ANTITHROMBOTIC DISINTEGRIN DOMAIN IN HEMORRHAGIC TOXINS

鳄鱼蛇毒和蛇毒具有高度的蛋白质分解能力，并具有 能够导致严重的局部和全身出血和坏死。这个 抗蛇毒血清不能很好地治疗蛇毒的局部效应，通常 受影响的附件会出现功能障碍或由于极端的 出血和坏死。我们已经分离并鉴定了这些毒素 在西部小菜蛾响尾蛇的毒液中， 它们是造成这些影响的主要原因。我们已经证明了 这些毒素是金属蛋白酶和基底膜的裂解 膜成分破坏毛细血管导致出血， 章鱼毒液中可见水肿和最终的坏死。从我们的 对这些毒素的结构研究我们已经确定所有的 出血性毒素具有同源的蛋白酶结构。我们有 从阿曲霉毒腺中创建了一个cdna文库，并进行了测序。 各种出血性毒素的代表性克隆。这些序列 揭示了几个非常有趣的特征。蛋白水解酶 合成为酶原，具有类似于 基质金属蛋白酶的半胱氨酸开关区。大的 出血性毒素，ht-a，具有三元结构，由一个 蛋白水解酶结构域、去整合素样结构域和含有高半胱氨酸的结构域 域。一个中等尺度的ht-e前兆缺少高的cys结构域，但 保留了蛋白水解酶和去整合素域，但在 去整合素结构域的蛋白质水平是从 毒素。小的ht-d克隆缺少这两个辅助结构域。 仅具有蛋白水解酶结构域。因此，我们现在有了遗传证据 毒液金属蛋白酶的三种大小类别。我们还有 有足够的序列数据表明该酶中的哪些残基 结构域可能是出血活动的关键。在这个项目中，我们希望 以确定在矩阵中观察到的CyS开关机制 金属蛋白酶在毒液酶中起作用。我们会调查 确定出血性毒素中的关键残留物是否事实上 它们在底物识别和切割中很重要。的作用 在第二类和第三类毒素中发现的非蛋白酶域将是 使用毒素的重组构建物进行评估以确定 它们通过底物等机制调节出血活动。 识别、抑制血小板聚集、毒素蛋白分解 这些研究将有助于扩大我们对 毒液产生的出血，基底膜蛋白分解破坏， 金属蛋白酶的结构和功能，以及去整合素与 整合素受体与抑制血小板聚集的结果。 我们认为这些都是非常重要的目标，具有广泛的适用性 除了毒液外，田野还会产生出血。此外，由于 最近在哺乳动物中发现了去整合素和高半胱氨酸结构域。 参与整合素相互作用的蛋白质我们认为研究提出了 变得更加相关和令人兴奋。

Crotalid and Viperid snake venoms are highly proteolytic and have the ability to cause severe local and systemic hemorrhage and necrosis. The local effects of envenomation are not well treated by antivenin and often the affected appendages suffer dysfunction or may be lost due to extreme hemorrhage and necrosis. We have isolated and characterized the toxins in the venom of the western diamondback rattlesnake, Crotalus atrox, which are primarily responsible for these effects. We have demonstrated that these toxins are metalloproteinases and the cleavage of basement membrane components disrupt the capillaries resulting in the hemorrhage, edema and eventual necrosis observed in crotalid envenomation. From our structural studies on these toxins we have determined that all the hemorrhagic toxins have homologous proteinase structures. We have created a cDNA library from C. atrox venom glands and have sequenced representative clones for various hemorrhagic toxins. These sequences revealed several very interesting features. The proteinases are synthesized as zymogens with a consensus sequence similar to the cysteine-switch region of the matrix metalloproteinases. The large hemorrhagic toxin, ht-a, has a tripartite structure which consists of a proteinase domain, a disintegrin-like domain and a high cys containing domain. A medium sized precursor for ht-e lacks the high cys domain but retains the proteinase and the disintegrin domains, however, at the protein level the disintegrin domain is proteolytically processed from the toxin. The small ht-d clone lacks both of these auxiliary domains possessing only the proteinase domain. Thus we now have genetic evidence for the three size classes of the venom metalloproteinases. We also have sufficient sequence data to suggest which residues in the proteinase domain may be crucial for hemorrhagic activity. In this project we hope to determine whether the cys-switch mechanism observed in the matrix metalloproteinases is operational in the venom enzymes. We will probe the critical residues in the hemorrhagic toxins to determine if in fact they are important in substrate recognition and cleavage. The role of the nonproteinase domains found in the class II and III toxins will be assessed using recombinant constructs of the toxins to determine whether they modulate the hemorrhagic activity via such mechanisms as substrate recognition, platelet aggregation inhibition, toxin proteolytic processing, etc. These studies will serve to expand our understanding of venom produced hemorrhage, basement membrane proteolytic disruption, metalloproteinase structure and function, and disintegrin binding to integrin receptors with the result of inhibition of platelet aggregation. We feel these are very significant goals with broad applicability to many fields in addition to venom produced hemorrhage. Furthermore, since disintegrin and high cys domains have recently been reported in mammalian proteins involved in integrin interaction we believe the studies proposed to be even more relevant and exciting.