Academic literature on the topic 'Cleaved serpin'
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Journal articles on the topic "Cleaved serpin"
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Pippel, Jan, E. Bartholomeus Kuettner, David Ulbricht, Jan Daberger, Stephan Schultz, John T. Heiker, and Norbert Sträter. "Crystal structure of cleaved vaspin (serpinA12)." Biological Chemistry 397, no. 2 (January 1, 2016): 111–23. http://dx.doi.org/10.1515/hsz-2015-0229.
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Abstract The adipokine vaspin (serpinA12) is mainly expressed in white adipose tissue and exhibits various beneficial effects on obesity-related processes. Kallikrein 7 is the only known target protease of vaspin and is inhibited by the classical serpin inhibitory mechanism involving a cleavage of the reactive center loop between P1 (M378) and P1′ (E379). Here, we present the X-ray structure of vaspin, cleaved between M378 and E379. We provide a comprehensive analysis of differences between the uncleaved and cleaved forms in the shutter, breach, and hinge regions with relation to common molecular features underlying the serpin inhibitory mode. Furthermore, we point out differences towards other serpins and provide novel data underlining the remarkable stability of vaspin. We speculate that the previously reported FKGx1Wx2x3 motif in the breach region may play a decisive role in determining the reactive center loop configuration in the native vaspin state and might contribute to the high thermostability of vaspin. Thus, this structure may provide a basis for future mutational studies.
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Patston, PA, RL Medcalf, Y. Kourteva, and M. Schapira. "C1-inhibitor-serine proteinase complexes and the biosynthesis of C1- inhibitor by Hep G2 and U 937 cells." Blood 82, no. 11 (December 1, 1993): 3371–79. http://dx.doi.org/10.1182/blood.v82.11.3371.3371.
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Abstract The biosynthesis of the serpin alpha 1-proteinase inhibitor is regulated by a feedback mechanism whereby complexes between alpha 1- proteinase inhibitor and serine proteinases bind to liver cells and monocytes, a reaction that activates alpha 1-proteinase-inhibitor gene transcription. Such a mechanism may form the basis for the development of new therapeutic strategies for serpin deficiency states with reduced levels of otherwise normally functioning serpins. This issue was addressed for C1-inhibitor, the missing serpin in hereditary angioedema. C1-inhibitor biosynthesis by Hep G2 hepatoma cells was assessed by enzyme-linked immunosorbant assay, by metabolic labeling followed by immunoprecipitation, and by Northern blotting. C1-inhibitor biosynthesis was stimulated by gamma-interferon (100 U/mL) but not by cell exposure to C1-inhibitor-kallikrein (1 mumol/L), C1-inhibitor-C1s (1 mumol/L), and C1-inhibitor-plasmin complexes (1 mumol/L) or to reactive site-cleaved C1-inhibitor (1 mumol/L). Moreover, radioiodinated C1s-C1-inhibitor complex did not bind to Hep G2 cells. C1-inhibitor-kallikrein complex was also without effect on C1-inhibitor mRNA in U 937 cells. Therefore, the proposed mechanism, by which serpin- enzyme complex or reactive site-cleaved serpin binding to a specific receptor provides a signal for the stimulation of the biosynthesis of that serpin, is not operative for the biosynthesis of C1-inhibitor by Hep G2 or U 937 cells.
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Patston, PA, RL Medcalf, Y. Kourteva, and M. Schapira. "C1-inhibitor-serine proteinase complexes and the biosynthesis of C1- inhibitor by Hep G2 and U 937 cells." Blood 82, no. 11 (December 1, 1993): 3371–79. http://dx.doi.org/10.1182/blood.v82.11.3371.bloodjournal82113371.
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The biosynthesis of the serpin alpha 1-proteinase inhibitor is regulated by a feedback mechanism whereby complexes between alpha 1- proteinase inhibitor and serine proteinases bind to liver cells and monocytes, a reaction that activates alpha 1-proteinase-inhibitor gene transcription. Such a mechanism may form the basis for the development of new therapeutic strategies for serpin deficiency states with reduced levels of otherwise normally functioning serpins. This issue was addressed for C1-inhibitor, the missing serpin in hereditary angioedema. C1-inhibitor biosynthesis by Hep G2 hepatoma cells was assessed by enzyme-linked immunosorbant assay, by metabolic labeling followed by immunoprecipitation, and by Northern blotting. C1-inhibitor biosynthesis was stimulated by gamma-interferon (100 U/mL) but not by cell exposure to C1-inhibitor-kallikrein (1 mumol/L), C1-inhibitor-C1s (1 mumol/L), and C1-inhibitor-plasmin complexes (1 mumol/L) or to reactive site-cleaved C1-inhibitor (1 mumol/L). Moreover, radioiodinated C1s-C1-inhibitor complex did not bind to Hep G2 cells. C1-inhibitor-kallikrein complex was also without effect on C1-inhibitor mRNA in U 937 cells. Therefore, the proposed mechanism, by which serpin- enzyme complex or reactive site-cleaved serpin binding to a specific receptor provides a signal for the stimulation of the biosynthesis of that serpin, is not operative for the biosynthesis of C1-inhibitor by Hep G2 or U 937 cells.
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Kousted, Tina, Karsten Skjoedt, Steen Petersen, Claus Koch, Lars Vitved, Maja Sochalska, Céline Lacroix, et al. "Three monoclonal antibodies against the serpin protease nexin-1 prevent protease translocation." Thrombosis and Haemostasis 111, no. 01 (2014): 29–40. http://dx.doi.org/10.1160/th13-04-0340.
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SummaryProtease nexin-1 (PN-1) belongs to the serpin family and is an inhibitor of thrombin, plasmin, urokinase-type plasminogen activator, and matriptase. Recent studies have suggested PN-1 to play important roles in vascular-, neuro-, and tumour-biology. The serpin inhibitory mechanism consists of the serpin presenting its so-called reactive centre loop as a substrate to its target protease, resulting in a covalent complex with the inactivated enzyme. Previously, three mechanisms have been proposed for the inactivation of serpins by monoclonal antibodies: steric blockage of protease recognition, conversion to an inactive conformation or induction of serpin substrate behaviour. Until now, no inhibitory antibodies against PN-1 have been thoroughly characterised. Here we report the development of three monoclonal antibodies binding specifically and with high affinity to human PN-1. The antibodies all abolish the protease inhibitory activity of PN-1. In the presence of the antibodies, PN-1 does not form a complex with its target proteases, but is recovered in a reactive centre cleaved form. Using site-directed mutagenesis, we mapped the three overlapping epitopes to an area spanning the gap between the loop connecting α-helix F with β-strand 3A and the loop connecting α-helix A with β-strand 1B. We conclude that antibody binding causes a direct blockage of the final critical step of protease translocation, resulting in abortive inhibition and premature release of reactive centre cleaved PN-1. These new antibodies will provide a powerful tool to study the in vivo role of PN-1’s protease inhibitory activity.
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Zhang, Weiqing, Richard Swanson, Gonzalo Izaguirre, Yan Xiong, Lester F. Lau, and Steven T. Olson. "The heparin-binding site of antithrombin is crucial for antiangiogenic activity." Blood 106, no. 5 (September 1, 2005): 1621–28. http://dx.doi.org/10.1182/blood-2005-02-0547.
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Abstract The heparin-binding site of antithrombin is shown here to play a crucial role in mediating the antiangiogenic activity of conformationally altered cleaved and latent forms of the serpin. Blocking the heparin-binding site of cleaved or latent antithrombin by complexation with a high-affinity heparin pentasaccharide abolished the serpin's ability to inhibit proliferation, migration, capillary-like tube formation, basic fibroblast growth factor (bFGF) signaling, and perlecan gene expression in bFGF-stimulated human umbilical vein endothelial cells. Mutation of key heparin binding residues, when combined with modifications of Asn-linked carbohydrate chains near the heparin-binding site, also could abrogate the anti-proliferative activity of the cleaved serpin. Surprisingly, mutation of Lys114, which blocks anticoagulant activation of antithrombin by heparin, caused the native protein to acquire antiproliferative activity without the need for conformational change. Together, these results indicate that the heparin-binding site of antithrombin is of crucial importance for mediating the serpin's antiangiogenic activity and that heparin activation of native antithrombin constitutes an antiangiogenic switch that is responsible for turning off the antiangiogenic activity of the native serpin.
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O'Reilly, M. S. "Antiangiogenic Activity of the Cleaved Conformation of the Serpin Antithrombin." Science 285, no. 5435 (September 17, 1999): 1926–28. http://dx.doi.org/10.1126/science.285.5435.1926.
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Wells, Michael, William Sheffield, and Morris Blajchman. "The Clearance of Thrombin-antithrombin and Related Serpin-enzyme Complexes from the Circulation: Role of Various Hepatocyte Receptors." Thrombosis and Haemostasis 81, no. 03 (1999): 325–37. http://dx.doi.org/10.1055/s-0037-1614472.
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IntroductionPeptide bond cleavage can herald the end of a protein’s active life, or its transformation from an inactive precursor to an active enzyme. If the newly activated protein is a proteinase, even a highly specific proteinase, then its activity must be regulated in order that unbridled cleavage and damage to the host organism do not ensue. Such regulation for many of the key serine proteinases of the coagulation, fibrinolytic, complement, and inflammatory pathways is provided by the inhibitory proteins of the serpin family.The serpins are a large family of over 100 proteins (1). Many are plasma proteins such as antithrombin (AT), α1-proteinase inhibitor (α-PI), α1-antichymotrypsin (α-AC), heparin cofactor II (HCII), plasminogen activator inhibitors (PAI) I and II, α2-antiplasmin (α2-AP) and proteinase nexin I (PN-1). While some serpins are readily recognizable as family members, solely by virtue of homology, others have been characterized in detail, particularly those that are suicide inhibitors of their cognate proteinases; enzymes that recognize and attack the reactive centre loop of the inhibitory serpins. The resulting serpin-enzyme complex (SEC) is comprised of the inhibitor, which is irreversibly inactivated by virtue of the cleavage of its reactive centre peptide bond, and the enzyme, which is reversibly inactivated by the formation of an acyl ester linkage between its active site serine and a serpin side chain. Thus, a stable, covalent, and stoichiometric complex resistant to denaturation is formed (2, 3).The reversible nature of the proteinase’s inactivation in the SEC means that while substantial regulation of the proteinase has been achieved, the organism has only prolonged the inevitable by forming the SEC. Because the SEC is only kinetically but not thermodynamically stable, given sufficient time it will break down, releasing cleaved serpin and active enzyme (4, 5). To prevent this, receptor-mediated mechanisms have evolved to effectively remove SECs from the circulation. Since the initial studies of Ohlsson, who investigated the clearance of α-PI-trypsin complexes in the circulation of dogs (6, 7), a large body of evidence has accumulated to indicate that SECs are cleared from the circulation more rapidly than their constituent serpins. This accelerated clearance seals the fate of the serpin-complexed proteinases, and prevents their release from SECs by sequestering the SECs inside cells, where they are catabolized. In this article, we review the available data with respect to the mechanisms involved in SEC removal from the circulation. Specifically, we address those proteins or molecules that have been reported to act as cellular receptors for SEC removal, and propose a model for SEC removal which includes several of the available candidate receptors. Where possible, we have focussed on the thrombin-antithrombin (TAT) complex, both because of our laboratory’s longstanding interest in antithrombin, and because of thrombin’s key role in haemostasis and thrombosis (8).
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Ellisdon, Andrew M., Qingwei Zhang, Michelle A. Henstridge, Travis K. Johnson, Coral G. Warr, Ruby HP Law, and James C. Whisstock. "High resolution structure of cleaved Serpin 42 Da from Drosophila melanogaster." BMC Structural Biology 14, no. 1 (2014): 14. http://dx.doi.org/10.1186/1472-6807-14-14.
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Onda, Maki, Kazuyo Nakatani, Sayaka Takehara, Mika Nishiyama, Nobuyuki Takahashi, and Masaaki Hirose. "Cleaved Serpin Refolds into the Relaxed State via a Stressed Conformer." Journal of Biological Chemistry 283, no. 25 (April 7, 2008): 17568–78. http://dx.doi.org/10.1074/jbc.m709262200.
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Hejgaard, Jørn, William A. Laing, Salla Marttila, Andrew P. Gleave, and Thomas H. Roberts. "Serpins in fruit and vegetative tissues of apple (Malus domestica): expression of four serpins with distinct reactive centres and characterisation of a major inhibitory seed form, MdZ1b." Functional Plant Biology 32, no. 6 (2005): 517. http://dx.doi.org/10.1071/fp04220.
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Most serpins irreversibly inhibit serine proteinases of the chymotrypsin family using a suicide-substrate-based mechanism. Serpins are present in all domains of life, but physiological functions in the plant kingdom are yet to be elucidated. Inhibitory properties of many abundant cereal grain serpins are well characterised, but serpins have not been identified in eudicot seeds. In apple (Malus domestica Borkh.), the origin of 88 serpin expressed sequence tags (ESTs) identified among 160 000 ESTs from 30 cultivar-, tissue- and time-specific libraries showed that serpin genes are expressed in a wide variety of tissues, including developing and mature fruits, seeds and vegetative buds as well as developing, mature and senescing leaves. Analysis of 46 sequences, most full-length, identified serpins with four distinct reactive centres belonging to two subfamilies (MdZ1 and MdZ2) with ~85% amino acid sequence identity. MdZ1 included three molecular forms with identical reactive centre loop (RCL) sequences except for three different, but related, residues at P2 (Asp, Asn or Glu). A major seed serpin, MdZ1b, with P2–P1′ Glu–Arg–Arg was purified from decorticated seeds and characterised kinetically. MdZ1b was a fast inhibitor of bovine and porcine trypsin (second-order association rate constant k a ~4 × 106 m –1 s–1 and stoichiometry of inhibition SI = 1). Human plasmin and urokinase-type plasminogen activator (u-PA), but not thrombin, were inhibited at lower rates (k a ~104 m –1 s–1). Chymotrypsin was inhibited at the same site (k a~4 × 103 m –1 s–1), but a significant part of MdZ1b was cleaved as substrate (SI > 2). Unexpectedly, the MdZ1b-trypsin complex was relatively short-lived with a first-order dissociation rate constant k d in the order of 10−4 s–1. The bulk of mature seed MdZ1b was localised to the cotyledons. The content of MdZ1b in ripe apples was 5–26 µg per seed, whereas MdZ1b could not be detected in the cortex or skin. Localisation and inhibitory specificity of serpins in monocot and eudicot plants are compared and putative functions are discussed.
Dissertations / Theses on the topic "Cleaved serpin"
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Pippel, Jan, E. Bartholomeus Kuettner, David Ulbricht, Jan Daberger, Stephan Schultz, John T. Heiker, and Norbert Sträter. "Crystal structure of cleaved vaspin (serpinA12)." De Gruyter, 2016. https://ul.qucosa.de/id/qucosa%3A33438.
Full textAbstract:
The adipokine vaspin (serpinA12) is mainly expressed in white adipose tissue and exhibits various beneficial effects on obesity-related processes. Kallikrein 7 is the only known target protease of vaspin and is inhibited by the classical serpin inhibitory mechanism involving a cleavage of the reactive center loop between P1 (M378) and P1′ (E379). Here, we present the X-ray structure of vaspin, cleaved between M378 and E379. We provide a comprehensive analysis of differences between the uncleaved and cleaved forms in the shutter, breach, and hinge regions with relation to common molecular features underlying the serpin inhibitory mode. Furthermore, we point out differences towards other serpins and provide novel data underlining the remarkable stability of vaspin. We speculate that the previously reported FKGx1Wx2x3 motif in the breach region may play a decisive role in determining the reactive center loop configuration in the native vaspin state and might contribute to the high thermostability of vaspin. Thus, this structure may provide a basis for future mutational studies.
Book chapters on the topic "Cleaved serpin"
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Jamil, George, and Sthefan Gabriel Berwanger. "Choosing a Business Model." In Advances in Business Strategy and Competitive Advantage, 1–20. IGI Global, 2019. http://dx.doi.org/10.4018/978-1-5225-7265-7.ch001.
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This chapter discusses the critical choice for a business manager, when a business model must be considered for its posterior design and implementation. Different modalities are available today and adaptations or changes are completely admissible, as competition evolves. A solid conceptualization of business models is studied, and some typical initiatives analyzed, observing their potentialities, requirements, and risks. As a market solution, several considerations are proposed at the end, regarding BM adoption in real cases, serving as a basic motivation for a clearer decision process to be pursued by business readers. This chapter contributions reach both scholars, researchers, managers, stakeholders and investors on increasing the comprehension about business models' propositions, characteristics and implementation aspects.
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Shaver, Lea. "Serving All Languages." In Ending Book Hunger, 46–63. Yale University Press, 2020. http://dx.doi.org/10.12987/yale/9780300226003.003.0004.
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This chapter clarifies how English is the most widely studied foreign language in the world according to David Crystal. Since World War II, it has emerged as the dominant language of global commerce and culture. The chapter emphasizes that being fluent in English greatly expands one's reading options. English accounts for 80 percent of the e-book titles available on Amazon.com, 80 percent of academic journals, and more than half of all content on the Internet. The chapter also discusses how several organizations are working to expand multilingual children's literature: the African Storybook Project, Books for Asia, the Global Book Alliance, Nabu.org, Worldreader, and myriad small publishers serving specific language communities. Their programs make clearer than ever before what it means to effectively promote the right to read. This requires the coordinated efforts of the United Nations, national governments, foundations, businesspeople, charities, publishers, authors, and illustrators.
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Klawans, Jonathan. "Innovation Asserted." In Heresy, Forgery, Novelty, 117–58. Oxford University Press, 2019. http://dx.doi.org/10.1093/oso/9780190062507.003.0004.
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This chapter traces the trajectory of early Christianity’s eventual embrace of the new, as articulated in the New Testament. Early sections probe the Gospels, illustrating how difficult it is to trace the word “new” back to the sayings of Jesus himself. Clearer evidence emerges in Paul, though he balances assertions of innovation with appeals to a prior covenant of faith. Other gospel traditions—above all, the Sermon on the Mount—seek to establish the novelty of Jesus’s teaching, a claim that sometimes entails denying earlier precedents for Jesus’s instruction. Going one important step further, the Letter to the Hebrews provides the earliest evidence for supersessionism, when the valorization of innovation is undergirded by a condemnation of the old. But an alternate discourse is also in evidence in texts like the Didache, which speak not of an old/new contrast but a timeless duality between good and evil.
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Stangl, Paul. "Wilhelmstrasse." In Risen from Ruins. Stanford University Press, 2018. http://dx.doi.org/10.11126/stanford/9781503603202.003.0006.
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Wilhelmstrasse evolved over several centuries from an upscale residential quarter to the center of German government. Its architecture was considered less culturally and artistically significant than Unter den Linden and more tainted by association with the Prussian-German state and the NSDAP in particular. In the late 1940s, Berlin planners intended for the area to continue serving as government center. They began to transform Wilhelmplatz into a larger square, Thälmannplatz, with a memorial to honor the fallen Communist leader. After the state founding of the GDR, Ulbricht and leading GDR planners shifted planning for the government center to Marx-Engels Square, leaving Wilhelmstrasse as an area of secondary concern. Socialist realism had limited impact here, as decisions over demolition and preservation hinged more on utilitarian spatial value than architectural merit or place-based meaning. Nazi structures were cleansed of iconography and preserved, while older residential structures were demolished.
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Wilken, Rowan. "Location Integration and Data Markets." In Cultural Economies of Locative Media, 66–88. Oxford University Press, 2019. http://dx.doi.org/10.1093/oso/9780190234911.003.0004.
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This chapter explores the still-evolving business and revenue models and geolocation data capture efforts of two commercial businesses now central to the contemporary settlement of locative media: Foursquare and Facebook. In Foursquare’s case, it underwent a quite dramatic series of transformations, evolving from a check-in based mobile social networking service, to a search and recommendation service, and now also serving as a firm offering location intelligence related enterprise services. In Facebook’s case, it set about further strengthening its grip on social media data markets by adding geolocation functionalities and geodata capture capabilities to its social networking operations. These two case studies provide a rich composite picture of the business ecologies of locational information. The aim in selecting these cases is to develop a clearer understanding of how both firms accrue location data and how they extract location value—that is, how this information is shared, harvested, valued, reused, and commodified.
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McNamara, John M., and Olof Leimar. "Future Perspectives." In Game Theory in Biology, 261–72. Oxford University Press, 2020. http://dx.doi.org/10.1093/oso/9780198815778.003.0011.
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Important areas for future developments of game theory in biology are put forward. These include several issues that are dealt with in the book, such as trait co-evolution, the consequences of variation, time structure, and the embedding of games into an ecological context and into the lives of individuals. New areas are also suggested, with Tinbergen’s four questions about the study of animal behaviour serving as a starting point. Game theory could be combined with phylogenetic analysis by examining how Evolutionarily Stable Strategies (ESSs) might change over evolutionary time, including major shifts between different ESSs, which might correspond to different species over evolutionary time. Concerning behavioural mechanisms in large worlds, the question of which mechanism parameters that are tuned by evolution is addressed, with a brief summary of the current knowledge about comparative cognition. The possible importance of limited flexibility in mechanisms is illustrated by outlining a model of a trust game. Finally, the potential for game theory to contribute to the study of cognitive development is discussed, using mutualism between cleaner fish and their client fish as an illustration.
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Moore, Cerwyn. "Friday Prayers and Sermon in the Grand Mosque of Mosul." In Al-Qaeda 2.0, edited by Donald Holbrook, 147–52. Oxford University Press, 2018. http://dx.doi.org/10.1093/oso/9780190856441.003.0010.
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On 4th July 2014 ‘Al-Furqan’, one of the media outlets used by ISIS, published ‘exclusive coverage’ of a Friday sermon delivered by the organization's leader Abu Bakr al-Baghdadi at the Grand Mosque in Mosul. It was with this confidence and sense of purpose, that the leadership of ISIS declared at the beginning of the holy month of Ramadan that it was establishing a worldwide Caliphate which would be called the Islamic State. Al-Baghdadi, the group announced, was to become Caliph Ibrahim, commander of the faithful. As he greeted worshipers at the Grand Mosque, he presented the Caliphate’s creation as a divine mission which all believers would be obliged to support. The split from Al-Qaeda could not be clearer or more public, the rejection of al-Zawahiri’s authority could not be more stark. The two separate jihadi entities were no longer just competitors locally, embroiled in the turmoil in Syria, but engaged in a global confrontation, vying for the hearts and minds of the same constituency.
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Soares, Rui M. G. Monge, Pedro Valério, Mariana Nabais, and António M. Monge Soares. "A Espada do Monte das Oliveiras (Serpa) - uma arma do Bronze Pleno do Sudoeste." In Arqueologia em Portugal 2020 - Estado da Questão - Textos, 1055–64. Associação dos Arqueólogos Portugueses e CITCEM, 2020. http://dx.doi.org/10.21747/978-989-8970-25-1/arqa76.
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In this paper, we present a detailed examination of a sword dating from the Southwestern Middle Bronze Age, which was found several years ago during farming activities near the town of Serpa, Portugal. The sword was apparently found out of an archaeological context. The finder of the sword, who kindly allowed us to study it, cleaned the artefact of its corrosion and kept it in good conditions. The sword is about 50 cm long and its handle show two rivets (another one is missing) which are kept housed in notches. The rivets’ heads are spherical caps covered with a golden leaf. The use of a p-EDXRF equipment allowed us to determine and quantify the elemental composition of the blade, as well as of the golden leaf covering the rivet heads. It was thus possible to establish that the blade was manufactured with arsenical copper, while the composition of the golden leaf refers to a natural alloy. Finally, the Monte das Oliveiras sword is compared with several other coeval examples of Southern Iberia weaponry. Its hilt design, namely the three peripheral notches, seems to be of an unique variant in swords, being relatively rare in similarly shaped weapons, such as daggers.
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"areas. In 1995 the Serbs in most of Croatia were ‘ethnically cleansed’, expelled by force to Bosnia or Serbia, while Bosnian Serbs captured two Muslim ‘safe’ areas. In September 1995 much of the area hitherto occupied by Serbs to the south of Banja Luka was captured by Bosnian and Croat forces with the (unrelated?) assistance of NATO air attacks FIGURE 6.3 Proposed improvements to existing transportation links in." In Geography of the World's Major Regions, 228. Routledge, 2003. http://dx.doi.org/10.4324/9780203429815-54.
Full textConference papers on the topic "Cleaved serpin"
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de Agostini, A., F. Barja, S. Carrel, P. C. Harpel, and M. Schapira. "C1 -INHIBITOR: STRUCTURE-ACTIVITY RELATIONSHIPS." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1642903.
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Cl-inhibitor [C1 -In] and other protease inhibitors of the serpin superfamily inactivate serine proteases by forming bimolecular enzyme-inhibitor complexes, a reaction that is associated with changes in the inhibitor conformation. To determine the significance of these changes, we have examined the influence of various treatments on the binding to C1-In of monoclonal antibody 4C3. This antibody was previously shown to bind to an epitope created during the reaction of C1-In with the Arg-specific protease plasma kallikrein [K]: the site for 4C3 was expressed on the K-C1-In complex, on C1-In cleaved at position Pi and released from K-C1-In [C1-In*], but not on unreacted C1-In. The binding of 4C3 to the various forms of C1-In was now measured by radioimmunoassay and Western blot. Following inactivation by C1-In of the Arg-specific enzymes factor XII active fragment [Xllf] or C1s, the binding site for 4C3 was detectable on XIIf-CI-In, C1s-C1-In and C1-In*. However, when K or Xllf were incubated with heat-inactivated C1-In, bo+h enzymes remained active, no complex was formed, and the site for 4C3 was not created. When C1-In was cleaved by neutrophil elastase [E] (a Met-orVal-specific protease that is not inhibited by C1 -In), the 1st cleavage product C1-In’ retained inhibitory activity (as shown by its ability to form a complex with Xllf) but did not bind 4C3. However, subsequent cleavage of C1-In’ by E at position P3 yielded C1-In’, a product which was inactive but bound 4C3. Thus, identical conformational changes of C1-In (as assessed by the emergence of the site for 4C3) are seen when Cl-In inactivates its target enzymes while being cleaved at Pi or when the inhibitor is catalytically inactivated by cleavage at P3. Therefore, these changes are necessary but not sufficient for observing enzyme inactivation.
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Ny, T., L. Hansson, and B. Åstedt. "ISOLATION OF cDNA FOR TYPE-2 PLASMINOGEN ACTIVATOR INHIBITOR." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1642855.
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The placental type plasminogen activator inhibitor (PAI-2) has been purified from extracts of human placenta and from a histiocytic lymphoma cell line. It is mainly an uPA inhibitor but it also inhibits the two-chain form of tPA.In order to determine the factors regulating PAI-2 gene expression and thereby clarify the physiological role of PAI-2 we have undertaken the molecular cloning of PAI-2 cDNA. A λgt11 expression library prepared from placental mRNA, was screened, immunologically using a monoclonal antibody probe developed against PAI-2 purified from human placenta. When 1.7×105 recombinant phages were screened six positive clones were obtained. Hybridization experiments and comparison of restriction enzyme cleavege pattern revealed that the DNA inserts of the six clones were, related. To identify the clones as coding for PAI-2, a lysogen made from one of them was induced, and the proteins were separated by SDS-PAGE. In immuno-blotting wxperiments the recombinant fusion protein and purified PAI-2 were recognized by the monoclonal antibody and a monospecific polyclonal antibody against PAI-2, revealing an immunological similarity. The nucleotide sequence of the largest cDNA was determined. It was found to code for a protein with extensive sequence homology with members of the serine protease inhibitor family (serpins) Alignment of the active center region with other serpins indicates that PAI-2 is an arg-serpin, as expected for an inhibitor of plasminogen activators.
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Teshima, G., R. Harris, R. Keck, A. Meunier, J. Burnier, and B. Keyt. "CHARACTERIZATION OF LIMITED PROTEOLYTIC DIGESTS OF TISSUE PLASMINOGEN ACTIVATOR." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1644375.
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Tissue plasminogen activator (tPA) is a single chain glycoprotein of 527 amino acids consisting of structural domains homologous to other plasma proteins ("finger","epidermal growth factor", "kringles" and "protease"). Unlike zymogens of other serine proteases, tPA in the single chain form (1-527), has amidolytic and fibrinolytic activity. However, the amidolytic activity is enhanced when tPA is cleaved by plasmin at the Arg275-Ile276 bond to yield the disulfide bonded two chain form. We used trypsin to study the structure and function of tPA by limited digestion. Aliquots of tPA (1 mg/ml) were digested at pH 7 with varying amounts of trypsin (1:10,000, 1:1000, 1:100 and 1:10; enzyme to substrate ratio). The dilute solutions of trypsin (1:10,000) were effective at completely converting one chain tPA to the two chain form, but little additional proteolysis was observed on SDS-PAGE. The proteolytic fragments of tPA were isolated by reduction and carboxymethylation (RCM), SDS gel electrophoresis and reversed phase HPLC. The RCM polypeptides were identified by amino acid composition and sequence. Specific antisera were prepared against peptide antigens of tPA including (1-27), (1-275), (276-527) and (502525). Immunoblotting experiments with the tryptic digests of tPA indicated that the region (1-275) is more susceptible to proteolytic attack than the protease (275-527). Specific cleavage sites were identified at positions 7, 10, 27 and 40. Partially digested tPA preparations were tested for enzymatic activity as determined by hydrolysis of the peptide substrate S-2288 or by clot lysis. Limited proteolysis at the amino terminus was correlated with significant loss of fibrinolytic . activity but minimal effect on the amidolytic activity. Increased tryptic digestion resulted in complete loss of amidolytic activity and significant reduction in antigenic activity as determined by polyclonal anti-tPA ELISA. These results areconsistent with the amino terminal "finger" domain being in part responsible for the fibrin-binding specificity of tPA. Limited tryptic digest of tPA, cleaves first at Arg-275, then subsequently cleaves the "finger" with associated loss of fibrinolytic activity.
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Thomas, Antony, Jifu Tan, Susan Perry, and Yaling Liu. "Characterization of Nanoparticle Distribution in Microcirculation Through a Microfluidics Device." In ASME 2011 Summer Bioengineering Conference. American Society of Mechanical Engineers, 2011. http://dx.doi.org/10.1115/sbc2011-53943.
Full textAbstract:
Various methods of targeted nano drug delivery have been developed in recent years to reduce side effects, toxicity, and lower drug doses [1]. The use of nanoparticles in drug delivery provides advantages in drug targeting, delivery and release along with serving in diagnosis and therapy [2]. Higher percentage of nanoparticle drug is uptaken by the target cells while larger drug particles are easily cleaned off by the human body. Nanoparticles also have large surface to volume ratio, which aids in attachment of many functional groups and thereby enhances targeting.
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Homes, W. E., H. R. Lijnen, L. Nelles, C. Kluft, and D. Collen. "AN ALANINE INSERTION IN α2-ANTIPLASMIN ‘ENSCHEDE’ ABOLISHES ITS PLASM IN INHIBITORY ACTIVITY." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1642897.
Full textAbstract:
Congenital deficiencies of the fibrinolytic inhibitor α2antiplasmin (α2AP) may result in bleeding disorders. An abnormal a AP (α2AP‘Enschede’) is known. 2 siblings with 3% functional activity and normal antigen level have parents with 50% activity and normal antigen. The protein interacts normally with the lysine-binding site(s) of plasmin(ogen) but does not inhibit plasmin irreversibly. α2AP Enschede is a plasmin substrate that like the normal protein releases a M 8,000 peptide upon reaction with plasmin. In the present study, Southern blot analysis, using an α2AP cDNA probe showed a restriction fragment length polymorphism within a small genomic DNA fragment of the Enschede family members. Cloning and sequencing of these fragments revealed a GCG inframe insertion that results in an alanine addition between amino acids 353 and 357, 7-10 positions NH -terminal to the reactive site PI residue, Arg364. This area is homologous to the A4 B-sheet of reactive site cleaved a -antitrypsin. Clones from each individual confirm the parents as true heterozygotes and the children as true homozygotes. A cloned genomic DNA sequence containing the insertion (V ) was exchanged for the normal sequence in a eukaryotic a AP expression plasmid. Recombinant α2AP‘Enschede’ (ra AfVAla) purified from the conditioned media of transfected Chinese Hamster Ovary Cells is analogous to plasma a α2AP‘Enschede’ with respect to interactions with plasmin and plasminogen. Preliminary analysis of the released Mr 8,000 recombinant peptide shows that its NH -terminus is the same as the peptide cleaved from normal a AP. Although ra α2APVAla does not inhibit plasmin irreversibly it does, however, act as a competitive inhibitor of hydrolysis of the chromogenic substrate S-2251 by plasmin.The K for this interaction is 25 nM. Thus, α2APAla retains a high affinity for the active center of plasmin. In conclusion, an Ala insertion near the reactive site of α2AP must have resulted in a structural perturbation that has abolished the plasmin inhibitory activity of a α2AP‘Enschede’. This variant may provide a model for further investigation of structure-function relationships in the serpins which determine the relative inhibitor vs. substrate properties.
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Dahiback, Bjorn, Ake Lundwall, Andreas Hillarp, Johan Malm, and Johan Stenflo. "STRUCTURE AND FUNCTION OF VITAMIN K-DEPENDENT PROTEIN S, a cofactor to activated protein C which also interacts with the complement protein C4b-binding protein." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1642960.
Full textAbstract:
Protein S is a single chain (Mr 75.000) plasma protein. It is a cofactor to activated protein C (APC) in the regulation of coagulation factors Va and Villa. It has high affinity for negatively charged phospolipids and it forms a 1:1 complex with APC on phospholipid surfaces, platelets and on endothelial cells. Patients with heterozygous protein S deficiency have a high incidence of thrombosis. Protein S is cleaved by thrombin, which leads to a loss of calcium binding sites and of APC cofactor activity. Protein S has two to three high affinity (KD 20uM) calcium binding sites - unrelated to the Gla-region - that are unaffected by the thrombin cleavage. In human plasma protein S (25 mg/liter) circulates in two forms; free (approx. 40%) and in a 1:1 noncovalent complex (KD 1× 10-7M) with the complement protein C4b-binding protein (C4BP). C4BP (Mr 570.000) is composed of seven identical 70 kDa subunits that are linked by disulfide bonds. When visualized by electron microscopy, C4BP has a spiderlike structure with the single protein S binding site located close to the central core and one C4b-binding site on each of the seven tentacles. When bound to C4BP, protein S looses its APC cofactor activity, whereas the function-of C4BP is not directly affected by the protein S binding. Chymotrypsin cleaves each of the seven C4BP subunits close to the central core which results in the liberation of multiple 48 kDa “tentacte” fragments and the formation of a 160 kDa central core fragment. We have successfully isolated a 160 kDa central core fragment with essentially intact protein S binding ability.The primary structure of both bovine and human protein S has been determined and found to contain 635 and 634 amino acids, respectively, with 82 % homology to each other. Four different regions were distinguished; the N-terminal Gla-domain (position 1-45) was followed by a region which has two thrombin-sensitive bonds positioned within a disulfide loop. Position 76 to 244 was occupied by four repeats homologous to the epidermal growth factor (EGF) precursor. In the first EGF-domain a modified aspartic acid was identified at position 95, B-hydroxaspartic acid (Hya), and in corresponding positions in the three following EGF-domains (positions 136,178 and 217) we found B-hydroxyasparagine (Hyn). Hyn has not previously been identified in proteins. The C-terminal half of protein S (from position 245) shows no homology to the serine proteases but instead to human Sexual Hormon Binding Globulin (SHBG)(see separate abstract). To study the structure-function relationship we made eighteen monoclonal antibodies to human protein S. The effects of the monoclonals on the C4BP-protein S interaction and on the APC cofactor activity were analysed. Eight of the antibodies were calciumdependent, four of these were against the Gla-domain, two against the thrombin sensitive portion and two against the region bearing the high affinity calcium binding sites. Three of the monoclonals were dependent on the presence of chelating agents, EDTA or EGTA, and were probably directed against the high affinity calcium binding region. Three other monoclonals inhibited the protein S-C4BP interaction. At present, efforts are made to localize the epitopes to gain information about functionally important regions of protein S.
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Edgington, T. S., J. H. Morrissey, and H. Fakhrai. "MOLECULAR CLONING OF HUMAN TISSUE FACTOR cDNA." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643740.
Full textAbstract:
Tissue factor (TF), the cell-surface receptor and allo-steric activator for factor Vll/VIIa, is important in hemostasis andinflammation. The TF apoprotein was purifiedfrom human brain using factor Vll-affinitychromatography and SDS gel electrophoresis,and was found to consist of a 47 kDa heavy chain plus a 12.5 kDa light chain. Approximately one-third of the heavy chain amino acid sequence was determined for four regions by microsequencing the intact protein and peptides derived from V8-protease digestion. A λgtll cDNA library, made from mRNA derived from the human fibroblastic cell line WI38,was screened with (a) affinity-purified rabbit antibodies to human tissue factor, and (b) a 45-mer oligonucleotide probe based on TF heavy chain amino acid sequence. Five overlapping cDNA clones were identifiedand sequenced which confirmed all four partial TF amino acid sequences. Together these clones span the entire heavy chain coding sequence as well as 5" and 3" nontranslated regions. The N-terminusof the TF heavy chain is preceded by an unusually long signal peptide which appears to be cleaved at alternative sites two amino acids apart. This results in two variants of TF heavy chains which differ slightly in length and amino-terminal sequence.The deduced protein sequence shows no major homology to known protein sequences. However,a relatively uncommon tripeptide sequence, Trp-Lys-Ser (WKS), appears three times in the TF heavy chain. This tripeptidesequence also occurs in HMW kininogen, factor VIII,von Willebrand"s factor andant ithrombin-III. Limited sequence similarity is observed in flanking sequences,andthis may indicate a possible functional domain for the recognition of members ofthe vitamin K-dependent serine protease famil.Supported by NIH grants HL-16411 andCA-41085.
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Tans, G., J. Rosing, M. Berrettini, B. Lammle, and J. H. Griffin. "AUTOACTIVATION OF HUMAN PLASMA PREKALLIKREIN." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1642898.
Full textAbstract:
Incubation of purified human plasma prekallikrein with sulfatides or dextran sulfate resulted in spontaneous activation of prekallikrein as judged by the appearance of amidolytic activity towards the chromogenic substrate H-D-pro-phe-arg-p-nitroanilide (S 2302). The time course of generation of amidolytic activity was sigmoidal with an apparent lag phase followed by a rapid activation until finally a plateau was reached. Soybean trypsin inhibitor completely blocked prekallikrein activation whereas corn, limabean and ovomucoid trypsin inhibitor did not. The Ki of the reversible inhibitor, benzamidine, for autoactivation (240 uM) was identical to the Ki of benzamidine for kallikrein. Thus, spontaneous prekallikrein activation and kallikrein showed the same specificity for a number of serine protease inhibitors, indicating that prekallikrein is activated by its own enzymatically active form, kallikrein. Immunoblotting analysis showed that, concomitant with the appearance of amidolytic activity, prekallikrein was cleaved. However, prekallikrein was not quantitatively converted into two-chain kallikrein since other polypeptide products were visible on the gels. This accounts for the observation that in amidolytic assays not all prekallikrein present in the reaction mixture was measured as active kallikrein. Kinetic analysis showed that prekallikrein activation can be described by a second-order reaction mechanism in which prekallikrein is activated kallikrein. The apparent second order rate constant was 27000 M-ls-1 (pH 7.2, 50 uM sulfatides, ionic strength 1=0.06, at 37°C). Autocatalytic prekallikrein activation was strongly dependent on the ionic strength, since there was a considerable decrease in the rate of the reaction at high salt concentrations. Our data support a prekallikrein autoactivation mechanism in which surface-bound kallikrein activates surface-bound prekallikrein. The rate constant of autoactivation is considerably lower than the rate constants reported for Factor Xlla dependent prekallikrein formation. Autocatalytic prekallikrein activation may, however, contribute to kallikrein formation during the initiating phase of contact activation.
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Govers-Riemslag, J. W. P., M. H. J. Knapen, G. Tans, R. F. A. Zwaal, and J. Rosing. "STRUCTURAL AND FUNCTIONAL PROPERTIES OF A PROTHROMBIN ACTIVATOR FROM THE VENOM OF BOTHROPS NEUWIDI." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1644321.
Full textAbstract:
The prothrombin activator from the venom of Bothrops neuwidi has been purified to homogeniety by gelfiltration on Sephadex G-100, ion-exchange chromatography on DEAE-Sephacel and metal-chelate affinity chromatography on an Epoxy-activated Sepharose 6B column loaded with ZnCl . The overall purification was about 200-fold, which indicates that the crude venom contains about 0.5 weight % of the prothrombin activator. The venom activator is a single chain protein with an apparent molecular weight of 60,000 dalton. It readily activated bovine prothrombin with a Km of 37.7 uM and a Vmax of 120 umoles prothrombin activated per min/mg of purified venom activator. Venom-catalyzed prothrombin activation was not accelerated by the accessory components of the prothrombinase complex i.e. phospholipids plus calcium-ions and Factor Va. The venom activator does not require added calcium-ions for the expression of its prothrombin-converting activity. Calcium ions do, however, affect the catalytic activity of the venom activator. At 2 mM CaCl there is a 2-fold increase of the rate of venom-catalyzed prothrombin activation. However, at higher CaCl concentrations there is a gradual decrease of the activity of the venom activator. Gelelectro-phoretic analysis of prothrombin activation indicated that the venom activator only cleaved the Arg 323-Ile 324 bond of bovine prothrombin since meizothrombin was the only product of prothrombin activation. The activator did not hydrolyze the chromogenic substrates S2222, S2337, S2238, S2366, S2302 or chromozym TH and its prothrombin converting activity was not inhibited by benza-midine, phenylmethylsulfonylfluoride, dansyl-glu-gly-arg-chloro-methylketone and soybean trypsin inhibitor. However, chelating agents such as EDTA, EGTA and o-phenanthroline strongly inhibited the enzymatic activity of the venom activator. The activity of chelator-treated venom activator could, however, be restored by the addition of an excess CaCl . These results indicate that the enzyme from Bothrops neuwidi does not belong to the serine proteases but has the properties of a metal proteinase. Thus, the activator differs remarkably from Factor Xa, but strongly resembles the prothrombin activator from the venom of Echis carinatus, both structurally and functionally.
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Suzuki, Koji, Yoshihiro Deyashiki, Junji Nishioka, Kazunori Toma, and Shuji Yamamoto. "THE INHIBITOR OF ACTIVATED PROTEIN C: STRUCTURE AND FUNCTION." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1642963.
Full textAbstract:
In the final step of protein C pathway, activated protein C (APC) is neutralized with a plasma inhibitor, termed protein C inhibitor (PCI). PCI was first described by Marlar and Griffin (1980) and then isolated from human plasma as a homogeneous form and characterized by the authors (1983). PCI is a single chain glycoprotein with M 57,000 and a plasma concentration of 5 ug/ml. Analysis of a cDNA nucleotide sequence has clarified that a precursor of human PCI consists of a mature protein of 387 amino acid residues (M 43,759) and a signal peptide of 19 amino acid residues. Only one cysteine residue is present in the entire protein as in α1antitrypsin (α1AT) and α1antichymotrypsin (α1ACT). Three Asn-X-Ser/Thr sequences and two Ser/Thr-X-X-Pro sequences are present as potential attachment sites of carbohydrate chains. Based on the amino acid sequence of the carboxyl-terminal peptide released from the inhibitor by APC digestion, the reactive site peptide bond of PCI was found to be Arg(354)-Ser(355). It is similar to the reactive sites of the other serine protease inhibitors which are located to their carboxyl-terminal Arg(393)-Ser (394), Met(358)-Ser(359) and Leu(358)-Ser(359) in antithrombin III, α1AT and α1ACT, respectively. The alignment of the amino acid sequence of PCI with heparin cofactor II, α1plasmin inhibitor, ovalbumin, angiotensinogen and the above noted plasma inhibitors showed that PCI is a member of serine protease inhibitor superfamily. PCI inhibits APC noncompetitively in a 1:1 stoichiometry and forms a covalent acyl-bond with a Ser residue in the active center of APC. The half life of APC in plasma approximately 30 min, which is rather slow compared with the other protease inhibitors. However, optimal concentrations of heparin, dextran sulfate and its derivatives potentiate the rate of inhibition 30-60 fold. PCI has Ki of 10-8m for APC, and can inhibit thrombin, Factor Xa, urokinase and tissue plasminogen activator as well in the presence of heparin or dextran sulfate, though the Ki for these enzymes is slightly higher. During the complex formation with APC, PCI is cleaved by the complexed APC to form a modified form with M 54,000. PCI is synthesized in several hepatoma cell lines and decreased in plasma of patients with liver cirrhosis. It is also decreased in patients with DIC or those during cardiopulmonary bypass in parallel with the decrease in protein C, suggesting that PCI participates in regulation of the protein C pathway in intravascular coagulation. Recently, we have obtained the recombinant PCI from COS-1 cells which were transfected with expression vector pSV2 containing the cDNA of PCI. The recombinant PCI had the same Mr and specific activity as the protein purified from plasma. It also had an affinity for heparin and dextran sulfate. Moreover, we have predicted a three dimentional structure of the proteolytically modified PCI with computer graphics based on its amino acid sequence homology with the modified α1AT whose structure had been elucidated with X-ray crystallography. All potential carbohydrate attachment sites were estimated to exist on the surface of the protein. Succesively we have constructed the interaction model between the intact PCI predicted from the modified form and the active center of APC which was simulated from that of trypsin. From the model, it was observed that the amino-group of Arg (354, PI site) of PCI could strongly interact with the carboxy1-group of Asp (88, SI site) of the heavy chain of APC at the base of the active center pocket of the enzyme.