Two distinct urokinase-serpin interactions regulate the initiation of cellsurface-associated plasminogen activation

Single-chain urokinase-type plasminogen activator (scu-PA) possesses enzyma tic 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 tha t plasminogen activator inhibitor type 3 (PAI-3) reversibly inhibits scu-PA in solution, maintaining the system "off." Because the scu-PA/PAT-3 intera ction is reversible, cellular expression of u-PAR allows partitioning of sc u-PA from PAI-3 to u-PAR with resultant expression of full enzymatic activi ty. PAI-3 that was originally complexed to scu-PA remains in solution, reta ining its functional activity. Importantly, the scu-PA on cell surface u-PA R 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 c leaves scu-PA to the mature protease, two-chain u-PA (tcu-PA), which is eff iciently and irreversibly inhibited by PAI-3 via the standard serpin mechan ism, 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 initiatin g 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.