Phospholipids and protein C inhibitor

Protein C inhibitor (PCI) inhibits serine proteases by forming stable, enzymatically inactive 1:1 complexes. Human PCI is expressed in many tissues and is present in many body fluids and secretions. It has initially been described as an inhibitor of the anticoagulant protease activated protein C (APC). However, in the meantime it has been shown that PCI has a very broad specificity. PCI is a heparin-binding serpin, and - depending on the protease - heparin can stimulate or abolish its inhibitory activity and thereby also regulate its specificity. In a previous project (P16093-B04) using purified synthetic phospholipids, we have shown that PCI binds to anionic/oxidized phospholipids (i.e. to unoxidized and oxidized phosphatidylserine and to oxidized phosphatidylethanolamine) and that these phospholipids have a similar effect on PCI activity as heparin. Binding to these phospholipids seems to involve the heparin-binding site of PCI. In this project we want to analyze the physiological relevance of the interaction of PCI with (phospho)lipids. Phosphatidylserine and phosphatidylethanolamine are localized in the inner leaflet of the plasma membrane and therefore not accessible for PCI. However, in certain situations such as apoptosis they become surface exposed. Oxidation of phospholipids takes place mainly at sites of inflammation. These phospholipids could therefore function as "inducible" regulators of PCI activity. It is our aim to determine if and which lipids are endogenously bound to plasma and urinary PCI. Furthermore we want to determine if membrane vesicles (microparticles) present in plasma contain PCI, which could contribute to their thrombogenic potential. We also want to determine if phospholipid vesicles derived from cell membranes have a similar effect on PCI activity as pure, synthetic phospholipids. Cell surface exposed phosphatidylserine is a major signal for clearance of apoptotic cells by phagocytosis. We therefore want to analyze the effect of PCI on phagocytosis of apoptotic, phosphatidylserine exposing cells. We will especially study the phagocytosis of murine male germ cells by Sertoli cells, since male PCI-knockout mice are infertile and many apoptotic cells can be detected in their testes, suggesting impaired phagocytosis in the absence of PCI. Results of these studies should allow us to define the biological relevance of the PCI-phospholipid interaction.

Protein C inhibitor (PCI) inhibits serine proteases by forming stable, enzymatically inactive 1:1 complexes. Human PCI is expressed in many tissues and is present in many body fluids and secretions. It has initially been described as an inhibitor of the anticoagulant protease activated protein C (APC). However, in the meantime it has been shown that PCI has a very broad specificity. PCI is a heparin-binding serpin, and - depending on the protease - heparin can stimulate or abolish its inhibitory activity and thereby also regulate its specificity. In a previous project (P16093-B04) using purified synthetic phospholipids, we have shown that PCI binds to anionic/oxidized phospholipids (i.e. to unoxidized and oxidized phosphatidylserine and to oxidized phosphatidylethanolamine) and that these phospholipids have a similar effect on PCI activity as heparin. Binding to these phospholipids seems to involve the heparin-binding site of PCI. In this project we want to analyze the physiological relevance of the interaction of PCI with (phospho)lipids. Phosphatidylserine and phosphatidylethanolamine are localized in the inner leaflet of the plasma membrane and therefore not accessible for PCI. However, in certain situations such as apoptosis they become surface exposed. Oxidation of phospholipids takes place mainly at sites of inflammation. These phospholipids could therefore function as "inducible" regulators of PCI activity. It is our aim to determine if and which lipids are endogenously bound to plasma and urinary PCI. Furthermore we want to determine if membrane vesicles (microparticles) present in plasma contain PCI, which could contribute to their thrombogenic potential. We also want to determine if phospholipid vesicles derived from cell membranes have a similar effect on PCI activity as pure, synthetic phospholipids. Cell surface exposed phosphatidylserine is a major signal for clearance of apoptotic cells by phagocytosis. We therefore want to analyze the effect of PCI on phagocytosis of apoptotic, phosphatidylserine exposing cells. We will especially study the phagocytosis of murine male germ cells by Sertoli cells, since male PCI-knockout mice are infertile and many apoptotic cells can be detected in their testes, suggesting impaired phagocytosis in the absence of PCI. Results of these studies should allow us to define the biological relevance of the PCI-phospholipid interaction.