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.