Tans G, Govers-Riemslag J W, van Rijn J L, Rosing J
J Biol Chem. 1985 Aug 5;260(16):9366-72.
The prothrombin activator present in the venom of the mainland tiger snake (Notechis scutatus scutatus) was purified to homogeneity by gel chromatography on Sephadex G-200 followed by ion-exchange chromatography on SP-Sephadex. The venom activator has an apparent molecular weight of 54,000. It consists of a heavy chain (Mr = 32,000) and a light chain (Mr = 23,000) held together by one or more disulfide bridges. The active site is located at the heavy chain region of the molecule. The venom activator contains 8 gamma-carboxyglutamic acid residues/molecule. Gel electrophoretic analysis of prothrombin activation indicates that the venom activator is capable of cleaving both the Arg 274-Thr 275 and Arg 323-Ile 324 bonds of bovine prothrombin. The order of bond cleavage appears to be random since prethrombin-2 and meizothrombin occur as intermediates during prothrombin activation. Prothrombin activation by the venom activator alone is very slow. This is explained by the unfavorable kinetic parameters for the reaction (Km for prothrombin = 105 microM, Vmax = 0.0025 nmol of prothrombin activated per min/microgram of venom activator). Phospholipids plus Ca2+ and Factor Va greatly stimulate venom-catalyzed prothrombin activation. In the presence of 50 microM phospholipid vesicles composed of 20 mol % phosphatidylserine and 80 mol % phosphatidylcholine, the Km drops to 0.2 microM, whereas there is hardly any effect on the Vmax. Factor Va causes a 3,500-fold increase of the Vmax (8.35 nmol of prothrombin activated per min/microgram of venom activator) and a 10-fold decrease of the Km (9.5 microM). The most favorable kinetic parameters are observed in the presence of both 50 microM phospholipid and Factor Va (Km = 0.16 microM, Vmax = 27.9 nmol of prothrombin activated per min/microgram of venom activator). These changes of the kinetic parameters explain the stimulatory effects of Factor Va and phospholipid on venom-catalyzed prothrombin activation. The venom activator slowly converts the Factor Xa-specific chromogenic substrates CH3SO2-D-leucyl-glycyl-L-arginine-p-nitroanilide and N-benzoyl-L-isoleucyl-L-glutamyl-(piperidyl)-glycyl-L-arginyl-p-nitroani lide hydrochloride. Factor Va causes a 7-fold stimulation of chromogenic substrate conversion by the venom activator. This stimulation appears to be the result of the formation of a tight 1:1 complex between the venom activator and Factor Va.
通过在葡聚糖凝胶G - 200上进行凝胶色谱，随后在SP - 葡聚糖凝胶上进行离子交换色谱，将大陆虎蛇（盾鼻蛇盾鼻亚种，Notechis scutatus scutatus）毒液中的凝血酶原激活剂纯化至同质。该毒液激活剂的表观分子量为54,000。它由一条重链（Mr = 32,000）和一条轻链（Mr = 23,000）通过一个或多个二硫键连接而成。活性位点位于分子的重链区域。该毒液激活剂每分子含有8个γ - 羧基谷氨酸残基。凝血酶原激活的凝胶电泳分析表明，该毒液激活剂能够切割牛凝血酶原的Arg 274 - Thr 275和Arg 323 - Ile 324键。键切割顺序似乎是随机的，因为凝血酶原-2和中凝血酶在凝血酶原激活过程中作为中间体出现。仅毒液激活剂对凝血酶原的激活非常缓慢。这可以通过该反应不利的动力学参数来解释（凝血酶原的Km = 105 microM，Vmax = 每分钟每微克毒液激活剂激活0.0025 nmol凝血酶原）。磷脂加Ca2 +和因子Va极大地刺激毒液催化的凝血酶原激活。在由20摩尔％磷脂酰丝氨酸和80摩尔％磷脂酰胆碱组成的50 microM磷脂囊泡存在下，Km降至0.2 microM，而对Vmax几乎没有影响。因子Va使Vmax增加3500倍（每分钟每微克毒液激活剂激活8.35 nmol凝血酶原），Km降低10倍（9.5 microM）。在同时存在50 microM磷脂和因子Va的情况下观察到最有利的动力学参数（Km = 0.16 microM，Vmax = 每分钟每微克毒液激活剂激活27.9 nmol凝血酶原）。动力学参数的这些变化解释了因子Va和磷脂对毒液催化的凝血酶原激活的刺激作用。该毒液激活剂缓慢转化因子Xa特异性显色底物CH3SO2 - D - 亮氨酰 - 甘氨酰 - L - 精氨酸 - 对硝基苯胺和N - 苯甲酰 - L - 异亮氨酰 - L - 谷氨酰 - （哌啶基） - 甘氨酰 - L - 精氨酰 - 对硝基苯胺盐酸盐。因子Va使毒液激活剂对显色底物的转化增加7倍。这种刺激似乎是毒液激活剂与因子Va之间形成紧密1:1复合物的结果。
