Effect of snake venoms on blood coagulation

Abstract

Snake venoms are rich collections of enzymes, proteins, peptides and other components that can cause a wide range of physiological, neurological and haemostatic effects on their prey in an attempt to immobilise them and aid in digestion. Among these effects, the venom components that affect mammalian haemostasis have been most well studied for more than 150 years. They have contributed to elucidation of the detailed mechanisms of the coagulation cascade (e.g. platelet aggregation and inhibition, mechanism of defibrination, DIC, various coagulopathies, etc), elucidation of various clinical disorders (e.g. congenital haemorrhagic disorder, various blood factor deficiencies, etc), development of many diagnostics (e.g. Styphen or ReptilaseB. time) and therapeutics (e.g. ancrod and batroxobin). Therefore venoms have been regarded as b gold minesb for researchers, pharmaceutical companies, clinical analysts as well as medical practitioners and surgeons.An anticoagulant proteinase, named agkistase, was isolated from the venom of pit viper, Agkistrodon halys halys, through successive ion-exchange and size exclusion liquid chromatography. Its purity was checked by high resolution HPLC, capillary electrophoresis and mass spectrophotometry. It is a serine protease with a,b-fibrinogenase activity, which cleaves plasmatic fibrinogen chain a and b rendering it unclottable by thrombin. Agkistase was found to be fibrinogenolytic with slight fibrinolytic activity and did not affect other coagulation factors nor cause platelet aggregation. This fibrinogenase activity is unaffected by pH 5 ~ 9 and salt concentrations up to 0.8 M NaCl but was inhibited by serine protease inhibitors (e.g. PMSF and aprotinin). It has an a-fibrinogenase and b-fibrinogenase activities of 15.1 and 0.25 mg min-1 mg enzyme-1, respectively. Thrombelastographic analyses revealed a prolongation in r and k values but unchanged MA, with extension in incubation with whole blood samples. This observation is usually seen for haemophilic blood samples, evident of abnormalities in one or more coagulation factors b in this case, fibrinogen.Human blood coagulation assays on agkistase showed that it has EC50 values of 5.1 B1 1.5, 0.26 B1 0.1 and 0.5 B1 0.2 ug/ml on prothrombin time, recalcification time and thrombin time, respectively, in vitro. In vivo studies on C57BL mice showed that it is not toxic, haemorrhagic or thrombogenic up to 1.67 mg/kg when injected intravenously. These mice also showed no signs of thrombocytopenia. Evaluation of its anti-thrombotic potential on a thrombotic mouse model exhibited positive results in both reductions in thrombi occurrence and size, at agkistase concentration of only 50 ng/kg mouse when injected intravenously. We concluded that it will be a promising new anti-thrombotic drug different from current available snake venom thrombin-like enzymes (SVTLEs) due to: (i) its low effective concentration with no observable negative effects, (ii) fast and specific fibrinogenolytic activities, (iii) presence of mild secondary fibrinolytic activities, and (iv) successful demonstration of its anti-thrombotic capability on a mouse model.