Purification and characterization of the anticoagulant principle of Trimeresurus mucrosquamatus venom.
By means of CM-Sephadex column chromatography, Trimeresurus mucrosquamatus venom was separated into 20 fractions. Fraction XX had the marked anticoagulant action. This fraction was refractionated three times on Sephadex G-75, 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 1.61 S was obtained by ultracentrifugation. It was a single peptide chain with a molecular weight of 11 700. The isoelectric point was higher than pH 10. The anticoagulant principle possessed phospholipase A activity and was calcium ion dependent. It did not possess proteolytic, tosyl-L-arginine methyl ester esterase, phosphodiesterase and alkaline phosphomonoesterase activities of the crude venom. The phospholipase A activity was heat-labile at pH 7.4, but was heat-stable at pH 5.6. The anticoagulant activity was more resistant to heat treatment as compared with phospholipase A activity. The anitoagulant action of the purified principle was competitively inhibited by platelet phospholid, tissue thromboplastin and cephalin, and was neutralized by antiserum. The anticoagulant principle inhibited platelet aggregation induced by ADP. It did not destroy fibrinogen, Factor X, prothrombin and thrombin; nor did it induce fibrinolysis nor interfere with the interaction between thrombin and fibrinogen. It is concluded that the anticoagulant action of this phospholipase A was due to the inhibition of the activations of Factors X and II through the inactivation of the procoagulant activity of phospholipids mediated partly by phospholipid-binding activity of this venom enzyme and partly by its enzymatic hydrolysis of phospholipids.
D000596:Amino Acids / Q000032:analysis; D000925:Anticoagulants / Q000302:isolation & purification / Q000378:metabolism*; D001777:Blood Coagulation / Q000187:drug effects; D001792:Blood Platelets / Q000378:metabolism; D005170:Factor X / Q000378:metabolism; D006358:Hot Temperature; D007106:Immune Sera; D010714:Phosphatidylethanolamines / Q000494:pharmacology; D010740:Phospholipases / Q000378:metabolism; D010974:Platelet Aggregation / Q000187:drug effects; D011516:Prothrombin / Q000378:metabolism; D012910:Snake Venoms; D013925:Thromboplastin / Q000494:pharmacology
D000596:Amino Acids; D000925:Anticoagulants; D007106:Immune Sera; D010714:Phosphatidylethanolamines; D012910:Snake Venoms; D011516:Prothrombin; D005170:Factor X; D013925:Thromboplastin; D010740:Phospholipases