Chaoho Ouyang

Fibrinogenolytic enzymes of Trimeresurus mucrosquamatus venom

By means of CM-Sephadex C-50 column chromatography, Trimeresurus mucrosquamatus venom was separated into twenty fractions. The fibrinogenolytic activity was concentrated in Fractions 8, 10, 12, 13 and 14. Fractions 8 adn 13 had the highest ratio of fibrinogenolytic and caseinolytic activities. Fraction 8 possessed tosyl-L-arginine methyl esterase activity, while the others did not. The caseinolytic activities of Fractions 10, 12, 13 and 14 were inhibited by EDTA, while that of Fraction 8 was not. Fractions 8 and 13 were further purified by CM-cellulose and gel filtration and were homogeneous as judged by electrophoresis on polyacrylamide gel and cellulose acetate membrane. The molecular weights of the purified Fractions 8 and 13 were 26 000 and 22 400, respectively. Both were single peptide chains. The specific fibrinogenolytic activity of Fraction 8 was 17 mg fibrinogen/min/mg protein, while that of Fraction 13was 100 mg fibrinogen/min/mg protein. Fraction 13 digested specifically the alpha(A) chain of monomeric fibrinogen to yield two cleavage products. Fraction 8 digested the beta(B) chain first to yield four cleavage products. When the incubation time was prolonged, the alpha(A) chain was also partially digested by Fraction 8 to yield two cleavage products.

Characterization of snake venom components acting on blood coagulation and platelet function

Snake venoms can affect blood coagulation and platelet function in various ways. The physicochemical properties and the mechanisms of actions of the snake venom components affecting blood coagulation and platelet function are discussed.

Antiplatelet effect of butylidenephthalide

Butylidenephthalide inhibited, in a dose-dependent manner, the aggregation and release reaction of washed rabbit platelets induced by collagen and arachidonic acid. Butylidenephthalide also inhibited slightly the platelet aggregation induced by PAF and ADP, but not that by thrombin or ionophore A23187. Thromboxane B2 formation caused by collagen, arachidonic acid, thrombin and ionophore A23187 was in each case markedly inhibited by butylidenephthalide. Butylidenephthalide inhibited the aggregation of ADP-refractory platelets, thrombin-degranulated platelets, chymotrypsin-treated platelets and platelets in the presence of creatine phosphate/creatine phosphokinase. Its inhibition of collagen-induced aggregation was more marked at lower Ca2+ concentrations in the medium. The aggregability of platelets inhibited by butylidenephthalide could be recovered after the washing of platelets. In human platelet-rich plasma, butylidenephthalide and indomethacin prevented the secondary aggregation and blocked ATP release from platelets induced by epinephrine. Prostaglandin E2 formed by the incubation of guinea-pig lung homogenate with arachidonic acid could be inhibited by butylidenephthalide, indomethacin and aspirin. It is concluded that the antiplatelet effect of butylidenephthalide is mainly due to an inhibitory effect on cyclo-oxygenase and may be due partly to interference with calcium mobilization.

Potent platelet aggregation inhibitor from Trimeresurus gramineus snake venom.

Using DEAE-Sephadex A-50 column chromatography and gel filtration, a potent platelet aggregation inhibitor from Trimeresurus gramineus venom was purified. It was an acidic phospholipase A, rich in aspartic acid, glutamic acid and half-cystine, with an isoelectric point of 3.6. At a concentration of 10 micrograms/ml, the purified inhibitor showed a marked inhibitory effect on platelet aggregations induced by adenosine diphosphate, collagen, sodium arachidonate and ionophore A-23187 in rabbit platelet-rich plasma, washed platelet suspension, as well as in thrombin-degranulated platelet suspension. The ID50 of this venom inhibitor was about 2.5-5 micrograms/ml in platelet aggregations induced by all these aggregation inducers. The action of this inhibitor could be partially antagonized by phosphatidylethanolamine. High concentration of Ca2+ (5 mM) did not reverse the inhibitory action even in the presence of ionophore A-23187. The [14C]serotonin release induced by sodium arachidonate and thrombin was unaffected. Malonic dialdehyde formation induced by these aggregation inducers remained unchanged. Basal and prostaglandin E1-stimulated cAMP levels were not altered by this inhibitor. No lactate dehydrogenase was released even at a concentration of 62.5 micrograms/ml. Polylysine-induced platelet agglutination was not affected. beta-Mercaptoethanol inactivated both its phospholipase A enzymatic and platelet inhibitory activities, while p-bromophenacyl bromide only inactivated the former activity. The possibility of acting on a common final step of platelet aggregation, i.e. the intercellular adhesion between the activated platelets, was proposed.

α- and β-fibrinogenases from Trimeresurus gramineus snake venom

Abstract By means of DEAE-Sephadex A-50 column chromatography, Trimeresurus gramineus venom was separated into twelve fractions. The fibrinogenolytic activities were distributed in Fractions 1 and 10. These enzymes were further purified by gel filtration and were homogeneous as judged by cellulose acetate membrane, sodium dodecyl sulfate polyacrylamide gel electrophoresis and ultracentrifugal analysis. Both of them were single peptide chains. The sedimentation constants of α- (Fraction 1) and β-fibrinogenases (Fraction 10) were 2.20 and 3.60, respectively. The molecular weights of α and β-fibrinogenases were 23 500 and 25 000 respectively. The contents of proline and glycine were higher in β-fibrinogenase than in α-fibrinogenase. The isoelectric points of α-fibrinogenase and β-fibrinogenase were pH > 10 and 4.5, respectively. The optimal pH of α-fibrinogenase was approx. 7.4 and that of β-fibrinogenase was approx. 9.0. The activity of α-fibrinogenase was completely destroyed after 30 min at 60°C, pH 5.4, 7.4 and 9.0, while that of β-fibrinogenase was much less affected by the same treatment. The specific fibrinogenolytic activity of α-fibrinogenase was 31 mg fibrinogen/min per mg protein, while that of β-fibrinogenase was 9 mg fibrinogen/min per mg protein. α-Fibrinogenase cleaved specifically the α(A) chain of monomeric fibrinogen without cleaving the β(B) chain and γ-chain. β-fibrinogenase preferentially cleaved the β(B) chain, and the α(A) chain was also partially cleaved by β-fibrinogenase, if the incubation time was prolonged. Both enzymes showed proteolytic activities toward fibrinogen, fibrin and casein, but were devoid of phospholipase A, alkaline phosphomonoesterase and phosphodiesterase activities found in the crude venom. The tosyl- l -arginine methylester esterase activity of β-fibrinogenase was about 14 times that of crude venom, while α-fibrinogenase was completely devoid of this activity. The fibrinogenolytic activity of α-fibrinogenase was markedly inhibited by EDTA and cysteine, while that of β-fibrinogenase was inhibited markedly by phenylmethanesulfonylfluoride. α- and β-fibrinogenases exert their fibrinogenolytic activity by a direct action on fibrinogen or fibrin without activation of plasminogen

Purification and characterization of the fibrinolytic principle of Agkistrodon acutus venom

By means of DEAE-Sephadex A-50 column chromatography, Agkistrodon acutus venom was separated into twelve fractions. The fibrinolytic activity was concentrated in Fraction 9. This fraction was rechromatographed on Sephadex G-75 three times and a single peak was obtained. The patterns of microzone and disc electrophoresis also showed a single band. A single, symmetrical boundary with a value of 2.44 S was obtained by ultracentrifugation, the molecular weight of which was estimated to be 24 100, and the isoelectric point 3.8. The specific activity was four times higher than that of crude venom. The optimal pH value on fibrinolysis was 7.4. In addition to fibrinolytic activity, the purified principle also had fibrinogenolytic and caseinolytic activities. The purified fibrinolytic principle had a specific action on the a(A) chain subunit of fibrinogen, leaving the beta(B) chain and the gamma chain unaffected.

Characterization of a potent platelet aggregation inhibitor from Agkistrodon rhodostoma snake venom

By means of CM-Sephadex C-50 column chromatography and gel filtration on Sephadex G-75 and G-50 columns, a potent platelet aggregation inhibitor was purified and characterized. It was a glycoprotein with a molecular weight of 31,000. It was devoid of phospholipase A, ADPase, esterase and fibrino(geno)lytic activities. It inhibited dose-dependently the aggregation of washed platelets induced by collagen, thrombin, sodium arachidonate, platelet activating factor and ionophore A23187 with a similar IC50 (5-10 micrograms/ml). It was also active in platelet-rich plasma, with an IC50 of 10-15 micrograms/ml. The venom inhibitor reduced the elasticity of whole blood clot and inhibited the thrombin-induced clot retraction of platelet-rich plasma. These activities were related to its inhibitory activity on platelet aggregation rather than blood coagulation. The venom inhibitor had various effects on [14C]serotonin release stimulated by aggregation agonists. It had no effect on thromboxane B2 formation of platelets stimulated by sodium arachidonate, collagen and ionophore A23187. The presence of this venom inhibitor prior to the initiation of aggregation was a prerequisite for the maintenance of its maximal activity. It showed a similar inhibitory effect on collagen or thrombin-induced aggregation even when it was added after the platelets had undergone the shape change. High fibrinogen levels partially antagonized its activity. The venom inhibitor completely inhibited the fibrinogen-induced aggregation of alpha-chymotrypsin-treated platelets. It is concluded that this venom inhibitor interferes with the interaction of fibrinogen with fibrinogen receptors, leading to inhibition of aggregation.

A potent platelet aggregation inhibitor purified from Agkistrodon halys (mamushi) snake venom.

By means of gel filtration on Sephadex G-75, DEAE-Sephadex A-50 column chromatography and three gel filtrations on Sephadex G-75, a potent platelet aggregation inhibitor was purified from Agkistrodon halys snake venom and shown to be a single peptide chain, as judged by SDS-polyacrylamide gel electrophoresis. The purified platelet aggregation inhibitor was an acidic protein with a molecular weight of 14,000 and possessed phospholipase A2 activity. Its inhibitory activity on platelet aggregation was heat stable (at 96 degrees C, 30 min) in an acidic medium (pH 5.5), while its phospholipase A enzymatic activity was heat labile under the same conditions. Its inhibitory activity on platelet aggregation induced by thrombin, sodium arachidonate, collagen or ionophore A-23187 was non-competitive and dose-dependent with a similar ID50 (approximately 11 micrograms/ml). It exerted its inhibitory action without pre-incubation with platelet suspension, however, its inhibitory effect could be moderately increased after longer incubation (30 min).

Biphasic effect on platelet aggregation by phospholipase a purified from Vipera russellii snake venom.

A basic phospholipase A was isolated from Vipera russellii snake venom. It induced a biphasic effect on washed rabbit platelets suspended in Tyrodes solution. The first phase was a reversible aggregation which was dependent on stirring and extracellular calcium. The second phase was an inhibitory effect on platelet aggregation, occurring 5 min after the addition of the venom phospholipase A without stirring or after a recovery from the reversible aggregation. The aggregating phase could be inhibited by indomethacin, tetracaine, papaverine, creatine phosphate/creatine phosphokinase, mepacrine, verapamil, sodium nitroprusside, prostaglandin E1 or bovine serum albumin. The venom phospholipase A released free fatty acids from synthetic phosphatidylcholine and intact platelets. p-Bromophenacyl bromide-modified venom phospholipase A lost its phospholipase A enzymatic and platelet-aggregating activities, but protected platelets from the aggregation induced by the native enzyme. The second phase of the venom phospholipase A action showed a different degree of inhibition on platelet aggregation induced by some activators in following order: arachidonic acid greater than collagen greater than thrombin greater than ionophore A23187. The longer the incubation time or the higher the concentration of the venom phospholipase A, the more pronounced was the inhibitory effect. The venom phospholipase A did not affect the thrombin-induced release reaction which was caused by intracellular Ca2+ mobilization in the presence of EDTA, but inhibited collagen-induced release reaction which was caused by Ca2+ influx from extracellular medium. The inhibitory effect of the venom phospholipase A and also lysophosphatidylcholine or arachidonic acid could be antagonized or reversed by bovine serum albumin. It was concluded that the first stimulatory phase of the venom phospholipase A action might be due to arachidonate liberation from platelet membrane. The second phase of inhibition of platelet aggregation and the release of ATP might be due to the inhibitory action of the split products produced by this venom phospholipase A.

Purification and properties of the anticoagulant principle of agkistrodon acutus venom

Abstract By means of DEAE-Sephadex column chromatography, Agkistrodon acutus venom was separated into 12 fractions. Fractions 6 and 7 had marked anticoagulant action in tests of whole blood clotting time, calcium clotting time and plasma prothrombin time. The activity of Fraction 6 was the highest. Fraction 6 was rechromatographed twice on Sephadex G-100, and a single peak was obtained. The patterns of microzone and disc electrophoresis also showed a single band. A single, symmetrical boundary with a value of 2.00 S was obtained by ultracentrifugation. The estimated molecular weight was 20 650. The isoelectric point was pH 4.7. Chemical analysis showed that the anticoagulant principle was a glycoprotein and that it was thermolabile. The anticoagulant activity of this purified principle was four times higher than that of the crude venom. This principle did not possess caseinolytic, tosyl- l -arginine methyl ester esterase, phospholipase A, phosphodiesterase, alkaline phosphomonoesterase or fibrinolytic activities.