Lloyd Waxman

[18] Purification and characterization of inhibitors of blood coagulation factor Xa from hematophagous organisms

Publisher Summary This chapter describes the purification and characterization of inhibitors of blood coagulation factor Xa from Hematophagous organisms. Hematophagous organisms, such as leeches and ticks possess in their salivas an extensive repertoire of biochemical factors that act in concert to maintain blood in a liquid state, both during the ingestion of a blood meal and during its prolonged storage in the gut. The therapeutic potential of leeches or leech-derived substances is recognized because of antiquity. The most extensively studied leech-derived substance, hirudin, the extremely potent anticoagulant found in the saliva of the leech Hirudo medicinalis , is a selective, stoichiometric inhibitor of thrombin. Activation of coagulation through either the intrinsic or extrinsic pathway results in the formation of factor Xa (fXa), which catalyzes the conversion of prothrombin to thrombin. Salivary gland extracts from the Mexican leech Haementeria officinalis are shown to contain a potent antimetastatic and anticoagulant activity. The protein responsible for both of these activities has been purified and named antistasin (ATS).

Comparison of the multicatalytic proteinases isolated from the nucleus and cytoplasm of chicken red blood cells

1. Two chromatographically distinct multicatalytic proteinases (MCPs) were isolated from the cytoplasm of chicken red blood cells and one MCP was purified from the nuclei. 2. The nuclear and the majority (97-99%) of the cytoplasmic multicatalytic proteolytic activity were chromatographically similar and differed from the minor cytoplasmic activity in their elution from hydroxylapatite, number of subunits on 2D-SDS-PAGE, and in their sensitivity to proteinase inhibitors. 3. Dichloroisocoumarin, a serine proteinase inhibitor, inhibited the hydrolysis of fluorogenic peptides but stimulated the degradation of casein by the multicatalytic proteinases suggesting that this enzyme has distinct active sites for protein and peptide hydrolysis.

Identification of a soluble enzyme from C3H/10T12 cells which is inhibited by the Bowman-Birk proteinase inhibitor

The anticarcinogenic Bowman-Birk proteinase inhibitor (BBI) inhibits a 70-kDa serine proteinase in C3H/10T1/2 transformed fibroblasts. Two serine proteinases, the proline endopeptidase and a novel neutral proteolytic activity, both having a mass of approximately 70-kDa, were isolated from the cytoplasm of C3H/10T1/2 cells. BBI did not inhibit diisopropylfluorophosphate binding to the proline endopeptidase or its ability to hydrolyze peptides. However, BBI blocked the binding of diisopropylfluorophosphate and inhibited the cleavage of peptides by the novel cytoplasmic enzyme. Thus BBI does not inhibit the proline endopeptidase but another soluble 70-kDa serine proteinase from C3H/10T1/2 cells.