Calloselasma Research Articles

Primary specificity of ancrod, the coagulating serine proteinase from the Malayan pit viper ( Agkistrodon rhodostoma) venom

Values of steady-state and pre-steady-state parameters for the hydrolysis of ZArgONp and ZLysONp catalysed by ancrod, the coagulating serine proteinase from the Malayan pit viper ( Agkistrodon rhodostoma) venom, have been determined, between pH 2.5 and 8 ( I = 0.1 M) at 21 ± 0.5°C, and analysed in parallel with those of bovine α-thrombin and porcine pancreatic β-kallikrein-B. In addition to the well-known coagulating behaviour, ancrod also shows catalytic properties, in the hydrolysis of ZArgONp and ZLysONp, reminiscent of those of porcine pancreatic β-kallikrein-B.

Clinical significance of venom antigen levels in patients envenomed by the Malayan pit viper (Calloselasma rhodostoma).

Serial venom antigen levels were measured by enzyme-linked immunosorbent assay (ELISA) in 46 patients with systemic envenoming by the Malayan pit viper (Calloselasma rhodostoma), a major cause of snake bite in Southeast Asia. The principal effects of the venom are defibrination, hemorrhage and local tissue necrosis. Admission venom levels, which varied between 0 and 595 ng/ml, correlated with the incidence of spontaneous systemic bleeding, blood incoagulability and concentrations of plasma fibrinogen and serum fibrin degradation products. The presence or absence of nonclotting blood also correlated with the time elapsed between the bite and hospital admission. The development of nonclotting blood may be delayed by up to 72 hr after the bite even though circulating venom and raised FDP may be detected at presentation. This is probably explained by a temporary equilibrium between synthesis and consumption of fibrinogen. Venom antigenemia recurred in 12 patients (26%) suggesting continuous absorption of venom from the wound or saturation of extravascular binding sites. Admission venom levels also correlated with the extent of local swelling and the occurrence of tissue necrosis at the site of the bite. Venom was detected in 87% of wound aspirates and 88% of urine specimens taken on admission. Tourniquets, of the type used in rural Thailand, did not delay the absorption of venom into the circulation.

Purification and characterization of a platelet aggregation inducer from Calloselasma rhodostoma (Malayan pit viper) snake venom

C. Ouyang, H.-I Yeh and T.-F. Huang. Purification and characterization of a platelet aggregation inducer from Calloselasma rhodostoma (Malayan pit viper) snake venom. Toxicon 24, 633 – 643, 1986. — A potent platelet aggregation inducer was purified from Calloselasma rhodostoma snake venom by Sephadex G-75, CM-Sephadex C-50 and Sephacryl S-300 column chromatography. It was homogeneous as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, with a molecular weight estimated to be 28,160 ± 1280. It was devoid of phospholipase A 2, fibrino(geno)lytic and thrombin-like activities. The venom inducer elicited platelet aggregation and the serotonin release reaction in rabbit platelet-rich plasma and platelet suspension. Exogenous calcium was required for its platelet activation. Creatine phosphate/creatine phosphokinase and indomethacin did not inhibit the venom inducer-induced aggregation and release reaction. Mepacrine and verapamil preferentially inhibited aggregation, while PGE 1 completely blocked both aggregation and release reaction. It is concluded that the venom inducer activates platelets through the activation of endogenous phospholipase A 2 or C, leading to intracellular calcium mobilization, but independent of the ADP release reaction or thromboxane A 2 formation.

Enzymatic activities of Calloselasma rhodostoma (Malayan pit viper) venom

The enzyme contents of four venom samples of Calloselasma rhodostoma were analyzed. The venoms contained phosphodiesterase, alkaline phosphomonoesterase, 5′-nucleotidase, protease, phospholipase A, l-amino acid oxidase, hyaluronidase, arginine ester hydrolase, arginine amidase, fibrinogenase and coagulant enzyme activities. There is significant variation in the contents of coagulant enzyme, arginine ester hydrolase, hyaluronidase, protease, phosphodiesterase, alkaline phosphomonoesterase and l-amino acid oxidase. DEAE-Sephacel ion exchange chromatography of the venom resolved it into eight major protein fractions. The eight fractions were heterogeneous and exhibited more than one type of enzymatic activity. The 5′-nucleotidase, alkaline phosphomonoesterase, protease, coagulant enzyme, arginine ester hydrolase, arginine amidase and fibrinogenase exist in multiple forms.

Detection of specific proenzyme activators in snake venoms by a new immunoabsorbant-chromogenic substrate method

In separate experiments, antibodies to plasminogen, factor X and protein C were applied to microtitre trays as commonly used in enzyme-linked immunoassays. After incubation with dilute normal human plasma as a source of the corresponding proenzyme antigen, the wells were exposed to dilutions of various snake venoms. After thorough washing, the microtitre tray wells were tested overnight with chromogenic tripeptide substrates known to be relatively specific for the activated forms of the above factors, i.e., plasmin, factor Xa and activated protein C. The immunochromometric assay described detected two new activators of protein C in Agkistrodon piscivorus and Agkistrodon contortrix venoms and a new factor X activator in Agkistrodon rhodostoma venom. Gel filtration of the latter venom indicated that the factor X activator eluted with high molecular weight, was clearly distinct from the peak fibrinogen clotting activity (Ancrod) and appeared to have no procoagulant activity. Although several Bothrops venoms appeared to contain plasminogen activator by this technique, the observed strong chormogenic activity was observed in microtitre wells independently of plasminogen and represented nonspecific amidase activity.

Ancrod: normalization of fibrinolytic enzyme abnormalities in patients with systemic lupus erythematosus and lupus nephritis.

Ancrod is a thrombinlike enzyme from Malayan pit viper (Agkistrodon rhodostoma) venom that has a selective enzyme substrate specificity for fibrinogen. Unlike thrombin, it splits only fibrinopeptide A from the fibrinogen molecule and does not activate factor XIII. Simultaneously with the occurrence of hypofibrinogenemia there is a reduction of plasma plasminogen and a rise in fibrin degradation products, suggesting secondary recruitment of the fibrinolytic enzyme system. Ancrod was given to 18 patients with systemic lupus erythematosus and glomerular and vascular microthrombi. Before treatment vascular plasminogen activator (VPA) was low or unmeasurable in 14, an inhibitor of urokinase-induced plasminogen activation (IPA) was elevated in 18, and an inhibitor of plasmin (PI) was elevated in five. Ancrod treatment resulted in prompt normalization of IPA levels in 13 patients; they were classified as fibrinolysis responders. In five patients IPA levels remained elevated throughout treatment with ancrod; they were classified as fibrinolysis nonresponders. In these five the PI level was elevated before treatment and decreased slowly toward the normal range during ancrod administration. The PI did not appear related to the nonspecific serine protease inhibitors, and was shown to be identical with alpha 2-antiplasmin. In the fibrinolysis responders serial histologic studies showed a striking decrease of disappearance of microvascular thrombosis; in the fibrinolysis nonresponders microvascular thrombosis persisted. The action of ancrod is discussed.

Variation in the composition of [formula omitted] (Malayan pit viper) venoms from different geographical origins : By Nget-Hong TAN and M.S. Kanthimathi, Department of Biochemistry, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia

Variation in the composition of [formula omitted] (Malayan pit viper) venoms from different geographical origins : By Nget-Hong TAN and M.S. Kanthimathi, Department of Biochemistry, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia

Interactions of Venoms and Venom Components with Blood Platelets

AbstractMany venoms demonstrate their main toxicity on systems other than blood. Despite this, many venoms also have effects on blood and blood components such as platelets. Crude venoms of snakes, scorpions and spiders have been shown to have indirect (through stimulation of coagulation or epinephrine release, e.g., Echis carinatus and Gentruroides sculpturatus venoms, respectively) and/or direct effects on platelets. For coagulation-dependent effects on platelets, thrombin- or prothrombin-like action (Agkistrodon rhodostoma or contortrix, Bothrops atrox, Crotalus species) may not affect platelets, while those acting at earlier steps may yield normal thrombin and thus activate platelets (Vipera russelli, Echis carinatus). Crude venom effects can be stimulatory (promote or cause aggregation) or inhibitory, with either predominating as a function of dose. Such variability, if observed, often indicates the presence of opposing activities in crude venom, but may also indicate biphasic activity of a single co...

α-Fibrinogenase from Agkistrodon rhodostoma (Malayan pit viper) snake venom

By means of DEAE-Sephadex A-50 column chromatography, Agkistrodon rhodostoma (Malayan pit viper) snake venom was separated into eleven fractions. Fraction II had fibrinogenolytic activity, and when further purified by gel filtration was homogeneous, as judged by sodium dodecylsulfate polyacrylamide gel electrophoresis. It had a single peptide chain with a molecular weight of 25,360 and an isoelectric point greater than 10. The fibrinogenolytic activity was completely destroyed after heating for 30 min at 60°C at pH 5.6, 7.4 or 8.8. This enzyme cleaved specifically the α(A) chain of monomeric fibrinogen, without cleaving the β(B) chain or γ chain. The specific fibrinogenolytic activity was 51 mg fibrinogen/min per mg protein. This enzyme showed proteolytic activities toward fibrinogen, fibrin and casein, but was devoid of phospholipase A and tosyl- l-arginine methylester esterase activities which are found in the crude venom. The fibrinogenolytic activity was inhibited by EDTA and cysteine, but not by ε-aminocaproic acid.

Coordinate accumulation of the mRNAs for the alpha, beta, and gamma chains of rat fibrinogen following defibrination.

Cell-free translation and hybridization to cloned cDNA probes were used to study variations in fibrinogen mRNA levels in the livers of rats injected with Malayan pit viper venom and glucocorticoids. Animals defibrinated with Malayan pit viper venom showed a rapid and substantial increase in the relative abundance of hepatic mRNAs for the A alpha, B beta, and gamma chains of fibrinogen. The onset, rate, and maximal extent of message accumulation were virtually identical for each of the three fibrinogen polypeptide chains. This response was detectable 1-2 h after injection of venom and, by 12-16 h, fibrinogen species made up approximately 10% of the polyadenylated RNA compared to less than 1% in noninjected animals. Physiologic levels of glucocorticoids appeared to be necessary for maximal levels of message; adrenalectomized animals had a median 4.8-fold increase in fibrinogen mRNA species after defibrination compared with a 12.5-fold increase in normal animals. Although it is uncertain whether the changes in mRNA levels are a function of increased transcription or message stabilization, the similar kinetics of accumulation suggest that a common mechanism is responsible for coordinating the levels of each fibrinogen mRNA.

Molecular cloning of cDNA for the alpha, beta, and gamma chains of rat fibrinogen. A family of coordinately regulated genes.

Recently, we have found that defibrination of rats with Malayan pit viper venom induces a 10-38-fold increase in the levels of translatable fibrinogen mRNA in the liver. We have used this response to obtain cDNA clones for the three polypeptide chains of rat fibrinogen. A large cDNA library was created in pBR322 from induced rat liver polyadenylated RNA by the poly(dG, dC)-tailing method. Part of this library was screened using colony hybridization with [32P]cDNA prepared from induced and noninduced rat liver RNA. Colonies consistently giving a more intense signal with the induced [32P]cDNA were considered possible fibrinogen recombinants and were used for hybrid selection and translation of mRNA. In this way, cDNA clones for each of the three fibrinogen mRNA's were identified. Analysis of polyadenylated RNA by Northern blotting indicates that the three chains are synthesized from mRNA's of 2300, 2050, and 1950 nucleotides for the alpha, beta, and gamma chains, respectively. The fact tha each of the three chains has a separate mRNA indicates that the highly coordinated regulation of the three messages for rat fibrinogen does not occur by translation of a common cytoplasmic RNA.

Effect of defibrinogenation on lung water accumulation after pulmonary microembolism in dogs.

We determined the effects of chronic fibrinogen depletion on the development of pulmonary edema after pulmonary microembolization. Dogs were defibrinogenated with a purified fraction of Malayan pitviper venom (ancrod). Studies were made in four groups: control untreated (group I); emboli untreated (group II); control defibrinogenated (group III); and emboli defibrinogenated (group IV). Fibrinogen decreased (P < 0.05) from 570.6 +/- 100.9 to 5.3 +/- 3.1 mg/100 ml in the ancrod-treated groups. Pulmonary arterial pressure was increased to similar levels in both embolized groups after infusion of 100-mug-diam nonsiliconized glass beads into the right atrium. Pulmonary vascular resistance and pulmonary perfusion pressure were initially increased to similar levels in both embolized groups, but by 75 min postembolization (PE) both parameters were higher (P < 0.05) in the defibrinogenated group. The extravascular lung water-to-bloodless dry lung ratio at 75 min PE of 4.53 +/- 0.24 in group II was greater than the control value of 2.84 +/- 0.22 in group I (P < 0.05). In contrast, the extravascular lung water-to-bloodless dry lung ratio of 3.64 +/- 0.09 in group IV was not different from the control value of 3.38 +/- 0.04 in group III, but was less than 4.53 +/- 0.24 in group II (P < 0.05). Therefore, chronic defibrinogenation in dogs prevented the development of pulmonary edema after pulmonary microembolization. The protective effect may be due to inhibition of the increase in lung vascular permeability and to a time-dependent reduction in pulmonary microvascular pressure.

In vitro studies on complement inactivation by snake venoms

Snake venoms of the families Elapidae, Viperidae and Crotalidae were investigated concerning their effects on the human complement system. Twenty-two of 26 venoms inhibited complement activity in normal human serum. Most efficient were the venoms from Naja naja, Agkistrodon rhodostoma, A. halys, Vipera lebetina and Trimeresurus okinawensis. Consumption of the complement factors C2, C3 and C4 was observed with most venoms. Factor B and C3 were transformed to physiological conversion products. Purified C3 was converted by most venoms, suggesting a direct effect on this factor. Conversion was inhibited by EDTA, but could in some cases be restored by the addition of Mg 2+ or Ca 2+. We conclude that viperid and crotalid venoms inhibit the complement system through proteolytic action on several of its components, in contrast to active elapid venoms of the genus Naja, which activate the system owing to ‘cobra venom factor’. In no other case could a clear specific attack on a single pathway be demonstrated. However, some distinctive features of the multipoint attack made possible a grouping of the venoms into three categories which might be valuable in future studies of the complex factors involved in complement activation.

Fragmentation of actin by thrombin-like snake venom proteases

The effect of thrombin-like snake venom proteases (Ancrod of Agkistrodon rhodostoma and Batroxobins of Bothrops moojeni and Bothrops marajoensis) on skeletal muscle actin was studied and compared to the thrombic cleavage of this protein. Only EDTA-pretreated G- and F-actin were split by thrombin and Ancrod, while Batroxobins hydrolyzed native G-actin, too. The time course of digestion was followed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. A split product of 37 500 daltons appeared first which was cleaved further resulting in three lower molecular weight fragments. The sodium dodecyl sulfate gel pattern of thrombic fragmentation was well distinguishable from those caused by Ancrod and Batroxobins. The first split products of Batroxobin digestion — a smaller peptide and the 37 500 dalton fragment — were isolated and by estimating their N-, and C-terminal end groups and amino acid compositions the peptide bond hydrolyzed first was located in the primary structure of actin. It was established that while thrombin split off two actinopeptides (at Arg(28)-Ala(29) and Arg(39)-His(40)) from the N-terminal end of the molecule only Arg(39)-His(40) was cleaved by Batroxobins.

Influence of snake venoms on blood coagulation and fibrinolysis

The influences of several kinds of snake venoms, which indicate thrombin-like actions on the blood coagulation and fibrinolysis were investigated with special reference to factor XIII, and the results that were obtained are as follows;1) Venoms are able to form cross-linkage of fibrin more slowly than thrombin can act.2) Ca-ion is necessary in the formation of the cross-linkage of fibrin by the use of venoms.3) When cysteine solutions are not added into the reaction mixture, the fibrin clot formed by thrombin does not dissolve in 1% monochlor acetic acid solution, but the fibrin clot formed by venoms dissolves in the acidic solution.4) When the reaction mixture containes cysteine, the fibrin clot formed by venoms becomes insoluble in 1% monochlor acetic acid solution, after 30min. or 1hr. incubation.5) The action of Defibrase prepared from Bothrops atrox marajoensis venom on the activation of factor XIII resembles tha of Venacil from Agkistrodon rhodostoma venom, but Defibrase from Bothrops atrox moojeni venom demonstrates a slightly stronger activity for the activation of factor XIII in comparison with the activities of the other two venoms.

Ancrod (arvin)

Since we reviewed ancrod (Arvin - Berk) 5 years ago1 it has been marketed and the reported indications for its use have widened. Ancrod is the partly purified fraction of the venom of the Malayan pit viper (Agkistrodon rhodostoma). The drug produces its anticoagulant effect by converting plasma fibrinogen into an unstable form of fibrin which is then rapidly removed from the circulation. This defibrination reduces the concentration of circulating fibrinogen without producing vascular occlusion. Ancrod does not seem to affect the other clotting factors directly but the high level of fibrin degradation products during the induction period may transiently impair platelet function. Fibrinogen depletion might be useful in the treatment of venous thrombo-embolism; loss of fibrinogen also reduces blood viscosity and may help in a variety of conditions, for example in peripheral arterial insufficiency.

Thrombin-like enzyme from the venom of [formula omitted] (Eastern Diamondback Rattlesnake)

The thrombin-like enzyme (crotalase) from the venom of the eastern diamondback rattlesnake has been purified to homogeneity and characterized (F.S. Markland and P.S. Damus, J. Biol. Chem. 246 , 6460, 1971). In the present report we describe the action of crotalase on fibrinogen. In contrast to thrombin, crotalase cleaves only the A peptides from human fibrinogen. Furthermore, crotalase slowly degrades the β (B) chains producing smaller fragments as evidenced by SDS polyacrylamide gel electrophoresis of the washed clot components. This activity and at least one other difference in specificity distinguish crotalase from ancrod, the thrombin-like enzyme from Malayan pit viper venom. The mechanism by which crotalase produces a hypocoagulable state is discussed.

Isolation of thrombin-like factors from Malayan pit viper venom by affinity chromatography

Affinity resins based on 4-aminobenzamidine (4-Bz), l-arginine methyl ester (AME), and its N-α-substituted acyl derivatives N-α-tosyl- l-arginine methyl ester (TAME), N-α-benzoyl- l-arginine methyl ester (BAME), N-α-acetyl- l-arginine methyl ester (AAME), and N-α-carbobenzoxy- l-arginine methyl ester (CAME) covalently linked to succinylated aminododecyl-cellulose (SADC) were used to purify thrombin-like factors from the venom of Agkistrodon rhodostoma. On the other hand, weaker basic effectors like l-histidine methyl ester (HISME) and l-lysine methyl ester (LYSME) adsorb only very small quantities of proteins from the crude venom. Furthermore, by gel-filtration on Sephadex G75 superfine the peak from AME-SADC was separated into two coagulant fractions, α-thrombin-like factor and ß-thrombin-like factor having molecular weights of about 45,000 and 12,000, respectively.

Thrombin-like enzymes of snake venoms: Actions on prothrombin

Five crotalid venoms were examined for thrombin-like action on prothrombin, as determined by dodecyl sulfate-polyacrylamide gel electrophoresis in conjunction with assays for thrombin and prothrombin. The thrombin-like venom enzymes of Crotalus adamanteus and Agkistrodon contortrix contortrix cleaved specifically the thrombin-vulnerable activation bonds of human prothrombin, but differed from each other and from thrombin in their requirement for phospholipid and Ca ++. In contrast, the thrombin-like venom enzymes of Agkistrodon rhodostoma, Bothrops moojeni , and Bothrops marajoensis had no proteolytic action on human prothrombin. These results are of pharmacologic interest and indicate also the suitability of snake venom enzymes for comparative structure-function studies on the clotting actions of thrombin.

The Clotting of a Snake (Crotalus Viridis Helleri) Plasma and its Interaction with Various Snake Venoms

The clotting of C. V. Helleri plasma is not accelerated by the factor X activator or thrombin-like enzymes from its own venom. Clotting of the plasma is accelerated by the factor Xactivator from Russell's viper venom, but not by the thrombin-like enzyme from Agkistrodon Rhodostoma venom ("Arvin"). The prothrombin activator from the Taipan venom clots C.V. Helleri plasma equally well as human plasma, but the thrombin which is produced has a marked specificity for its own fibrinogen, and clots bovine fibrinogen more slowly. C.V. Helleri plasma contains an inhibitor which progressively inactivates bovine factor Xa and thrombin, but the inhibitor is not potentiated by heparin. The slow, protracted clotting of the snake plasma either alone or when mixed with human plasma or bovine fibrinogen suggests that this inhibitor may interfere with the polymerisation of fibrin monomer.