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1. Cleavage of the human antithrombin III--thrombin complex with [14C]methoxyamine hydrochloride results in inactive thrombin and 14C-labelled antithrombin III. 2. Discontinuous polyacrylamide-gel electrophoresis of the reduced dissociation fragments of the complex in the presence of sodium dodecyl sulphate reveals two antithrombin III bands that do not resolve during electrophoresis without reduction. The heavy band has the electrophoretic mobility of the native protein. The light band has an apparent mol.wt. that is approx. 4000 less than the molecular weight of native antithrombin III. 3. Treatment of the cleavage products of the complex with carboxypeptidase B yields 1 mumol of arginine, a new C-terminal amino acid, per mumol of thrombin dissociated. The results indicate that during formation of the antithrombin III--thrombin complex, the inhibitor is cleaved at an arginine--X bond; this arginine residue forms a carboxylic ester with the enzyme, while the excised polypeptide remains bound through a disulphide bridge(s).

ABSTRACT. al-Antichymotrypsin (al-ACHY) and al-an-(I). al-AT and aI-ACHY play major roles in the host response titrypsin (al-AT) are closely related glycoprotein protease to trauma and inflammation. The major function of aI-AT, inhibitors, present in plasma and other extracellular fluids, which is present at high levels in human plasma (1 5 3 . 5 mg/ that neutralize proteases released by leukocytes in re-mL), is to inhibit elastases released by leukocytes at sites of sponse to trauma and inflammatory stimuli. Both inhibitors inflammation. al-ACHY, present in plasma at lower levels (0.3-are synthesized primarily by hepatocytes, although lower 0.6 mg/mL), is thought to neutralize ch~motry~sin-like Proteases levels of synthesis by monocytes and breast and intestinal S U C~ aS cathepsin G also released by leukocytes at inflammatory epithelial cells have been demonstrated. Recently, the im-sites. a,-ACHY and (UI-AT belong to the serpin supergene family m u n o~i s t o c~e m i c a~ localization of a l -~~ and aI-~~-~~ in as does CBG, the major glucocorticoid camer in the circulation intrauterine and extrauterine human trophoblastic tissue (2). CBG is cleaved by elastase at a unique site in its C-terminal has been reported.the present study, we have sought to region resulting in decreased glucocorticoid binding (3). Because determine whether human trophoblast is also able to syn-local elastase levels depend to a large degree on local levels of althesize al-AT andMessenger RNA for both AT, delivery of glucocorticoids to inflammatory sites may be inhibitors was found by Northern blotting in chorionic villi regulated by a mechanism involving the interaction of elastase and aI-AT. obtained from first trimester and term placenta. SubstanaI-AT and are synthesized primarily by hepatocytes tial differences in messenger levels for both inhibitors and to a lesser degree by monocytes (4). We have shown that among individual placentas were noted' al-ACHY and al-MCF-7 human breast cancer cells, a cell line of epithelial AT messenger was present in trophoblast in can also synthesize these protease inhibitors and that their synprimary culture. Synthesis of al-AT and al-ACHY protein thesis can be stimulated by steroid hormones, cytokines, and was demonstrated by SDS-PAGE after immuno~reci~ita-growth factors (5). ~l t h o u g h the amount of al-AT and aI-ACHY tion of 135S1-labeled ~I -A T and a]-ACHY from conditioned produced by nonhepatic cells is small in comparison to that media of trophoblast cells in culture metabolically labeled by hepatocytes, local synthesis may be important at with [35S]-methionine. It is of some interest that the M, of sites of inflammation, particularly at locations not directly in the al-AT and ffl-ACHY secreted by trophob1ast contact with the circulation where levels of al-AT and aI-ACHY 50 000 and 49 000, respectively, compared with 54 000 and may only be a fraction of that in plasma (6). Implantation of the 6 8 000 for these proteins in plasma (or secreted by H~P G~ embryo in the uterus elicits an inflam...