Characterization of a cDNA for Rhesus Monkey Protein C Inhibitor – Evidence for N-Terminal Involvement in Heparin Stimulation

SummarycDNAs for protein C inhibitor (PCI) were cloned from human and rhesus monkey 1 liver RNAs by reverse transcription and polymerase chain reaction (PCR) amplification. Sequencing showed that rhesus monkey and human PCI cDNAs were 93% identical. Predicted amino acid sequences differed at 26 of 387 residues. Pour of these differences (T352M, N359S, R362K, L3631) were in the reactive center loop that is important for inhibitory specificity, and two were in the N-terminal helix (M8T, E13K) that is implicated in glycosaminoglycan binding. PCI in human or rhesus monkey plasma showed comparable inhibitory activity towards human activated protein C in the presence of 10 U/ml heparin. However, maximal acceleration of the inhibition of activated protein C required 5-fold lower heparin concentration for rhesus monkey than for human plasma, consistent with the interpretation that the additional positive charge (E13K) in a putative-heparin binding region increased the affinity for heparin.

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Activated Protein C−Protein C Inhibitor Complex Formation: Characterization of a Neoepitope Provides Evidence for Extensive Insertion of the Reactive Center Loop

Biochemistry ◽

10.1021/bi001640h ◽

2000 ◽

Vol 39 (51) ◽

pp. 15713-15720 ◽

Cited By ~ 17

Author(s):

Karin Strandberg ◽

Margareta Kjellberg ◽

Eva-Maria Erb ◽

Ulla Persson ◽

Deane F. Mosher ◽

...

Keyword(s):

Complex Formation ◽

Protein C ◽

Activated Protein C ◽

C Protein ◽

Inhibitor Complex ◽

Reactive Center Loop ◽

Reactive Center ◽

Protein C Inhibitor

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The Polymerase Chain Reaction with Sequence Specific Primers for the Detection of the Factor V Mutation Associated with Activated Protein C Resistance

Thrombosis and Haemostasis ◽

10.1055/s-0038-1649840 ◽

1995 ◽

Vol 74 (03) ◽

pp. 874-878 ◽

Cited By ~ 49

Author(s):

Nancy E Kirschbaum ◽

Paul A Foster

Keyword(s):

Protein C ◽

Activated Protein C ◽

Pcr Amplification ◽

Factor V ◽

Activated Protein C Resistance ◽

Chain Reaction ◽

Specific Primers ◽

Allele Specific ◽

Fv Leiden ◽

Polymerase Chain

SummaryThe prevalence of the Factor V (FV) mutation associated with activated protein C resistance (FV Leiden) and its significance as a genetic risk factor for venous thrombosis have necessitated the development of a simple, rapid, and accurate assay for its detection. The polymerase chain reaction with sequence specific primers (PCR-SSP) provides a powerful technique for the discrimination of alleles resulting from single base substitutions. PCR amplification was performed using a sense primer complementary to both FV alleles coupled with either of two antisense allele specific primers, one complementary to the normal FV allele and one complementary to the FV Leiden allele. PCR conditions were developed that favored amplification only in the case of perfect complementation between template DNA and allele specific primer. The FV genotype was assigned based on whether or not each allele specific primer set produced an amplified product. Assignment of genotypes correlated 100% with those determined by the method of PCR amplification followed by MnII digestion. PCR-SSP allows the rapid and accurate identification of carriers of the Factor V Leiden mutation by a simple PCR reaction without the need for the usual post-amplification specificity step.

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Role of the A+ Helix in Heparin Binding to Protein C Inhibitor

Thrombosis and Haemostasis ◽

10.1055/s-0038-1650363 ◽

1996 ◽

Vol 75 (05) ◽

pp. 760-766 ◽

Cited By ~ 21

Author(s):

Marc G L M Elisen ◽

Machiel H H Maseland ◽

Frank C Church ◽

Bonno N Bouma ◽

Joost C M Meijers

Keyword(s):

Protein C ◽

Electrostatic Interactions ◽

Deletion Mutant ◽

Activated Protein C ◽

Structural Features ◽

Heparin Binding ◽

Wild Type ◽

Protein C Inhibitor ◽

Positively Charged

SummaryInteractions between proteins and heparin(-like) structures involve electrostatic forces and structural features. Based on charge distributions in the linear sequence of protein C inhibitor (PCI), two positively charged regions of PCI were proposed as possible candidates for this interaction. The first region, the A+ helix, is located at the N-terminus (residues 1-11), whereas the second region, the H helix, is positioned between residues 264 and 280 of PCI. Competition experiments with synthetic peptides based on the sequence of these regions demonstrated that the H helix has the highest affinity for heparin. In contrast to previous observations we found that the A+ helix peptide competed for the interaction of PCI with heparin, but its affinity was much lower than that of the H helix peptide.Recombinant PCI was also used to investigate the role of the A+ helix in heparin binding. Full-length (wild-type) rPCI as well as an A+ helix deletion mutant of PCI (rPCI-Δ2-l 1) were expressed in baby hamster kidney cells and both had normal inhibition activity with activated protein C and thrombin. The interaction of the recombinant PCIs with heparin was investigated and compared to plasma PCI. The A+ helix deletion mutant showed a decreased affinity for heparin in inhibition reactions with activated protein C and thrombin, but had similar association constants compared to wild-type rPCI. The synthetic A+ helix peptide competed with rPCI-Δ2-11 for binding to heparin. This indicated that the interaction between PCI and heparin is fairly non-specific and that the interaction is primarily based on electrostatic interactions.In summary, our data suggest that the H helix of PCI is the main heparin binding region of PCI, but the A+ helix increases the overall affinity for the PCI-heparin interaction by contributing a second positively charged region to the surface of PCI.

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Involvement of Lys 62[217] and Lys 63[218] of Human Anticoagulant Protein C in Heparin Stimulation of Inhibition by the Protein C Inhibitor

Thrombosis and Haemostasis ◽

10.1055/s-0037-1614632 ◽

1999 ◽

Vol 82 (07) ◽

pp. 72-79 ◽

Cited By ~ 19

Author(s):

Lei Shen ◽

Bruno Villoutreix ◽

Björn Dahlbäck

Keyword(s):

Rate Constants ◽

Protein C ◽

Double Mutant ◽

Activated Protein C ◽

Human Kidney ◽

Peptide Bond ◽

Dimensional Structure ◽

Heparin Binding ◽

Protein C Inhibitor ◽

Stimulation Of

SummaryInhibition of activated protein C (APC) by protein C inhibitor (PCI) is stimulated by heparin, whereas inhibition by α1-antitrypsin (AAT) is heparin-independent. Three lysine residues located in a positively charged cluster in the serine protease domain of protein C (PC) were mutated to probe their involvement in the heparin stimulation of inhibition by PCI. These mutations were selected after analysis of the three-dimensional structure of APC and of molecular models for PCI and the APC-PCI complex. A double mutant, K62[217]N/K63[218]D, a single mutant, K86[241]S, and wild-type PC were expressed in embryonic human kidney 293 cells. Heparin stimulated the rate of inhibition of wt-APC by PCI approximately 400-fold, with second order rate constants (k 2 ) in the absence and presence of heparin of 0.72 × 103 M–1s–1 and 2.87 × 105 M–1s–1, respectively. In contrast, heparin only yielded a 52-fold stimulation of the rate of inhibition of the double mutant APC by PCI as the rate constants in the absence and presence of heparin were k 2 = 2.44 × 103 M–1s–1 and k 2 = 1.26 × 105 M–1s–1, respectively. The double mutant K62N/K63D eluted at approximately 10% lower NaCl concentration from a heparin Sepharose column than the K86S mutant or wt-APC. These data suggest K62 and K63 in APC to be part of a heparin binding site which is important for heparin-mediated stimulation of inhibition of APC by PCI. Abbreviations: APC, activated protein C; PC, protein C; PCI, protein C inhibitor, AAT, α1-antitrypsin also called α1-proteinase inhibitor, AT, antithrombin; TM, thrombomodulin. The chymotrypsinogen nomenclature for APC (1) is used in the text while the PC numbering is indicated between brackets whenever appropriate. P1, P2... and P1’, P2’.. designate inhibitor residues amino- and carboxy-terminal to the scissile peptide bond, respectively, and S1, S2.. and S1’, S2’.. the corresponding subsites of the protease (2). The antitrypsin numbering (3) for PCI is used along this article while the PCI numbering is mentioned between brackets whenever appropriate.

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

Blood ◽

10.1182/blood-2006-09-046953 ◽

2007 ◽

Vol 109 (11) ◽

pp. 4769-4776 ◽

Cited By ~ 26

Author(s):

Julia M. Malleier ◽

Olga Oskolkova ◽

Valery Bochkov ◽

Ingrid Jerabek ◽

Barbora Sokolikova ◽

...

Keyword(s):

Binding Site ◽

Protein C ◽

Aqueous Suspension ◽

Activated Protein C ◽

Annexin V ◽

Dependent Manner ◽

Heparin Binding ◽

Heparin Binding Site ◽

Protein C Inhibitor

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 heparin-binding 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.

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Protein C Inhibitor from Human Plasma: Characterization of Native and Cleaved Inhibitor and Demonstration of Inhibitor Complexes with Plasma Kallikrein

Thrombosis and Haemostasis ◽

10.1055/s-0038-1651022 ◽

1989 ◽

Vol 62 (03) ◽

pp. 885-891 ◽

Cited By ~ 20

Author(s):

Martin Laurell ◽

Johan Stenflo

Keyword(s):

Sequence Analysis ◽

Human Plasma ◽

Protein C ◽

Activated Protein C ◽

Immunoaffinity Chromatography ◽

Plasma Kallikrein ◽

Cleavage Sites ◽

Protein C Inhibitor ◽

Secondary Cleavage

SummaryProtein C inhibitor (PCI) was purified from human plasma using immunoaffinity chromatography and heparin Sepharose chromatography, a method that allowed the purification of active and inactive inhibitor. PCI purified from outdated plasma was inactive and either in complex with plasma kallikrein or proteo-lytically degraded. Sequence analysis of cleaved PCI and of complexes between PCI and activated protein C or urokinase identified the previously recognized inhibitor cleavage site Arg354-Ser355. Two additional cleavage sites were observed in the modified inhibitor i.e. Arg357-Leu358 and Arg362-Leu363 which probably represent secondary cleavage of the inhibitor. Furthermore the sequence analysis of the inhibitor, whether purified from fresh or outdated plasma, revealed that it was microheterogeneous in the NH2-terminus as a result of cleavage by a trypsin like enzyme(s).

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New lipid interaction partners stimulate the inhibition of activated protein C by cell-penetrating protein C inhibitor

Thrombosis and Haemostasis ◽

10.1160/th13-06-0478 ◽

2014 ◽

Vol 111 (01) ◽

pp. 41-52 ◽

Cited By ~ 8

Author(s):

Felix Christof Wahlmüller ◽

Barbora Sokolikova ◽

Daniela Rieger ◽

Margarethe Geiger

Keyword(s):

Protein C ◽

Activated Protein C ◽

Direct Interaction ◽

Heparin Binding ◽

Cellular Membranes ◽

Akt Activation ◽

Lipid Signalling ◽

Lipid Species ◽

Protein C Inhibitor

SummaryProtein C inhibitor (PCI, SerpinA5) is a heparin-binding serpin which can penetrate through cellular membranes. Selected negatively charged phospholipids like unsaturated phosphatidylserine and oxidised phosphatidylethanolamine bind to PCI and stimulate its inhibitory activity towards different proteases. The interaction of phospholipids with PCI might also alter the lipid distribution pattern of blood cells and influence the remodelling of cellular membranes. Here we showed that PCI is an additional binding partner of phosphatidic acid (PA), cardiolipin (CL), and phosphoinositides (PIPs). Protein lipid overlay assays exhibited a unique binding pattern of PCI towards different lipid species. In addition PA, CL, and unsaturated, monophosphorylated PIPs stimulated the inhibitory property of PCI towards activated protein C in a heparin like manner. As shown for kallistatin (SerpinA4) and vaspin (SerpinA12), the incubation of cells with PCI led to the activation of protein kinase B (AKT), which could be achieved through direct interaction of PCI with PIPs. This model is supported by the fact that PCI stimulated the PIP-dependent 5-phosphatase SHIP2 in vitro, which would result in AKT activation. Hence the interaction of PCI with different lipids might not only stimulate the inhibition of potential target protease by PCI, but could also alter intracellular lipid signalling.

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