Protease nexin I is a 43-50 kDa glycoprotein capable of inhibiting a number of serine proteases. In cultured differentiated human skeletal muscle (myotubes), we previously found that protease nexin I was localized in patches at their surface where it was active and able to inhibit thrombin. To understand the role of skeletal muscle protease nexin I after injury or in inflammatory conditions where thrombin might be extravasated by blood vessels, we examined the role of inflammatory factors on protease nexin I synthesis and secretion by myotubes in culture. By enzyme-linked immunosorbent assay (ELISA) and Western blotting, we found that this serine protease inhibitor is secreted by cultured human myotubes. Protease nexin I secretion is stimulated by tumor necrosis factor-alpha, transforming growth factor-beta and interleukin-1. Complex formation experiments with labeled thrombin reveal active protease nexin I bound to the surface of the treated cells. Secreted protease nexin I-thrombin complex was enhanced in the presence of transforming growth factor-beta and tumor necrosis factor-alpha. Protease nexin I mRNA was detected by reverse transcription-polymerase chain reaction (RT-PCR) and Northern blot analysis. Whatever the conditions, no significantly different levels were observed, indicating that the changes in cell and media protease nexin I concentration are elicited at the translational/posttranslational levels. Immunocytochemical studies on human skeletal muscle biopsies of patients suffering from inflammatory myopathies showed an overexpression of protease nexin I together with the above inflammatory factors. These findings suggest that skeletal muscle protease nexin I might play a role after injury or inflammatory pathologies.