Two distinct urokinase-serpin interactions regulate the initiation of cell surface-associated plasminogen activation.

Single-chain urokinase-type plasminogen activator (scu-PA) possesses enzymatic activity that increases by 2-3 orders of magnitude upon binding to its cellular cofactor, the u-PA receptor (u-PAR), hence activating an enzymatic cascade initially composed of zymogens. The present study demonstrates that plasminogen activator inhibitor type 3 (PAI-3) reversibly inhibits scu-PA in solution, maintaining the system "off." Because the scu-PA/PAI-3 interaction is reversible, cellular expression of u-PAR allows partitioning of scu-PA from PAI-3 to u-PAR with resultant expression of full enzymatic activity. PAI-3 that was originally complexed to scu-PA remains in solution, retaining its functional activity. Importantly, the scu-PA on cell surface u-PAR is protected from PAI-3 inhibition, remaining an effective activator in a PAI-rich environment. Plasmin formed as a result of scu-PA activity then cleaves scu-PA to the mature protease, two-chain u-PA (tcu-PA), which is efficiently and irreversibly inhibited by PAI-3 via the standard serpin mechanism, even on u-PAR. This data generates a new hypothesis which, in contrast to the previous paradigm, holds that receptor bound scu-PA is the initiating enzyme and that tcu-PA is generated not to augment enzymatic activity but rather to allow for inhibition and therefore appropriate regulation of the process.