Effect of Protobothrops mucrosquamatus venom and its fractions on bIood coaguIation / 中国药理学与毒理学杂志

ABSTRACT

OBJECTIVE To investigate the effect of Protobothrops mucrosquamatus venom (PMV) and its fractions on functions of the circulatory system in vitro in order to better understand its toxicity mechanism. METHODS PMV was isolated to three fractions FⅠ, FⅡ and FⅢ with a different molecular mass range by Sephadex G-75 gel filtration chromatography. Platelet rich plasma was adjusted to 3×1011 L-1 by platelet poor plasma. Platelet suspension was incubated with PMV and its fractions 0.03 g.L-1 for 5 min, respectively, and platelet aggregation was determined on an LBY-NJ4 aggregometer. PMV and its fractions 0.05 g.L-1 were preincubated with plasminogen 0.1 U.L-1 for 10 min before chromogenic substrate cleavage activity was measured by endpoint and enzyme kinetics determination. PMV and its fractions 1.0 g.L-1 were incubated with rat plasma for 5 or 30 min, and thrombin time (TT), activated partial thromboplastin time (APTT), prothrombin time (PT) and fibrinogen (FlB) content were assayed. The microvascular endothelial cells were exposed to PMV and its fractions 10, 50 and 250 mg.L-1 , respectively, for 24 h, while the morphological change was observed using an inverted phase contrast microscope, and the cell viability was determined by MTT method. PMV and its fractions were incubated with guinea pig red blood cell suspension in the presence or absence of lecithin for different time, and hemolysis was measured. RESULTS Compared with normal control, platelet aggregation rate was significantly increased by PMV and FⅠ (>71 ku)〔(12.4±4.1)%,(61.0±5.8)% and (56.9±5.9)%〕(P<0.01). PMV and FⅡ (18-37 ku) significantly hydrolyzed chromogenic substrate S-2251(P<0.01). PMV and FⅠ caused plasma coagulation. Compared with normal control, FⅡ and FⅢ (<10 ku) remarkably prolonged TT, APTT and PT( P<0.01). Morphological observation revealed that PMV, FⅠ and FⅡdetached the adherent cells. Compared with normal control group, PMV, F Ⅰ and F Ⅱ inhibited cell viability, and the survival rate of the cells decreased to (56.8±3.6)%,(71.6±3.8)% and(58.2±5.5)%, respectively. PMV and FⅡ slowly caused slight hemolysis in absence of lecithin. PMV and FⅡ caused significant hemolysis in the presence of lecithin, and the hemolytic rate increased to (81.0±4.0)% and (81.0±1.0)%( P <0.01) in 0.5 min, respectively, compared with (17.7±1.0)% of the control group. CONCLUSION PMV possesses different activities that affect the functions of the circulatory system in vitro, and the fractions play different roles in toxicity mechanisms.