Initiation of blood coagulation - evaluating the relevance of specific surface functionalities using self assembled monolayers [Elektronische Ressource] / von Marion Fischer

Fischer-Marion

Sujets

• Biologie

Informations

• Publié par: technische_universitat_dresden
• Publié le: 01 janvier 2010
• Nombre de lectures: 18
• Langue: English
• Poids de l'ouvrage: 4 Mo

Extrait

Initiation of blood coagulation –
Evaluating the relevance of specific surface functionalities using self assembled
monolayers



D I S S E R T A T I O N

zur Erlangung des akademischen Grades

Doctor rerum naturalium
(Dr. rer. nat.)




vorgelegt

der Fakultät Mathematik und Naturwissenschaften
der Technischen Universität Dresden







von

Dipl. Ernährungswissenschaftlerin Marion Fischer

geboren am 16.10.1979 in Dresden







Eingereicht am 05.06.2010 in Dresden.



Die Dissertation wurde in der Zeit von 03/2007 bis
05/2010 im Leibniz Institut für Polymerforschung Dresden angefertigt

I











































Gutachter: Prof. Dr. Carsten Werner
Prof. Dr. Brigitte Voit
IITable of contents

Table of contents


Acknowledgements ...................................................................................................... III
Preface ............................................................................................................................ V


1. Theoretical background ......................................................................................... 1
1.1. Hemocompatibility of medical devices 1

1.2. Self assembled monolayers as model surfaces 1

1.3. Initial processes of coagulation 3
1.3.1 Protein adsorption 4
1.3.2 Activation of coagulation via contact activation (intrinsic pathway) 7
1.3.3 Activation of coagulation via tissue factor (extrinsic pathway) 9
1.3.4 Sources and activity of TF 9
1.3.5 Cellular responses upon material-blood contact focussing on
platelet adhesion 11

2. Experimental part ................................................................................................ 13
2.1. Preparation of gold substrates 13

2.2. Preparation and characterisation of self assembled monolayers 14
2.2.1 Preparation and characterisation of C -COOH/ C -CH 14 15 15 3
2.2.2 Preparation and characterisation of C -COOH/ C -CH 15 10 10 3
2.2.3 Preparation and characterisation of C -COOH/ 10
C -(O-CH CH ) -O-CH ) 17 11 2 2 3 3

2.3. Characterisation of protein adsorption and enzyme activation 17
2.3.1 Human fibrinogen/ fibrin 17
2.3.2 Adsorption of complement fragments C3b and release of C5a 19
2.3.3 Contact activation: factor XIIa and kallikrein activity 20
2.3.4 Thrombin 21

2.4. Surface incubation with human blood plasma or platelet rich plasma
(PRP) 22
2.4.1 LDH assay on COOH/CH and COOH/CH /OH 22 3 3
2.4.2 Detection of platelets after immunostaining using fluorescence scanner 22

2.5. Whole blood incubation assay 22

2.6. Western blot of leukocyte isolates 26
2.6.1 Optimisation of leukocyte lysis 26
2.6.2 Optimisation of gel-loading conditions 27
®2.6.3 Optimisation of leukocyte isolation using Polymorphprep 27
ITable of contents


3. Results .................................................................................................................... 28
3.1. Preparation of gold substrates 28

3.2. Preparation and characterisation of self assembled monolayers 30
3.2.1 Preparation and characterisation of C -COOH/ C -CH 30 15 15 3
3.2.2 Preparation and characterisation of C -COOH/ C -CH 33 10 10 3
3.2.3 Preparation and characterisation of C -COOH; C -OH 40 10 11
3.2.4 Preparation and characterisation of C -COOH/ 10
C -(O-CH CH ) -O-CH ) 42 11 2 2 3 3

3.3. Characterisation of protein adsorption and enzyme activation 46
3.3.1 Human fibrinogen/ fibrin 46
3.3.2 Adsorption of complement fragment C3b 51
3.3.3 Contact activation: factor XIIa and kallikrein activity 52
3.3.4 Thrombin 58

3.4. Surface adhesion of platelets 61
3.4.1 LDH assay on SAMs after incubation with PRP 61
3.4.2 Detection of platelets after immunostaining using fluorescence scanning 62

3.5. Analysis of TF in leukocyte lysates 64
3.5.1 Isolation of leukocytes using ERL-kit 64
3.5.2 Optimisation: using standard-TF and different gel-loading conditions 66
®
3.5.3 Isolation of leukocytes using Polymorphprep 67

3.6. Whole blood incubation 68
3.6.1 Whole blood incubation of -CH /-COOH terminated SAMs 68 3
3.6.2 Whole blood incubation of -CH /-COOH and -COOH/-OH 3
terminated SAMs 75

4. Discussion .............................................................................................................. 84

5. Summary and conclusion ..................................................................................... 91

List of abbreviations ..................................................................................................... 93
References...................................................................................................................... 95

IIAcknowledgements
Acknowledgements

I am deeply grateful to my adviser Dr. Claudia Sperling - not only for giving me the
opportunity to perform this thesis work in her lab but also for many, many hours of
great supervision and discussion…also in hard times. Thanks for letting me participate
in the project that you brought into being and that presented an excellent scientific base
for the present thesis. Thanks for your experience both scientifically and personally, for
critical interpretation of results and for finding time to solve problems. Thank you for
everything.

Special thanks to Prof. Dr. Carsten Werner for the opportunity of joining his group. I
appreciate his essential project supervision, scientific discussions and indispensable
motivation for the projects process. Thanks also to Prof. Dr. Brigitte Voit for her
scientific support and for reviewing the thesis.

Many thanks also to Dr. Manfred Maitz, who acted like a co-adviser for my work.
Manfred contributed significantly to this thesis and to my knowledge about lab work in
general. Thanks for your uncounted proof-readings and all the fruitful discussions.

I also want to thank the group of Prof. Pentti Tengvall, which quickly integrated me and
supported me with help, guidance and suggestions. I thank Pentti for his kindness at any
time and for having created a nice working atmosphere as well as for scientific
discussions and proof-reading of our publication.

Lots of thanks also to Grit Eberth and Martina Franke for their considerable support in
lab works, preparing blood incubation assay and numerous gold surfaces.

Thanks to Babette Lanfer, Marina Prewitz, Andrea Zieris, all from the MBC, for lab
support, advice with several techniques, and for being great colleagues and friends.
Thanks for valuable support in difficult moments and for helpful comments and
discussions.

All this work would not have been possible without the constant support of my family,
especially my parents and my friends. Most of all, I would I like to thank my mother for
IIIAcknowledgements
the uncounted hours of childcare and Tim and Paula for helping me through this time
and for providing encouragement.


Thanks also to the DFG for funding my project under grant SP 966 2.


Marion Fischer
April 30. 2010
IVPreface
Preface
The surface of biomaterials can induce contacting blood to coagulate, similar to the
response initiated by injured blood vessels to control blood loss. This poses a challenge
to the use of biomaterials as the resulting coagulation can impair the performance of
hemocompatible devices such as catheters, vascular stents and various extracorporeal
tubings [1], what can moreover cause severe host reactions like embolism and
infarction.
Biomaterial induced coagulation processes limit the therapeutic use of medical
products, what motivates the need for a better understanding of the basic mechanisms
leading to this bio-incompatibility [2] in order to define modification strategies towards
improved biomaterials [3]. Several approaches for the enhancement of hemocompatible
surfaces include passive and active strategies for surface modifications. The materials’
chemical-physical properties like surface chemistry, wettability and polarity are
parameters of passive modification approaches for improved hemocompatibility and are
the focus of the present work.
In the present study self assembled monolayers with different surface functionalities
(-COOH, -OH, -CH ) were applied as well as two-component-layers with varying 3
fractions of these, as they allow a defined graduation of surface wettability and charge.
The ease of control over these parameters given by these model surfaces enables the
evaluation of the influence of specific surface-properties on biological responses.
To evaluate the effects of different surface chemistry on initial mechanisms of
biomater