Figure 5 Bovine EP cleaves human PCI and complex formation is observed. Figure 6 Inhibition of bovine EP by PCI in the absence or presence of UFH and phospholipid vesicles. A dose-response curve for the effect of UFH on the inhibition of bEP by PCI was determined. Ivacaftor EC50 Increasing concentrations of UFH strongly reduced the inhibitory activity of PCI towards bEP (Figure 6B). The interfering effect of UFH on bEP inhibition by PCI was even stronger as compared with the inhibition of human recombinant EP. Interaction of Other Serpins with Human EP To investigate if other serpins also interact with EP, activity assays were performed in the presence of either A1AT or AT. After incubation of EP with A1AT for 60 min, no significant reduction of EP activity was observed, even at a molar [A1AT]:[EP] ratio of 10001 (not shown).

When EP was incubated with AT for the same time, there was no reduction in EP activity. However, AT slightly inhibited EP when either UFH or LMWH were present (Figure 7). Heparin alone had no effect on the activity of EP. Inhibitory activity of A1AT and AT was assured by experiments studying inhibition of trypsin by A1AT and of thrombin by AT, respectively. Figure 7 Inhibition of EP by antithrombin. Western Blotting of Pancreas Lysate Human pancreas lysate was applied to SDS-PAGE and Western blotting. Monoclonal anti-serpinA5 IgG was used to detect PCI. A band at about 57 kDa was seen in pancreas lysate, corresponding to the molecular mass of PCI purified from human plasma. Additionally, two faint bands at about 30 and 37 kDa (possible degradation products) were seen (not shown).

Discussion In this study, we can demonstrate that PCI is a fairly strong inhibitor of EP, with a kapp comparable to most protease-PCI interactions which range from 8.00��102 M?1 s?1 for APC inhibition in the absence of heparin to 5.60��107 M?1 s?1 for acrosin inhibition in the presence of heparin [9], [45]. It was the first time that an interaction of EP with a serpin-type inhibitor was shown. Additionally, it was also the first time that inhibition rate constants and the stoichiometry of inhibition were calculated for the interaction of a transmembrane serine protease with PCI. It has been shown previously that heparin and phospholipids are able to stimulate or to reduce the inhibitory activity of PCI towards several proteases [43], [46].

Glycosaminoglycans like heparin seem to regulate the inhibitory activity of PCI by binding to GSK-3 the target protease as well as to the serpin [13]. In case of PCI, this bridging mechanism is strongly protease-dependent and often leads to enhancement of protease inhibition [47]. Interestingly, the inhibition of plasma kallikrein by PCI is not stimulated by heparin [3], factor Xa inhibition shows only a slight stimulation [48], and the interaction of PCI with tissue kallikrein is completely abolished in the presence of glycosaminoglycans [6].