PCI (SERPINA5, PAI3) is a non-specific, secreted serine protease inhibitor (serpin) with affinity for heparin (review: Geiger 2007, Suzuki 2008). First described as an inhibitor of activated protein C (aPC) an anticoagulant serine protease in human plasma, it has been shown that PCI inactivates a variety of other proteases and has a wide tissue distribution.

As PCI can be internalized by cells and translocated to the nucleus, the PCI sequence was inspected for sequences homologous to the nuclear localizing sequence (NLS). 2 homologous sequences could be identified, located on the A⁺ and on the H-helix. Further investigations showed that the NLS homologous sequence localized in the H-Helix could be functional.

Phosphatidylserine (PS), oxidized PS (OxPS) or oxidized phosphatidylethanolamine (OxPE) interact with PCI and stimulate its activity towards aPC in a heparin-like manner (Malleier et al., 2007; Nishioka et al., 1998). The head group of PE fits into the helix A gap and the acyl chains in the hydrophobic D' and H' channels of PCI. In addition, the internalization of PCI by cells is supported by PE, and internalized PCI promotes phagocytosis of bacteria (Baumgärtner et al. 2007).

The interaction of PCI with various glycerophospholipids in particular phosphoinositides and derived second messengers was analyzed, focusing on differences in fatty acid composition and headgroup phosphorylation.

Binding was studied by protein overlay assays (dot blot analysis) and native PAGE. Stimulation of PCI activity was analyzed by modulation of aPC-inhibition.

Inositol-1,4,5-trisphosphate and inositol-1,3,4,5-tetrakisphosphate did not interact with PCI. Mono- and diacylglycerols showed interaction in dot blot analysis but no stimulatory activity on aPC-inhibition. Among the different phosphatidic-acids (PAs), oxidized PA exhibited the lowest binding to PCI, but highest stimulatory activity, while saturated or lyso-PA did not stimulate aPC inhibition. From all studied lipids phosphatidylglycerol (PG) had the strongest stimulatory effect on aPC-inhibition. Oxidation of PG led to a decrease and saturation to a complete loss in stimulatory activity. Cardiolipin, PG and oxidized PA showed the highest stimulatory effect on aPC inhibition, similar to heparin.

Among the PI-monophosphates, PI(5)P showed the strongest binding to PCI. A mobility shift of PCI antigen was observed when PCI was incubated with phosphatidylinositol-3,5-diphosphate and phosphatidylinositol-4,5-diphosphate (PI(4,5)P₂).

Therefore we conclude that phosphoinositides and other glycerophospholipids may function as additional intracellular interaction partners of PCI.