Snake venom serine proteases are complex and multifunctional enzymes that primarily affect haemostasis. Different groups of serine proteases in the venom can have either anticoagulant or procoagulant effects. Molecules that have the latter property are rare. From previous studies on the venom of the nose-horned viper (Vipera ammodytes ammodytes) we already had the cDNA sequence of the protease VaaSP-6 and thus its amino acid sequence, but its biological and molecular mechanisms were not yet known. Due to the high sequence similarity, we speculate that its molecular mechanism of action is similar to that of VaaSP-VX, which can activate the blood coagulation factors V and X (FV and FX). It therefore exerts a procoagulant effect in the blood system. In this Master's thesis, we successfully isolated the VaaSP-6 protease using size exclusion chromatography, cation exchange chromatography and reversed-phase high-pressure liquid chromatography. Mass spectroscopy was used to identify the protease. Using two-dimensional gel electrophoresis, we determined several isoelectric points in the pH range of 4.5–7.5 and an apparent molecular mass of 35 kDa. We investigated its influence on the process of blood coagulation by measuring the activated partial thromboplastin time, the prothrombin time and the thrombin time. We were able to show that VaaSP-6, in contrast to VaaSP-VX, prolongs the activated partial thromboplastin time and prothrombin time and thus acts as an anticoagulant. We have also shown that it does not degrade fibrinogen and does not activate FX.
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