Isolation and partial characterization of a prothrombin-activating enzyme from the venom of the Australian rough-scaled snake (Tropidechis carinatus).

A prothrombin activator from the venom of Tropidechis carinatus has been isolated by means of gel filtration and benzamidine-based affinity chromatography, a novel use of the latter technique. Two bands possessing prothrombinase activity were obtained from the affinity chromatography procedure and designated A1 and A2. The bulk of the enzyme activity was recovered in peak A2 which represented 27-31% of the starting activity and a 14-16-fold purification. The venom contained, in total, around 5% by weight of the two isoforms of the prothrombin activator. The two fractions were electrophoretically similar on polyacrylamide electrophoresis, migrating with a mol. wt of 64,500 under native conditions and as a single band of 41,500 under reducing conditions. The prothrombinase was dependent on factor Va, phospholipid and calcium ions for its activity and is, thus, a member of the type II class of prothrombinases requiring such co-factors. The enzyme did not possess any phospholipase activity nor did it cleave the substrates N-alpha-benzoyl-L-arginine-p-nitroanilide (BAPNA), N-benzoyl-L-tyrosine ethyl ester (BTEE), azocollagen or azocasein, indicating a lack of amidolytic, esterolytic and broad-spectrum protease activity.

Practical applications of snake venom toxins in haemostasis.

Snake venom toxins have an established role in the coagulation laboratory for the assay of haemostatic parameters and a potential role for therapeutic treatment of thrombotic disorders. In the laboratory, snake venom thrombin-like enzymes (SVTLEs) are used for the assay of fibrinogen and detection of fibrinogen breakdown products and dysfibrinogenaemias. Importantly, because SVTLEs are not inhibited by heparin, they can be used for assaying antithrombin III and other parameters in samples which contain heparin. Prothrombin activators occur in many snake venoms and these have become established in the assay of prothrombin, in the study of dysprothrombinaemias and in the preparation of meizothrombin and non enzymic forms of prothrombin. Russell's viper (Daboia russelli) venom contains a number of useful compounds including toxins which can be used to assay blood clotting factors V, VII, X, platelet factor 3 and lupus anticoagulants (LA). More recently, activators from the taipan, Australian brown snake and saw-scaled viper have been used to assay LA. Proteins C and S can be measured by means of protac, a fast acting inhibitor from Southern copperhead snake venom and von Willebrand factor can be studied with botrocetin from Bothrops jararaca venom. The disintegrins, a large family of Arg-Gly-Asp (RGD)-containing proteins found in snake venoms, show great potential for the study of platelet glycoprotein receptors, notably, GPIIb/IIIa and Ib, and in the treatment of arterial thrombotic disease. Established SVTLEs used in clinical practice include ancrod and defibrase although success with these agents has been limited. A further group of enzymes under consideration as thrombolytic agents are the fibrinogenases.

Isolation and characterisation of two haemorrhagic proteins (HTa and HTb) from the venom of Bitis gabonica (Gaboon viper).

Two distinct haemorrhagic proteinases, HTa and HTb, were isolated from the venom of Bitis gabonica by gel filtration and ion-exchange chromatography with native mol. wts of 180,000 and 111,000, respectively. After reduction with dithiothreitol, smaller mol. wts of 77,600 and 69,200 were recorded for HTa and HTb, suggesting that under native conditions the haemorrhagins exist as dimeric molecules. Both toxins possessed caseinolytic and collagenase activity although HTa was 15-36 times more potent than HTb with respect to collagenase activity. No zinc could be detected in the toxins; however, dialysis against ethylenediamine tetracetic acid (EDTA) reduced caseinolytic activity, suggesting the dependence of the latter on other metal ions. HTa and HTb had a marked effect on the intrinsic cascade coagulation mechanism (factors IX, XI and XII) but no effect on the final common coagulation pathway (factor X and prothrombin). Light and electron microscopical studies demonstrated that both HTa and HTb caused organ-specific lesions, with the lungs, diaphragm and body wall muscle being most affected. HTa caused widespread haemorrhage whilst HTb caused discrete focal lesions near the site of injection and elsewhere. However, both toxins appeared to cause capillary rupture by the separation of cells from one another and both caused cell detachment and cell death of bovine endothelial cells cultured in vitro, consonant with the massive disruption of capillaries seen in vivo.