Functionally inactive protein C inhibitor in seminal plasma may be associated with infertility.
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Protein C inhibitor (PCI) has been found in seminal plasma and is considered to protect intact surrounding cells and seminal plasma proteins from possible proteolytic damage. In the present study, we showed that although the antigenic levels of PCI in two seminal plasma samples from patients with infertility were normal or slightly elevated, their inhibitory activities toward urokinase plasminogen activator (uPA) and tissue-type plasminogen activator (tPA) were absent. In contrast, uPA and tPA proteolytic activities in these two samples were 20-60-fold higher than that from normal volunteers. A time-course analysis of PCI-uPA complex formation showed that >80% of the complex had been formed within 15 min in normal seminal plasma in the presence of heparin, compared with the total complex formed after 150 min incubation, whereas no response to heparin stimulation was observed in the assays with the two patient samples. Similarly, >90% of PCI-tPA complex was formed after 30 min of heparin stimulation in normal seminal plasma but no response was observed in the two patient samples. Kinetic assays of PCI inhibitory function in the presence of activated protein C (APC) showed that PCI inhibitory activity in the two patient samples was absent and not stimulated by heparin. Western blotting also showed that most of the intact PCI molecules, in normal samples, formed complexes with either uPA or tPA but there was no complex formed in one of the two patient samples and very little complex was observed in the other, suggesting that PCI in the two patient samples is inactive. These results suggest that the presence of functionally inactive PCI in seminal plasma may be associated with infertility. (+info)
Protein C inhibitor is expressed in keratinocytes of human skin.
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Protein C inhibitor is a member of the serpin family that inhibits a variety of serine proteases. Protein C inhibitor is present in numerous body fluids and is produced in the liver and by various epithelial cells. To determine if this epithelial serpin is present in skin, immunohistochemical studies were performed that showed strong staining for protein C inhibitor antigen in the epidermis. Protein C inhibitor mRNA was detected in the keratinocyte cell line HaCaT and the epidermoid carcinoma cell line A431 using reverse transcription-polymerase chain reaction suggesting that also in normal skin protein C inhibitor is derived from keratinocytes. Conditioned media from these cell lines were analyzed on immunoblots, which revealed a protein C inhibitor-antigen band that comigrated with protein C inhibitor derived from the hepatoma cell line HepG2. Using an enzyme-linked immunosorbent assay specific for total protein C inhibitor antigen the accumulation of protein C inhibitor in the cell culture supernatants of HaCaT keratinocytes was found to be 0.3 ng per h per 1 million cells. This is similar to the amount of plasminogen activator inhibitor-1 produced by these cells, which also produce tissue plasminogen activator and urokinase. Fluorescence-activated cell sorter analysis revealed similar expression of intracellular protein C inhibitor antigen in proliferating and confluent HaCaT cells. These findings demonstrate that protein C inhibitor antigen is present in the normal epidermis and that protein C inhibitor is constitutively expressed by keratinocytes in culture. Therefore, protein C inhibitor may provide protease inhibitory activity not only to internal, but also to the external surface of the body. Additionally, protein C inhibitor could contribute to the regulation of retinoid supply in the epidermis, as we have shown recently that retinoic acid binds specifically to protein C inhibitor. (+info)
Synthesis and ultrastructural localization of protein C inhibitor in human platelets and megakaryocytes.
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The occurrence of protein C inhibitor (PCI) in human platelets and megakaryocytes was analyzed. As judged from enzyme-linked immunosorbent assays (ELISAs), PCI was present in platelets at a concentration of 160 ng/2 x 10(9) cells. Its specific activity was 5 times higher than that of plasma PCI. Consistently, mainly the 57-kD form (active PCI) and some high molecular weight (M(r)) forms, but no bands corresponding to cleaved PCI, were detected when platelet lysates were immunoprecipitated with monoclonal anti-PCI-IgG and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. The localization of PCI in platelets was studied by immunofluorescence histochemistry and immunotransmission electron microscopy: PCI was detected in alpha granules, in the open canalicular system, and on the plasma membrane. At these sites, colocalization with plasminogen activator inhibitor-1 was seen. Studies were performed to clarify whether platelet PCI is endogenously synthesized or taken up from plasma. Internalization of biotinylated-PCI was analyzed using platelets in suspension and gold-labeled streptavidin for visualization of incorporated biotin. Dose- and time-dependent uptake of PCI was found. PCI mRNA was detected in platelets by reverse transcriptase-polymerase chain reaction (RT-PCR) and Southern blotting, as well as in megakaryocytes by in situ hybridization of human bone marrow cryosections. We therefore conclude that platelets contain a functionally active PCI pool that is derived from both endogenous synthesis as well as internalization. (+info)
Role of activated protein C in Helicobacter pylori-associated gastritis.
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The protein C (PC) pathway has recently been suggested to play a role in the regulation of the inflammatory response. To further extend the anti-inflammatory effect of activated PC (APC) in vivo, particularly its biological relevance to human disease, the activity of APC in the mucosa of patients with Helicobacter pylori-associated gastritis and the effect of vacuolating cytotoxin (VacA), cytotoxin-associated antigen (CagA), and H. pylori lipopolysaccharide (LPS) on PC activation were evaluated. This study comprised 35 patients with chronic gastritis. There were 20 patients with and 15 without H. pylori infection. The levels of PC and APC-PC inhibitor (PCI) complex were measured by immunoassays. The level of PC was significantly decreased and the level of APC-PCI complex was significantly increased in biopsy specimens from gastric corpus and antrum in patients with H. pylori-associated gastritis as compared to H. pylori-negative subjects. The concentrations of VacA, CagA, and LPS were significantly correlated with those of the APC-PCI complex in biopsy mucosal specimens from the gastric corpus and antrum. H. pylori LPS, VacA, and CagA induced a dose-dependent activation of PC on the surface of monocytic cells. APC inhibited the secretion of tumor necrosis factor alpha (TNF-alpha) induced by H. pylori LPS. Overall, these results suggest that H. pylori infection is associated with increased APC generation in the gastric mucosa. The inhibitory activity of APC on TNF-alpha secretion may serve to protect H. pylori-induced gastric mucosal damage. (+info)
Identification of the protein C/activated protein C binding sites on the endothelial cell protein C receptor. Implications for a novel mode of ligand recognition by a major histocompatibility complex class 1-type receptor.
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The endothelial cell protein C receptor (EPCR) is an endothelial cell-specific transmembrane protein that binds both protein C and activated protein C (APC). EPCR regulates the protein C anticoagulant pathway by binding protein C and augmenting protein C activation by the thrombin-thrombomodulin complex. EPCR is homologous to the MHC class 1/CD1 family, members of which contain two alpha-helices that sit upon an 8-stranded beta-sheet platform. In this study, we identified 10 residues that, when mutated to alanine, result in the loss of protein C/APC binding (Arg-81, Leu-82, Val-83, Glu-86, Arg-87, Phe-146, Tyr-154, Thr-157, Arg-158, and Glu-160). Glutamine substitutions at the four N-linked carbohydrate attachment sites of EPCR have little affect on APC binding, suggesting that the carbohydrate moieties of EPCR are not critical for ligand recognition. We then mapped the epitopes for four anti-human EPCR monoclonal antibodies (mAbs), two of which block EPCR/Fl-APC (APC labeled at the active site with fluorescein) interactions, whereas two do not. These epitopes were localized by generating human-mouse EPCR chimeric proteins, since the mAbs under investigation do not recognize mouse EPCR. We found that 5 of the 10 candidate residues for protein C/APC binding (Arg-81, Leu-82, Val-83, Glu-86, Arg-87) colocalize with the epitope for one of the blocking mAbs. Three-dimensional molecular modeling of EPCR indicates that the 10 protein C/APC binding candidate residues are clustered at the distal end of the two alpha-helical segments. Protein C activation studies on 293 cells that coexpress EPCR variants and thrombomodulin demonstrate that protein C binding to EPCR is necessary for the EPCR-dependent enhancement in protein activation by the thrombin-thrombomodulin complex. These studies indicate that EPCR has exploited the MHC class 1 fold for an alternative and possibly novel mode of ligand recognition. These studies are also the first to identify the protein C/APC binding region of EPCR and may provide useful information about molecular defects in EPCR that could contribute to cardiovascular disease susceptibility. (+info)
Disruption of the protein C inhibitor gene results in impaired spermatogenesis and male infertility.
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Protein C inhibitor (PCI) is a nonspecific, heparin-binding serpin (serine protease inhibitor) that inactivates many plasmatic and extravascular serine proteases by forming stable 1:1 complexes. Proteases inhibited by PCI include the anticoagulant activated protein C, the plasminogen activator urokinase, and the sperm protease acrosin. In humans PCI circulates as a plasma protein but is also present at high concentrations in organs of the male reproductive tract. The biological role of PCI has not been defined so far. However, the colocalization of high concentrations of PCI together with several of its target proteases in the male reproductive tract suggests a role of PCI in reproduction. We generated mice lacking PCI by homologous recombination. Here we show that PCI(-/-) mice are apparently healthy but that males of this genotype are infertile. Infertility was apparently caused by abnormal spermatogenesis due to destruction of the Sertoli cell barrier, perhaps due to unopposed proteolytic activity. The resulting sperm are malformed and are morphologically similar to abnormal sperm seen in some cases of human male infertility. This animal model might therefore be useful for analyzing the molecular bases of these human conditions. (+info)
Structural and energetic characteristics of the heparin-binding site in antithrombotic protein C.
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Human activated protein C (APC) is a key component of a natural anticoagulant system that regulates blood coagulation. In vivo, the catalytic activity of APC is regulated by two serpins, alpha1-antitrypsin and the protein C inhibitor (PCI), the inhibition by the latter being stimulated by heparin. We have identified a heparin-binding site in the serine protease domain of APC and characterized the energetic basis of the interaction with heparin. According to the counter-ion condensation theory, the binding of heparin to APC is 66% ionic in nature and comprises four to six net ionic interactions. To localize the heparin-binding site, five recombinant APC variants containing amino acid exchanges in loops 37, 60, and 70 (chymotrypsinogen numbering) were created. As demonstrated by surface plasmon resonance, reduction of the electropositive character of loops 37 and 60 resulted in complete loss of heparin binding. The functional consequence was loss in heparin-induced stimulation of APC inhibition by PCI, whereas the PCI-induced APC inhibition in the absence of heparin was enhanced. Presumably, the former observations were due to the inability of heparin to bridge some APC mutants to PCI, whereas the increased inhibition of certain APC variants by PCI in the absence of heparin was due to reduced repulsion between the enzymes and the serpin. The heparin-binding site of APC was also shown to interact with heparan sulfate, albeit with lower affinity. In conclusion, we have characterized and spatially localized the functionally important heparin/heparan sulfate-binding site of APC. (+info)
Binding of retinoic acid by the inhibitory serpin protein C inhibitor.
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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 (3)H-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-[(3)H]retinoic acid bound in a specific dose-dependent and time-dependent way to PCI (apparent K(d) = 2.43 microm, 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. (+info)