[Viper (Vipera berus) snake bites]. / Ustknutí zmijí obecnou (Vipera berus).

Cases of snake bites (Vipera berus) have as compared with past years a rising trend in the Czech Republic. This ensues among other factors from a higher prevalence of snakes due to the improving ecological situation. The morbidity of snake bites is of no epidemiological importance, the frequency of snake bites amounts to several tens per year and in some clinically manifest intoxication does not develop. Nevertheless in individual cases, in children weakened subjects, a viper bite may be manifested by a serious and in exceptional instances fatal affection. Within the framework of first aid the authors do not recommend application of a tourniquet or dissecting of the wound because of undesirable potentiation of tissue traumatization. Non-specific treatment involves the administration of corticoids and antihistaminics. Specific immunotherapy, administration of horse antiserum (Ipser Europe, Pasteur Mérieux, France) is indicated only in case of systemic or very severe local symptoms and is associated with the risk of a severe allergic reaction. In case of severe systemic symptoms, symptomatic treatment in a health institution of the appropriate type is of fundamental importance. In all cases observation of the affected subject is recommended to rule out intoxication or the development of possible complications.

Severe coagulopathy after a bite of a green bush viper (Atheris squamiger): case report and biochemical analysis of the venom.

A 34 year old male bitten by an adult Atheris squamiger snake developed symptoms of nausea, vomiting, diarrhea which were followed by drowsiness and impaired breathing. Local hemorrhage, edema and pain at the bite-site occurred, but no systemic bleeding or hemorrhagic diathesis developed. All clinical and laboratory parameters were in the normal range except for afibrinogenemia, thrombocytopenia and slight proteinuria. Replacement therapy (fibrinogen and platelet concentrates) and treatment of shock stabilized the patient within 2d and coagulation returned to normal. Atheris squamiger venom was subjected to biochemical and biological analysis. The LD50 of the venom was 5 mg/kg (mice, s.c.). It produced local hemorrhage corresponding to about 25% of the activity of puff adder venom (Bitis arietans). In vitro the venom had a fibrinogen-converting activity, it did not activate purified prothrombin but very likely contained a F V and Ca2+-dependent prothrombin activator. The venom exhibited strong platelet-aggregating activity, which was not inhibited by protease inhibitors and by EDTA or EGTA. The venom also aggregated acetylsalicylic acid treated platelets indicating, that the arachidonic acid pathway was not essential for activation. Rat serum rapidly inhibited the platelet-aggregating activity of the venom; human serum, however, had only a partial inhibitory effect. Preliminary experiments showed that platelet-aggregating activity may be separated from fibrinogen-converting activity by anion-exchange chromatography.

Endothelial desquamating activity of rat synthetic fibrinopeptide B and its analogues "in vivo": identification of responsible sequence.

The endothelial desquamating activity of the synthetic rat fibrinopeptide B (ATTDSDKVDLSIAR-OH), and its analogues was studied "in vivo" after intravenous administration to rats. Rat fibrinopeptide B (FPB) caused a significant increase in the count of circulating endothelial cell carcasses at the dose of 100 nmol/kg. Maximal effect reaching about 270% of the normal value was achieved with the dose of 600 nmol/kg in 30 min. after the injection. No significant thrombocytopenia, no hemolysis and no other life-threatening complications were observed. The same endothelial desquamating effect was achieved with N-terminal FPB(1-7) peptide ATTDSDK-OH, but very low activity exhibited the two FPB mutant peptides: ATDSDKVDLSIAR-OH and ATTNSNK-OH. Our results indicate that N-terminal sequence (1-7) consisting of N-terminal "pig tail" (ATT), acid region (DSD) and basic amino acid (K) is responsible for endothelial desquamating activity of rat FPB. Similar corresponding sequences may be recognized in FPB of different species. The conservation of this common "active site" sequence is less apparent in primates.

Heparan sulfate as a venostatic endothelial stabilizing factor.

A humoral transfer of a factor inducing a decrease of circulating endothelial cells (CEC) released during venostasis was demonstrated in rats. The possibility to block its activity by an in-vitro addition of protamine suggests its identity with an endogenous heparan sulfate possessing an inhibitory effect on endothelial turnover. This was supported by an analogous effect of intravenously administered heparan sulfate-related agent.

[Venomous snake bites]. / Ustknutí jedovatými hady.

Although venomous snakebites are not a serious world wide problem from the epidemiological point of view and are rare under our conditions, it is frequently a very serious and life threatening matter. The patient's prognosis depends among others also on early adequate and comprehensive therapeutic measures. The authors give a list of poisonous snakes and types of toxins and their action on man. Next they describe principles of first aid, specific and non-specific therapy of snakebites and complications resulting from intoxication. The article is supplemented by a special chapter on Viper berus and several clinical cases of snakebites by tropical poisonous snakes.

Antithrombotic effects of heparin and related agents.

Native heparin was very effective in models of arterial and venous thrombosis as well as in a model demonstrating the effect on endothelial stability in rats. The effect on venous thrombosis was particularly prominent. The activities of LMW-heparin were about the same in all three models, the absolute effective dose against arterial thrombosis being much lower than with native heparin. A heparan sulfate-related preparation (suleparoide) was much less effective in both thrombosis models, especially in the venous one, while the activity was most specifically directed to the maintenance of endothelial stability.

Antithrombotic activity of an unsaturated fatty acid preparation.

An unsaturated fatty acid preparation from fish oil, "Epavit", completely prevented arterial thrombosis induced in the rat aorta by a combined stenosis, extensive endothelial perturbation and i.v. serotonin, at an optimum dose of 0.1 ml/200 g orally. A partial effect lasted more than 18 hours. After repeated administrations the acute completely inhibitory effect was decreased but the partial and chronic one remained unchanged. Aspirin (ASA) showed no synergic activity. Venous thrombosis (one-sided ligature of the caval vein combined with extensive endothelial perturbation) was inhibited to a similar degree as arterial thrombosis. Epavit also prevented the destabilizing effect of i.v. citrate on the endothelial lining.

Non-specific therapy of a hemorrhagic diathesis after a bite by a young Bothrops asper (barba amarilla): a case report.

Hemorrhagic diathesis developed 4 hr after a bite by one fang of a two-month-old specimen of Bothrops asper. Severe allergy to horse serum contraindicated the use of horse antivenom, and a substitution therapy was started 20 hr after the bite. During the following 4 days the patient was treated with infusions of 8 g human fibrinogen, 2500 U of cryoprecipitate, 1000 ml of human plasma and vitamin K in several portions. By means of plasmapheresis 1800 ml of plasma was exchanged. Until plasmapheresis on the third day the treatment resulted in short remission and diminution of the spontaneous bleeding, which ceased on the 5th day. Coagulation tests relevant to disseminated intravascular coagulation and consumption coagulopathy were performed for 12 days. Fibrinogen levels started to rise on the 8th day and normalized 12 days after the bite. Analysis of the venoms from juvenile and adult Bothrops asper snakes revealed that the former has a strong prothrombin-converting activity, the latter contained mainly a thrombin-like, fibrinogen-converting enzyme.

The action of a fibrin-promoting enzyme from the venom of Agkistrodon contortrix contortrix on rat fibrinogen and plasma.

The action of a fibrin-promoting enzyme isolated from the venom of A.c.contortrix was investigated. The ratio of fibrinopeptides A and B released was similar in isolated human and rat fibrinogen and human plasma, fibrinopeptide B always being released preferentially. However, no clotting occurred in rat plasma, and no fibrinopeptides were released even after prolonged incubation. The results suggest strong, fast-acting irreversible neutralization of the enzyme activity in rat plasma.

Cross reactivity of mono- and polyvalent antivenoms with Viperidae and Crotalidae snake venoms.

The lethal, local and defibrinating effects of two African Viperidae and four Crotalidae were assayed. Two commercial antivenoms (Behringwerke Nord Africa and Wyeth anticrotalic) and gamma-globulins from hyperimmune rabbit serum were tested for their ability to neutralize the toxic effects of the venoms. Cross neutralization of the lethal effect (antiviperidae antivenom against Crotalidae venom and vice versa) was evident with Behringwerke against Crotalidae as well as with Wyeth against Viperidae. No cross-reactivity was observed with monovalent antivenoms. Practically complete cross-reactivity was observed with polyvalent, but less with monovalent antivenoms in the neutralization of skin hemorrhage. The local effect was neutralized to 0-20% if the antivenom was mixed with the venom. An i.v. injection of antivenom had no effect after intradermally injected venom. Only Echis and Bothrops venoms cause defibrination in vivo. The ability of the two polyvalent antivenoms to neutralize defibrination was very weak. One half milliliter of antivenom neutralized 1 microgram (Echis carinatus) or 10 micrograms (Bothrops asper) of defibrinating venom if injected simultaneously. If antivenom was administered 3 hr after the venom, the fibrinogen level increased to about 50% of the normal values after 24 hr. Cross-reactivity in the neutralization of the defibrinating activity was observed with both polyvalent antivenoms.

Conversion of fibrinogen to fibrin induced by preferential release of fibrinopeptide B.

Fibrin clot-promoting enzyme preferentially releasing fibrinopeptide B from fibrinogen was isolated from the crude venom of Agkistrodon contortrix and its mode of action was studied in detail. A purification procedure involving affinity chromatographies on immobilized lectin and arginine removed plasmin-like and kallikrein-like activities towards low-molecular-weight chromogenic substrates. Fibrin-promoting enzyme cleaved off only fibrinopeptides A and B from fibrinogen. The initial relative rate of release of fibrinopeptide B/fibrinopeptide A depended strongly on the presence of Ca2+. In the absence of Ca2+ the amount of fibrinopeptides released by fibrin-promoting enzyme at the gel point was greater. Fibrinopeptide B was no longer released before fibrinopeptide A from the non-polymerizing N-terminal disulphide knot of fibrinogen. Catalyzed by activated factor XIII, complete gamma-dimer and alpha-polymer formation was observed in fibrin from which only 23% of fibrinopeptide A, but 100% of fibrinopeptide B was released by fibrin-promoting enzyme. gamma-dimer formation markedly preceded alpha-polymer formation. These results strongly imply a similar overall arrangement of monomer molecules in this fibrin when compared with a thrombin-induced fibrin in which fibrinopeptide A is released before fibrinopeptide B. These observations support the concept that on fibrinopeptide B release from fibrinogen polymerization sites are exposed which reinforce the sites exposed on the release of fibrinopeptide A.

Fibrinopeptide-releasing enzymes in the venom from the southern copperhead snake (Agkistrodon contortrix contortrix).

Protein fractionation techniques utilizing the different properties of the sample (size, charge, sugar moiety) were employed to characterize the crude A. c. contortrix venom. Gel filtration chromatography resolved about six to eight peaks, high performance liquid ion exchange chromatography and chromatofocusing about 12-14 peaks exhibiting hydrolytic activity. Two fibrin clot-promoting enzymes--both releasing fibrinopeptides A and fibrinopeptides B, but with different fibrinopeptide A/fibrinopeptide B relative rates were observed. The two enzymes (or enzyme isoforms) were serine proteinases with essentially the same hydrolytic activity towards low molecular chromogenic substrate for thrombin. They were of approximately the same size (one peak of 68,000 relative mol.wt on gel filtration chromatography), had apparent isoelectric points of about 6.4 and 5.4, respectively, and were glycoproteins.

Evaluation of individual variability in the composition of Agkistrodon contortrix contortrix venom by means of HPLC and two-dimensional PAGE.

Qualitative and quantitative differences of coagulant enzymes in venom samples from individuals of the Agkistrodon c.c. were studied by means of 2D-PAGE and high performance anion-exchange chromatography. A great diversity was found among individual venoms. The two most similar venoms had an identical composition in only about 90%, the least similar ones in about 45% spots. All venoms studied contained more than two fractions with fibrinolytic activity. In three out of the nine analysed venoms two different thrombic proteases were present, one venom contained only one of these enzymes, whereas in five venoms no thrombic activity was detectable.

Assessment of a complex mixture of interacting proteins in the coagulant active venom from Agkistrodon contortrix contortrix.

The crude venom of Agkistrodon contortrix contortrix was characterized by means of 2D-PAGE (using various separation principles in the respective directions) and high performance gel filtration chromatography. It was found that the venom presents a rich and remarkably stable mixture of proteins, mostly glycoproteins, which may interact each other. High stability of the venom in spite of the presence of many proteolytic enzymes, must most likely be attributed to the sugar moieties of venom proteins. Carbohydrate composition also causes considerable heterogeneity in charge and the presence of wide range of charge isomers. The intricate complexity of the venom makes it a real difficult-to-separate mixture.

Ecarin test in diagnosis of dicoumarol therapy, liver diseases and DIC.

The conversion of prothrombin by ecarin is independent of the presence of gamma carboxyglutamic residues on the N terminal of the molecule. Ecarin converts therefore also the acarboxylated precursor-PIVKA II. In a group of 347 patients under dicoumarol therapy of different intensity and duration PIVKA was detected in BaSO4 adsorbed PPP only in samples where prothrombin level (Quick's test) was lower than 50% of normal values. Increase in PIVKA did not correspond to the decrease of prothrombin. In some cases where the treatment was longer than two years no PIVKA was detected even when prothrombin was under 20%. This is explained by synthesis of partially carboxylated prothrombin molecules, which can be adsorbed. In liver diseases the ecarin test and Quick's time in native, untreated PPP showed about identical decrease of prothrombin level. This indicates that no PIVKA is released to plasma. The functional defect of the hepatocyte does not involve gamma carboxylation as long as vitamin K is provided. In patients with DIC ecarin test showed significantly higher level of thrombin activity than those obtained with Quick's test. It is known that during hypercoagulation stage of DIC prethrombin 1 and 2 are formed by the excess of thrombin. These split products of prothrombin are convertible by ecarin to meizothrombin 1 and 2. A positive ecarin test can be also due to acarboxylated precursors which are released during increased proteosynthesis triggered by consumption coagulopathy.