ANTITHROMBOTIC DISINTEGRIN DOMAIN IN HEMORRHAGIC TOXINS

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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.