Characterization of recombinant human protein C inhibitor expressed in Escherichia coli

Réhault, Sophie; Zechmeister-Machhart, Margareta; Fortenberry, Yolanda M.; Malleier, Julia M.; Binz, Nikki M.; Cooper, Scott; Geiger, Margarethe; Church, Frank C.

doi:10.1016/j.bbapap.2004.12.003

Cited by 22 publications

(17 citation statements)

References 64 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Second-order rate constants were calculated from 3 independent experiments performed in duplicates. 7,18,31 Control experiments verified that peptide AA264-283 influenced neither the amidolytic activity of aPC nor the inhibitory activity of PCI in the absence of heparin and phospholipids. Also, phospholipids did not significantly affect the amidolytic activity of aPC.…”

Section: Inhibition Of Apc By Pcimentioning

confidence: 96%

“…31 Deletion of bases 962 to 1004 32 of the cDNA of human PCI coding for amino acids 264-277 (H-helix) was done according to the QuickChange Site-directed Mutagenesis Kit procedure (Stratagene, Amsterdam, the Netherlands), using the following primers respectively: 5Ј-GTG GAG AAT GGA CTG AGG CAG CTC GAG CTT-3Ј (forward) and 5Ј-AAG CTC GAG CTG CCT CAG TCC ATT CTC CAC-3Ј (reverse). The plasmids were purified and sequenced using ABI PRISM Big Dye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems, Foster City, CA) on a 310 Genetic Analyzer from Perkin Elmer (Wellesley, MA) to verify the deletion in the mutant construct.…”

Section: Mutagenesis Of Pcimentioning

confidence: 99%

“…The plasmids were purified and sequenced using ABI PRISM Big Dye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems, Foster City, CA) on a 310 Genetic Analyzer from Perkin Elmer (Wellesley, MA) to verify the deletion in the mutant construct. The expression and purification of the mutated protein (⌬H-PCI) was done according to the protocol described 31 except that the heparin-Sepharose step was omitted.…”

Section: Mutagenesis Of Pcimentioning

confidence: 99%

See 2 more Smart Citations

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

Malleier

Oskolkova

Bochkov

et al. 2007

Blood

Self Cite

View full text Add to dashboard Cite

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...

show abstract

Section: Inhibition Of Apc By Pcimentioning

confidence: 96%

Section: Mutagenesis Of Pcimentioning

confidence: 99%

Section: Mutagenesis Of Pcimentioning

confidence: 99%

See 1 more Smart Citation

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

Malleier

Oskolkova

Bochkov

et al. 2007

Blood

Self Cite

View full text Add to dashboard Cite

show abstract

“…Heparin binding increases the rate of APC inhibition by PCI about 400 fold [10] and thrombin inhibition 30 fold [11], [12]. PCI also binds oxidized and unoxidized negatively charged phospholipids like phosphatidylethanolamine (PE) and phosphatidylserine (PS), which also modulate its inhibitory activity [13].…”

Section: Introductionmentioning

confidence: 99%

Protein C Inhibitor (PCI) Binds to Phosphatidylserine Exposing Cells with Implications in the Phagocytosis of Apoptotic Cells and Activated Platelets

et al. 2014

Self Cite

View full text Add to dashboard Cite

Protein C Inhibitor (PCI) is a secreted serine protease inhibitor, belonging to the family of serpins. In addition to activated protein C PCI inactivates several other proteases of the coagulation and fibrinolytic systems, suggesting a regulatory role in hemostasis. Glycosaminoglycans and certain negatively charged phospholipids, like phosphatidylserine, bind to PCI and modulate its activity. Phosphatidylerine (PS) is exposed on the surface of apoptotic cells and known as a phagocytosis marker. We hypothesized that PCI might bind to PS exposed on apoptotic cells and thereby influence their removal by phagocytosis. Using Jurkat T-lymphocytes and U937 myeloid cells, we show here that PCI binds to apoptotic cells to a similar extent at the same sites as Annexin V, but in a different manner as compared to live cells (defined spots on ∼10–30% of cells). PCI dose dependently decreased phagocytosis of apoptotic Jurkat cells by U937 macrophages. Moreover, the phagocytosis of PS exposing, activated platelets by human blood derived monocytes declined in the presence of PCI. In U937 cells the expression of PCI as well as the surface binding of PCI increased with time of phorbol ester treatment/macrophage differentiation. The results of this study suggest a role of PCI not only for the function and/or maturation of macrophages, but also as a negative regulator of apoptotic cell and activated platelets removal.

show abstract

“…The interactions of rMan-KPI, rErs-KPI and their domains with chymotrypsin and elastase were analyzed by SDS-PAGE using the method described by Rehault [39], with some modifications.…”

Section: Gel Analysis Of Recombinant Inhibitors Upon Interaction Withmentioning

confidence: 99%

Two Kazal-type protease inhibitors from Macrobrachium nipponense and Eriocheir sinensis: Comparative analysis of structure and activities

Qian

Yang

et al. 2012

Fish & Shellfish Immunology

View full text Add to dashboard Cite

Characterization of recombinant human protein C inhibitor expressed in Escherichia coli

Cited by 22 publications

References 64 publications

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

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

Protein C Inhibitor (PCI) Binds to Phosphatidylserine Exposing Cells with Implications in the Phagocytosis of Apoptotic Cells and Activated Platelets

Two Kazal-type protease inhibitors from Macrobrachium nipponense and Eriocheir sinensis: Comparative analysis of structure and activities

Contact Info

Product

Resources

About