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Purification and preliminary characterization of Bothrops moojeni venom components active on haemostasis (Botmo Thesis) Inauguraldissertation zur Erlangung der Würde eines Doktors der Philosophie vorgelegt der Philosophisch-Naturwissenschaftlichen Fakultät der Universität Basel von Anna Maria Perchu ć aus Warschau, Polen Referent: Prof. Dr. Beat Ernst Korreferent: Prof. Dr. phil. Jürg Meier Basel, 2010 Genehmigt von der Philosophisch-Naturwissenschaftlichen Fakultät auf Antrag von Herrn Prof. Dr. Beat Ernst und Herrn Prof. Dr. phil. Jürg Meier Basel, den 16. September 2008 Prof. Dr. Eberhard Parlow Dekan Wissenschaft ist der gegenwärtige Stand unseres Irrtums Jakob Franz Kern (1897-1924) Anna Maria Perchuć – Botmo Thesis I Acknowledgements To my Doktorvater Prof. Dr. Beat Ernst for his patience, support and scientific advice. To Prof. Dr. phil. Jürg Meier (my Korreferent) and to Prof. Dr. Reto Brun for their input to my PhD exam. To Pentaphatm Ltd. for giving me the opportunity to work on the “Bothrops moojeni Venom Proteomics Project”. To Marianne and Bea for helpful discussions, scientific, practical and personal support, their enthusiasm and friendship. To Reto for giving me the encouragement and guidance and for coordinating the Botmo Project. To the whole Hämostase und Test-Kit-Entwicklung Team for their friendly and helpful cooperation. To Laure, Reto, Philou and the whole Atheris Team for their scientific assistance. To Marc and his Synthesis Team for their involvement in the Botmo Project, their help, support and sense of humour. To Uwe and Andre for the fruitful cooperation. To Andreas for his great assistance and many valuable suggestions. To Remo and Martin for their support in the cell culture lab. To Garth, Markus and Franco for reading the manuscript. To everyone else who gave me scientific guidance and helpful comments I offer my gratitude and thanks. You know who you are and so do I! On a personal level I must thank Bernadette and George for their superb support and the very special friendship, which I hope will last for long. II Anna Maria Perchuć – Botmo Thesis Also, my many dear Basler and Genfer Friends who gave me constant encouragement, real friendship and support. I must also acknowledge a particular debt of gratitude to my Sprüngli-Tante Danka (Ciotencja) and Bruno for their guidance and unfailing generosity. Finally, to my Parents and my Sister, I say with utmost sincerity ‘Thank you for making this possible!’. Anna Maria Perchuć – Botmo Thesis III Abstract For almost every factor involved in haemostasis there exists a venom protein that can mimic, activate or deactivate it. Many of these components are insensitive to the physiological and therapeutically used coagulation inhibitors and, because of their unique features, are applied as molecular tools for diagnosis and therapy of haemostatic disorders. Batroxobin, purified from the venoms of Bothrops moojeni and B. atrox snakes, commonly known under its trade names Reptilase® and Defibrase® is still widely used in pharmaceutical and diagnostic applications. However, the venom material needed for its production is not further analyzed, although it still contains a large number of potentially active compounds. The goal of this Botmo Thesis was the investigation of the crude B. moojeni venom for the identification of new active ingredients affecting haemostasis. Various protein purification methods, mass spectrometry and biocomputing, as well as newly developed coagulation screening tools were applied. B. moojeni crude venom and its fractions were used as model substances for the development of different screening tools applying mass spectrometry (MS) technology. The complexity of the venom provides a good selectivity assessment for the developed methods. By means of the novel MS approaches, 15 new sequences of Bradykinin-potentiating peptides (BPPs) were revealed, as well as the presence of acetylcholinesterase inhibiting venom component. To our knowledge, it is the first time acetylcholinesterase inhibiting properties were described in the venom of B. moojeni snake. Further, two novel Lys49 phospholipases A2 (PLA2) have been purified and fully sequenced, showing a characteristic sequence in the C-terminal region known in the literature to interact with heparin. Both PLA2s were able to interact in vitro with unfractionated heparin (UFH) and low molecular weight heparin (LMWH) neutralizing their anticoagulant properties. The revealed sequences, possessing the LMWH neutralization properties, may be of medical interest, because there is no LMWH antidote used in clinical applications. Moreover, a procoagulant protein resembling the coagulation FVIIa could be identified in the B. moojeni venom. Such activity has never been reported for any snake venom until now and further investigations would be of medical interest. IV Anna Maria Perchuć – Botmo Thesis In addition a new method was developed for the identification of active ingredients acting on blood platelets. It can be used for screening complex protein mixtures, such as B. moojeni venom, containing platelet activating prothrombin activators or thrombin like-enzymes. By means of this methodology, a platelet aggregation activator and platelet antagonist with broad inhibitory activity, which is most likely a disintegrin, could be identified. Up to now, it could be shown that B. moojeni venom is a highly complex mixture of active ingredients and still remains a huge “source of undiscovered potential”. Further purifications and characterization steps are needed in order to get a complete activity profile of the venom, also in fields other than haemostasis. Nevertheless, in the framework of this Botmo Thesis, some active ingredients, also such of medical interest, were identified. Anna Maria Perchuć – Botmo Thesis V Preface The work described in this thesis (Botmo Thesis) was conducted in the framework of “Bothrops moojeni venom proteomics” project under the supervision of Prof. Dr. Beat Ernst, Institute of Molecular Pharmacy at the University of Basel. It was performed at Pentapharm Ltd. in Aesch under the guidance of Dr. Marianne Wilmer and Dr. Reto Schöni. Parts of the Botmo Thesis have already been published in peer reviewed journals or will be submitted for publication. These manuscripts are presented in the corresponding paragraphs the way they were prepared for submission to the corresponding journals. The posters are included in Appendix. Manuscripts published or prepared: • Liesener A., Perchuc A.M., Schöni R., Wilmer M. and Karst U. “Screening for proteolytic activities in snake venom by means of a multiplexing electrospray ionization mass spectrometry assay scheme.” Rapid Commun Mass Spectrom. 2005; 19 (20): 2923-8. • Perchuc A.M., Menin L., Stöcklin R., Bühler B. and Schöni R. “The Potential of Bothrops moojeni Venom in the Field of Hemostasis. Established Use and New Insights.” Pathophysiol Haemost Thromb. 2005; 34: 241-245 • Liesener A., Perchuc A.M., Schöni R., Schebb N.H., Wilmer M. and Karst U. “Screening of acetylcholinesterase inhibitors in snake venom by electrospray mass spectrometry.” Pure Appl. Chem. 2007; 79: 2339-2349 • Menin L., Perchuc A.M., Favreau P., Perret F., Schöni R. and Stöcklin R. “Precursor ion mass spectra for efficient screening of Pyroglutamate and Bradykinin potentiating peptide (BPP)-type substances in Bothrops moojeni snake venom using liquid chromatography/tandem mass spectrometry.” Toxicon 2008 Jun 1; 51 (7): 1288-302 • Perchuc A.M., Menin L., Favreau P., Bühler B., Schöni R., Wilmer M. Ernst B. and Stöcklin R. “Isolation and characterization of two new Lys49 PLA2s with heparin neutralizing properties from Bothrops moojeni snake venom.” In preparation for Toxicon VI Anna Maria Perchuć – Botmo Thesis • Ritz V., Bühler B., Zaborosch Ch., Wilmer M., Ernst B. and Perchuc A.M. “A protease possessing blood coagulation factor VIIa-like activity isolated from venom of Bothrops moojeni snake.” In preparation for Thrombosis Research • Demler C., Bühler B., Menin L., Stöcklin R., Wilmer M., Ernst B. and Perchuc A.M. “Platelet-active substances in the venom of Bothrops moojeni snake – a novel evaluation method using whole blood aggregometry.“ In preparation for Platelets • Perchuc A.M. and Wilmer M. “Schlangengifte” in Hämostaseologie 2nd Edition In press Parts of this work were presented at conferences: Oral presentations: • Perchuc A.M., Menin L., Stöcklin R., Bühler B. and Schöni R. “The Potential of Bothrops moojeni Venom in the Field of Hemostasis. Established Use and New Insights.” 3rd International Conference on Exogenous Factors Affecting Thrombosis and Hemostasis (EFATH 2005); Sydney, Australia; August 13th- 14th, 2005 • Perchuc A.M., Menin L., Favreau P., Bühler B., Bulet P., Schöni R. and Stöcklin R. “Structural and heparin-binding properties of two Bothrops moojeni phospholipases A2 and their derived C-terminal synthetic fragments.” NP2D - Natural Peptides to Drug; Zermatt, Switzerland; April 18th-21st, 2006 • Perchuc A.M., Menin L., Favreau P., Bühler B., Bulet P., Schöni R. and Stöcklin R. “Heparin-neutralizing properties of two novel Lys49 PLA2 variants from the Bothrops moojeni venom.” 15th World Congress on Animal, Plant & Microbial Toxins; Glasgow, Scotland; July 23rd- 28th, 2006 Posters: • Perchuc A.M., Menin L., Stöcklin R., Bühler B. and Schöni R. “Bothrops moojeni Proteomics – A Second Look at the Venom.” 15th European Symposium on Animal, Plant and Microbial Toxins; Brdo Estate (Kranj), Slovenia; June 19th- 23rd, 2004 Anna Maria Perchuć – Botmo Thesis
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  • Enhancement of the Expression of Urokinase-Type Plasminogen Activator from PC-3 Human Prostate Cancer Cells by Thrombin'

    Enhancement of the Expression of Urokinase-Type Plasminogen Activator from PC-3 Human Prostate Cancer Cells by Thrombin'

    ICANCERRESEARCH54,3300-3304,June15,1@94I Enhancement of the Expression of Urokinase-Type Plasminogen Activator from PC-3 Human Prostate Cancer Cells by Thrombin' Etsuo Yoshida,2 Elaine N. Verrusio, Hisashi Mihara,2 Doyeun Oh, and Hau C. Kwaan3 Department of Medicine, Hematology/Oncology Division, Northwestern University Medical Schoo4 and VA Lakeside Medical Ce,Uer, Chicago, Illinois 6@11 [E. V., E. N. V., D. 0., H. C. K./, and Department ofPhysiology, Miyazaki Medical College, Miyazaki 889-16, Japan [H. M.J ABSTRACT extracellular matrix. This is supported by the fact that anti-uPA could reduce tumor invasion and metastatic ability (7). uPA has been also The presence of procoagulants and fibrin deposition have been dem found to have a mitogenic effect on several cell lines (8, 9). onstrated in malignant tumors. Although thrombin, a key enzyme in In view of these findings, it is possible that thrombin may affect coagulation, has other various biological functions, the significance of its tumor cell behavior by modulating the expression of the components presence in tumors is not known. We studied the effects of thrombin on of the plasminogen-plasmin system. In the present study, we focused the expression of urokinase-type plasminogen activator (uPA) which is on the effect of thrombin on uPA expression in cancer cells and known to play a role in tumor invasion, using a human prostate cancer cell line PC-3. Human a-thrombin added to cultures of PC-3 produced a examined three prostate cancer cell lines: PC-3, a high uPA producer; dose-dependent and time-dependent increased secretion of uPA that was DU-145, which is a low uPA producer; and LNCaP, a non-uPA greatest at 3—6h after exposure to thrombin.