Purification and characterization of L-amino acid oxidase from king cobra (Ophiophagus hannah) venom and its effects on human platelet aggregation.
Venoms of several snake species contain large amounts of L-amino acid oxidase but its effects on human plasma coagulation and platelet aggregation have not been explored. We have purified L-amino acid oxidase from king cobra venom through CM-Sephadex C-25, Sephadex G-100 and DEAE Sephadex A-50 chromatographies. The purified enzyme has a mol. wt of 135,000 as determined by gel filtration and 65,000 by SDS-PAGE under non-reducing and reducing conditions. Incubation of plasma with L-amino acid oxidase at 200 micrograms/ml did not affect prothrombin time, activated partial thromboplastin time, or thrombin time. Upon addition of L-amino acid oxidase, platelets in platelet-rich plasma were aggregated. The enzyme-induced aggregation was abolished by catalase. The aggregation was also inhibited by indomethacin, aspirin, ethylenediaminetetraacetate, sodium nitroprusside, prostaglandin E1, mepacrine and verapamil, but not by heparin, hirudin, creatine phosphate/creatine phosphokinase or antimycin/2-deoxy-D-glucose. These results suggest that L-amino acid oxidase induces human platelet aggregation through the formation of H2O2, and subsequent thromboxane A2 synthesis requiring Ca2+ but independent of ADP release. The platelet aggregation caused by L-amino acid oxidase is likely to contribute to toxicity inflicted by cobra venom.
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Hemophilia and Thrombosis Center/Sylvester Cancer Center, University of Miami School of Medicine, FL.
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D000527:Alprostadil / Q000494:pharmacology; D000594:Amino Acid Oxidoreductases / Q000009:adverse effects / Q000737:chemistry / Q000302:isolation & purification*; D000818:Animals; D000968:Antimycin A / Q000494:pharmacology; D001241:Aspirin / Q000494:pharmacology; D001780:Blood Coagulation Tests; D001792:Blood Platelets / Q000187:drug effects*; D002374:Catalase / Q000378:metabolism; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004347:Drug Interactions; D004492:Edetic Acid / Q000494:pharmacology; D004546:Elapid Venoms / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D006493:Heparin / Q000494:pharmacology; D006629:Hirudins / Q000494:pharmacology; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism; D007213:Indomethacin / Q000494:pharmacology; D007526:Isoelectric Point; D050830:L-Amino Acid Oxidase; D008970:Molecular Weight; D009599:Nitroprusside / Q000494:pharmacology; D010974:Platelet Aggregation / Q000187:drug effects*; D011796:Quinacrine / Q000494:pharmacology; D013928:Thromboxane A2 / Q000096:biosynthesis; D014700:Verapamil / Q000494:pharmacology
D004546:Elapid Venoms; D006629:Hirudins; D009599:Nitroprusside; D013928:Thromboxane A2; D000968:Antimycin A; D006493:Heparin; D004492:Edetic Acid; D006861:Hydrogen Peroxide; D014700:Verapamil; D002374:Catalase; D000594:Amino Acid Oxidoreductases; D050830:L-Amino Acid Oxidase; D000527:Alprostadil; D011796:Quinacrine; D001241:Aspirin; D007213:Indomethacin