INHIBITORS OF UROKINASE AND THROMBIN IN CULTURED NEURAL CELLS

Recent studies have suggested important roles for certain proteases and protease inhibitors in the growth and development of the CNS. In the present studies, inhibitors of urokinase or thrombin in cultured neural cells and serum-free medium from the cells were identified by screening for components that formed sodium dodecyl sulfate-stable complexes with I-125-urokinase or I-125-thrombin. Rinsed glioblastoma cells possessed two components that complexed I-125-urokinase. One was type 1 plasminogen activator inhibitor (PAI-1), because the I-125-urokinase-containing complexes were immunoprecipitated with anti-PAI-1 antibodies. The other component formed complexes with I-125-urokinase that were not recognized by antibodies to PAI-1 or protease nexin-1 (PN-1). Its identity is unknown. In addition to these cell-bound components, the glioblastoma cells also secreted two inhibitors that formed complexes with I-125-urokinase; one was PAI-1, and the other was PN-1, and the other was PN-1. The secreted PN-1 also formed complexes with I-125-thrombin. It was the only thrombin inhibitor detected in these studies. Human neuroblastoma cells did not contain components that formed detectable complexes did not contain components that formed detectable complexes with either I-125-urokinase or I-125-thrombin. However, human neuroblastoma cells did contain very low levels of PN-1 mRNA and PN-1 protein. Added PN-1 bound to the surface of both glioblastoma and neuroblastoma cells. This interaction accelerated the inhibition of thrombin by PN-1 and blocked the ability of PN-1 to form complexes with I-125-urokinase. Thus, cell-bound PN-1 was a specific thrombin inhibitor. Together, these studies show that urokinase and thrombin inhibition is mediated primarily by nonneuronal cells of the CNS, although neuroblastoma cells can regulate both the activity and target protease specificity of PN-1.