title: Hementin: anticoagulant protease from the salivary gland of the leech Haementeria ghilianii.
abstract: Leech Haementeria ghilianii has an anticoagulant in its salivary glands that renders ingested blood incoagulable by thrombin. The mechanism of blood incoagulation is associated with cleavage of peptide bonds in fibrinogen, and thus the active agent, called hementin, is a proteolytic enzyme. Hementin was isolated and purified 16-fold from the anterior salivary glands by anion-exchange chromatography, ammonium sulfate fractionation, and cation exchange chromatography. Pure material obtained by slab gel electrophoresis contained a single polypeptide chain with approximate Mr of 120,000. Hementin was stable for many hours at room temperature, but on incubation at 60 degrees C for 15 min all activity was lost. At 4 degrees, hementin had no activity, but this inhibition was fully reversible. Complete inactivation occurred in the presence of EDTA, cysteine, DTT, sodium phosphate and at extreme pH (greater than 11 or less than 5), whereas citrate, Tris, glycine, and EGTA caused only partial loss of activity. DFP, PMSF, iodoacetic acid, and leupeptin had no effect on hementin activity. The data indicated hementin to be a neutral metalloprotease with optimum pH of 7.5. The enzyme had high affinity for cleaving fibrinogen and calculations of kinetic data from a double-reciprocal plot gave a Km of hementin for fibrinogen of 1.0 +/- 0.1 microM. Normal human citrated plasma or fresh blood were rendered incoagulable after incubation with hementin, indicating that the enzyme activity was not affected by plasma protease inhibitors. Plasma levels of coagulation factors II, V, VII, VIII, IX, X, XI, XII, prekallikrein, and high-molecular-weight kininogen were not altered by the enzyme. Hementin, a neutral metalloprotease resistant to plasma protease inhibitors, executes its anticoagulant effect on blood by selective cleavage of fibrinogen.
mesh_terms: D000818:Animals; D000925:Anticoagulants / Q000302:isolation & purification*; D001779:Blood Coagulation Factors / Q000378:metabolism; D001780:Blood Coagulation Tests; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000302:isolation & purification*; D005340:Fibrinogen / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007865:Leeches / Q000502:physiology*; D008666:Metalloendopeptidases; D008970:Molecular Weight; D010447:Peptide Hydrolases / Q000302:isolation & purification*; D012469:Salivary Glands / Q000201:enzymology
chemical_list: D000925:Anticoagulants; D001779:Blood Coagulation Factors; D005340:Fibrinogen; D010450:Endopeptidases; D010447:Peptide Hydrolases; D008666:Metalloendopeptidases; C040192:hementin