Characterization of a thrombin-like enzyme, grambin, from the venom of Trimeresurus gramineus and its in vivo antithrombotic effect.
A thrombin-like enzyme, grambin, was purified to homogeneity by gel filtration, affinity and ion-exchange chromatography from the venom of Trimeresurus gramineus. Its mol. wt was estimated to be 45,400 by SDS-PAGE under reduced conditions. The mass of neutral sugars in grambin is estimated to be 20.7% of total mass. Grambin's NH2-terminal ten amino acid residues show a high homology to other venom thrombin-like enzymes. It clots human fibrinogen with a specific activity of 220-250 NIH thrombin-equivalent units/mg protein. It preferentially releases fibrinopeptide A accompanied by a slow release of trace amounts of fibrinopeptide B as monitored by HPLC following enzyme treatment of fibrinogen. EDTA, aprotinin, hirudin and heparin did not affect the fibrinogen-clotting activity of grambin in purified human fibrinogen solution. Diisopropyl fluorophosphate, phenylmethylsulfonyl fluoride and leupeptin inhibited the clotting activity of grambin whereas iodoacetamide did not affect its activity, indicating that grambin is a serine protease rather than a cysteine protease. In addition, it caused defibrinogenation and showed a marked antiplatelet effect when administered intravenously to mice. It also significantly prolonged the time lapse of platelet-rich thrombus formation in the irradiated mesenteric venules of fluorescein sodium-treated mice. Therefore, grambin may be used as a therapeutic agent not only in treatment of venous thrombosis but also in prevention of arterial thrombosis.
Pharmacological Institute, College of Medicine, National Taiwan University, Taipei.
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Chang
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Huang
D000595:Amino Acid Sequence; D000818:Animals; D000925:Anticoagulants / Q000627:therapeutic use; D003435:Crotalid Venoms / Q000201:enzymology*; D005343:Fibrinolytic Agents / Q000627:therapeutic use*; D006801:Humans; D008297:Male; D051379:Mice; D008813:Mice, Inbred ICR; D008969:Molecular Sequence Data; D010975:Platelet Aggregation Inhibitors / Q000627:therapeutic use; D013917:Thrombin / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000627:therapeutic use; D017841:Trimeresurus
D000925:Anticoagulants; D003435:Crotalid Venoms; D005343:Fibrinolytic Agents; D010975:Platelet Aggregation Inhibitors; C097268:grambin; D013917:Thrombin