INHIBITION OF SERINE PROTEASES BY REACTIVE-SITE MUTANTS OF PROTEIN-C INHIBITOR (PLASMINOGEN-ACTIVATOR INHIBITOR-3)

Protein C inhibitor (PCI), also known as plasminogen activator inhibit or-3, is a heparin-dependent serine protease inhibitor. Heparin and ot her sulphated polysaccharides act as template, thereby increasing the rate of inhibition by PCI. PCI can inhibit various serine proteases in blood coagulation, fibrinolysis and reproduction. However, its physio logical target enzyme is still unknown. Reactive site mutants of PCI w ere used to elucidate its target specifity, which may provide clues to wards its function in vivo. We have studied the importance of the regi on P3-P3' of PCI in reactivity towards acrosin, kallikrein, factor XIa and urokinase. The kinetic analysis was performed using both pseudo f irst-order kinetics and slow-binding kinetics. The results demonstrate d the importance of the P3-P3' region and in particular the P2 and P3 residues of PCI in protease recognition. The data showed the preferenc e of a hydrophobic residue at P2 for the inhibition of kallikrein and acrosin, whereas alanine at P2 benefits the inhibition of urokinase in hibition. In addition to the preference for a hydrophobic P2 residue f or serpin reactivity towards kallikrein and acrosin, it was also shown that a positively charged residue at P3 also benefits serpin reactivi ty towards these proteases. The results further support the general id ea that reactive site residues are important for the specifity of the serpin, but that these are not solely responsible for serpin specifici ty. We observed that heparin affected the specific activity of acrosin but not of kallikrein. In addition, acrosin inhibition by PCI was str ongly enhanced by heparin, whereas PCI reactivity towards kallikrein w as unaffected by this glycosaminoglycan. Since heparin acts as templat e, the mechanism of stimulation of PCI reactivity by heparin is most l ikely the result of bringing both protease and PCI in close proximity and not by inducing major changes in the conformation affecting the re active site loop. Therefore, the binding of PCI to the heparin templat e affects the orientation of the reactive site towards the active site of the protease and this suggests that the heparin-binding domains of PCI are also determinants in target specifity of PCI.