Gordon S G, Cross B A
J Clin Invest. 1981 Jun;67(6):1665-71. doi: 10.1172/jci110203.
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.
在人和动物肿瘤提取物以及培养的恶性细胞中已鉴定出一种蛋白水解促凝剂。它可直接激活因子X，但与其他激活因子X的丝氨酸蛋白酶的相似性尚不清楚。本研究描述了为确定这种酶，即癌促凝剂，是丝氨酸蛋白酶还是半胱氨酸蛋白酶所开展的工作。从兔V2癌中纯化的癌促凝剂与对氯汞苯甲酸琼脂糖亲和柱结合，并用二硫苏糖醇洗脱。癌促凝剂引发的重新钙化的枸橼酸盐血浆凝固活性受到5 mM二异丙基氟磷酸酯、1 mM苯甲基磺酰氟、0.1 mM HgCl2和1 mM碘乙酰胺的抑制。5 mM二硫苏糖醇可使二异丙基氟磷酸酯、苯甲基磺酰氟和HgCl2抑制的样品恢复活性；碘乙酰胺的抑制作用是不可逆的。作为对照的激活因子X的丝氨酸蛋白酶——罗素蝰蛇毒，不受0.1 mM HgCl2或1 mM碘乙酰胺的抑制。在两阶段试验中，二异丙基氟磷酸酯和碘乙酰胺抑制了癌促凝剂对因子X的直接激活。二异丙基氟磷酸酯抑制丝氨酸蛋白酶，大多数商业制剂中一种未明确的杂质抑制半胱氨酸蛋白酶。用硫酸铜和咪唑水解二异丙基氟磷酸酯几乎消除了对凝血酶的抑制，但对癌促凝剂的抑制仍然完全，这表明癌促凝剂是被这种未明确的杂质所抑制。这些结果表明癌促凝剂是一种半胱氨酸内肽酶，这使其有别于包括组织因子在内的其他凝血因子。这一结果及其他数据表明肿瘤细胞产生这种独特的半胱氨酸蛋白酶，它可能启动血液凝固。
