Purification and properties of the anticoagulant principle of Trimeresurus gramineus venom.
By means of DEAE-Sephadex A-50 column chromatography, Trimeresurus gramineus venom was separated into 12 fractions. Fraction 8 had marked anticoagulant action in the tests of whole blood clotting time, calcium clotting time and plasma prothrombin time. Fraction 8 was rechromatographed on Sephadex G-100, then on DEAE-Sephadex A-50 again, and finally 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 1.70 Svedberg units was obtained by ultracentrifugation. The estimated molecular weight was 19 500. The isoelectric point was pH 4.5. Chemical analysis showed that the anticoagulant principle was a glycoprotein and that it was thermolabile. The anticoagulant activity of this purified principle was 3.5 times higher than that of the crude venom. Fraction 5 potentiated its anticoagulant activity to 10 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, fibrinolytic, hemorrhagic or local irritating activities. The purified anticoagulant principle did not destroy fibrinogen, induce fibrinolysis, inactivate thrombin nor interfere with the interaction between thrombin and fibrinogen. However, a marked inhibition of prothrombin activation was caused by the anticoagulant principle. The inhibition of prothrombin activation was not due to the destruction of prothrombin or its activation factors, but due to an interference in the interaction between prothrombin and its activation factors because of the reversible binding of these factors with the anticoagulant principle of the venom.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D000925:Anticoagulants / Q000302:isolation & purification / Q000494:pharmacology*; D001777:Blood Coagulation / Q000187:drug effects; D001780:Blood Coagulation Tests; D002850:Chromatography, Gel; D005340:Fibrinogen / Q000378:metabolism; D005342:Fibrinolysis / Q000187:drug effects; D007928:Lethal Dose 50; D051379:Mice; D008970:Molecular Weight; D011517:Prothrombin Time; D012910:Snake Venoms / Q000032:analysis*; D013696:Temperature; D013917:Thrombin / Q000378:metabolism; D013925:Thromboplastin / Q000378:metabolism; D014688:Venoms / Q000032:analysis*
D000596:Amino Acids; D000925:Anticoagulants; D012910:Snake Venoms; D014688:Venoms; D005340:Fibrinogen; D013925:Thromboplastin; D013917:Thrombin