Characterization of the receptor for protease nexin-I: Protease complexes on human fibroblasts

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Abstract
Fibroblasts as well as several other cell types, secrete a number of protease inhibitors into their culture media. Among these inhibitors are the protease nexins, a class of proteins which covalently bind serine proteases, thereby inactivating their specific targets. Protease nexin-l, first discovered in human foreskin fibroblasts, binds thrombin, plasmin, and urokinase with high affinity, forming covalently linked complexes. Human fibroblasts bind complexes of protease nexin-l and its target protease via a cell-surface, high-affinity receptor. We have analyzed a number of characteristics of this receptor, and found them to be typical of class II receptors in general. At 4°C binding of PN-l:protease complexes was competable by heparin. In addition, binding was independent of the particular protease bound to the PN-l; purified complexes of PN-l with thrombin or urokinase competed equipotently for [125]l-thrombin:PN-l binding. As the pH of the binding buffer was lowered, binding to cells increased. A twofold increase in binding was attained by lowering the pH from 7.5 to 4.5. This phenomenon was not due to irreversible, pH-induced changes to either the cell surface or the labeled complexes. At 37°C, the removal of labeled complexes from culture medium was rapid; approximately 80% was removed by 4 hours under given conditions. The internalization of complexes was also very rapid, with an estimated ke (endocytic rate cor stant) of 1.0 min−1 At neutral pH, fibroblasts bind complexes in a saturable manner. Scatchard analysis yields areceptor number of 250,000 per cell and a Kd of 1 nM.