable fibrinolytic ability of purified serine protease in vitro and in vivo. First, purified serine protease was analyzed for its hydrolytic activities toward mimics of plasminogen and fibrin. Fibrinolytic activity of purified serine protease was compared with those of known fibrinolytic enzymes such as thrombin, prothrombin or coagulation factor Xa. The fibrin/fibrinogen degradation pattern demonstrated that purified serine protease preferentially degraded the Aa- and Bb-chains of fibrin and fibrinogen, suggesting it as alpha,beta-fibrinogenase. Further, purified serine protease is devoid of acute toxicity in a mice model and is non-toxic to mammalian cells, suggesting suitable candidate for development of a thrombolytic drug to prevent and combat the various thrombosis-associated diseases and cardiovascular disorders


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