[Haemostaseologic effects of fractionated snake venoms]. / Wirkungen von Schlangengiftfraktionen auf das Gerinnungssystem.

Comparative investigation concerning gelfiltration as well as haemostaseologic analysis of venoms and venom fractions of some snakes (elapidae and viperidae) have shown that in elapidae an inhibition of coagulation is dominant whilst in viperidae the stimulation of coagulation is of importance. Our investigations produce a basis to select substances for activation of coagulation and substances for inhibition of coagulation. Under pharmacological viewpoints the data may produce information to use snake fractions for anticoagulation or for procoagulant therapy in bleeding tendency.

Involvement of (Na+ + K+)-ATPase in binding and actions of palytoxin on human erythrocytes.

Palytoxin (about 1 pM) increases the permeability of human erythrocytes. We now report its radiolabeling with 125I, followed by affinity purification on porcine kidney membranes. The resulting ligand binds fast and reversibly to intact erythrocytes. The Kd from velocity and equilibrium measurements is 2 X 10(-11) M, and the number of binding sites about 200 per cell. Binding is promoted by divalent cations (Ca2+ greater than Sr2+ greater than Ba2+) and by borate. It is inhibited by K+ (IC50 2 mM), ouabain (IC50 3 X 10(-9) M) and ouabagenin (IC50 6 X 10(-6) M). Conversely, [3H]ouabain is displaced by the substances and concentrations mentioned, and also by palytoxin (Ki 3 X 10(-11) M). Dog erythrocytes, which are known to possess a very low (Na+ + K+)-ATPase activity, are resistant to and lack specific binding sites for palytoxin. Binding of 125I-palytoxin, like that of [3H]ouabain, depends on the state of (Na+ + K+)-ATPase. ATP depletion decreases binding of both ligands to erythrocytes. Binding of 125I-palytoxin and [3H]ouabain to red cell stroma is partially restored by ATP. In contrast to [3H]ouabain, binding of 125I-palytoxin to red cell stroma is not promoted by Mg2+ and Pi. The data show that (a) all known promoters and inhibitors of palytoxin action on human red cells do so by enhancing or decreasing its binding, (b) (Na+ + K+)-ATPase serves as a receptor for palytoxin, and (c) the antagonism by ouabain is competitive at the receptor level. They support our previous hypothesis that palytoxin increases human erythrocyte permeability by formation of pores through (Na+ + K+)-ATPase or its close vicinity.

Palytoxin-induced permeability changes in excitable membranes.

Palytoxin, a toxin isolated from the Caribean corrall Palythoa caribaeorum, increases the cation permeability of excitable membranes in vitro. Three membrane systems have been investigated: axonal membranes from crayfish walking leg nerves, membranes rich in nicotinic acetylcholine receptor isolated from Torpedo californica electric tissue and, for control, artificial liposomes. Ion permeability of the latter was not affected by palytoxin, but with both biological membranes an increase in cation permeability was observed at a palytoxin concentration of 0.14 microM. Palytoxin-induced cation flow through the axonal membrane was not inhibited by tetrodotoxin, indicating that the voltage-dependent sodium channels were not involved. The effect of palytoxin on the receptor-rich membranes was not blocked by alpha-bungarotoxin, a competitive antagonist of the nicotinic acetylcholine receptor, nor by triphenylmethylphosphonium, a blocker of the receptor-ion channel. But with both the axonal and the receptor-rich membranes ouabain was an inhibitor of the palytoxin-induced cation flow. Evidence is presented that it is not the (Na+ + K+)-ATPase which is affected by palytoxin as has been postulated for similar observations with non-neuronal membranes (Chhatwal, G.S., Hessler, H.-J. and Habermann, E. (1983) Naunyn-Schmiedeberg's Arch. Pharmacol. 323, 261-268).

Delayed haemolytic action of palytoxin. General characteristics.

1. Palytoxin is a haemolysin. The erythrocytes from various species can be classified into a sensitive and a hardly sensitive group. The former contain potassium as their main inside cation and are arranged according to their sensitivity as hog greater than or equal to rat, mouse greater than rabbit greater than guinea-pig greater than man. The latter, comprising those from sheep and cattle, have sodium as their main inside cation. In addition, chicken erythrocytes are relatively insensitive. 2. Haemolysis of rat erythrocytes is preceded by a lag period of 1--2 h. With increasing temperature the haemolysis proceeds more quickly but reaches the same final range between 25 and 42 degrees C. The pH optimum in Britton-Robinson buffer supplemented with saline is between 7 and 8. Washing off palytoxin during the prelytic period reduces the haemolytic power. 3. The sensitivity of rat erythrocytes decreases with increase of osmolarity between 235 and 415 mosM. Accordingly, their osmotic resistance is lowered by palytoxin in a concentration-dependent manner. 4. With both rat and sheep erythrocytes, potassium loss by far precedes the haemolysis due to palytoxin. Potassium loss is measurable already after 1 min and increases with time. After 2 hours the quotient between the ED50 of haemolysis and that of potassium loss is around 200. Thus palytoxin is an unusually strong but slow haemolysin of the osmotic type. The extreme prelytic potassium loss and the correlation between susceptibility and potassium content of erythrocytes points towards the relevance of ionic fluxes.

Structure of cobra cardiotoxin CTX I as derived from nuclear magnetic resonance spectroscopy and distance geometry calculations.

The structure of cardiotoxin CTX I from Naja naja atra has been investigated by NMR spectroscopy. Sequence specific resonance assignments have been obtained for all backbone protons as well as for most side-chain protons. Distance geometry calculations were carried out using a metric matrix DG program. A total of 715 NOE constraints, 27 phi angle constraints and a list of the hydrogen bond donors were used for the metric matrix DG calculations and refinement. The average pairwise r.m.s.d. of the resulting structures was 1.01 A for the backbone heavy atoms, and 1.69 A for all heavy atoms. The protein is rich in beta structure and consists of a large triple-stranded, antiparallel beta sheet as well as a short double-stranded, antiparallel beta sheet. Non-regular hydrogen bonding is found between side-chains of the carboxy-terminal end and the rest of the core region. The structure is discussed in terms of evolutionary aspects as well as recent investigations about the biological function and active site.

The influence of the rate of electrical stimulation on the effects of the Anemonia sulcata Toxin ATX II in guinea pig papillary muscle.

In guinea pig papillary muscle, the rate of electrical stimulation (0.1-2 Hz) strongly influenced the effects of the Anemonia sulcata toxin ATX II on action potential duration (APD) and contractile force. In the concentration range studied (10-8-10-7 M), ATX II always produced a larger prolongation in APD at low rates of stimulation. At 0.1 Hz there was a temporal dissociation between the onset of the APD-prolonging and the positive inotropic effect. However, under equilibrium conditions there was a positive relationship between the APD expressed as a fraction of the time during which the membrane was depolarized, and the contractile force irrespective of the change in experimental conditions being variation of stimulation frequency or the addition of ATX II. The results suggest that the positive inotropic effects of both ATX II and increased stimulation frequency could be induced by a similar mechanism, e.g. an increase in sodium of the heart muscle.

Anticoagulant fucoidan fractions from Fucus vesiculosus induce platelet activation in vitro.

Anticoagulant fucoidan fractions of different molecular weight and sulfate content were prepared and investigated for their effects on platelet function in vitro. The fucoidan fractions were incubated with human platelet rich plasma (PRP) at concentrations of 5, 10 and 50 micrograms/ml. Platelet activation was subsequently studied by a standard aggregation assay and flow cytometric determination of the activation dependent platelet-surface markers CD62p (P-selectin, GMP-140) and CD63 (GP53). All fucoidan fractions induced irreversible platelet aggregation in a dose-dependent manner. Comparing fractions of identical molecular weight (100 kDa) the low sulfate content fucoidan FF5 (S = 7.6%) exerted a significantly greater effect than the highly sulfated fucoidan FF7 (S = 10.2%) over the whole concentration range (n = 5, P < 0.05). Among fractions of identical sulfate content fucoidan-induced platelet aggregation was also found to depend on the molecular weight of the fucoidan. At concentrations of 10 and 50 micrograms/ml the high molecular weight fraction FF7/1 (150 kDa) showed a significantly greater effect than the 50 kDa fraction FF7/3 (24.8 +/- 6.7 vs. 7.0 +/- 3.5 and 54.6 +/- 13.5 vs. 15.0 +/- 9.0%, respectively; mean +/- SD, n = 5, P < 0.05). The molecular weight dependence of the fucoidan effect was also reflected by the flow cytometric data. Coincubation of FF7/1 and FF7/3 (10 micrograms/ml) with PRP increased the number of CD62p and CD63 positive platelets by 9.0 +/- 3.3 vs. 2 +/- 1.9 and 7.1 +/- 2.4 vs. 3.2 +/- 2.6% over control values, respectively (n = 5, P < 0.05). In conclusion, our results show that the low molecular weight fucoidan FF7/3 combines potent anticoagulant and fibrinolytic properties with only minor platelet activating effects and is therefore a suitable substance for further pharmacological studies.

Iodine labelling of sea anemone toxin II, and binding to normal and denervated diaphragm.

1. Sea anemone toxin II (ATX II) which keeps the activated sodium channels open, can be labelled at its histidine residues with 125I up to a specific radioactivity of 500 Ci/mmole. Upon intraventricular injection in mice, ATX II causes acute, short-lasting hyperexcitation and convulsions. Its LD50 in mice is between 25 and 50 ng of the native peptide, and between 50 and 100 ng of the radioactive material per animal. 2. The labelled peptide is bound to mouse diaphragm from where it can be displaced by ATX II and, even better, by scorpion neurotoxin but not by other basic peptides, e.g., histone or aprotinin. Binding is not significantly influenced by 50 mM potassium, by replacing sodium with choline, by veratridine or tetrodotoxin. In contrast to binding of alpha-bungarotoxin, binding of ATX II is not changed by denervation of the diaphragm. ATX II binds not only to the muscular but also to the tendinous moiety of the mouse diaphragm. 3. ATX II lowers the surface tension of water. Further experiments are needed to establish the usefulness of 125I-ATX for labelling sodium channels in excitable membranes.

The action of a toxin from the sea anemone Anemonia sulcata upon Mammalian heart muscles.

The cardiac activity of toxin II, a basic polypeptide (m.w.: 4770) from the sea anemone Anemonia sulcata, was investigated in isolated electrically driven guinea-pig and rat auricles, Langendorff heart preparations of guinea-pigs and cat heart-lung preparations. Low concentrations of toxin II (2-100 nM) evoked a dose-dependent positive inotropic effect in the three different heart muscle preparations investigated. Higher concentrations of toxin II produced toxic symptoms like contracture and arrhythmia in auricles and atria (about 25 nM). In isolated cat hearts high toxin II concentrations (about 160 nM) caused unusual toxic symptoms such as long periods of ventricular fibrillation alternating with periods of normal cardiac activity. In rat and guinea-pig auricles as well as in Langendorff hearts of guinea-pigs the extent and rate of the positive inotropic effect induced by toxin II depended on the extracellular calcium concentration (0.45 to 2.7 mM). Toxin II did not alter the heart rate in spontaneously beating isolated cat hearts. In electrically driven guinea-pig auricles, the rate of the inotropic effect induced by toxin II was accelerated by increasing stimulation frequencies. Toxin II did not change the coronary flow in Langendorff heart preparations of guinea-pigs.

Preparation and properties of fluorescence labeled neuro- and cardiotoxin II from the sea anemone (Anemonia sulcata).

Labeling of the sea anemone toxin II with fluoresceinisothiocyanate is described. The native toxin and low molecular weight fluorescent compounds were completely separated from the derivative. Compared to fluorescein, the fluorescence of the toxin-derivative has a red shift and a decreased quantum yield. Like the native toxin, the fluorescent derivative affects the voltage-dependent sodium channel in nerve membranes. Under voltage-clamp conditions, the effect of the modified toxin on the inactivation of sodium channels was 45% of that of the unlabeled toxin.

A simple biochemical method in the search for bioactive polypeptides in a sea anemone (Anemonia sulcata).

The sea anemone Anemonia sulcata is a well-known natural source of supply of biologically active polypeptides. So far, five toxins, ATX I, II, III, IV and AS V, several polyvalent protease inhibitors, an elastase inhibitor, two blood pressure-depressive polypeptides and very recently peptides that inhibit competitively the binding of 125I-dendrotoxin to rat brain membranes and block the voltage-sensitive K+ channels, have been isolated from it. The sea anemone toxins (especially toxin II of A. sulcata, ATX II) are very important tools in neurophysiological and pharmacological research, and their structure-function relationship has been investigated. Because of the great scientific value of the sea anemone toxins a simplification of their purification procedure was elaborated.

A method for the isolation of the caribbean palytoxin (C-PTX) from the coelenterate (zooanthid) Palythoa caribaeorum.

Chromatographically pure toxin was isolated from the zooanthid Palythoa caribaeorum. Toxin isolation was achieved by extraction with 50% ethanol from the homogenized specimens, gel filtration on Sephadex G-50 and ion exchange chromatography on QAE- and SP-Sephadex. Final purification was obtained by gel filtration on Biogel P-6. The LD50 tested on the shore crab Carcinus maenas was 62.5 ng/kg.

Isolation and characterisation of five neurotoxic and cardiotoxic polypeptides from the sea anemone Anthopleura elegantissima.

Five toxins (APE 1 to APE 5) of the sea anemone species Anthopleura elegantissima (Brandt) have been isolated from a toxic by-product fraction of its concentrated crude watery-methanolic extract, prepared previously for the isolation of a neuropeptide (the head-activator) by Schaller and Bodenmüller (Proc. Natl. Acad. Sci. USA 78 (1981) 7000) from 200kg sea anemones. Toxin purification was performed by desalting of the starting material by dialysis (MWCO 3500) against distilled water, anion exchange chromatography on QAE-Sephadex A25 at pH 8, twice gel filtration on Sephadex G50 m, repeated chromatography on QAE-Sephadex at pH 10 and chromatography on the cation exchanger Fractogel EMD SO(3)(-)-650 M.Final purification of the toxins was achieved by HPLC on MN SP 250/10 Nucleosil 500-5 C(18) PPN and MN SP 250/21 Nucleosil 300-7 C(18). Each toxin was composed of at least two isotoxins of which APE 1-1, APE 1-2, APE 2-1, APE 2-2 and APE 5-3 were isolated in preparative scale. With exception of APE 5-3 the sequences of the isotoxins have been elucidated. They resemble the 47 residue type-I long polypeptide toxins native to Anemonia sulcata (Pennant). All isotoxins paralyse the shore crab (Carcinus maenas) by tetanic contractions after i.m. application. The toxins modify current passing through the fast Na(+) channel in neuroblastoma cells, leading to delayed and incomplete inactivation. APE 1-1, APE 2-1 and APE 5-3 produce a positive inotropic effect in mammalian heart muscle, although they differ in potency. The order of potency is APE 2-1>APE 1-1>APE 5-3 (i.e. threshold concentrations are 1, 10 and 300nM, respectively). In addition, they enhance the spontaneous beating frequency in isolated right atria (guinea pig). The most potent cardiotoxic isotoxin is APE 2-1, its sequence is identical with that of AP-C, a toxin isolated and characterised previously by Norton et al. (Drugs and Foods from the Sea, 1978, University of Oklahoma Press, p. 37-50).LD50 APE 2-1:1 micro g/kg b.w. C. maenas (i.m.). LD50 APE 1-1:10 microg/kg b.w. C. maenas (i. m.). LD50 APE 5-3:50 microg/kg b.w. C. maenas (i.m.).

Improved method for the isolation, characterization and examination of neuromuscular and toxic properties of selected polypeptide fractions from the crude venom of the Taiwan cobra Naja naja atra.

An improved chromatographic method was developed to isolate and purify polypeptides and proteins from the crude venom of the Taiwan cobra Naja naja atra. The procedure devised is simple, easy to reproduce, and enables large scale isolation of almost all polypeptides and proteins in this cobra venom. Six pure polypeptide fractions of the venom were isolated and characterized using gel filtration on Sephadex G50 (medium), ion exchange chromatography on SP-Sephadex C25, desalting on Sephadex G25 (fine) and preparative HPLC on a RPC 18 column. The neuromuscular activity of these fractions was tested on the chick biventer cervicis nerve-muscle preparation and their toxicity (LD(50)) was determined after i.v. administration in mice. Their antinociceptive activity was tested in the mouse abdominal test by i.v. application. Two of these polypeptide samples had major physiological effects: one acted as a cardiotoxin causing reversible myocardial contractures with no effect on muscle twitches elicited by nerve stimulation (NS); another was a neurotoxin that blocked muscle contractions in response to NS and exogenously added acetylcholine. The cardiotoxic fraction was identified as CTX I, a well-known cardiotoxin present in this venom, and the neurotoxin was identified as neurotoxin-α with an LD50 in mice of 0.075 mg/kg.