Activation of trypsinogen is an obligatory step in the pathogenesis of acute necrotizing pancreatitis. So far, it is not fully understood how trypsinogen is activated prematurely in vivo. This function might be executed intracellularly by cathepsin B. Some authors also suggest a role of EP by reflux of duodenal fluid into the pancreatic duct. However, this theory remains controversial. Outside the digestive system, EP and its substrate trypsinogen are present in keratinocytes during their terminal differentiation and might be involved in the regulation of desquamation. They are also expressed by oral squamous cell carcinoma and prostate cancer cell lines. Active trypsin can increase tumor cell invasiveness. Regulation of EP by protease inhibitors may therefore be not only important in the digestive system, but also in epidermal differentiation and tumor invasion. 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 PD325901 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 GDC-0879 Raf inhibitor inhibitory activity of PCI towards several proteases. Glycosaminoglycans like heparin seem to regulate the inhibitory activity of PCI by binding to the target protease as well as to the serpin. In case of PCI, this bridging mechanism is strongly protease-dependent and often leads to enhancement of protease inhibition. Interestingly, the inhibition of plasma kallikrein by PCI is not stimulated by heparin, factor Xa inhibition shows only a slight stimulation, and the interaction of PCI with tissue kallikrein is completely abolished in the presence of glycosaminoglycans. Heparin slightly reduced the inhibition of recombinant human EP by PCI and this effect was even more pronounced using bovine EP purified from calf intestine. This could be explained by the fact that the recombinant EP carries a positively charged His-tag at the Cterminus which might counteract the repulsive effect of the negatively charged heparin. AT, on the other hand, inhibited EP only when heparin was present. So, heparin stimulated the inhibition of EP by AT, but reduced the inhibition of EP by PCI. To our knowledge, this is the first demonstration that heparin led to a reduced inhibition of a particular protease by PCI, but an increased inhibition by AT. This may be due to differences in regulation of serpin activity by heparin, as AT undergoes a conformational change when bound to glycosaminoglycans compared to the bridging mechanism of PCI. Furthermore the different heparin-binding sites of PCI and AT may also contribute to this opposed effect. As mentioned above, native EP is a type II transmembrane serine protease. It contains an N-terminal hydrophobic segment from position 18 to 44, predicted to span the membrane. The recombinant EP used is a mixture of two forms, in which the heavy chain is truncated and starts either at Leu41 or Ser118. Nterminal sequence analysis by Edman degradation revealed that also the bEK contains a mixture of two heavy chains starting at Gly53 and Ser118 respectively. Phospholipids did not influence EP inhibition by PCI. Assuming a heparinlike bridging mechanism for the stimulatory effect of phospholipids on PCI-protease interactions, these results are not surprising, since it has been shown previously that a truncated EP lacking the transmembrane domain does not interact with phospholipid vesicles. Supporting this data, a commercially available protein-lipid overlay assay containing membrane phospholipids was performed.