In this work we prepared rabbit antisera containing functional antibodies (Ab) specific for each class of pathology-inducing Vipera ammodytes ammodytes (Vaa) venom constituents, haemorrhagic metalloproteinases (H) and neurotoxic ammodytoxins (Atxs), separately and for both together. The involvement of these Ab in neutralising the toxicity of whole Vaa venom in mice was assessed using the ED50 assay, with the primary aim of quantifying the active compound neutralising venom-induced pathology of the antivenom. The ED50 assay in mice is the only regulatory approved assay for estimating antivenom potency. Fully functional anti-Atx Ab were shown to be responsible for neutralising the venom toxicity, while anti-H Ab are not protective in this assay. Thus, the mouse ED50 assay, intended to measure the active principle of the antivenom, does not measure Ab specific for Vaa venom haemorrhagins, and consequently does not fulfil its primary task from the regulatory point of view.
COBISS.SI-ID: 25699367
We have characterized a novel component of the nose-horned viper venom, termed here VaH3, as a potently hemorrhagic snake venom metalloproteinase (SVMP). Its proteolytic activity and overall stability depend on the presence of Zn2+ and Ca2+ ions. The molecular mass of this slightly acidic molecule, determined by MALDI/TOF analysis, is 104 kDa. Chemical reduction and S-carbamoylmethylation result in a single monomer of 53.7 kDa. N-deglycosylation decreased this mass by 4.6 kDa. The complete amino acid sequence of VaH3 was determined by protein and cDNA sequencing, showing that each of the identical glycoprotein subunits comprise a metalloproteinase, a disintegrin-like domain and a cysteine-rich domain, VaH3 belongs to the P-IIIc class of SVMPs. It shows strong sequence similarity to vascular endothelial cell apoptosis-inducing reprolysins. Anti-ammodytagin antibodies strongly crossreacted with VaH3 and completely neutralized its hemorrhagic activity in rat, despite the fact that the two hemorrhagic P-III SVMPs from V. a. ammodytes venom do not share a very high degree of amino acid sequence identity. In spite of its narrow proteolytic specificity, VaH3 rapidly cleaved some basal membrane and extracellular matrix proteins, such as collagen IV, fibronectin and nidogen. Moreover, it also hydrolyzed plasma proteins involved in blood coagulation. It is an effective alpha-fibrinogenase that cleaves prothrombin and factor X without activating them. The degradation of these proteins likely contributes to the hemorrhagic activity of VaH3. A three-dimensional model of VaH3 was built to help explain structure-function relationships in ADAM/ADAMTS, a family of proteins having significant therapeutic potential and substantial sequence similarity to VaH3.