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 inhibitor-3, is a heparin-dependent serine protease inhibitor. Heparin and other 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 physiological target enzyme is still unknown. Reactive site mutants of PCI were used to elucidate its target specifity, which may provide clues towards its function in vivo. We have studied the importance of the region P3-P3' of PCI in reactivity towards acrosin, kallikrein, factor XIa and urokinase. The kinetic analysis was performed using both pseudo first-order kinetics and slow-binding kinetics. The results demonstrated the importance of the P3-P3' region and in particular the P2 and P3 residues of PCI in protease recognition. The data showed the preference of a hydrophobic residue at P2 for the inhibition of kallikrein and acrosin, whereas alanine at P2 benefits the inhibition of urokinase inhibition. In addition to the preference for a hydrophobic P2 residue for serpin reactivity towards kallikrein and acrosin, it was also shown that a positively charged residue at P3 also benefits serpin reactivity towards these proteases. The results further support the general idea that reactive site residues are important for the specifity of the serpin, but that these are not solely responsible for serpin specificity. We observed that heparin affected the specific activity of acrosin but not of kallikrein. In addition, acrosin inhibition by PCI was strongly enhanced by heparin, whereas PCI reactivity towards kallikrein was unaffected by this glycosaminoglycan. Since heparin acts as template, the mechanism of stimulation of PCI reactivity by heparin is most likely the result of bringing both protease and PCI in close proximity and not by inducing major changes in the conformation affecting the reactive site loop. Therefore, the binding of PCI to the heparin template 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.