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...
The serpin superfamily includes inhibitors of serine proteases and noninhibitory members with other functions (e.g. the hormone precursor angiotensinogen and the hormone carriers corticosteroid‐binding globulin and thyroxine‐binding globulin). It is not known whether inhibitory serpins have additional, noninhibitory functions. We studied binding of 3H‐labeled hydrophobic hormones (estradiol, progesterone, testosterone, cortisol, aldosterone, and all‐trans‐retinoic acid) to the inhibitory serpins antithrombin III, heparin cofactor II, plasminogen activator inhibitor‐1, and protein C inhibitor (PCI). All‐trans‐[3H]retinoic acid bound in a specific dose‐dependent and time‐dependent way to PCI (apparent Kd = 2.43 µm, 0.8 binding sites per molecule of PCI). We did not observe binding of other hormones to serpins. Intact and protease‐cleaved PCI bound retinoic acid equally well, and retinoic acid did not influence inhibition of tissue kallikrein by PCI. Gel filtration confirmed binding of retinoic acid to PCI in purified systems and suggested that PCI may also function as a retinoic acid‐binding protein in seminal plasma. Therefore, our present data, together with the fact that PCI is abundantly expressed in tissues requiring retinoic acid for differentiation processes (e.g. the male reproductive tract, epithelia in various organs), suggest an additional biological role for PCI as a retinoic acid‐binding and/or delivering serpin.
The types of kinins released from purified native, single chain human high and low molecular mass kininogens (HMMKs and LMMKs, respectively) by purified human urinary kallikrein were separated by reverse-phase HPLC and quantitated by the rat uterus bioassay.[Hy@]-lysyl-bradykinin, a recently discovered kinin, represented up to 58% of the biological activity released from 4 individual HMMK preparations purified from 4 different healthy volunteers. In contrast, the majority of the biological activity released from LMMKs purified from pooled plasma was identified as Lys-bradykinin and [Hy@]-lysyl-bradykinin represented only 6.4 f 3.8%. These findings indicate posttranslational hydroxylation of human kininogens and suggest a preference of HMMKs for this modification.
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