Regulation of protein C inhibitor (PCI) activity by specific oxidized and negatively charged phospholipids

Abstract

Protein C inhibitor (PCI) is a serpin with affinity for heparin and phosphatidylethanolamine (PE). We analyzed the interaction of PCI with different phospholipids and their oxidized forms. PCI bound to oxidized PE (OxPE), and oxidized and unoxidized phosphatidylserine (PS) immobilized on microtiter plates and in aqueous suspension. Binding to OxPE and PS was competed by heparin, but not by the aminophospholipid-binding protein annexin V or the PCI-binding lipid retinoic acid. PS and OxPE stimulated the inhibition of activated protein C (aPC) by PCI in a Ca ؉؉ -dependent manner, indicating that binding of both, aPC (Ca ؉؉ dependent) and PCI (Ca ؉؉ independent), to phospholipids is necessary. A peptide corresponding to the heparin-binding site of PCI abolished the stimulatory effect of PS on aPC inhibition. No stimulatory effect of phospholipids on aPC inhibition was seen with a PCI mutant lacking the heparinbinding site. A heparin-like effect of phospholipids (OxPE) was not seen with antithrombin III, another heparin-binding serpin, suggesting that it is specific for PCI. PCI and annexin V were found to be endogenously colocalized in atherosclerotic plaques, supporting the hypothesis that exposure of oxidized PE and/or PS may be important for the local regulation of PCI activity in vivo. IntroductionProtein C inhibitor (PCI) is a member of the serpin (serine protease inhibitor) family of protease inhibitors. It belongs to the subgroup of alpha-1-antitrypsin-like serpins (clade A) and its gene symbol is SERPINA5. 1 PCI, originally described in plasma as an inhibitor of the anticoagulant serine protease activated protein C (aPC), 2,3 inactivates many other serine proteases including blood coagulation factors, 4,5 fibrinolytic enzymes, 4,6 tissue kallikrein, 7 and the sperm protease acrosin. 8,9 Human PCI is expressed in many organs and tissues [10][11][12][13] and is present in many body fluids and secretions. 11 Complexes of PCI with proteases are present in body fluids, 6,14 indicating that PCI interacts with the respective proteases not only in vitro but also in vivo.Human PCI demonstrates modest efficiency as a protease inhibitor, and for some of its target proteases more efficient inhibitors have been described. 15,16 However, its specific environment may change the activity and target enzyme specificity of PCI in vivo. PCI is a heparin-binding serpin, [17][18][19][20][21] and heparin can stimulate the interactions of PCI with many of its target proteases. In vivo heparin is present mainly intracellularly, 22 while on the cell surface membrane-associated heparin sulfate-containing proteoglycans can substitute for heparin. 18 In contrast to their activating action on aPC inhibition by PCI, glycosaminoglycans interfere with the inhibition of tissue kallikrein by PCI. 7,23 Thus, local factors such as glycosaminoglycans and proteoglycans could play an important role in regulating both the activity of PCI and its selectivity with respect to different target proteases. 23 It has been shown by Nishioka et al 24 that...

Circulating erythrocyte-derived microparticles are associated with coagulation activation in sickle cell disease

“…Recently, it was shown that oxidized and unoxidized phospholipids have different effects on inhibition of coagulation. 25 In our thrombin generation experiments, we observed an almost 50% reduction in thrombin generation by anti-human factor XI. Factor XI plays an important role in enhancing thrombin generation, since trace amounts of thrombin can activate factor XI to factor XIa, which then augments thrombin generation via the tenase complex.…”

N-Glycans and the N Terminus of Protein C Inhibitor Affect the Cofactor-enhanced Rates of Thrombin Inhibition

“…A potential pH dependence may be important for the role of PCI at inflammatory sites, if PCI is targeted to sites of inflammation, as indicated by the presence of sialyl-Le a/x epitopes and by the functions of PCI in e.g. atherosclerosis and wound healing (31,32). Future investigations are required to investigate whether these findings reflect different functions of the two forms in vivo.…”

Alboserpin, a Factor Xa Inhibitor from the Mosquito Vector of Yellow Fever, Binds Heparin and Membrane Phospholipids and Exhibits Antithrombotic Activity

“…Studies have also demonstrated that PE enhances the PS-mediated sensitivity of factor VIIa-tissue factor activity (53) and induces high affinity binding sites for factor VIII on surfaces containing PS (54). Moreover, PE has been reported in tumor cell membranes (55) and is known to regulate the activation of blood coagulation by enhancing APC inhibition by PCI (50,51). It is possible that occupation of these sites by Alboserpin interferes with the inhibitory function of PCI toward APC, which displays particularly important anticoagulant and anti-inflammatory activities in vivo (7,56).…”

“…However, PCI was later demonstrated to interact with oxidized PE or oxidized PS but not with PC (47,51). Therefore, binding of Alboserpin to phospholipids vesicles in solution was investigated.…”