A factor X-activating cysteine protease from malignant tissue.
A proteolytic procoagulant has been identified in extracts of human and animal tumors and in cultured malignant cells. It directly activated Factor X but its similarity to other Factor S-activating serine proteases was not clear. This study describes work done to determine whether this enzyme, cancer procoagulant, is a serine or cysteine protease. Purified cancer procoagulant from rabbit V2 carcinoma was bound to a p-chloromercurialbenzoate-agarose affinity column and was eluted with dithiothreitol. The initiation of recalcified, citrated plasma coagulation activity by cancer procoagulant was inhibited by 5 mM diisopropylfluorophosphate, 1 mM phenylmethylsulfonylfluoride, 0.1 mM HgCl2, and 1 mM iodoacetamide. Activity was restored in the diisopropylfluorophosphate-, phenylmethylsulfonylfluoride-, and HgCl2-inhibited samples by 5 mM dithiothreitol; iodoacetamide inhibition was irreversible. Russell's viper venom, a control Factor X-activating serine protease, was not inhibited by either 0.1 mM HgCl2 or 1 mM iodoacetamide. The direct activation of Factor X by cancer procoagulant in a two-stage assay was inhibited by diisopropylfluorophosphate and iodoacetamide. Diisopropylfluorophosphate inhibits serine proteases, and an undefined impurity in most commercial preparations inhibits cysteine proteases. Hydrolysis of diisopropylfluorophosphate with CuSO4 and imidazole virtually eliminated inhibition of thrombin, but cancer procoagulant inhibition remained complete, suggesting that cancer procoagulant was inhibited by the undefined impurity. These results suggest that cancer procoagulant is a cysteine endopeptidase, which distinguishes it from other coagulation factors including tissue factor. This and other data suggest that neoplastic cells produce this unique cysteine protease which may initiate blood coagulation.
D000818:Animals; D001777:Blood Coagulation / Q000187:drug effects; D002846:Chromatography, Affinity; D003546:Cysteine Endopeptidases; D010450:Endopeptidases / Q000032:analysis / Q000302:isolation & purification*; D004789:Enzyme Activation; D005170:Factor X / Q000378:metabolism*; D007460:Iodoacetamide / Q000494:pharmacology; D007531:Isoflurophate / Q000494:pharmacology; D009363:Neoplasm Proteins; D009374:Neoplasms, Experimental / Q000201:enzymology*; D010664:Phenylmethylsulfonyl Fluoride / Q000494:pharmacology; D011480:Protease Inhibitors; D011817:Rabbits
D009363:Neoplasm Proteins; D011480:Protease Inhibitors; D007531:Isoflurophate; D010664:Phenylmethylsulfonyl Fluoride; D005170:Factor X; D010450:Endopeptidases; D003546:Cysteine Endopeptidases; C030910:cancer procoagulant; D007460:Iodoacetamide