ANTITHROMBOTIC DISINTEGRIN DOMAIN IN HEMORRHAGIC TOXINS

响尾蛇科和蝰蛇科蛇毒具有高度的蛋白水解作用， 能够引起严重的局部和全身出血和坏死。 的 毒液蛰入的局部影响不能用抗蛇毒血清很好地治疗， 受影响的附件遭受功能障碍或可能会失去由于极端 出血和坏死。 我们已经分离并鉴定了 西部菱背响尾蛇的毒液中 这是造成这些影响的主要原因。 我们已经证明 这些毒素是金属蛋白酶， 膜成分破坏毛细血管导致出血， 水肿和最终的坏死观察到响尾蛇envenomation。 从我们 对这些毒素的结构研究表明， 出血性毒素具有同源蛋白酶结构。 我们有 构建了C. atrox毒腺，并已测序 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。 蛋白酶是 作为酶原合成，其共有序列与 基质金属蛋白酶的半胱氨酸转换区。 大 出血性毒素ht-a具有三重结构， 蛋白酶结构域、去整合素样结构域和高含半胱氨酸的 域 ht-e的中等大小的前体缺乏高cys结构域， 保留蛋白酶和去整合素结构域，然而， 去整合素结构域被蛋白水解加工的蛋白质水平 毒素 小ht-d克隆缺乏这两个辅助结构域 仅具有蛋白酶结构域。 因此我们现在有了基因证据 毒液金属蛋白酶的三种大小类别。 我们也有 有足够的序列数据表明 结构域可能对出血活性至关重要。 在这个项目中，我们希望 为了确定在矩阵中观察到的cys开关机制是否 金属蛋白酶在毒液酶中起作用。 我们会调查 出血性毒素中的关键残留物来确定 它们在底物识别和切割中是重要的。 的作用 在II类和III类毒素中发现的非蛋白酶结构域将被 使用毒素的重组构建体进行评估，以确定是否 它们通过底物等机制调节出血活性， 识别，血小板聚集抑制，毒素蛋白水解 这些研究将有助于扩大我们的理解， 毒液引起的出血，基底膜蛋白水解破坏， 金属蛋白酶的结构和功能，以及去整合素与 整合素受体，结果抑制血小板聚集。 我们认为这些目标非常重要，对许多人都有广泛的适用性。 除了毒液外还产生出血。 此外，由于 去整合素和高Cys结构域最近在哺乳动物中被报道 我们认为，这些研究提出的蛋白质参与整合素相互作用， 变得更有意义更令人兴奋

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.