Snake bites and hemostasis/thrombosis

Snake venom toxins have evolved to affect many prey physiological systems including hemostasis and thrombosis. These toxins belong to a diverse array of protein families and can initiate or inhibit multiple stages of the coagulation pathway or platelet aggregation with incredible specificity. Such specificity toward vertebrate molecular targets has made them extremely useful for diagnosis of human diseases or as molecular scalpels in physiological studies. The large number of yet-to-be characterized venoms provides a vast potential source of novel toxins and subsequent cardiovascular therapeutics and diagnostic agents.     

Structure–function relationships and mechanism of anticoagulant phospholipase A2 enzymes from snake venoms

Phospholipase A₂ (PLA₂) enzymes from snake venom are toxic and induce a wide spectrum of pharmacological effects, despite similarity in primary, secondary and tertiary structures and common catalytic properties. Thus, the structure–function relationships and the mechanism of this group of small proteins are subtle, complex and intriguing challenges. This review, taking the PLA₂ enzymes from spitting cobra (Naja nigricollis) venom as examples, describes the mechanism of anticoagulant effects. The strongly anticoagulant CM-IV inhibits both the extrinsic tenase and prothrombinase complexes, whereas the weakly anticoagulant PLA₂ enzymes (CM-I and CM-II) inhibit only the extrinsic tenase complex. CM-IV binds to factor Xa and interferes in its interaction with factor Va and the formation of prothrombinase complex. In contrast, CM-I and CM-II do not affect the formation of prothrombinase complex. In addition, CM-IV inhibits the extrinsic tenase complex by a combination of enzymatic and nonenzymatic mechanisms, while CM-I and CM-II inhibit by only enzymatic mechanism. These functional differences explain the disparity in the anticoagulant potency of N. nigricollis PLA₂ enzymes. Similarly, human secretory enzyme binds to factor Xa and inhibits the prothrombinase complex. We predicted the anticoagulant region of PLA₂ enzymes using a systematic and direct comparison of amino acid sequences. This region between 54 and 77 residues is basic in the strongly anticoagulant PLA₂ enzymes and neutral or negatively charged in weakly and non-anticoagulant enzymes. The prediction is validated independently by us and others using both site directed mutagenesis and synthetic peptides. Thus, strongly anticoagulant CM-IV binds to factor Xa (its target protein) through the specific anticoagulant site on its surface. In contrast, weakly anticoagulant enzymes, which lack the anticoagulant region fail to bind specifically to the target protein, factor Xa in the coagulation cascade. Thus, these studies strongly support the target model which suggests that protein–protein interaction rather than protein–phospholipid interaction determines the pharmacological specificity of PLA₂ enzymes.     

Molecular isoforms of cobra venom factor-like proteins in the venom of Austrelaps superbus.

Cobra venom factor (CVF) is characteristic of the elapid cobras and has not been reported from venoms of any other families of snakes. During our search for novel proteins, we isolated a polypeptide from the venom of the snake Austrelaps superbus (Lowland Copperhead) that showed structural similarity to C-terminal segment of the alpha-chain of CVF and hence named as AVFalphac (AVF-A. superbus venom factor). cDNA sequence of AVFalphac and its precursor indicated the presence of two isoforms of CVF-like proteins in A. superbus venom gland. This is the first report of molecular isoforms of CVF-like proteins in the venom of an Australian elapid snake. We have determined the complete cDNA sequence of both the isoforms (AVF-1 and AVF-2). They differ in their potential glycosylation sites and the characteristic thioester bond sequence. They display the overall domain structure of CVF and complement C3 proteins. By real-time quantitative analysis, we show that there is a 140-fold difference in the mRNA expression levels of the two isoforms in the venom gland of A. superbus. We also show the presence of AVF-1 and its variant (not AVF-2) in A. superbus venom by partial purification, dot blots, Western blots and peptide mapping using mass spectrometry. Partially purified proteins activate human Factor B in the presence of Factor D and Mg(2+), and deplete the complement activity in human and guinea pig serum. The bimolecular complex (AVFBb) formed activates complement C3 but not complement C5. Thus, AVF proteins may serve as potential candidates for therapeutic complement depletion without side effects. Thus, the discovery of CVF-like proteins in the venom of this Australian elapid snake provides an alternative source of research tools, and contributes to our understanding of the structure-function relationships and evolution of new members of CVF-like proteins.     

Molecular moulds with multiple missions: functional sites in three-finger toxins

1. Snake venoms are complex mixtures of pharmacologically active peptides and proteins. 2. These protein toxins belong to a small number of superfamilies of proteins. The present review describes structure-function relationships of three-finger toxins. 3. All toxins share a common structure of three beta-stranded loops extending from a central core. However, they bind to different receptors/acceptors and exhibit a wide variety of biological effects. 4. Thus, the structure-function relationships of this group of toxins are complicated and challenging. 5. Studies have shown that the functional sites in these "sibling" toxins are located on various segments of the molecular surface.     

Titrimetric micro-determination of therapeutically active phenothiazines using periodate.

Two new simple, accurate and precise titrimetric micro-procedures are described for the analysis of phenothiazines in pure sample, tablets, injections and syrup using periodate as the oxidant. The first method is based on the oxidation of phenothiazines with periodate in acid medium and the iodate formed in the reaction is determined by reacting it with iodide and titrating the liberated iodine with thiosulfate after masking the excess of periodate with molybdate. In the second procedure, the unreacted (excess) periodate is determined iodometrically under basic conditions. The reaction conditions have been optimised and the stoichiometry of the reaction has been evaluated. A linear relationship exists between the amount of the drug and the titration end-point as shown by the values of correlation coefficient, r (0.9991-0.9999). The slope of the linear relationship has been calculated and found to be in the range, 0.1457-0.3120. The methods were applied to the analysis of dosage forms with results comparable to those given by the official methods. Both the methods are indirect visual titration methods, and are simpler than, and superior to, many existing methods for the assay of phenothiazines.