Hemorrhagic activity and mechanism of FIIa, a fibrinolytic enzyme from Agkistrodon acutus venom.
OBJECTIVE
To study the local hemorrhagic activity of a fibrinolytic enzyme (FII(a)) from Agkistrodon acutus venom and its mechanism.


METHODS
The local hemorrhagic activity was determined by subcutaneous injection on the back of mouse. The effects of FIIa on factor X, prothrombin, gelatin, and collagen were visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Platelet aggregation assays were performed in rat platelet-rich plasma (PRP). Human umbilical vein endothelial cells (HUVEC) were cultured and passaged in complete M199 medium. Cell viability and nuclear morphology change were determined by fluorescein diacetate (FDA) staining and Hoechst 33258 staining, respectively.


RESULTS
The minimum hemorrhagic dose (MHD) of FIIa was 89 microg. In vitro, FII(a) (0.25 g/L) degraded factor X, prothrombin, collagen, and gelatin, and dose-dependently (0.25, 0.50, 0.75, and 1.00 g/L) inhibited the platelet aggregation induced by ADP in rat PRP. When HUVEC in culture treated with FII(a), HUVEC showed detachment in a dose-dependent manner, but no apoptosis sign was observed.


CONCLUSIONS
FIIa had local hemorrhagic activity, and the mechanism was related to the degradation of factor X, prothrombin, gelatin, and collagen, the inhibition of ADP-induced platelet aggregation, and inducement of HUVEC detachment.
Wang
Qing-qing
QQ
Department of Pharmacology, Zhongshan Medical College, Sun Yat-Sen University, Guangzhou 510080, China.
Chen
Jia-shu
JS
Liang
Xiu-xia
XX
Qiu
Peng-xin
PX
Wang
Yi-wen
YW
Yan
Guang-mei
GM
D017836:Agkistrodon; D000818:Animals; D002478:Cells, Cultured; D003094:Collagen / Q000378:metabolism; D003435:Crotalid Venoms / Q000737:chemistry* / Q000302:isolation & purification / Q000494:pharmacology*; D042783:Endothelial Cells / Q000166:cytology / Q000187:drug effects*; D005170:Factor X / Q000378:metabolism; D005260:Female; D005343:Fibrinolytic Agents / Q000494:pharmacology*; D006801:Humans; D008297:Male; D008666:Metalloendopeptidases; D051379:Mice; D010974:Platelet Aggregation / Q000187:drug effects; D011516:Prothrombin / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D014471:Umbilical Veins / Q000166:cytology
D003435:Crotalid Venoms; D005343:Fibrinolytic Agents; D011516:Prothrombin; D005170:Factor X; D003094:Collagen; C478420:FIIa; D008666:Metalloendopeptidases