Francischetti I M, Castro H C, Zingali R B, Carlini C R, Guimarães J A
Departamento de Bioquímica Médica-ICB, Universidade Federal do Rio de Janeiro, Brazil.
Comp Biochem Physiol C Pharmacol Toxicol Endocrinol. 1998 Jan;119(1):21-9. doi: 10.1016/s0742-8413(97)00163-1.
Procoagulant, proteolytic, phospholipase and platelet pro-aggregating and inhibiting activities were screened for pooled venoms of seven Bothrops species as well as Crotalus durissus terrificus and Lachesis muta snakes typical of the Brazilian territory. As reported by other authors, we also found that examination of the electrophoretic and gel filtration patterns of Bothrops snakes venoms could not be used for identification of the species of a given venom because of the lack of marked interspecific differences within the same genus. Our data indicated that B. cotiara, B. alternatus and B. atrox possess no platelet pro-aggregating activity, low inhibitory effect on platelet aggregation and very low or intermediate levels for the other activities. B. moojeni, B. neuwiedi and B. jararacussu whose venoms possess high procoagulant, platelet pro-aggregating and phospholipase activities are low in both proteolytic and platelet inhibitory activities. B. jararaca venom showed the highest inhibitory effect on platelet aggregation and very low platelet pro-aggregating activity. Compared with the Bothrops venoms studied, L. muta venom showed that highest proteolytic activity while C. d. terrificus venom presented remarkable high platelet pro-aggregating and phospholipase activities. In all venoms, proteolytic activity could be completely inhibited by EDTA (2 mM) alone. In contrast, the presence of phenylmethylsulfonyl fluoride (5 mM) inhibited partially the caseinolytic activity of all venoms, except that L. muta venom, which was almost completely blocked by this reagent. Altogether, these data confirm the presence of high levels of metalloproteinases in the venoms of Crotalinae snakes. Most of these enzymes are dependent of the availability of Ca2+, being much less the same concerning the presence of serine residues in their active sites. The data indicated that the presence and levels of procoagulant, azocaseinolytic and phospholipase A2 activities alone could not differentiated the species of the Bothrops venoms studied, particularly in the cases of B. jararaca, B. moojeni and B. atrox. However, the platelet inhibiting property of low doses of B. jararaca venom can be useful to differentiate it from B. moojeni venom. In the same way, the platelet pro-aggregating activity of high doses of B. jararaca venom may be used to distinguish it from B. atrox crude venom, otherwise very similar but incapable to activate platelets. In conclusion, our comparative screening of biological properties has indicated that platelet studies may serve as a tool to distinguish among venoms that otherwise behave biochemically in a very similar way. Although promising, the general applicability of platelet activation studies by snake venoms for classification or taxanomical purposes has yet to be extended to other family of snakes to be proven useful.
对七种矛头蝮属物种以及巴西本土特有的三色矛头蝮和巨蝮蛇的混合毒液进行了促凝、蛋白水解、磷脂酶以及血小板促聚集和抑制活性的筛选。正如其他作者所报道的，我们还发现，由于同一属内种间差异不明显，对矛头蝮属蛇毒的电泳和凝胶过滤图谱进行检测不能用于鉴定特定毒液的种类。我们的数据表明，科氏矛头蝮、交替矛头蝮和矛头蝮不具有血小板促聚集活性，对血小板聚集的抑制作用较低，其他活性水平极低或处于中等水平。穆氏矛头蝮、诺维迪矛头蝮和巴西矛头蝮的毒液具有高促凝、血小板促聚集和磷脂酶活性，但其蛋白水解和血小板抑制活性较低。巴西矛头蝮毒液对血小板聚集的抑制作用最强，血小板促聚集活性极低。与所研究的矛头蝮属毒液相比，巨蝮毒液的蛋白水解活性最高，而三色矛头蝮毒液具有显著的高血小板促聚集和磷脂酶活性。在所有毒液中，单独使用2 mM的乙二胺四乙酸（EDTA）可完全抑制蛋白水解活性。相比之下，5 mM的苯甲基磺酰氟的存在部分抑制了所有毒液的酪蛋白水解活性，但巨蝮毒液几乎被该试剂完全阻断。总之，这些数据证实了蝰蛇亚科蛇毒中存在高水平的金属蛋白酶。这些酶中的大多数依赖于Ca2+的存在，而在其活性位点中丝氨酸残基的存在则相对较少。数据表明，仅促凝、偶氮酪蛋白水解和磷脂酶A2活性的存在和水平不能区分所研究的矛头蝮属毒液的种类，特别是对于巴西矛头蝮、穆氏矛头蝮和矛头蝮的情况。然而，低剂量巴西矛头蝮毒液的血小板抑制特性可用于将其与穆氏矛头蝮毒液区分开来。同样，高剂量巴西矛头蝮毒液的血小板促聚集活性可用于将其与矛头蝮粗毒液区分开来，否则它们非常相似但无法激活血小板。总之，我们对生物学特性的比较筛选表明，血小板研究可作为区分在生化行为上非常相似的毒液的一种工具。尽管很有前景，但蛇毒诱导的血小板激活研究在分类或分类学目的方面的普遍适用性还有待扩展到其他蛇类家族以证明其有用性。
