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Protease nexin-1 (PN-1) is a protease inhibitor that is secreted by fibroblasts and several other cultured cells. PN-1 forms complexes with certain serine proteases in the extracellular environment including thrombin, urokinase, and plasmin. The complexes then bind to the cells and are rapidly internalized and degraded. This report demonstrates that PN-1 is present on the surface of fibroblasts, bound to the extracellular matrix. Immunofluorescent studies showed that PN-1 colocalized with fibronectin on both intact cells and in preparations of extracellular matrix made from these cells. In contrast, PN-1 did not colocalize with the epidermal growth factor receptor, a plasma membrane marker. An enzyme-lined immunosorbent assay was developed which showed that the extracellular matrix contained at least 60-80% of the cellular immunoreactive PN-1. Extraction of the matrix with 2 M NaCl removed PN-1 in a form which reacted with 125I-thrombin to form complexes which were immunoprecipitated by anti-PN-1 IgG and were of identical size as complexes made from soluble PN-1 and 125I-thrombin. These data indicate that in addition to its role as a soluble protease inhibitor, PN-1 is also a component of the extracellular matrix and might control its proteolysis.

This review summarizes the mechanisms by which several serine proteases, particularly urokinase, thrombin, and elastase, interact with cultured fibroblasts. Many of these studies were prompted by findings that interactions of these proteases with cells and the extracellular matrix are important in a number of physiologic and pathologic processes. Two main pathways have been identified for specific interactions of these proteases with fibroblasts. One involves surface binding sites for the free protease that appear to bind only one particular protease. An unusual feature collectively shared by the binding sites for urokinase, thrombin, and elastase is that the bound protease is not detectably internalized by the fibroblasts. The other pathway by which serine proteases interact with fibroblasts involves proteins named protease nexins (PNs). Three PNs have been identified. They are secreted by fibroblasts and inhibit certain serine proteases by forming a covalent complex with the protease catalytic site serine. The complexes then bind back to the fibroblasts via the PN portion of the complex and are internalized and degraded. Recent studies showing that the fibroblast surface and extracellular matrix accelerate the inactivation of thrombin by PN-1 support the hypothesis that the PNs control protease activity at and near the cell surface. The PNs differ from plasma protease inhibitors in their molecular properties, absence in plasma, site of synthesis, and site of clearance of the inhibitor:protease complexes.

Protease nexin-1 (PN-1) is a proteinase inhibitor that is secreted by human fibroblasts in culture. PN-1 inhibits certain regulatory serine proteinases by forming a covalent complex with the catalytic-site serine residue; the complex then binds to the cell surface and is internalized and degraded. The fibroblast surface was recently shown to accelerate the rate of complex-formation between PN-1 and thrombin. The present paper demonstrates that the accelerative activity is primarily due to cell-surface heparan sulphate, with a much smaller contribution from chondroitin sulphate. This conclusion is supported by the effects of purified glycosaminoglycans on the second-order rate constant for the inhibition of thrombin by PN-1. Also, treatment of 35SO4(2-)-labelled cells with heparitin sulphate lyase or chondroitin sulphate ABC lyase demonstrated two discrete pools of 35S-labelled glycosaminoglycans; subsequent treatment of plasma membranes with these glycosidases showed that heparitin sulphate lyase treatment abolished about 80% of the accelerative activity and chondroitin sulphate ABC lyase removed the remaining 20%. These results show that two components are responsible for the acceleration of PN-1-thrombin complex-formation by human fibroblasts. Although dermatan sulphate is also present on fibroblasts, it did not accelerate the inhibition of thrombin by PN-1.

Protease nexin (PN) is a protein protease inhibitor secreted by human fibroblasts in culture that complexes and inhibits certain regulatory serine proteases. The PN-protease complexes then bind to these cells and are rapidly internalized and degraded. This report shows that the fibroblast surface accelerates the formation of PN-thrombin complexes. In contrast, it did not accelerate the formation of complexes between thrombin and antithrombin III, a closely related protease inhibitor found in plasma. These results support a role for PN in the regulation of certain proteases in the extravascular compartment at and near the surface of tissue cells. The activity that accelerated PN-thrombin complex formation was membrane-associated, since fixed cells, purified membranes, and extracellular matrix preparations all contained this activity. The ability of cells to accelerate the reaction between PN and thrombin was inhibited by protamine, suggesting that the activity was similar to that of heparin. Heparitinase digestion of plasma membranes prior to assay reduced the activity by about 80%, suggesting that heparan sulfate may account for most of the accelerative activity.