Investigations of the interactions between human plasma proteinase inhibitors (PPI) and snake venom metalloproteinases will be continued. The bond cleaved during inactivation of human alpha 1-antichymotrypsin (alpha 1-AC) by venom metalloproteinases will be established by structural studies of the reaction products. Detailed studies will be initiated, based upon the recent finding in this laboratory that opossum (Didelphis virginiana) PPI differ from known mammalian PPI and resist digestion by venom proteinases. These opossum PPI will be purified and characterized: oprin (opossum proteinase inhibitor), HTI (hemorrhagic toxin inhibitor), alpha1-PI (alpha1-proteinase inhibitor), alpha1-AC and TI (trypsin inhibitor). Inhibition of venom hemorrhagic toxins by HTI will be studied, and structural analyses will establish the reactive site of HTI and its relationship to known mammalian PPI. The antihemorrhagic factor will be isolated from Crotalus adamanteus serum, and studied in terms of inhibitor/enzyme complex formation with venom hemorrhagic factors. Resistance of opossum alpha1-PI to digestion by venom metalloproteinases under conditions in which human alpha1-PI is totally inactivated will be examined. Structural studies of opossum alpha1-PI will delineate the factors in the reactive site loop and amino-terminal regions which account for the resistance. Similar investigations will be done on opossum alpha1-AC. Cloning and sequencing of the cDNA's coding for oprin and HTI are in progress and will be continued. Investigations will be initiated, aimed at cloning and sequencing the cDNA's coding for two metalloproteinases from C. adamanteus venom, namely Proteinase H (the hemorrhagic factor from this venom) and Proteinase II (which inactivates human PPI). Deduced amino acid sequences will be compared with known protein sequences to homologous proteins, and to establish evolutionary relationships. The results with oprin and other opossum PPI will provide insights into the defense mechanisms involved in resistance to the localized and systemic effects of envenomation. The studies with HTI and snake serum antihemorrhagic factors will enhance understanding of mechanisms regulating localized hemorrhage. Structural analyses of the naturally occurring model (opossum alpha1-PI) will be applicable to the development, by genetic engineering of proteins-resistant human Alpha1-PI variants which may be of therapeutic use in diseases such as emphysema.
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