Human protease nexin-I. Further characterization using a highly specific polyclonal antibody.

We have used purified protease nexin-I (PN-I) from human fibroblasts to develop a polyclonal antibody that specifically blocks the PN-I-mediated cellular binding of thrombin and urokinase.Anti-PN-I IgG did not inhibit the binding of l2'1-epidermal growth factorbinding protein to fibroblasts, which is mediated by protease nexin-11, another cell-secreted, serine protease inhibitor that is distinct from PN-I.This furthers the belief that the protease nexins are distinct from one another.In addition, while anti-PN-I IgG immunoprecipitated PN-Iathrombin complexes, it did not do so with antithrombin-III*thrombin. Metabolically labeled PN-I was also immunoprecipitated by IgG, indicating that the protein can be labeled in vivo.The antibody also recognized primarily one band on Western transfers of conditioned medium from fibroblast cultures.These results suggest that anti-PN-I will be useful in probing the physiological role of PN-I as well as its biosynthesis.Human fibroblasts in culture secrete three known protease inhibitors referred to as protease nexins (1).PN-I' binds thrombin and urokinase (2), whereas PN-I1 binds EGF-BP (3) and PN-I11 binds y-nerve growth factor (4). Protease nexins are similar to plasma protease inhibitors, such as AT-111, al-antitrypsin, and C1 inactivator.Inhibition of the protease occurs when it binds to the inhibitor, cleaves a portion of the inhibitor, and remains covalently attached through an acyl linkage (2).The complexes formed between plasma protease inhibitors and active proteases are cleared from the circulation, presumably by binding to receptors found in liver (5).Protease nexins are found in nonvascular areas and are produced and taken up by various cells, including those derived from lung, kidney, embryo, and connective tissue (2,6).Like plasma protease inhibitors, they are cleared from their