Thrombin‐mediated mitogenesis: The role of secreted protease nexin

Protease nexin (PN) is a cell‐secreted protein that links to thrombin (Th) and certain other serine proteases. PN mediates the binding, internalization, and degradation of these proteases by cells (Baker et al., 1980; Low et al., 1981). Here we show that binding of Th‐PN complexes to human foreskin fibroblasts (HF cells) accounted for 90% of the specific cellular Th binding at certain mitogenic doses of the protease. However, cell‐associated Th‐PN complexes were likely to be inactive mitogenically because heparin (170 units/ml) inhibited cellular binding of 125‐Th‐PN by about 95% (a reduction from 1.3 × 105 to 6 × 103 125I‐Th‐PN complexes per cell) but did not influence Th‐mediated mitogenic stimulation. In experiments with mouse embryo cells, heparin also markedly decreased cellular binding of 125I‐Th‐PN without changing the mitogenic response to Th. The lack of mitogenic activity of cell‐associated Th‐PN complexes suggested that PN might inhibit the mitogenically essential proteolytic activity of Th. This possibility is supported by the following findings. First, amounts of serum‐free conditioned culture medium that contained enough PN to complex a large fraction of added Th inhibited the clotting activity of Th. Second, heparin increased the formation of 125I‐Th‐PN complexes and also increased this inhibitory effect of conditioned medium. We conclude that PN acts as a negative modulator of thrombin mitogenic activity. It is shown that like other fibroblastic cells HF cells bound free 125I‐Th specifically (although with relatively low affinity, Kass < 108 M‐1). Specific binding of free 125I‐Th to HF cells increased fourfold in the presence of heparin (50 IU/ml).