Hemorrhagin VaH4, a covalent heterodimeric P-III metalloproteinase from Vipera ammodytes ammodytes with a potential antitumour activity.

In the envenomation caused by a bite of Vipera ammodytes ammodytes, the most venomous snake in Europe, hemorrhage is usually the most severe consequence in man. Identifying and understanding the hemorrhagic components of its venom is therefore particularly important in optimizing medical treatment of patients. We describe a novel high molecular mass hemorrhagin, VaH4. The isolated molecule is a covalent dimer of two homologous subunits, VaH4-A and VaH4-B. Complete structural characterization of A and partial characterization of B revealed that both belong to the P-III class of snake venom metalloproteinases (SVMPs), comprising a metalloproteinase, a disintegrin-like domain and a cysteine-rich domain. However, neither VaH4-A nor VaH4-B possess the Cys174 involved in the inter-subunit disulphide bond of P-III SVMPs. A three-dimensional model of the VaH4 dimer suggests that Cys132 serves this function. This implies that dimers in the P-III class of SVMPs can be formed either between their Cys132 or Cys174 residues. The proteolytic activity and stability of VaH4 depend on Zn²âº and Ca²âº ions and the presence of glycosaminoglycans, which indicates physiological interaction of VaH4 with the latter element of the extracellular matrix (ECM). The molecular mass of VaH4, determined by MALDI/TOF mass spectrometry, is 110.2 kDa. N-deglycosylation reduced the mass of each monomer by 8.7 kDa. The two possible N-glycosylation sites in VaH4-A are located at completely different positions from those in homodimeric P-IIIc VaH3 from the same venom, however, without any evident functional implications. The hemorrhagic activity of this slightly acidic SVMP is ascribed to its hydrolysis of components of the ECM, particularly fibronectin and nidogen, and of some blood coagulation proteins, in particular the α-chain of fibrinogen. VaH4 is also significant medically as we found it cytotoxic against cancer cells and due to its substantial sequence similarity to ADAM/ADAMTS family of physiologically very important human proteins of therapeutic potential.

Pharmacological aspects of Vipera xantina palestinae venom.

In Israel, Vipera xantina palestinae (V.x.p.) is the most common venomous snake, accounting for several hundred cases of envenomation in humans and domestic animals every year, with a mortality rate of 0.5 to 2%. In this review we will briefly address the research developments relevant to our present understanding of the structure and function of V.x.p. venom with emphasis on venom disintegrins. Venom proteomics indicated the presence of four families of pharmacologically active compounds: (i) neurotoxins; (ii) hemorrhagins; (iii) angioneurin growth factors; and (iv) different types of integrin inhibitors. Viperistatin, a α1ß1selective KTS disintegrin and VP12, a α2ß1 selective C-type lectin were discovered. These snake venom proteins represent promising tools for research and development of novel collagen receptor selective drugs. These discoveries are also relevant for future improvement of antivenom therapy towards V.x.p. envenomation.

Acutolysin C, a weak hemorrhagic toxin from the venom of Agkistrodon acutus with leucoagglutination activity

The properties and agglutination activity of acutolysin C, a hemorrhagic metalloproteinase obtained from Agkistrodon acutus venom, were studied herein. Acutolysin C is a basic glycoprotein consisting of a single polypeptide chain with a molecular weight of 23.1 kDa and pI 8.7, containing one Zn2+ and one Ca2+ per molecule. It possesses caseinolytic, weak lethal (LD50 = 7.6 mg/kg) and weak hemorrhagic (MHD = 12.0 µg) activities, but does not present fibrinolytic, fibrinogenolytic, arginine esterase and phospholipase A2 actions. In addition, it revealed agglutination activity on some animal lymphocytes, including five species of mammals, six of birds, three of reptiles and one of amphibians, but had no effect on lymphocytes from two species of reptiles, one amphibian and nine species of fish. It had no effects on the erythrocytes and platelets of all 26 animal species tested. Both leucoagglutination and caseinolytic activities were inhibited by EDTA; while cysteine, 2-mercaptoethanol, 1,4-dithiothreitol, glutathione, serum against acutolysin C and serum against homologous snake venom as well as glucose, sucrose, mannose, lactose and galactose had no effects on inhibition. The lowest concentration of acutolysin C that induced mouse lymphocyte agglutination was 2.5 µg/mL. Acutolysin C is an interesting substance since it is the first member of the hemorrhagin family to be shown to have leucoagglutination activity. (AU)

Serum kinetics of Calloselasma rhodostoma (Malayan pit viper) venom components in a rabbit

The serum kinetics of Calloselasma rhodostoma (Malayan pit viper) venom - specifically two of its components, the major hemorrhagin (rhodostoxin) and a thrombin-like enzyme - was examined in a rabbit by double-sandwich enzyme-linked immunosorbent assay (ELISA). The animal received intramuscularly a 1.0-mg/kg dose of C. rhodostoma venom. The venom level in serum peaked 12 hours after the injection, followed by a gradual decline and finally reached low rates 72 hours after administration. The serum kinetic profile of venom components, however, did not correspond to the profile of the whole C. rhodostoma venom. The serum levels of the C. rhodostoma thrombin-like enzyme increased slowly and peaked only 48 hours post-injection. Then both thrombin-like enzyme and rhodostoxin remained at relatively high levels 72 hours after administration. Data suggest that various venom components bind to tissue at the injection site with different affinities and that conjugated venom components were continuously released into circulation at different rates. The prolonged high serum levels of both thrombin-like enzyme and hemorrhagin are consistent with the clinical picture of prolonged clotting deficiency in severe cases of C. rhodostoma envenomation. Our results also suggest that since venom components are being released into and eliminated from the circulation at different rates, the "average composition" of the venom antigen in the circulation changes over time. This implies that data from ELISA quantification of antigen levels from serum venom employing "whole venom" as reagent must be interpreted with care.(AU)