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1.
Adv Exp Med Biol ; 966: 93-101, 2017.
Article in English | MEDLINE | ID: mdl-28639251

ABSTRACT

It is generally accepted that the phospholipid bilayer of the cell membrane is impermeable for proteins and peptides and that these molecules require special mechanisms for their transport from the extra- to the intracellular space. Recently there is increasing evidence that certain proteins/peptides can also directly cross the phospholipid membrane. SERPINA5 (protein C inhibitor) is a secreted protease inhibitor with broad protease reactivity and wide tissue distribution. It binds glycosaminoglycans and certain phospoholipids, which can modulate its inhibitory activity. SERPINA5 has been shown to be internalized by platelets, granulocytes, HL-60 promyelocytic leukemia cells, and by Jurkat lymphoma cells. Once inside the cell it can translocate to the nucleus. There are several indications that SERPINA5 can directly cross the phospholipid bilayer of the cell membrane. In this review we will describe what is known so far about the conditions, as well as the cellular and molecular requirements for SERPINA5 translocation through the cell membrane and for its penetration of pure phospholipid vesicles.


Subject(s)
Cell Membrane Permeability , Cell Membrane/metabolism , Protein C Inhibitor/metabolism , Animals , Humans , Protein C Inhibitor/chemistry , Protein Conformation , Protein Transport , Structure-Activity Relationship
2.
PLoS One ; 10(11): e0143137, 2015.
Article in English | MEDLINE | ID: mdl-26580551

ABSTRACT

Protein C inhibitor is a secreted, non-specific serine protease inhibitor with broad protease reactivity. It binds glycosaminoglycans and anionic phospholipids, which can modulate its activity. Anionic phospholipids, such as phosphatidylserine are normally localized to the inner leaflet of the plasma membrane, but are exposed on activated and apoptotic cells and on plasma membrane-derived microparticles. In this report we show by flow cytometry that microparticles derived from cultured cells and activated platelets incorporated protein C inhibitor during membrane blebbing. Moreover, protein C inhibitor is present in/on microparticles circulating in normal human plasma as judged from Western blots, ELISAs, flow cytometry, and mass spectrometry. These plasma microparticles are mainly derived from megakaryocytes. They seem to be saturated with protein C inhibitor, since they do not bind added fluorescence-labeled protein C inhibitor. Heparin partially removed microparticle-bound protein C inhibitor, supporting our assumption that protein C inhibitor is bound via phospholipids. To assess the biological role of microparticle-bound protein C inhibitor we performed protease inhibition assays and co-precipitated putative binding partners on microparticles with anti-protein C inhibitor IgG. As judged from amidolytic assays microparticle-bound protein C inhibitor did not inhibit activated protein C or thrombin, nor did microparticles modulate the activity of exogenous protein C inhibitor. Among the proteins co-precipitating with protein C inhibitor, complement factors, especially complement factor 3, were most striking. Taken together, our data do not support a major role of microparticle-associated protein C inhibitor in coagulation, but rather suggest an interaction with proteins of the complement system present on these phospholipid vesicles.


Subject(s)
Blood Platelets/chemistry , Cell Membrane/chemistry , Cell-Derived Microparticles/chemistry , Megakaryocytes/chemistry , Protein C Inhibitor/chemistry , Protein C/antagonists & inhibitors , Adult , Blood Platelets/cytology , Cell Membrane/metabolism , Cell-Derived Microparticles/metabolism , Female , Heparin/chemistry , Humans , Immunoglobulin G/chemistry , Immunoglobulin G/metabolism , Jurkat Cells , Male , Megakaryocytes/cytology , Middle Aged , Phospholipids/chemistry , Phospholipids/metabolism , Platelet Factor 3/chemistry , Platelet Factor 3/metabolism , Protein Binding , Protein C/metabolism , Protein C Inhibitor/metabolism , Thrombin/chemistry , Thrombin/metabolism
3.
J Biol Chem ; 290(5): 3081-91, 2015 Jan 30.
Article in English | MEDLINE | ID: mdl-25488662

ABSTRACT

Protein C inhibitor (PCI) is a serpin with broad protease reactivity. It binds glycosaminoglycans and certain phospholipids that can modulate its inhibitory activity. PCI can penetrate through cellular membranes via binding to phosphatidylethanolamine. The exact mechanism of PCI internalization and the intracellular role of the serpin are not well understood. Here we showed that testisin, a glycosylphosphatidylinositol-anchored serine protease, cleaved human PCI and mouse PCI (mPCI) at their reactive sites as well as at sites close to their N terminus. This cleavage was observed not only with testisin in solution but also with cell membrane-anchored testisin on U937 cells. The cleavage close to the N terminus released peptides rich in basic amino acids. Synthetic peptides corresponding to the released peptides of human PCI (His(1)-Arg(11)) and mPCI (Arg(1)-Ala(18)) functioned as cell-penetrating peptides. Because intact mPCI but not testisin-cleaved mPCI was internalized by Jurkat T cells, a truncated mPCI mimicking testisin-cleaved mPCI was created. The truncated mPCI lacking 18 amino acids at the N terminus was not taken up by Jurkat T cells. Therefore our model suggests that testisin or other proteases could regulate the internalization of PCI by removing its N terminus. This may represent one of the mechanisms regulating the intracellular functions of PCI.


Subject(s)
Cell-Penetrating Peptides/chemistry , Cell-Penetrating Peptides/metabolism , Protein C Inhibitor/chemistry , Protein C Inhibitor/metabolism , Animals , Cell Line, Tumor , Cell Membrane Permeability/physiology , GPI-Linked Proteins/metabolism , Humans , Mice , Serine Endopeptidases/metabolism , U937 Cells
4.
PLoS One ; 7(6): e39262, 2012.
Article in English | MEDLINE | ID: mdl-22723979

ABSTRACT

The serine protease inhibitor protein C inhibitor (PCI) is expressed in many human tissues and exhibits broad protease reactivity. PCI binds glycosaminoglycans and certain phospholipids, which modulate its inhibitory activity. Enteropeptidase (EP) is a type II transmembrane serine protease mainly found on the brush border membrane of epithelial cells in the duodenum, where it activates trypsinogen to initiate the digestion of food proteins. Some active EP is also present in duodenal fluid and has been made responsible for causing pancreatitis in case of duodeno-pancreatic reflux. Together with its substrate trypsinogen, EP is furthermore present in the epidermis and in some cancer cells. In this report, we show that PCI inhibited EP with an apparent 2nd order rate constant of 4.48 × 10(4) M(-1) s(-1). Low molecular weight (LMWH) and unfractionated heparin (UFH) slightly reduced the inhibitory effect of PCI. The SI (stoichiometry of inhibition) value for the inhibition of EP by PCI was 10.8 in the absence and 17.9 in the presence of UFH (10 U/ml). By inhibiting trypsin, chymotrypsin, and additionally EP, PCI might play a role in the protection of the pancreas from autodigestion. Furthermore the interaction of PCI with EP may influence the regulation of epithelial differentiation.


Subject(s)
Enteropeptidase/metabolism , Protein C Inhibitor/metabolism , Serine Proteinase Inhibitors/metabolism , Animals , Antithrombins/metabolism , Antithrombins/pharmacology , Cattle , Dose-Response Relationship, Drug , Enteropeptidase/antagonists & inhibitors , Heparin/pharmacology , Humans , Mice , Protein Binding , Protein C Inhibitor/pharmacology , Serine Proteinase Inhibitors/pharmacology , Serpins/metabolism , Serpins/pharmacology
5.
Fertil Steril ; 88(4 Suppl): 1049-57, 2007 Oct.
Article in English | MEDLINE | ID: mdl-17434507

ABSTRACT

OBJECTIVE: To investigate the mechanisms responsible for the testicular abnormalities and infertility of previously generated male protein C inhibitor (PCI)-deficient mice. DESIGN: Determination of the localization of PCI in the reproductive organs of wild-type males. Generation of double knockout mice lacking the protease inhibitor PCI and one plasminogen activator, either urokinase (uPA) or tissue plasminogen activator (tPA), both of which are PCI-target proteases. SETTING: Animal research and histologic analysis. ANIMAL(S): Male mice of desired genotype. INTERVENTION(S): Fertility testing of double knockout mice. MAIN OUTCOME MEASURE(S): Infertility of PCI(-/-)uPA(-/-) and PCI(-/-)tPA(-/-) double knockout mice. RESULT(S): In the testes of wild-type males PCI was detected in spermatocytes of prophase I, as well as in late spermatids and mature spermatozoa, but absent from somatic cells. All PCI(-/-) uPA(-/-) and PCI(-/-) tPA(-/-) male mice were infertile and histologic analysis of testis showed similar alterations as previously described for PCI(-/-) mice. CONCLUSION(S): The abnormal spermatogenesis of PCI (plasminogen activator inhibitor-3)-deficient mice cannot be rescued by single plasminogen activator knockout.


Subject(s)
Fertility , Protein C Inhibitor/analysis , Protein C Inhibitor/genetics , Spermatogenesis , Testis/chemistry , Animals , Cell Differentiation/genetics , Female , Fertility/genetics , Infertility, Male/genetics , Infertility, Male/metabolism , Male , Mice , Mice, Knockout , Protein C Inhibitor/deficiency , Spermatogenesis/genetics , Testis/cytology , Testis/metabolism
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