REPOSITORY > RESULTS

Abstract

Cardiovascular diseases are the leading cause of death worldwide (31%) according to the latest World Health Organization report. Intensive research is being conducted to obtain new drugs, as current therapies cause serious side effects. Venoms from the snake family Viperidae are particularly rich in pharmacologically interesting proteins, which mainly influence the blood clotting process. In an attempt to discover new natural components suitable for the further development of safer drugs for the treatment of cardiovascular diseases or for haematological diagnostics, we studied toxins from two local vipers, nose-horned viper, Vipera ammodytes ammodytes (Vaa), and the common viper or adder, Vipera berus berus (Vbb), the most poisonous and the most common European snake, respectively.
The proteomic comparison of the Vaa and Vbb venoms has provided us with an explanation at the molecular level for the differences in the toxicity of these two venoms. The first and most important distinction is that the Vaa venom contains more diverse toxic components. We have also observed higher concentrations of P-III class metalloproteinases in Vaa venom, which are the cause of more severe bleeding in humans upon envenomation by Vaa. There is also a noticeable difference in the activity of both venoms, namely Vbb venom has a procoagulant effect, while Vaa venom has an anticoagulant effect. Among molecules responsible for the anticoagulant effect of the Vaa venom are amodytoxins, which we have identified only in this venom. Amodytoxins are also responsible for neurotoxic effects, which have been rarely observed in the case of Vbb venom poisoning, probably because they are not present in this venom. Because of the larger pool of haemostatically active components in Vaa venom, we have used this venom for further studies. Special attention was paid to the isolation of anticoagulant components. Therefore, we have developed complex chromatographic methods to purify haemostatically active molecules from the venom. The effect of the isolated proteins on blood coagulation was tested using standard clinical tests on commercially available plasmas. Based on the effect that the isolated toxins had on the homogeneous coagulation factors, we also determined their molecular mechanism of action. We discovered a metalloproteinase with fibrinogenolytic activity, VaF1, which specifically cleaves an α-chain of fibrinogen. As such, it has therapeutic potential as a defibrinogenating agent. It belongs to the P-IIIa class of metalloproteinases, which, in contrast to related enzymes, has no significant haemorrhagic activity. We have also identified and characterized a protein that inhibits the intrinsic pathway of blood coagulation, VaaSPH-1, which belongs to the family of serine proteases but is devoid of enzymatic activity. The anticoagulant activity of VaaSPH-1 is a consequence of its binding to FVIIIa antagonizing in this way the binding of a natural partner molecule FIXa. The identified structural features of VaaSPH-1 that are responsible for its interaction with FVIIIa provided an excellent basis for the development of safe medically useful anticoagulants, which are greatly missed in the treatment of venous thromboembolism. Simultaneously with the isolation of VaaSPH-1, another serine protease was isolated, VaaSP-VX. The approach described above helped us to define its mechanism of action, i.e. activation of FV and FX. VaaSP-VX is a suitable substance for the development of a new drug to promote blood clot formation. Due to its potentially dual, FV and FX, activation activity, VaaSP-VX is also interesting for lupus anticoagulant testing to replace the unreliable diluted Russell's viper venom. Additionally, our intention was to improve the current therapy after Vaa and Vbb poisoning by identifying cardiotoxins in their venoms. We identified ammodytin L, an enzymatically inactive myotoxic secreted phospholipase A2, as the most important cardiotoxin in both venoms.