Inhibition of platelet aggregation by a fibrinogenase from Naja nigricollis venom is independent of fibrinogen degradation

Fibrinogenases, proteinases which release peptides from the carboxy-terminal end of fibrinogen, are classified as alpha-fibrinogenases or beta-fibrinogenases, based on their ability to preferentially attack the A alpha or B beta chain, respectively, of fibrinogen. alpha-Fibrinogenases have been shown to inhibit platelet aggregation whereas beta-fibrinogenases do not. We have studied the inhibition of platelet aggregation by proteinase F1, an alpha-fibrinogenase from Naja nigricollis venom. This proteinase inhibits whole blood aggregation in a dose-dependent manner, with an IC50 value of 145 micrograms. However, the proteinase fails to inhibit aggregation in washed platelet suspensions. Thus, proteinase F1 appears to require a plasma factor to cause inhibition. Since fibrinogen acts as an adhesive protein which links platelets during aggregation, and since proteinase F1 cleaves fibrinogen, we investigated the role of fibrinogen in the inhibition of platelet aggregation by proteinase F1. The degradation products of fibrinogen formed by the proteinase did not cause significant inhibition. Thus, the inhibition of platelet aggregation appears to be independent of the formation of fibrinogen degradation products. We also studied the effect of proteinase F1 on aggregation of platelets that were reconstituted with defibrinogenated plasma. The proteinase inhibited aggregation of platelets even in the absence of plasma fibrinogen. Proteinase F1 was about 4-fold more potent in inhibiting platelet aggregation in defibrinogenated blood. From these results, we conclude that the inhibition of platelet aggregation by proteinase F1 from N. nigricollis venom is independent of its action on fibrinogen.     

Basic phospholipase from the venom of Naja nigricollis is cytotoxic

The basic phospholipase A2 purified from the venom of Naja nigricollis (Institut Pasteur), possesses an intense cytotoxic activity toward fetal cells from FL strain. At a concentration equal to 1.6 x 10(-6) M, the PLA2 lyses 50% of the cells present in a suspension containing 3.5 x 10(6) cells per millilitre. Other PLA2 from various origins do not exhibit such cytotoxic activity.     

Purification of a Class B1 platelet aggregation inhibitor phospholipase A2 from Indian cobra (Naja Naja) venom

A platelet aggregation inhibitor phospholipase A(2) (NND-IV-PLA(2)) was isolated from Naja naja (Eastern India) venom by a combination of cation and anion exchange chromatography. NND-IV-PLA(2) is the most catalytically active enzyme isolated from the Indian cobra venom. The acidic PLA(2) profile of Eastern regional Indian cobra venom is distinctly different from that of the western regional venom. However the acidic PLA(2)s from both the regions follow the pattern of increasing catalytic activity with increase in acidic nature of the PLA(2) isoform. NND-IV-PLA(2) is a Class B1 platelet aggregation inhibitor and inhibits platelet aggregation induced by ADP, collagen and epinephrine. Modification of active site histidine abolishes both catalytic activity and platelet aggregation inhibition activities while aristolochic acid, a phospholipase A(2) inhibitor has only partial effect on the two activities.     

Cardiotoxic effects of Naja nigricollis venom phospholipase A2 are not due to phospholipid hydrolytic products

N. nigricollis phospholipase A2 (200 micrograms) has marked cardiotoxic actions on the perfused rat heart including the induction of arrhythmias, increases in ventricular thresholds, conduction and resting tension, and decreases in contractile tension. In contrast, perfusion with lysophosphatidyl choline and oleic acid, in concentrations comparable to those estimated to be formed during N. nigricollis treatment, has little effect on cardiac function. The less toxic N. n. atra phospholipase A2 also has little effect on cardiac function even though it causes approximately the same low percentage of phospholipid hydrolysis as produced by N. nigricollis phospholipase A2. Perfusion with albumin did not alter the phospholipase A2 induced changes in cardiac function. Lysophosphatidyl choline in concentrations higher than expected to be formed during N. nigricollis phospholipase A2 treatment, increased conduction time to a greater extent than ventricular threshold whereas the reverse was true for phospholipase A2. We conclude that the cardiotoxic effects of N. nigricollis phospholipase A2 are not due to the accumulation of phospholipid hydrolytic products, and on the basis of prior studies with chemically modified phospholipase A2 enzymes we suggest that N. nigricollis phospholipase A2 has a direct, non-enzymatic, cardiotoxic action.     

Isolation and characterization of a cytotoxin P4 from the venom of Naja nigricollis nigricollis preferentially active on tumor cells.

Cytotoxin P4 was isolated from the venom of Naja nigricollis nigricollis in three steps and contained 55% of the crude cytotoxic activity. It had a molecular weight of 8 KD, was stable over a pH range of 1-11 and in boiling water for at least 15 min. It had no measurable enzymatic activities, but was destroyed by proteases. Concentrations of 0.8, 1, 1.2, 25. 20 and 45 ug/ml, were needed to destroy murine melanoma B16 and WEHI 3B leukemia, rat chondrosarcoma, mouse erythrocytes and spleen cells, and human erythrocytes, respectively, thereby showing preferential cytotoxicity to the examined tumor cells. It also prevented the development of the melanoma, leukemia and chondrosarcoma tumors in vivo when mixed with the cells prior to the injection into the animal.     

Role of tyrosine and tryptophan residues in the structure-activity relationships of a cardiotoxin from Naja nigricollis venom

This paper is an attempt to localize the critical area determining toxicity in a snake cardiotoxin. Toxin gamma is a single-chain polypeptide of 60 amino acids, which has been isolated from the venom of the African spitting cobra, Naja nigricollis. Three aromatic residues, namely, Trp-11, Tyr-22, and Tyr-51, have been individually modified by chemical means. The structure of the native toxin and of each derivative has been carefully investigated by circular dichroism, fluorescence, proton magnetic resonance spectroscopy, and two specific monoclonal antibodies. None of the chemical modifications alters the overall structure of the toxin, which in all cases remains folded into three adjacent loops (I, II, and III) rich in beta-pleated sheet emerging from a small globular region containing four disulfide bridges. A number of subtle changes, however, have been detected in the structure of each derivative compared with that of the native toxin. In particular, nitration of Tyr-51 provoked a structural perturbation in the globular region. Nitration of Tyr-22 induces a more substantial change in the beta-sheet area of the molecule. Thus, the strong inter-ring NOE that is observed in the native toxin between Tyr-22 and Tyr-51 vanishes in the Tyr-22 derivative, and significant changes are observed in the globular region. In contrast, no alteration of the beta-sheet structure of loops II and III has been detected after modification of Trp-11. All changes observed for this derivative remain located in the vicinity of the indole side chain of Trp-11 in loop I. The biological consequences of the modifications were measured: the lethal potency in vivo in mice and the cytotoxic activities in vitro on FL-cells. Lethal activities correlate with cytotoxicity: Tyr-51 modified toxin is equally potent as native toxin, whereas Tyr-22 and Trp-11 derivatized toxins are characterized by substantially lesser activities, the Trp-11 derivatized toxin being the least potent. We conclude that (1) Tyr-51 is not involved in the functional site of the toxin, although it is in interaction with the core of the molecule, (2) Tyr-22 may play a dual structural and functional role, and (3) Trp-11 is in, or in close proximity to, the functional site of the toxin. These data indicate the importance of loop I in determining toxicity of the cardiotoxin.     

Do cardiotoxins possess a functional site? Structural and chemical modification studies reveal the functional site of the cardiotoxin from Naja nigricollis

Examination of the literature has revealed that regarding the amino acid sequences, cardiotoxins constitute a family of homogeneous compounds. In contrast, cardiotoxins appear heterogeneous as far as their biological and spectroscopic properties are concerned. As a result, comparison between these molecules with a view to establishing structure-activity correlations is complicated. We have therefore reviewed recent works aiming at identifying the functional site of a defined cardiotoxin, ie toxin gamma from the venom of the spitting cobra Naja nigricollis. The biological and structural properties of toxin gamma are first described. In particular, a model depicting the 3-dimensional structure of the toxin studied by NMR spectroscopy is proposed. The toxin polypeptide chain is folded into 3 adjacent loops rich in beta-sheet structure connected to a small globular core containing the 4 disulfide bonds. A number of derivatives chemically modified at a single aromatic or amino group have been prepared. The structure of each derivative was probed by emission fluorescence, circular dichroism and NMR spectroscopy. Also tested was the ability of the derivatives to kill mice, depolarize excitable cell membranes and lyse epithelial cells. Modification of some residues in the first loop, in particular Lys-12 and at the base of the second loop substantially affected biological properties, with no sign of concomitant structural modifications other than local changes. Modifications in other regions much less affected the biological properties of the toxin. A plausible functional site for toxin gamma involving loop I and the base of loop II is presented. It is stressed that the functional site of other cardiotoxins may be different.     

Targeting of venom phospholipases: the strongly anticoagulant phospholipase A(2) from Naja nigricollis venom binds to coagulation factor Xa to inhibit the prothrombinase complex.

The strongly anticoagulant basic phospholipase A(2) (CM-IV) from Naja nigricollis venom has previously been shown to inhibit the prothrombinase complex of the coagulation cascade by a novel nonenzymatic mechanism (S. Stefansson, R. M. Kini, and H. J. Evans Biochemistry 29, 7742-7746, 1990). That work indicated that CM-IV is a noncompetitive inhibitor and thus it interacts with either factor Va or factor Xa, or both. We further examined the interaction of CM-IV and the protein components of the prothrombinase complex. Isothermal calorimetry studies indicate that CM-IV does not bind to prothrombin or factor Va, but only to factor Xa. CM-IV has no effect on the cleavage of prothrombin by factor Xa in the absence of factor Va. However, in the presence of factor Va, CM-IV inhibits thrombin formation by factor Xa. With a constant amount of CM-IV, raising the concentration of factor Va relieved the inhibition. The phospholipase A(2) enzyme inhibits by competing with factor Va for binding to factor Xa and thus prevents formation of the normal Xa-Va complex or replaces bound factor Va from the complex. Thus factor Xa is the target protein of this anticoagulant phospholipase A(2), which exerts its anticoagulant effect by protein-protein rather than protein-phospholipid interactions.     

The basic phospholipase A2 from Naja nigricollis venom inhibits the prothrombinase complex by a novel nonenzymatic mechanism

The three phospholipase A2 isoenzymes from Naja nigricollis venom inhibit blood coagulation with different potencies. The strongly anticoagulant basic isoenzyme CM-IV inhibits the prothrombinase complex, whereas the weakly anticoagulant isoenzymes CM-I and CM-II do not. To determine the role of enzymatic activity of the phospholipases in the inhibition of prothrombinase, we varied the time of incubation of each of these isoenzymes with the prothrombinase complex. The inhibition by CM-IV did not increase with time of incubation. CM-I and CM-II failed to inhibit the complex, even with complete hydrolysis of phospholipids in the assay mixture. After alkylation of its active-site histidine, CM-IV lost 97% of its enzymatic activity but retained 60% of its inhibitory potency on prothrombinase. CM-IV also inhibited prothrombinase activity in the absence of phospholipids, whereas CM-I and CM-II did not. The inhibition of the prothrombinase complex by CM-IV is thus not due to its binding to or hydrolysis of phospholipids. The kinetics of CM-IV inhibition of the prothrombinase complex in both the presence and absence of phospholipids was noncompetitive. This inhibition can be explained by binding of CM-IV to either factor Va or Xa, or both, to inhibit the complex. CM-IV differs from previously described nonenzymatic anticoagulants that are proteinase inhibitors or that inhibit the coagulation complexes by interfering with the binding of clotting factors to phospholipids. We conclude that the basic enzyme, CM-IV, inhibits the prothrombinase complex by a novel mechanism independent of enzymatic activity.     

Characterization of two platelet aggregation inhibitor-like polypeptides from viper venom

Two polypeptides, eristocophins I and II, have been characterized from leaf-nosed viper (Eristocophis macmahoni) venom. They contain 10 half-Cys residues of a total of 61/62 residues, have 72% residue identity, and exhibit similarities to platelet aggregation inhibitors and segments of adhesive proteins. Eristocophin I contains the sequence Arg-Gly-Asp, known to inhibit fibrinogen interaction with the platelet receptor. Eristocophin II has Met instead of Arg in this sequence, and an adjacent Trp-Asn-Asp segment. The latter is also typical of adhesive proteins, thus linking two potentially functional segments in one molecule. Exchanges are maximal in these segments, suggesting that the polypeptides exhibit functional divergence with isoform differences in important regions.     

Anticoagulants and inhibitors of platelet aggregation derived from leeches

Increased life expectancy is associated with aging populations in the developed countries, and we can expect an increased incidence of cardiovascular and inflammatory diseases and cancers. A priority for medical research is to reduce such morbidity. Leeches have been demonstrated to be a useful source of drugs to treat cardiovascular diseases, as they have evolved highly specific mechanisms to feed on their hosts by blocking blood coagulation. Powerful molecules acting at different points in the coagulation cascade or in the inhibition of platelet aggregation have been purified from these animals. Moreover, clinical trials confirm their potential to treat cardiovascular diseases.     

The complete covalent structure of a cardiotoxin from the venom of Naja nigricollis (African black-necked spitting cobra).

The complete covalent structure of a small, basic protein with cardiotoxic activity is described. This has been isolated from the venom of Naja nigricollis by gel filtration on Sephadex G-75 and gradient ion exchange chromatography on Bio-Rex 70. The cardiotoxin, molecular weight 6806 from amino acid composition, consists of 60 amino acids, cross-linked by four disulfide bridges, connecting 3-21, 14-38, 42-53, and 54-59. The protein contains one residue of tryptophan, phenylalanine, and glutamic acid, two residues of arginine and tyrosine, four residues of methionine, and nine residues of lysine. Histidine is absent. The chymotryptic peptides of the oxidized and S-carboxymethylated protein were isolated by gel filtration on Sephadex G-25 and zone electrophoresis on a cellulose column. The sequence was determined by Edman degradation, using the (manual) direct phenylthiohydantoin method and with the use of carboxypeptidase A. Disulfide pairing was determined on thermolysin cleaved peptides from the native protein. The sequence is shown to be homologous to other cardiotoxins and a lytic factor from snake venoms and also shows homology, both in sequence and disulfide pairing to neurotoxins. A partial reduction experiment in the absence of denaturing agent using 14-C-labeled iodoacetic acid as S-carboxymethylating agent shows that disulfide bonds 14-38 and 42-53 were reduced fastest followed marginally by 54-59, and then bond 3-21.     

Three-dimensional solution structure of a curaremimetic toxin from Naja nigricollis venom: a proton NMR and molecular modeling study.

The solution conformation of toxin alpha from Naja nigricollis (61 amino acids and four disulfides), a snake toxin which specifically blocks the activity of the nicotinic acetylcholine receptor (AcChoR), has been determined using nuclear magnetic resonance spectroscopy and molecular modeling. The solution structures were calculated using 409 distance and 73 dihedral angle restraints. The average atomic rms deviation between the eight refined structures and the mean structure is approximately 0.5 A for the backbone atoms. The overall folding of toxin alpha consists of three major loops which are stabilized by three disulfide bridges and one short C terminal loop stabilized by a fourth disulfide bridge. All the disulfides are grouped in the same region of the molecule, forming a highly constrained structure from which the loops protrude. As predicted, this structure appears to be very similar to the 1.4-A resolution crystal structure of another snake neurotoxin, namely, erabutoxin b from Laticauda semifasciata. The atomic rms deviation for the backbone atoms between the solution and crystal structures is approximately 1.7 A. The minor differences which are observed between the two structures are partly related to the deletion of one residue from the chain of toxin alpha. It is notable that, although the two toxins differ from each other by 16 amino acid substitutions, their side chains have an essentially similar spatial organization. However, most of the side chains which constitute the presumed AcChoR binding site for the curaremimetic toxins are poorly resolved in toxin alpha.(ABSTRACT TRUNCATED AT 250 WORDS)     

A potent platelet aggregation inducer from Trimeresurus gramineus snake venom

A potent platelet aggregation inducer (platelet aggregoserpentin) was purified from Trimeresurus gramineus snake venom by DEAE-Sephadex A-50 and Sephacryl S-300 column chromatography. It was homogeneous as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It elicited dose-dependently platelet aggregation and serotonin release reaction in rabbit platelet-rich plasma and platelet suspension. Exogenous calcium was required for its activity. Creatine phosphate/creatine phosphokinase and apyrase showed no significant inhibitory effect on aggregoserpentin-induced platelet aggregation in platelet suspension. Aggregoserpentin induced aggregation in ADP-refractory platelet-rich plasma. It caused no detectable malonic dialdehyde formation in the process of platelet aggregation. Indomethacin did not inhibit aggregoserpentin-induced platelet aggregation. Mepacrine abolished preferentially its aggregating activity, while prostaglandin E1 completely blocked both aggregoserpentin-induced aggregation and release reaction. Furthermore, platelet aggregoserpentin lowered basal and prostaglandin E1-stimulated cAMP levels in platelet suspension. Nitroprusside inhibited both its aggregating and releasing activity, while verapamil preferentially blocked its aggregating activity. It is concluded that aggregoserpentin activated platelets through lowering cAMP levels or the activation of endogenous phospholipase A2, resulting in the formation of platelet activating factor, but not of prostaglandins.