Differential inhibition of soluble and cell surface receptor-bound single-chain urokinase by plasminogen activator inhibitor type 2. A potential regulatory mechanism.

Urokinase (u-PA)-mediated cell surface plasminogen activation is required for cellular tissue invasion. This invasion occurs in environments rich in plasminogen activator inhibitors (PAIs), which efficiently inhibit receptor-bound two-chain u-PA. Single-chain u-PA (scu-PA) was recently found to efficiently initiate cell surface plasminogen activation, and we herein describe the interaction of scu-PA with PAI type 2 (PAI-2). In the fluid phase (no cells) the plasminogen-activating activities of both scu-PA and Glu158-scu-PA (a plasmin non-activatable variant of scu-PA) were inhibited in a concentration-dependent manner by recombinant human PAI-2. This inhibition occurred with both forms of scu-PA remaining as single-chain molecules throughout the interactions. Although scu-PA did not form SDS-stable complexes with PAI-2, preincubation of scu-PA with 125I-PAI-2 demonstrated a dose-dependent inhibition of SDS-stable complex formation between 125I-PAI-2 and subsequently added two-chain u-PA. This indicates that although a "stable intermediate" type complex between scu-PA and PAI-2 was not detected, there was a physical association between the two molecules that shared at least some determinants with the two-chain u-PA-PAI-2 complex. In contrast, Glu158-scu-PA bound to u-PA receptors on monocytes was only minimally inhibited by a large molar excess of PAI-2. These data suggest that the initiation of cell surface plasminogen activation may involve the partitioning of scu-PA between PAI-2 (a "negative modulator") and the u-PA receptor (a "positive modulator") and that the enzymatic activity of receptor-bound scu-PA may allow initiation of cell surface proteolysis even in PAI-2-rich environments. A model along these lines is presented.