Regulation of platelet-type 12-lipoxygenase activity in platelets with special consideration of gender effects [Elektronische Ressource] = Regulation der Aktivität der Platelet-type-12-Lipoxygenase in Thrombozyten unter besonderer Berücksichtigung geschlechtsspezifischer Einflüsse / vorgelegt von Ulrike Bühring

Max Reusner

Sujets

• Naturwissenschaften

Informations

• Publié par: eberhard_karls_universitat_tubingen
• Publié le: 01 janvier 2010
• Nombre de lectures: 35
• Langue: Deutsch
• Poids de l'ouvrage: 2 Mo

Extrait

Regulation of platelet-type 12-lipoxygenase activity
in platelets with special consideration of gender
effects


Regulation der Aktivität der platelet-type 12-
Lipoxygenase in Thrombozyten unter besonderer
Berücksichtigung geschlechtsspezifischer Einflüsse




Dissertation



der Fakultät für Chemie und Pharmazie
der Eberhard Karls Universität Tübingen


Zur Erlangung des Grades eines Doktors
der Naturwissenschaften



2010



vorgelegt von
Ulrike Bühring





























Tag der mündlichen Prüfung: 14. April 2010


Dekan: Prof. Dr. L. Wesemann
1. Berichterstatter: Prof. Dr. O. Werz
2. Berichterstatter: PD Dr. M. Düfer Danksagung
Danksagung
Die vorliegende Arbeit wurde unter der Leitung von Prof. Oliver Werz am
pharmazeutischen Institut der Eberhard Karls Universität Tübingen angefertigt.

Ich möchte mich herzlich bedanken bei

Prof. Dr. Oliver Werz für die Vergabe des Themas, die exzellente Betreuung, die
optimalen Arbeitsbedingungen für die Forschung, hervorragende und erfolgreiche
Zusammenarbeit mit fruchtbaren Diskussionen und die Unterstützung bei der
Weiterentwicklung neuer Ansätze.

PD Dr. Martina Düfer für die Erstellung des Zweitgutachtens.

Prof. Dr. Hinnak Northoff für die Bereitstellung des Zellmaterials.

Dr. Carlo Pergola für ein immer offenes Ohr für große und kleine Probleme und
stete Unterstützung bei der praktischen Umsetzung neuer Ideen.

Hanne Braun für ihre unkomplizierte Hilfe und Erfahrung im Kampf mit
organisatorischen und verwaltungstechnischen Belangen.

Bianca Jazzar für die exzellente Unterstützung bei der praktischen Durchführung
verschiedenster Untersuchungen und die hervorragende Sicherstellung eines
steten Stroms aller Labormaterialien.

Moritz Verhoff und Dr. Ulf Siemoneit für die unvergessliche Co-Betreuung des
DAB- Praktikums.

dem gesamten AK Werz für die wunderbare Atmosphäre bei der Arbeit und die
tolle Zeit in Tübingen. List of publications
List of publications/ presentations

“Licofelone suppresses prostaglandin E2 formation by interference with the
inducible microsomal prostaglandin E2 synthase-1.” Koeberle A, Siemoneit U,
Bühring U, Northoff H, Laufer S, Albrecht W, Werz O. J Pharmacol Exp Ther.
Sept 2008.

“Structural Optimization and Biological Evaluation of 2-Substituted 5-
Hydroxyindole-3-carboxylates as Potent Inhibitors of Human 5-Lipoxygenase”
Karg E-M, Luderer S, Pergola C, Bühring U, Rossi A, Northoff H, Sautebin L,
Troschütz R, Werz O, JMedChem, May 2009

“Sulindac sulfide suppresses 5-lipoxygenase at clinically relevant concentrations”
Steinbrink S, Pergola C, Buehring U, George S, Metzner J, Fischer A, Haefner A-
K, Wisniewska J, Geisslinger G, Werz O, Steinhilber D, Maier T, Cell Mol Life
Sci, November 2009

Coauthor „Hunnius“ Pharmaceutical lexicon, 10th edition, in press

„12-Lipoxygenase: Expression and Purification“, Bühring U, Poeckel D, Werz O,
poster presentation, DphG Jahrestagung 2007, Erlangen

“Novel potent inhibitors of human 5-lipoxygenase: Structural modification of the
hitherto existing 5-LO inhibitors of 2-amino-5-hydroxyindole-3-cyrboxylate
type”, Karg E-M, Jazzar B, Bühring U, Werz O, Troschütz R, poster presentation,
DphG Jahrestagung 2007, Erlangen

„Progesterone regulates 12-Lipoxygenase in human platelets”, Bühring U, Pergola
C, Verhoff M, Werz O, oral presentation, DphG- Jahrestagung 2009, Jena
Table of Contents
Table of Contents
Abbreviations IX
1. Introduction

1.1. Platelet physiology 1
1.2. Platelet intracellular kinase signalling cascades 4
1.3. Cytosolic phospholipase A (cPLA ) 8 2 2
1.4. Cyclooxygenase-1 (COX-1) 9
1.5. Platelet-type- 12-lipoxygenase: occurrence and (patho-) physiology 11
1.5.1. Human 12-lipoxygenases 11
1.5.2. Platelet-type 12-lipoxygenase (p12-LO) 13
1.6. Gender bias in inflammatory and cardiovascular disease 20
1.7. Progesterone and progesterone receptors 21
1.8. Cinnamyl-3,4-hydroxy-α-cyanocinnamate (CDC) 25
1.9. MK-886 27
1.10. Aim of the study 28
2. Materials and Methods

2.1. Materials 30
2.2. Methods 31
2.2.1. Isolation of human platelets from venous blood 31
2.2.2. Isolation of polymorphonuclear leukocytes (PMNL)
from venous blood 32
2.2.3. Preparation of platelet rich plasma (PRP) and plasma
from venous blood 32
2.2.4. Determination of p12-LO and COX-1 product formation
in intact human platelets 32
2.2.5. Determination of p12-LO and COX-1 activity in human
whole blood and PRP 33
2.2.6. Determination of p12-LO and COX-1 product formation
in platelet homogenates 34
2.2.7. Determination of cell-free COX-1/2 activity 34
2.2.8. Determination of TXB levels in human platelets 34 2
2.2.9. Determination of arachidonic acid (AA) release in human
platelets and cell-free cPLA activity 35 2
2.2.10. Determination of reversibility of inhibition 36
2.2.11. Determination of p12-LO translocation 36
2.2.12. Platelet aggregation (turbidimetric) 37
2+2.2.13. Measurement of intracellular [Ca ] levels in platelets i
and PMNL 37
2.2.14. Measurement of ROS-formation in human platelets 38
2.2.15. Determination of kinase phosphorylation status 38
2.2.16. Sample preparation for p12-LO protein amount determination
and progesterone receptor blots 39
2.2.17. Cell culture of PC-3 cells 39
2.2.18. SDS-PAGE 39
2.2.19. Western blot 39
2.2.20. Statistics 40
3. Results

3.1. p12-LO and gender influence on p12-LO activity 41
3.1.1. Determination of p12-LO activity in washed platelets 41
3.1.2. Determination of p12-LO activity in platelet rich plasma
(PRP) from male and female donors 42
3.1.3. Determination of p12-LO product formation in platelet
homogenates 44
3.1.4. p12-LO protein expression in male and female platelets 45
3.1.5. Subcellular localisation of p12-LO in male and
female platelets 45
3.1.6. AA release in male and female platelets 47
3.1.7. Determination of COX-1 product formation in
washed platelets and PRP 48

3.2. Effects of progesterone on p12-LO activity 49
3.2.1. Spontaneous 12-HETE formation in washed platelets treated
with sex hormones 49
3.2.2. 12-HETE formation in stimulated washed platelets treated
with sex hormones 50
3.2.3. p12-LO activity in female platelets resuspended in plasma
from male and female donors 54
3.2.4. p12-LO activity in plasma and PRP from pregnant donors 57
Table of Contents
3.2.5. AA release in intact platelets from male and female donors
treated with low hormone concentrations 62
3.2.6. AA release in platelets from female donors treated with
high hormone concentrations 63
3.2.7. Influence of progesterone on cPLA activity in a 2
cell free system 65
3.2.8. Determination of p12-LO activity in platelet homogenates
treated with sex hormones 65
3.2.9. Reversibility of the progesterone effect 67
3.2.10. Influence of progesterone on p12-LO subcellular localisation 68
3.2.11. Platelet aggregation after progesterone treatment 69
3.2.12. Influence of progesterone on kinase activation status 70
2+
3.2.13. Influence of hormone addition on [Ca ] influx in i
intact platelets 71
3.2.14. Influence of protein kinase A (PKA) activators and inhibitors
on p12-LO activity and on the effect of progesterone 74
3.2.15. Influence of cAMP analogue 8-bromo-cAMP on
p12-LO activity 75
3.2.16. Occurence of classical progesterone receptors in platelets 76

3.3. p12-LO inhibitor cinnamyl-3,4-dihydroxy-α-cyanocinnamate (CDC)
and its pharmacological profile in platelets 79
3.3.1. Influence of CDC on p12-LO and COX-1 in intact platelets 79
3.3.2. Inhibition of p12-LO and COX-1 by CDC in platelet
homogenates 80
3.3.3. Influence of CDC on COX-1 /2 and cPLA in a 2
cell free system 81
3.3.4. Influence of CDC on AA release in intact platelets 82
2+3.3.5. Influence of CDC on [Ca ] mobilisation in platelets 83 i
3.3.6. Influence of CDC on ROS- formation in platelets 85
3.3.7. Inhibition of p12-LO and COX-1 by CDC in whole blood
and PRP 85
3.3.8. Influence of CDC on platelet aggregation induced with
different stimuli 87

3.4. Effects of MK-886 on p12-LO in intact platelets 90
3.4.1. Influence of MK-886 on p12-LO and COX-1 activity in
was