A factor X-activating cysteine protease from malignant tissue. S G Gordon, B A Cross J Clin Invest. 1981;67(6):1665-1671. https://doi.org/10.1172/JCI110203. Research Article 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. […] Find the latest version: https://jci.me/110203/pdf A Factor X-Activating Cysteine Protease from Malignant Tissue STUART G. GORDON and BARBARA A. CROSS, Department of Medicine, University of Colorado Health Sciences Center, Denver, Colorado 80262 A B S T R A C T A proteolytic procoagulant has been is converted to its activated form (Factor Xa) by the identified in extracts of human and animal tumors and proteolytic removal of an activation glycopeptide with in cultured malignant cells. It directly activated Factor a molecular weight of -11,000 (1, 2). This activa- X but its similarity to other Factor X-activating serine tion can be facilitated by either Factors IXa and VIII, proteases was not clear. This study describes work done Factor VIIa and tissue factor, the autolytic conversion to determine whether this enzyme, cancer procoagu- by Factor Xa, or by an enzyme in the venom of Vipera lant, is a serine or cysteine protease. Purified cancer Russelli (Russell's viper venom) (1-4). The active procoagulant from rabbit V2 carcinoma was bound to component within all of these Factor X-activating a p-chloromercurialbenzoate-agarose affinity column systems is a serine protease (1-5). In addition, and was eluted with dithiothreitol. The initiation of Factor X can be activated by other serine proteases recalcified, citrated plasma coagulation activity by can- such as trypsin (1, 2, 6), cathepsin C (7), and the plant cer procoagulant was inhibited by 5 mM diisopropyl- cysteine protease, papain (8). fluorophosphate, 1 mM phenylmethylsulfonylfluoride, Increased vascular thrombosis and the deposition of 0.1 mM HgCl2, and 1 mM iodoacetamide. Activity was fibrin associated with malignant disease led us to look restored in the diisopropylfluorophosphate-, phenyl- for an abnormal coagulation factor associated with neo- methylsulfonylfluoride-, and HgCl2-inhibited samples plastic tissue. A proteolytic enzyme, cancer pro- by 5 mM dithiothreitol; iodoacetamide inhibition was coagulant, has been purified from rabbit V2 carcinoma irreversible. Russell's viper venom, a control Factor X- and some of its physical, chemical, and enzymatic activating serine protease, was not inhibited by either properties have been determined.' In addition, 0.1 mM HgCl2 or 1 mM iodoacetamide. The direct acti- cancer procoagulant activity has been identified in vation of Factor X by cancer procoagulant in a two-stage extracts of several human malignant tumors (9) and in assay was inhibited by diisopropylfluorophosphate and cultured malignant cells2 (10-13): This enzyme is iodoacetamide. Diisopropylfluorophosphate inhibits inhibited by diisopropylfluorophosphate (DFP)3 and serine proteases, and an undefined impurity in most initiates coagulation in the absence of Factor VII commercial preparations inhibits cysteine proteases. by directly activating Factor X (14). Thus, we assumed Hydrolysis of diisopropylfluorophosphate with CuS04 that cancer procoagulant was a $erine protease, and imidazole virtually eliminated inhibition of throm- similar to the other enzymes that activate Factor X. bin, but cancer procoagulant inhibition remained com- These studies provide data which indicate that plete, suggesting that cancer procoagulant was in- cancer procoagulant is not a serine protease but is a hibited by the undefined impurity. These results cysteine protease. suggest that cancer procoagulant is a cysteine endo- peptidase, which distinguishes it from other coagula- METHODS tion factors including tissue factor. This and other data Materials. A 0.5-M stock solution of DFP (Aldrich, suggest that neoplastic cells produce this unique cys- Chemical Co., Milwaukee, Wis.) was prepared in dry iso- teine protease which may initiate blood coagulation. INTRODUCTION 1 Gordon, Stuart G. Manuscript submitted for publication. 2 Gordon, Stuart G., and B. Lewis. Manuscript submitted Factor X is the point of confluence of the intrinsic and for publication. extrinsic pathways in the coagulation cascade. Factor X 3Abbreviations used in this paper: DFP, diisopropyl- fluorophosphate; DTT, dithiothreitol; MES, 2[N-morpholine]- Received for publication 6 October 1980 and in revised ethane sulfonic acid; PCMB, p-chloromercurialbenzoate- form 8 January 1981. agarose; PMSF, phenylmethylsulfonylfluoride. J. Clin. Invest. a The American Society for Clinical Investigation, Inc. * 0021-9738181/0611665107 $1.00 1665 Volume 67 June 1981 1665-1671 propanol and diluted 1: 100 in the sample to facilitate The protein peaks were collected separately and concen- inhibition. A stock solution of 0.01 M HgCl2 was prepared in trated -10-fold on a PM-10 ultrafiltration membrane and water and added to samples in a 1:100 ratio to a final con- dialyzed against MES buffer to remove the DTT. These centration of 0.1 mM. A stock solution of 0.1 M phenyl- samples were assayed for protein and procoagulant activity methylsulfonylfluoride (PMSF) (Sigma Chemical Co., St. as described above. Louis, Mo.) was prepared in dry dioxane and added to samples Inhibition studies. Samples purified by the method de- in a 1:100 ratio to a final concentration of 1 mM. A 1 x 20-cm scribed above and those obtained from the PCMB affinity p-chloromercurialbenzoate-agarose (PCMB) (Bio-Rad Labo- column were tested for their inhibition by 5 mM DFP, 1 mM ratories, Richmond, Calif.) affinity column was prepared in PMSF, 1 mM iodoacetamide, and 0.1 mM HgCl2. In each case, 20 mM 2[N-morpholino]ethane sulfonic acid (MES) buffer samples ofcancer procoagulant were assayed for procoagulant (pH 6.8) to test the efficiency of cancer procoagulant activity by measuring the single-stage recalcification clotting binding and for the fractionation of the protease. time of citrated human plasma with a fibrometer. The percent Citrated bovine and human plasma were obtained fresh decrease in the clotting time of samples containing cancer and prepared as described previously (14) for use in the single- procoagulant or controls compared to the appropriate 25 mM stage recalcified coagulation time assay. Pure bovine Factor X MES buffer blank was used to quantify coagulant activity. was kindly provided by Dr. Earl Davie and used in the two- Russell's viper venom was used as a coagulation control and as stage clotting assay as described previously (14). Russell's a Factor X-activating serine protease control. Portions of the viper venom (Sigma Chemical Co.) was used as a control samples and controls were treated with the appropriate in- Factor X-activating serine protease. Partially purified bovine hibitors, incubated for 30 min at 37°C, and reassayed for thrombin (Parke Davis and Co., Detroit, Mich.) was diluted procoagulant activity. Portions of each sample were made 5 to 0.1 U/ml in 10 mM veronal-buffered saline (pH 7.6). mM with DTT, incubated overnight at 5°C, dialyzed for 3 h Methods. General: Cancer procoagulant was purified from against two changes of 25 mM MES buffer, and reassayed for rabbit V2 carcinoma tumor tissue.4 Briefly, tumor tissue was procoagulant activity to determine if the inhibition had been removed surgically before the animal's death, extracted in effectively reversed by the reducing agent. In all studies, the three changes of 20 mM veronal buffer (pH 7.8) for 3 h each, activity of the untreated samples was compared with the same and the pooled extracts were concentrated about 10-fold over a sample after inhibition and reactivation by a paired t-test PM-10 ultrafiltration membrane. The concentrated extract analysis. was applied to a benzamidine affinity chromatography To determine whether cancer procoagulant was inhibited by column, unbound protein was washed from the column with DFP or by