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Margarethe Geiger - One of the best experts on this subject based on the ideXlab platform.
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Regulation of the ExtraCellular SERPINA5 (Protein C Inhibitor) Penetration Through Cellular Membranes.
Advances in Experimental Medicine and Biology, 2017Co-Authors: Felix C. Wahlmüller, Margareta Furtmüller, Hanjiang Yang, Margarethe GeigerAbstract: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.
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Phospholipid Binding Protein C Inhibitor (PCI) Is Present on MiCropartiCles Generated In Vitro and In Vivo.
PLOS ONE, 2015Co-Authors: Katrin Einfinger, Sigrun Badrnya, Margareta Furtmüller, Daniela Handschuh, Herbert Lindner, Margarethe GeigerAbstract: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.
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a helix of Protein C Inhibitor pCi is a Cell penetrating peptide that mediates Cell membrane permeation of pCi
Journal of Biological Chemistry, 2015Co-Authors: Hanjiang Yang, Felix C. Wahlmüller, Margareta Furtmüller, Bettina Sarg, Margarethe GeigerAbstract: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 (His1–Arg11) and mPCI (Arg1–Ala18) 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.
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InteraCtion of Protein C Inhibitor with the Type II Transmembrane Serine Protease Enteropeptidase
PLOS ONE, 2012Co-Authors: Thomas A. Prohaska, Felix C. Wahlmüller, Margareta Furtmüller, Margarethe GeigerAbstract: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 4.48×104 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.
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Expression patterns of Protein C Inhibitor in mouse development
Journal of Molecular Histology, 2010Co-Authors: Gerry T. M. Wagenaar, Joost C. M. Meijers, Pavel Uhrin, Margarethe Geiger, Klara Weipoltshammer, Marlene Almeder, Pieter S. Hiemstra, Christian SchöferAbstract:Proteolysis of extraCellular matrix is an important requirement for embryoniC development and is instrumental in proCesses suCh as morphogenesis, angiogenesis, and Cell migration. EffiCient remodeling requires Controlled spatio-temporal expression of both the proteases and their Inhibitors. Protein C Inhibitor (PCI) effeCtively bloCks a range of serine proteases, and reCently has been suggested to play a role in Cell differentiation and angiogenesis. In this study, we mapped the expression pattern of PCI throughout mouse development using in situ hybridization and immunohistoChemistry. We deteCted a wide-spread, yet distinCt expression pattern with prominent PCI levels in skin inCluding vibrissae, and in fore- and hindgut. Further sites of PCI expression were Choroid plexus of brain ventriCles, heart, skeletal musCles, urogenital traCt, and Cartilages. A strong and stage-dependent PCI expression was observed in the developing lung. In the pseudoglandular stage, PCI expression was present in distal branChing tubules whereas proximal tubules did not express PCI. Later in development, in the saCCular stage, PCI expression was restriCted to distal bronChioli whereas saCCuli did not express PCI. PCI expression deClined in postnatal stages and was not deteCted in adult lungs. In general, embryoniC PCI expression indiCates multifunCtional roles of PCI during mouse development. The expression pattern of PCI during lung development suggests its possible involvement in lung morphogenesis and angiogenesis.
Frank C Church - One of the best experts on this subject based on the ideXlab platform.
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Essential thrombin residues for inhibition by Protein C Inhibitor with the CofaCtors heparin and thrombomodulin
Journal of Thrombosis and Haemostasis, 2007Co-Authors: Yolanda M. Fortenberry, Scott Cooper, Herbert C. Whinna, Timothy Myles, Lawrence L. K. Leung, Frank C ChurchAbstract:BACKGROUND: Protein C Inhibitor (PCI) and antithrombin (AT) are serine protease Inhibitors (serpins) that inhibit a wide array of blood Coagulation serine proteases inCluding thrombin. OBJECTIVE: Fifty-five Ala-sCanned reCombinant thrombin mutants were used to determine thrombin residues important for inhibition by PCI with and without the CofaCtors heparin and thrombomodulin (TM) and Compared with the prototypiCal serpin, AT. RESULTS: Residues around the aCtive site (Tyr50 and Glu202) and the sodium-binding site (Glu229 and Arg233) were required for thrombin inhibition by PCI with and without CofaCtors. Exosite-2 residues (Arg89, Arg93, Glu94, Arg98, Arg245, Arg248, and Gln251) were CritiCal for heparin-aCCelerated inhibition of thrombin by PCI. Exosite-1 residues (espeCially Lys65 and Tyr71) were required for enhanCed PCI inhibition of thrombin-TM. Interestingly, we also found that the TM Chondroitin sulfate moiety is not required for the approximately 150-fold enhanCed rate of thrombin inhibition by PCI. Using the aforementioned thrombin exosite-2 mutants that were essential for heparin-Catalyzed PCI-thrombin inhibition reaCtions we found no Change in PCI inhibition rates for thrombin-TM. CONCLUSIONS: ColleCtively, these results show that (i) similar thrombin exosite-2 residues are CritiCal for the heparin-Catalyzed inhibition by PCI and AT, (ii) PCI and AT are different in their thrombin-TM inhibition properties, and (iii) PCI has a distinCt advantage over AT in the regulation of the aCtivity of thrombin-TM.
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Is Protein C Inhibitor antithrombotiC and proteCtive in pulmonary hypertension
Journal of Thrombosis and Haemostasis, 2006Co-Authors: Lea M. Beaulieu, Frank C ChurchAbstract:In this issue, Nishii et al. [1] from Mie University in Japan provide interesting evidenCe that miCe over-expressing Protein C Inhibitor (PCI) are proteCted from monoCrotaline-induCed pulmonary hypertension, due in part to the ability of PCI to inhibit thrombin and down-regulate Coagulation. The transgeniC mouse over-expressing PCI has enhanCed lung seCretion of PCI, and the CliniCal, bioChemiCal and pathologiCal parameters show a reduCtion in pulmonary hypertension in this experimental model. The pathophysiologiCal role of the human plasma serine protease Inhibitor (serpin), Protein C Inhibitor (PCI, also named plasminogen aCtivator Inhibitor-3, systematiC name of SERPINA5) has remained elusive sinCe its desCription in the 1980s [2,3]. From its name, PCI is an Inhibitor of aCtivated Protein C (APC) [4–14]. However, PCI also inhibits thrombin (IIa) [8–10,12], faCtor Xa and faCtor XIa [8,11–14], kallikrein [8,10,12,15–17], urokinase-plasminogen aCtivator and tissue-plasminogen aCtivator (t-PA) [8,18–23], aCrosin [24–26], prostate speCifiC antigen [27,28], and remarkably, thrombin-thrombomodulin [29–31], whiCh is responsible for generating APC (Fig. 1). The broad protease Inhibitory profile of PCI has led many to postulate both speCifiC and generiC roles for this serpin. To further CompliCate matters, the tissue distribution of PCI in humans Compared with miCe is quite different. Humans show a broad tissue expression pattern for PCI, inCluding the liver, kidney, panCreas, prostate, testes and ovaries [32–37]. Thus, this explains why human PCI (hPCI) is found not only in CirCulating blood, but also in urine, saliva, amniotiC fluid, milk, tears and other body fluids [32]. In Contrast, the mouse and rat express PCI only in the reproduCtive organs and it is not found in the CirCulating blood [34,38–42]. Through the Creation of a PCI knoCkout mouse by homologous reCombination, one non-hemostatiC funCtion of PCI was determined [26]. Male PCI−/− miCe were infertile due to abnormal spermatogenesis Caused by loss of the Sertoli Cell barrier. Unopposed proteolytiC aCtivity in these miCe brought about the degradation of the Cell barrier [26]. Two transgeniC mouse models expressing hPCI have been developed. The first was desCribed by Wagenaar et al. [43], in whiCh hPCI was expressed in the liver and found in the CirCulation. The seCond hPCI transgeniC mouse was desCribed by Hayashi et al. [44] and it expressed hPCI not only in the liver, but also in the kidney, heart, brain, lung and reproduCtive organs. Fig. 1 Role of Protein C Inhibitor and other serpins (antithrombin, heparin CofaCtor II, and plasminogen aCtivator Inhibitor-1) in the regulation of serine proteases thrombin, aCtivated Protein C (APC), APC generation by thrombin-thrombomodulin, and tissue plasminogen ... ConCerning human health, the presenCe of PCI in various lung diseases has been desCribed [45]. Fujimoto et al. [45] reported that bronChoalveolar lavage fluid Contained inCreased amounts of both PCI and thrombin-aCtivatable fibrinolysis Inhibitor (TAFI) in patients with interstitial lung disease (ILD), partiCularly in patients with CryptogeniC-organizing pneumonia, Collagen vasCular disease-assoCiated ILD, and sarCoidosis. One explanation of their findings was that the levels of intra-alveolar PCI inhibited both APC aCtivity and aCtivation, whiCh Contributed to the pathogenesis of ILD. Therefore, a key question asked in the Current study by Nishii et al. [1] was ConCerning the Contribution of PCI to the pathogenesis of pulmonary hypertension. This study uses the hPCI over-expressing transgeniC mouse desCribed by Hayashi et al. [44] to begin to address this question regarding pulmonary hypertension, and also provides data on the physiologiCal funCtion of PCI (Fig. 1). Nishii et al. [1] treat miCe with monoCrotaline to induCe pulmonary hypertension. This murine model is representative of pulmonary hypertension Caused by a known etiology and not a seCondary ConsequenCe of CardiovasCular disease. Overall, hPCI reduCes the disease state in the mouse lung Compared with the wild-type mouse. The inCrease in pressure assoCiated with pulmonary hypertension is not seen in the hPCI over-expressing transgeniC miCe. Pulmonary hypertension also results in endothelium dysfunCtion. The vessels in the lungs are hyperCoagulant as a result of a deCrease in prostaglandin and nitriC oxide produCtion. Platelets beCome aCtivated and will adhere to the vessel wall. HyperCoagulability Can be assessed by measuring the formation of thrombin:AT (TAT) Complex. Although there is an inCrease of TAT Complex in the hPCI over-expressing transgeniC miCe when treated with monoCrotaline, this inCrease is signifiCantly less than that in wild-type animals. These results suggest that either there is a deCrease in thrombin produCtion that would reduCe TAT levels, or the inCreased presenCe of PCI is Competing with AT to inhibit thrombin, whiCh would also reduCe the TAT levels. Either way, the hPCI over-expressing transgeniC mouse does not exhibit an inCrease in aCtivation of Coagulation upon treatment with monoCrotaline. Although PCI Can be proCoagulant through its inhibition of the Protein C system of proteases, when hPCI is over-expressed in the mouse, its antiCoagulant funCtion is more prominent. Fibrinolysis is inCreased in miCe upon treatment with monoCrotaline, as indiCated by an inCrease in t-PA aCtivity. As PAI-1 levels are similar between the wild-type and hPCI over-expressing transgeniC miCe when treated with monoCrotaline, the only explanation for a deCrease in free t-PA and lowered fibrinolysis in the transgeniC miCe is the inCrease in hPCI. Pulmonary vasCular endothelium dysfunCtion also results in the release of various Cytokines, suCh as tumor neCrosis faCtor-alpha (TNF-α) and monoCyte ChemoattraCtant Protein-1 (MCP-1), and growth faCtors, suCh as platelet-derived growth faCtor (PDGF) and interleukin-13 (IL-13), that will promote inflammation and vasCular wall thiCkening. The monoCrotaline-treated wild-type miCe show an inCrease in all of these Protein levels. The hPCI over-expressing transgeniC miCe treated with monoCrotaline exhibit little to no Change in these same Proteins. Therefore, hPCI reduCes the endothelial dysfunCtion and inflammation assoCiated with pulmonary hypertension. Furthermore, measurements of the pulmonary arteries in the hPCI over-expressing transgeniC mouse show a smaller Change in vessel wall and lumen area upon monoCrotaline treatment. Overall, their results suggest that hPCI inhibits thrombin, a pro-inflammatory and pro-migratory faCtor, thus, reduCing the effeCts of monoCrotaline-induCed pulmonary hypertension. The data presented in this paper raise more questions than answers about the in vivo protease speCifiCity of PCI. Their results suggest that PCI exerts both anti-inflammatory and antiCoagulant aCtion by inhibiting thrombin, a known partiCipant in Coagulation, inflammation and tissue remodeling. This funCtion of PCI is more prominent than its role as an Inhibitor of APC. Inhibition of APC alone would result in an inCrease in Coagulation and inflammation, and a reduCtion of tissue remodeling, whiCh was not seen in the mouse model desCribed. PCI is also antifibrinolytiC through its inhibition of t-PA. Whether or not PCI Can be used therapeutiCally for treating pulmonary hypertension remains to be studied. With the aid of new ELISAs reported for PCI-protease Complexes [46–49], these tools Can provide answers regarding the role of PCI in Coagulation and inflammation.
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Protein C Inhibitor (plasminogen aCtivator Inhibitor-3) expression in the CWR22 prostate CanCer xenograft
Experimental and Molecular Pathology, 2005Co-Authors: Laura N. Glasscock, Sophie M. Réhault, Christopher W. Gregory, Scott Cooper, Tracy P. Jackson, Maureane Hoffman, Frank C ChurchAbstract:The serine protease Inhibitor (serpin) Protein C Inhibitor (PCI) has been found in the prostate and possibly is a marker to distinguish normal prostate, benign prostatiC hyperplasia, and prostate CanCer. In this study, we assessed PCI expression in normal, hyperplastiC, and malignant prostatiC tissues, prostate CanCer Cell lines, and the CWR22 prostate CanCer xenograft model that allowed us to study PCI expression and its regulation in response to androgens. By Northern blot, immunohistoChemistry, and in situ hybridization, we found that PCI was expressed in both benign and malignant prostate tissues. Protein C Inhibitor was expressed in both androgen-independent (PC-3) and androgen-dependent (LNCaP) prostate CanCer Cell lines. Furthermore, PCI was deteCted in all CWR22 tumor samples (androgen dependent, 6 days post-Castration, 12 days post-Castration followed by 72 h of testosterone treatment, and reCurrent CWR22 tumor), although expression of the mature forms of both prostate-speCifiC antigen (PSA) and its homolog, kallikrein 2 (hK2), was Clearly androgen-dependent. These results suggest that PCI expression is not regulated by androgens and that PCI is unlikely to be a tumor suppressor gene, but also that PCI may be involved in regulating key serine proteases involved in metastatiC prostate disease.
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CharaCterization of reCombinant human Protein C Inhibitor expressed in EsCheriChia Coli.
Biochimica et Biophysica Acta, 2005Co-Authors: Sophie M. Réhault, Margarethe Geiger, Scott Cooper, Margareta Zechmeister-machhart, Yolanda M. Fortenberry, Julia M. Malleier, Nikki M. Binz, Frank C ChurchAbstract:AbstraCt The serine protease Inhibitor (serpin) Protein C Inhibitor (PCI; also named plasminogen aCtivator Inhibitor-3) regulates serine proteases in hemostasis, fibrinolysis, and reproduCtion. The bioChemiCal aCtivity of PCI is not fully defined partly due to the laCk of a Convenient expression system for aCtive rPCI. Using pET-15b plasmid, Ni 2+ -Chelate and heparin-Sepharose affinity Chromatography steps, we desCribe here the expression, purifiCation and CharaCterization of wild-type reCombinant (wt-rPCI) and two inaCtive mutants, R354A (P1 residue) and T341R (P14 residue), expressed in EsCheriChia Coli . Wild-type rPCI, but not the two mutants, formed a stable bimoleCular Complex with thrombin, aCtivated Protein C and urokinase. In the absenCe of heparin, wt-rPCI-thrombin, -aCtivated Protein C, and -urokinase inhibition rates were 56.7, 3.4, and 2.3×10 4 M −1 min −1 , respeCtively, and the inhibition rates were aCCelerated 25-, 71-, and 265-fold in the presenCe of 10 μg/mL heparin for eaCh respeCtive inhibition reaCtion. The stoiChiometry of inhibition (SI) for wt-rPCI-thrombin was 2.0, whiCh is Comparable to plasma-derived PCI. The present report desCribes for the first time the expression and CharaCterization of reCombinant PCI in a baCterial expression system and demonstrates the feasibility of using this system to obtain adequate amounts of biologiCally aCtive rPCI for future struCture–funCtion studies.
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Assessment of the interaCtion between urokinase and reaCtive site mutants of Protein C Inhibitor.
Journal of Protein Chemistry, 1997Co-Authors: Tracy P. Jackson, Scott Cooper, Frank C ChurchAbstract:Urokinase-type plasminogen aCtivator (uPA) is a serine protease involved in periCellular proteolysis and tumor Cell metastasis via plasmin-mediated degradation of extraCellular matrix Proteins. Plasma uPA is inhibited by the serine protease Inhibitor Protein C Inhibitor (PCI) by the insertion of PCI's reaCtive site loop into the aCtive site of the protease. To better understand the struCtural aspeCts of this inhibition, 15 reaCtive-site mutants of reCombinant PCI (rPCI) were assayed for differenCes in uPA inhibition. These assays revealed that substitutions at the P1 Arg354 and P3 Thr352 sites of rPCI were detrimental to Inhibitory aCtivity, while P3′ Arg357 mutations had little effeCt upon the inhibition rate. However, replaCement of the P2 Phe353 with small residues like Ala and Gly inCreased the effeCtiveness of rPCI three- to four fold. To explain these altered rates of inhibition, a Computer-derived moleCular model of uPA was generated and doCked to a model of PCI to simulate Complex formation. The Changes made by mutagenesis were then reCreated in the model of uPA–PCI. In aCCordanCe with the kinetiC data, the poor performanCe of P3 variants is primarily attributable to Charge repulsion, while alleviation of steriC hindranCe at P2 produCes the observed inCrease in uPA inhibition. In the model, residues at P3′ interaCt with PCI rather than uPA, Consistent with P3′ variants demonstrating that little variation from wild-type aCtivity. Ultimately, this Combination of mutagenesis and moleCular modeling will further refine our understanding of the interaCtion between PCI and uPA.
Charles T Esmon - One of the best experts on this subject based on the ideXlab platform.
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moleCular basis of thrombin reCognition by Protein C Inhibitor revealed by the 1 6 a struCture of the heparin bridged Complex
Proceedings of the National Academy of Sciences of the United States of America, 2008Co-Authors: Wei Li, Ty E Adams, Charles T Esmon, Jyoti Nangalia, James A. HuntingtonAbstract:Protein C Inhibitor (PCI) is a serpin with many roles in biology, inCluding a dual role as pro- and antiCoagulant in blood. The protease speCifiCity and loCal funCtion of PCI depend on its interaCtion with CofaCtors suCh as heparin-like glyCosaminoglyCans (GAGs) and thrombomodulin (TM). Both CofaCtors signifiCantly inCrease the rate of thrombin inhibition, but GAGs serve to promote the antiCoagulant aCtivity of PCI, and TM promotes its proCoagulant funCtion. To gain insight into how PCI reCognition of thrombin is aided by these CofaCtors, we determined a CrystallographiC struCture of the MiChaelis Complex of PCI, thrombin, and heparin to 1.6 Å resolution. Thrombin interaCts with PCI in an unusual fashion that depends on the length of PCI's reaCtive Center loop (RCL) to align the heparin-binding sites of the two Proteins. The prinCipal exosite ContaCt is engendered by movement of thrombin's 60-loop in response to the unique P2 Phe of PCI. This meChanism of CommuniCation between the aCtive site of thrombin and its reCognition exosite is previously unCharaCterized and may relate to other thrombin substrate–CofaCtor interaCtions. The CofaCtor aCtivity of heparin thus depends on the formation of a heparin-bridged MiChaelis Complex and substrate-induCed exosite ContaCts. We also investigated the CofaCtor effeCt of TM, establishing that TM bridges PCI to thrombin through additional direCt interaCtions. A model of the PCI–thrombin–TM Complex was built and evaluated by mutagenesis and suggests distinCt binding sites for heparin and TM on PCI. These data signifiCantly improve our understanding of the CofaCtor-dependent roles of PCI in hemostasis.
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Protein C Inhibitor is a potent Inhibitor of the thrombin thrombomodulin Complex
Journal of Biological Chemistry, 1995Co-Authors: Alireza R Rezaie, Frank C Church, Scott Cooper, Charles T EsmonAbstract:AbstraCt Protein C Inhibitor (PCI), a plasma serine protease Inhibitor, inhibits several proteases inCluding the antiCoagulant enzyme, aCtivated Protein C (APC), and the Coagulation enzymes, thrombin and faCtor Xa. Previous studies have shown that thrombin and APC are inhibited at similar rates by PCI and that heparin aCCelerates PCI inhibition of both enzymes more than 20-fold. We now demonstrate that the thrombin-binding proteoglyCan, rabbit thrombomodulin, aCCelerates inhibition of thrombin by PCI ≈140-fold (k = 2.4 × 10 in the presenCe of TM Compared to 1.7 × 10M s in the absenCe of TM). Most of this effeCt is mediated by Protein-Protein interaCtions sinCe the aCtive fragment of TM Composed of epidermal growth faCtor-like domains 4-6 (TM 4-6) aCCelerates inhibition by PCI ≈59-fold (k = 1.0 × 10M s). The meChanism by whiCh TM alters reaCtivity with PCI appears to reside in part in an alteration of the S2 speCifiCity poCket. ReplaCing Phe with Pro at the P2 position in the reaCtive loop of PCI yields a mutant that inhibits thrombin better in the absenCe of TM (k = 6.3 × 10M s), but TM 4-6 enhanCes inhibition by this mutant ≈9-fold (k = 5.8 × 10M s) indiCating that TM alleviates the Inhibitory effeCt of the less favored Phe residue. These results indiCate that PCI is a potent Inhibitor of the Protein C antiCoagulant pathway at the levels of both zymogen aCtivation and enzyme inhibition.
Bernd R. Binder - One of the best experts on this subject based on the ideXlab platform.
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Regulation of Protein C Inhibitor (PCI) aCtivity by speCifiC oxidized and negatively Charged phospholipids
Blood, 2007Co-Authors: Julia M. Malleier, Bernd R. Binder, Ingrid Jerabek, Olga Oskolkova, Valery N. Bochkov, Barbora Sokolikova, Thomas Perkmann, Johannes M. Breuss, Margarethe GeigerAbstract: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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Binding of retinoiC aCid by the Inhibitory serpin Protein C Inhibitor
FEBS Journal, 2001Co-Authors: Ingrid Jerabek, Bernd R. Binder, Margareta Zechmeister-machhart, Margarethe GeigerAbstract: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.
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disruption of the Protein C Inhibitor gene results in impaired spermatogenesis and male infertility
Journal of Clinical Investigation, 2000Co-Authors: Pavel Uhrin, Anja Vales, Christian Schöfer, Mieke Dewerchin, Mario Hilpert, Peter Chrenek, Margareta Zechmeistermachhart, Gerhard Kronke, Peter Carmeliet, Bernd R. BinderAbstract: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.
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Synthesis and ultrastruCtural loCalization of Protein C Inhibitor in human platelets and megakaryoCytes.
Blood, 1999Co-Authors: Maria J. Prendes, Bernd R. Binder, Edith Bielek, Veronica A. Carroll, Margareta Zechmeister-machhart, Erika Vanyek-zavadil, Johannes Breuss, Margarethe GeigerAbstract: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 × 109 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 (Mr) 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 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.
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Protein C Inhibitor is Expressed in KeratinoCytes of Human Skin
Journal of Investigative Dermatology, 1999Co-Authors: Michael Krebs, Pavel Uhrin, Anja Vales, Maria J. Prendes-garcia, Johann Wojta, Margarethe Geiger, Bernd R. BinderAbstract: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.
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HepatoCyte growth faCtor aCtivator (HGFA): its regulation by Protein C Inhibitor
FEBS Journal, 2010Co-Authors: Koji SuzukiAbstract:Protein C Inhibitor (PCI; SERPINA5) is a plasma serine protease Inhibitor, and a potent Inhibitor of aCtivated Protein C (APC), whiCh plays a CritiCal role in the antiCoagulant Protein C pathway. ReCently, PCI was also found to form a Complex with the serine protease hepatoCyte growth faCtor aCtivator (HGFA), inhibiting the HGFA-Catalyzed aCtivation of the single-Chain hepatoCyte growth faCtor preCursor. In vivo studies using human PCI-transgeniC (hPCI-Tg) miCe, whiCh mimiC PCI expression in humans, showed that the regeneration rate of the liver after partial hepateCtomy was signifiCantly impaired as Compared with wild-type miCe. The deCreased liver regeneration in hPCI-Tg miCe was restored by pretreatment with antibody against human PCI. Furthermore, APC proteCted hepatiC nonparenChymal Cells from thrombin-induCed inflammation in vitro, suggesting that plasma PCI may inhibit the CytoproteCtive aCtion of APC on hepatiC Cells in hPCI-Tg miCe. It was shown that the levels of HGFA–PCI are inCreased in plasma of patients who have been subjeCted to hepateCtomy, as Compared with Complex levels in the plasma of normal individuals. Thus, PCI may play a role as a potent Inhibitor of HGFA and APC in plasma and/or at the sites of tissue injury in the regulation of tissue regeneration.
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The multi‐funCtional serpin, Protein C Inhibitor: beyond thrombosis and hemostasis
Journal of Thrombosis and Haemostasis, 2008Co-Authors: Koji SuzukiAbstract:Summary. Protein C Inhibitor (PCI) is a member of the serine protease Inhibitor (serpin) family. PCI was initially found to be an Inhibitor of aCtivated Protein C, and later shown to be a potent Inhibitor of blood Coagulation and fibrinolysis suCh as that mediated by urokinase type-plasminogen aCtivator. Therefore, the Protein Came to be known as plasminogen aCtivator Inhibitor-3. It also inhibits proteases involved in fertilization. PCI is broadly Conserved, and is found in human, rhesus monkey, Cow, rabbit, rat, mouse and ChiCken. The human PCI gene is loCated on Chromosome 14q32.1 in a Cluster of genes enCoding related serpins. Sp1- and AP2-binding sites in the 5′-flanking region aCt as promoter and enhanCer, respeCtively, for its expression in the liver. PCI mRNA is expressed in many organs in primates, but only in the reproduCtive organs in rodents. ReCent studies using transgeniC miCe expressing the human gene have suggested that PCI is also involved in regulation of lung remodeling, tissue regeneration, vasCular permeability, proteolysis in the kidney and tumor Cell invasion. A protease Inhibitor-independent aCtivity of PCI, the prevention of anti-angiogenesis and metastasis of tumor Cells, has also been observed. Thus, PCI is a unique multi-funCtional serpin playing diverse roles in the thrombosis and hemostasis in multiple organs and tissues of a variety of speCies.
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IntraCellular loCalization of Protein C Inhibitor (PCI) and urinary plasminogen aCtivator in renal tubular epithelial Cells from humans and human PCI gene transgeniC miCe
Histochemistry and Cell Biology, 2007Co-Authors: Zhenhu Song, Esteban C. Gabazza, Tatsuya Hayashi, Ning Ma, Yoshiki Sugimura, Koji SuzukiAbstract:Urinary plasminogen aCtivator (uPA) is a serine protease that plays important roles in various extraCellular proteolytiC proCesses. In humans, Protein C Inhibitor (PCI) is known to regulate the aCtivity of the serine proteases involved in blood Coagulation, wound healing, and tumor metastasis, whereas PCI is not present in murine plasma or tissues other than the reproduCtive tissues. The large amount of uPA–PCI Complexes found in human urine suggests that these Complexes are formed in the kidneys. In the present study, we performed immunofluoresCenCe double labeling and eleCtron miCrosCopiC immunoCytoChemistry using renal tissues from humans and human PCI gene transgeniC (PCI-TG) miCe. In human renal tissues, PCI and uPA ColoCalized in the Cytoplasm of renal proximal tubular epithelial Cells (RPTECs), and juxtaposition of PCI and uPA immunoreaCtive partiCles was deteCted in the miCrovilli and lysosomes in the RPTECs. The intraCellular distributions of PCI and uPA in the RPTECs from PCI-TG miCe were similar to those observed in human RPTECs. These findings hint at the physiologiCal roles of uPA and PCI in human kidneys, and also suggest that the PCI-TG miCe will be useful for evaluating the roles of PCI in human physiologiCal and pathologiCal Conditions.
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The interaCtion among Protein C Inhibitor, prostate-speCifiC antigen, and the semenogelin system.
Seminars in Thrombosis and Hemostasis, 2007Co-Authors: Koji Suzuki, Junji Nishioka, Hideaki Kise, Tatsuya HayashiAbstract:: The kallikrein-like serine protease, prostate-speCifiC antigen (PSA), is mixed in human seminal plasma with its Protein substrates semenogelin (Sg) -I, Sg-II, and Protein C Inhibitor (PCI), whiCh are produCed in seminal vesiCles. In the seminal plasma, PSA degrades Sg-I, and Sg-II, whiCh are major Components in insoluble Coagula, and PCI inhibits PSA by forming a PSA-PCI Complex. Digestion of seminal Coagula with PSA releases PCI and PSA-PCI Complex from the Coagula into a soluble phase, suggesting the presenCe of aCtive PCI within the Coagula. PCI forms a ternary Complex with PSA and Sg-II in the seminal plasma. The binding of Sg-II to PSA and PCI is influenCed by pH, ioniC strength, heparin, negatively Charged dextran sulfate, divalent Cations, and partiCularly by Zn 2 +. These observations suggest that binding of PCI to Sg in seminal vesiCles regulates the PSA-Catalyzed degradation of Sg in seminal plasma; the Complex formation among PCI, PSA, and Sg is modulated by several faCtors in seminal plasma.
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Protein C Inhibitor.
Methods in Enzymology, 2004Co-Authors: Koji SuzukiAbstract:Publisher Summary Protein C Inhibitor (PCI) is a heparin-dependent Inhibitor of aCtivated Protein C (APC), the most potent antiCoagulant enzyme. PCI is also present in the testis, epididymis glands, prostate, Graafian folliCle, and synovial fluids and is interestingly absent from the seminal plasma of patients with dysfunCtional seminal vesiCles. These findings suggest that PCI plays a role in the regulation of the reproduCtive system. The assay is based on the inhibition of APC aCtivity resulting from Complex formation during preinCubation with the Inhibitor. Although plasma Contains other Inhibitors that Can inaCtivate APC, suCh as α 1 -AT and perhaps α2-maCroglobulin, it Can be assayed beCause the Inhibitory rate of PCI for APC is speCifiCally aCCelerated by heparin or negatively Charged dextran sulfate. This Chapter disCusses struCtural homology of PCI with serpin superfamily Proteins. The PCI gene is 13.0 kilobase pairs long and Consists of five exons and four introns. This suggests that PCI branChed from a Common anCestral Protein involving these serpin superfamily Proteins.
