The Procoagulant Snake Venom Serine Protease Potentially Having a Dual, Blood Coagulation Factor V and X-Activating Activity.

A procoagulant snake venom serine protease was isolated from the venom of the nose-horned viper (Vipera ammodytes ammodytes). This 34 kDa glycoprotein, termed VaaSP-VX, possesses five kDa N-linked carbohydrates. Amino acid sequencing showed VaaSP-VX to be a chymotrypsin-like serine protease. Structurally, it is highly homologous to VaaSP-6 from the same venom and to nikobin from the venom of Vipera nikolskii, neither of which have known functions. VaaSP-VX does not affect platelets. The specific proteolysis of blood coagulation factors X and V by VaaSP-VX suggests that its blood-coagulation-inducing effect is due to its ability to activate these two blood coagulation factors, which following activation, combine to form the prothrombinase complex. VaaSP-VX may thus represent the first example of a serine protease with such a dual activity, which makes it a highly suitable candidate to replace diluted Russell's viper venom in lupus anticoagulant testing, thus achieving greater reliability of the analysis. As a blood-coagulation-promoting substance that is resistant to serpin inhibition, VaaSP-VX is also interesting from the therapeutic point of view for treating patients suffering from hemophilia.

The First Intrinsic Tenase Complex Inhibitor with Serine Protease Structure Offers a New Perspective in Anticoagulant Therapy.

Components of the intrinsic blood coagulation pathway, among them factor VIIIa (FVIIIa), have been recognized as suitable therapeutic targets to treat venous thromboembolism, pathological process behind two very serious cardiovascular diseases, deep vein thrombosis and pulmonary embolism. Here, we describe a unique glycoprotein from the nose-horned viper (Vipera ammodytes ammodytes [Vaa]) venom, Vaa serine proteinase homolog 1 (VaaSPH-1), structurally a serine protease but without an enzymatic activity and expressing potent anticoagulant action in human blood. We demonstrated that one of its targets in the blood coagulation system is FVIIIa of the intrinsic tenase complex, where it antagonizes the binding of FIXa. Anticoagulants with such characteristics are intensively sought, as they would be much safer for medical application as the contemporary drugs, which frequently induce excessive bleeding and other complications. VaaSPH-1 is unlikely to be orally available for chronic usage as it has molecular mass of 35 kDa. However, it represents a very promising template to design low molecular mass FVIIIa-directed anticoagulant substances, based on structural features of the interaction surface between VaaSPH-1 and FVIIIa. To this end, we constructed a three-dimensional model of VaaSPH-1 bound to FVIIIa. The model exposes the 157-loop and the preceding α-helix as the most appropriate structural elements of VaaSPH-1 to be considered as a guideline to synthesize small FVIIIa-binding molecules, potential new generation of anticoagulants.

Ammodytin L is the main cardiotoxic component of the Vipera ammodytes ammodytes venom.

Venom of the nose-horned viper (V. a. ammodytes) as also venoms of some related European viperids can induce also cardiotoxic effects in mammals. In this work we demonstrated that the protein in the V. a. ammodytes venom acting on heart is a myotoxic secreted phospholipase A2 analogue ammodytin L (AtnL). In the isolated perfused rat heart AtnL induced significant and irreversible cardiotoxicity characterized by atrioventricular (AV) blockade. This venom protein induced appearance of high levels of creatine kinase, lactate dehydrogenase, aspartate aminotransferase and troponin I in the sinus effluent of the isolated heart, indicative for myocardial damage, which is obviously the primary cause of its cardiotoxic action. Gel filtration chromatography subfractions C1 and C2 of the V. a. ammodytes venom harboured most of the venom cardiotoxicity. As we showed, just these two subfractions contained also AtnL. Subfraction C1 in the final CF concentration 11.3 µg/mL (containing 3.1 µg/mL AtnL) induced a complete cardiac arrest while subfraction C2 in the final CF concentration 6.0 µg/mL (containing 0.8 µg/mL AtnL) and the pure AtnL (1.0 µg/mL) did not. Contrary to AtnL, subfraction C1 at 11.3 µg/mL was not able to induce the AV blockade. This exposed the only other cardiotoxic subfractions-specific venom protein, a cysteine-rich secretory protein (CRISP), as an additional venom component potentially involved in modulation of the heart activity. Cardiotoxicity reported in some cases of the adder (V. berus) venom and the asp viper (V. aspis) venom poisonings may be assigned to AtnL in these venoms.

Disintegrins from the Venom of Vipera ammodytes ammodytes Efficiently Inhibit Migration of Breast Cancer Cells.

Integrins are plasma membrane proteins, whose dysfunction frequently results in cancer pathology, and therefore they represent important targets of anti-tumour therapy. Snake venoms are a rich source of disintegrins (Dis), proteins that specifically bind integrins and thus interfere with their functions. In an attempt to discover new molecules for treatment of breast cancer, the major type of cancer in women, we isolated a dimeric Dis (Vaa-Dis) from the venom of the nosehorned viper. By cell viability testing we demonstrated that 50 nM and higher concentrations of Vaa-Dis were toxic to highly invasive human breast adenocarcinoma cell line MDA-MB-231. Wound-healing assay revealed that already at one order of magnitude lower concentrations Vaa-Dis efficiently inhibited MDA-MB-231 cell migration. This exposed a promising anti-metastatic potential of Vaa-Dis and a good perspective of these natural snake venom proteins for further research and development towards the application in breast cancer treatment.

Cardiotoxic effects of the Vipera ammodytes ammodytes venom fractions in the isolated perfused rat heart.

The nose-horned viper (Vipera ammodytes ammodytes) is the most venomous European snake. Its venom is known as haematotoxic, myotoxic and neurotoxic but it exerts also cardiotoxic effects. To further explore the cardiotoxicity of the venom we separated it into four fractions by gel filtration chromatography. Three fractions that contain polypeptides (A, B, and C) were tested for their effects on isolated rat heart. Heart rate (HR), incidence of arrhythmias (atrioventricular (AV) blocks, ventricular tachycardia, ventricular fibrillation, and asystolia), coronary flow (CF), systolic, developed and diastolic left ventricular pressure (LVP) were measured before, during, and after the application of venom fractions in three different concentrations. Fraction A, containing proteins of 60-100 kDa, displayed no effect on the rat heart. Fractions B and C disturbed heart functioning in similar way, but with different potency that was higher by the latter. This was manifested by significant decrease of HR and CF, the increase of diastolic, and the decrease of systolic and developed LVPs. All hearts treated with fraction C in the final CF concentrations 22.5 and 37.5 µg/mL suffered rapid and irreversible asystolia without AV blockade. They underwent also ventricular fibrillation and ventricular tachycardia. Fraction B affected hearts only at the highest dose inducing asystolia in all hearts, ventricular fibrillation in 80% and ventricular tachycardia in 70% of the hearts. Venom fraction C induced 71% of all recorded heart rhythm disturbances, significantly more than fraction B, which induced 29%. Most abundant proteins in fraction C were secreted phospholipases A2 among which the venom component acting on the heart is most probably to be looked for.

Venomics of Vipera berus berus to explain differences in pathology elicited by Vipera ammodytes ammodytes envenomation: Therapeutic implications.

UNLABELLED: Vipera berus berus (Vbb) is the most widely distributed and Vipera ammodytes ammodytes (Vaa) the most venomous viper in Europe. In particular areas of the Old continent their toxic bites constitute a considerable public health problem. To make the current envenomation therapy more effective we have analysed the proteome of Vbb venom and compared it with that of Vaa. We found the proteome of Vbb to be much less complex and to contain smaller levels of particularly snaclecs and sPLA2s. Snaclecs are probably responsible for thrombocytopenia. The neurotoxic sPLA2s, ammodytoxins, are responsible for the most specific feature of the Vaa venom poisoning - induction of signs of neurotoxicity in patients. These molecules were not found in Vbb venom. Both venoms induce haemorrhage and coagulopathy in man. As Vaa and Vbb venoms possess homologous P-III snake venom metalloproteinases, the main haemorrhagic factors, the severity of the haemorrhage is dictated by concentration and specific activity of these molecules. The much greater anticoagulant effect of Vaa venom than that of Vbb venom lies in its higher extrinsic pathway coagulation factor-proteolysing activity and content of ammodytoxins which block the prothrombinase complex formation. BIOLOGICAL SIGNIFICANCE: Envenomations by venomous snakes constitute a considerable public health problem worldwide, and also in Europe. In the submitted work we analysed the venom proteome of Vipera berus berus (Vbb), the most widely distributed venomous snake in Europe and compared it with the venom proteome of the most venomous viper in Europe, Vipera ammodytes ammodytes (Vaa). We have offered a possible explanation, at the molecular level, for the differences in clinical pictures inflicted by the Vbb and Vaa venoms. We have provided an explanation for the effectiveness of treatment of Vbb envenomation by Vaa antiserum and explained why full protection of Vaa venom poisoning by Vbb antiserum should not be always expected, especially not in cases of severe poisoning. The latter makes a strong case for Vaa antiserum production as we are faced with its shortage due to ceasing of production of two most frequently used products.

Structural and biochemical characterisation of VaF1, a P-IIIa fibrinogenolytic metalloproteinase from Vipera ammodytes ammodytes venom.

A high molecular mass metalloproteinase with α-fibrinogenolytic activity, termed VaF1, was purified from nose-horned viper (Vipera ammodytes ammodytes) venom. Subcutaneous injection of 9 µg of VaF1 did not induce bleeding in rats. Nevertheless, in vitro it degraded collagen IV, nidogen and fibronectin, components of the extracellular matrix, although with low efficacy and narrow specificity. VaF1 would be expected to exert anti-coagulant action, due to its hydrolysis of fibrinogen, factor X, prothrombin and plasminogen, plasma proteins involved in blood coagulation. The enzyme is a single-chain glycoprotein with a molecular mass of 49.7 kDa, as determined by mass spectrometry, and multiple isoelectric points centred at pH 5.8. The complete amino acid sequence of the precursor of VaF1 was deduced by cloning and sequencing its cDNA. Composed of metalloproteinase, disintegrin-like and cysteine-rich domains, VaF1 is a typical P-IIIa subclass snake venom metalloproteinase. Although it possesses a collagen-binding sequence in its disintegrin-like domain, VaF1 displayed no effect on collagen-induced platelet aggregation in vitro. Two consensus N-glycosylation sites are present in the sequence of VaF1, however, the extent of its glycosylation is low, only 5.2% of the total molecular mass. Interestingly, in standard experimental conditions VaF1 is not recognised by antiserum against the whole venom, so it can contribute to post-serotherapy complications, such as ineffective blood coagulation, in the envenomed patient.