Download Evaluation of blood interactions with a drug loaded protein matrix Master thesis

Master thesis
Evaluation of blood interactions with a drug loaded protein
matrix
Maria Wallstedt
2011
LITH-IFM-A-EX-11/2458-SE
Department of Physics and Measurement Technology, Biology and Chemistry
Linköping University
AddBIO AB
Master’s thesis
Evaluation of blood interactions with a drug loaded protein
matrix
Maria Wallstedt
2011
LITH-IFM-A-EX-11/2458-SE
Supervised by:
M.Sc. Henrik Aronsson, Ph.D. Trine Vikinge,
AddBIO AB
Ph.D. Lars Faxälv
Linköping University
Division of Clinical Chemistry,
Department of Clinical and Experimental Medicine
Examiner:
Prof. Pentti Tengvall
Linköping University
Division of Applied Physics
Department of Physics and Measurment Technology, Biology and Chemistry
Department of Physics and Measurment
AddBIO AB
Technology, Biology and Chemistry
Teknikringen 7
Linköping University
SE-583 30 Linköping Sweden
SE-581 83 Linköping, Sweden
Abstract
Many things might happen in the body when a titanium implant is inserted into bone.
Examples are activation of the immune system and imbalance between bone formation
and bone resorption, which might lead to damaged bone around the implant and at
worse, loosening of the implant.
Bisphosphonates, BP’s, is a class of drugs that is able to decrease the osteoclast (bone
resorption cell) activity and thereby strengthen the bone. FibMat2.0 is a fibrinogen
matrix and consists of a thin protein layer which can be applied on an implant and act as
a local drug delivery system.
The work in this thesis was divided into two parts where aim of the first part was to
study FibMat2.0 with integrated BP’s, and their effect in the presence of blood. The aim
for the second part was to determine whether it was possible to incorporate
antithrombotic drugs into the fibrinogen matrix. No detection method for the amount of
drugs incorporated into the fibrinogen matrix was used but the fact that the drugs gave
effect was verifying that it is possible to integrate other drugs than BP’s into FibMat2.0.
Methods that have been used in the experiments in presence of blood are imaging of
coagulation, fluorescence microscopy and cone-and-plate.
For the first part, the results showed that surfaces incubated with fibrinogen and
fibrinogen with integrated BP’s act alike in regard to coagulation and platelet adhesion.
Compared to titanium, which is known to be a biocompatible material, the surfaces with
fibrinogen and fibrinogen with BP’s behave similar in regard to platelet adhesion. When
it comes to coagulation, the surfaces coated with fibrinogen with or without an addition
of BP’s have shown a longer coagulation time compared to the clean titanium surface.
For the second part, some conclusions have been drawn according to the results.
Heparin and hirudin have shown anticoagulant effects when integrated in the matrix.
The platelet inhibitor cangrelor seemed to have better effect when added in blood and
incubated compared to incubation with the platelet inhibitor on the surface before
incubation in blood. Finally, when combining heparin and cangrelor, very clear
differences in regard to formation of fibrin network could be seen. It seems promising to
be able to load different kind of drugs in FibMat2.0.
Contents
Abbrevations ............................................................................................................. 1
1.
2.
Introduction........................................................................................................... 2
1.1
Background .................................................................................................... 2
1.2
Aim ................................................................................................................ 3
Theory .................................................................................................................. 4
2.1
2.1.1
Alternative pathway ................................................................................. 5
2.1.2
Lectin pathway ........................................................................................ 5
2.1.3
Classical pathway .................................................................................... 5
2.1.4
Membrane attack complex ....................................................................... 5
2.2
3.
The complement system.................................................................................. 4
Haemostasis .................................................................................................... 6
2.2.1
Plasma ..................................................................................................... 7
2.2.2
Platelets ................................................................................................... 7
2.2.3
The coagulation cascade .......................................................................... 8
2.2.4
Fibrinolysis ............................................................................................ 10
2.3
Fibrinogen .................................................................................................... 11
2.4
Drugs ............................................................................................................ 12
2.4.1
Bisphosphonates .................................................................................... 12
2.4.2
Heparin .................................................................................................. 13
2.4.3
Hirudin .................................................................................................. 13
2.4.4
Melagatran............................................................................................. 13
2.4.5
Cangrelor ............................................................................................... 14
2.4.6
Apyrase ................................................................................................. 14
2.5
Protein adsorption ......................................................................................... 15
2.6
Testing methods............................................................................................ 16
2.6.1
Ellipsometry .......................................................................................... 16
2.6.2
Imaging of coagulation .......................................................................... 17
2.6.3
Fluorescence microscopy ....................................................................... 17
2.6.4
Cone-and-plate setup ............................................................................. 18
Materials and methods......................................................................................... 19
3.1
Preparations of titanium surfaces .................................................................. 19
3.2
FibMat2.0 ..................................................................................................... 19
3.3
FibMat2.0 with bisphosphonates ................................................................... 20
3.4
FibMat2.0 with antithrombotic drugs ............................................................ 20
3.5
Platelet and coagulation experiments ............................................................ 21
3.5.1
Blood collection and plasma preparation ................................................ 21
3.5.2
Imaging of coagulation .......................................................................... 22
3.5.3
Platelet adhesion .................................................................................... 22
3.6
4.
Results ................................................................................................................ 24
4.1
Measurement of protein film thickness with ellipsometry.............................. 24
4.2
FibMat2.0 with bisphosphonates ................................................................... 25
4.2.1
Coagulation ........................................................................................... 25
4.2.2
Low shear platelet adhesion ................................................................... 27
4.3
5.
FibMat2.0 with antithrombotic drugs ............................................................ 28
4.3.1
Coagulation ........................................................................................... 28
4.3.2
Low shear platelet adhesion ................................................................... 33
4.3.3
High shear platelet adhesion .................................................................. 38
Discussion ........................................................................................................... 44
5.1
FibMat2.0 with bisphosphonates ................................................................... 44
5.1.1
Coagulation ........................................................................................... 44
5.1.2
Low shear platelet adhesion ................................................................... 45
5.2
6.
Statistics ....................................................................................................... 23
FibMat2.0 with antithrombotic drugs ............................................................ 46
5.2.1
Coagulation ........................................................................................... 46
5.2.2
Low shear platelet adhesion ................................................................... 47
5.2.3
High shear platelet adhesion .................................................................. 48
Conclusions......................................................................................................... 50
6.1
FibMat2.0 with bisphosphonates ................................................................... 50
6.2
FibMat2.0 with antithrombotic drugs ............................................................ 50
6.2.1
Anticoagulants integrated in the fibrinogen matrix ................................. 50
6.2.2
Platelet inhibitors integrated in the fibrinogen matrix ............................. 50
6.2.3
Anticoagulant and platelet inhibitor in the fibrinogen matrix .................. 51
7.
Future aspects ..................................................................................................... 52
8.
Acknowledgements ............................................................................................. 53
References .................................................................................................................. 54
Abbreviations
ADP
Adenosine diphosphate
BP’s
Bisphosphonates
C1-C9
Complement factor 1-9
FibMat2.0/X
Fibrinogen matrix version 2.0 with an integrated
antithrombotic drug
FibMat2.0/Z
Fibrinogen matrix version 2.0 with integrated zoledronate
MilliQ
Ultrapure water that is filtered and purified by reversed
osmosis
rpm
revolutions per minute
PBS
Phosphate buffered saline
PFA
Paraformaldehyde
PFP
Platelet- free plasma
PPP
Platelet- poor plasma
PRP
Platelet- rich plasma
TF
Tissue factor
Å
Ångström (1 Å = 10-10 m)
1
1. Introduction
1.1 Background
When a titanium implant is inserted into bone, many things happen in the body. The
biomaterial may for example activate the immune system. If the body sees the implant as a
foreign material, the complement cascade might be activated by proteins in the blood that
adsorbs to the surface of the implant. Directly after an operation, inflammation is part of the
normal healing process. Problems occur if the inflammation becomes chronic, which
happens if the body continues to see the biomaterial as foreign [1]. Another implantation
issue occurs if the balance between bone formation and bone resorption is disturbed.
When an implant is inserted, the activity of the osteoclasts, bone resorbing cells,
increases which leads to an over-representation of cells that break down bone. The
osteoblasts, which are responsible for bone formation, do not keep up with the increased
resorption made by the osteoclasts This leads to weakened bone and can cause implant
instability, or at worse, loosening of the implant [2].
Bisphosphonates, BP’s, are a class of drugs that is able to decrease the osteoclast
activity. Studies have shown that implant fixation in human cancellous bone have been
improved by treatment with BP’s [3]. When taking the BP’s orally, a high dose is
required. This might lead to unwanted side-effects, for example, gastrointestinal
problems. To avoid this dilemma, Prof. Pentti Tengvall together with Prof. Per
Aspenberg at Linköping University, Sweden, developed a fibrinogen multilayer for
local delivery of BP’s from an implant surface [4]. This drug delivery concept is now
property of the life-science company AddBIO AB, who has developed the fibrinogen
multilayer technology (named FibMat) and are now commercializing FibMat2.0.
FibMat stands for fibrinogen matrix and consists of a thin protein layer which can be
applied on an implant. It acts like a local drug delivery system and can effectively
release drugs near the implant. FibMat can be used to load different kinds of drugs, e.g.
BP’s, and have the advantages of being biocompatible, resorbable and invisible [5].
2
1.2 Aim
The aim of the first part of this thesis was to study titanium surfaces and compare them
with titanium surfaces coated with fibrinogen and fibrinogen with integrated BP’s. In
experiments made in presence of blood, the three types of surfaces will hopefully give
similar results with regard to coagulation and platelet adhesion. That will prove that
fibrinogen coated surfaces, with or without BP’s, can be used in the same way as
surfaces with titanium, which is known to be a biocompatible biomaterial for usage in
contact with bone. Even though this kind of modification is relevant to bone
applications, the implant will have contact with blood during the insertion and that is
why it is interesting to find out the effects of the coating in presence of blood.
The primary aim for the second part was to determine whether it was possible to
incorporate antithrombotic drugs into the fibrinogen matrix. If the integrated drugs gave
effect, it could be verified that it is possible to integrate other drugs than BP’s into
FibMat2.0. The secondary aim was to study the effects of the antithrombotic drugs and
also for how long time they stayed in the fibrinogen matrix in experiments made in
presence of blood. Such drug delivery systems could be applicable to cardiovascular
biomaterials where thrombus formation on the material is to be avoided.
3
2. Theory
2.1 The complement system
The complement system consist of around 30 soluble and membrane bound proteins [6]
and the role of the system is to be a major innate defense system against different kinds
of pathogenic agents like bacteria and viruses [7].
The system is activated by three complement pathways; the alternative, the lectin and
the classical complement pathway. Although the ways differ, the defense advantages
and end results are the same. Examples of functions that are performed by the proteins
produced by the complement pathways are opsonization, the ability to trigger
inflammation and remove harmful immune complexes from the body [8].
There are nine types of complement factors (C1-C9) involved in the complement
system [9].
Figure 1. A simplified overview of the complement system with the alternative, lectin and classical
pathways [10].
4
2.1.1 Alternative pathway
The alternative complement pathway is activated by C3b binding to microbial surfaces
and to antibody molecules [8]. C3 is cleaved all the time so there is always C3b
circulating in small amounts. Factor B binds to C3b. Factor B is cleaved into Ba and Bb
in presence of factor D. Bb includes the active site for a C3 convertase [11].
2.1.2 Lectin pathway
The lectin pathway activates complement through the mannose-binding lectin protein
which binds to carbohydrates found of many pathogens [12]. Activation of the lectin
pathway starts when the mannose-binding protein (MBP) binds to the mannose groups
of microbial carbohydrates. There are two other proteins involved in the lectin pathway;
MASP1 and MASP2 which are equivalent to C1r and C1s in the classical pathway.
MASP1 and MASP2 binds to MBP and forms an enzyme like C1 that can cleave C4
and C2 to C3 convertase which in turn can split C3 into C3a and C3b [10].
2.1.3 Classical pathway
The classical complement pathway is activated by antigen-antibody complexes [10].
C1q binds to an antibody that is bound to an antigen and activates C1r. C1r cleaves C1s
to activate the protease function. C1s cleaves C2 and C4. C4a mediates inflammation
and C4b binds C2 for cleavage by C1s. C4b also binds cell surfaces for opsonization.
C2b is an active enzyme that cleaves C3 and C5. C3b binds C5 for cleavage by C2b. It
also binds cell surfaces for opsonization and activation of the alternative pathway [11].
2.1.4 Membrane attack complex
C5b starts the assembly of the membrane attack complex (MAC). C6 binds C5b and
forms the acceptor for C7. C7 binds C5b6, inserts into a membrane and forms an
acceptor for C8. C8 binds C5b67 which initiates polymerization for C9. C9 polymerizes
around C5b678 and forms a channel that causes cell lysis [11].
5
2.2 Haemostasis
The originally greek word haemostasis means blood-stop, where haemo refers to blood
and stasis means stop or stagnant [13].
Haemostasis is a complex physiological mechanism in the body, which activates when
blood vessels are ruptured and keeps the blood from leaking out from the cardiovascular
system unhindered [14]. Figure 2a shows a schematic illustration of the events
following an injury or rupture of a blood vessel.
Figure 2. The sequential events following vessel injury: a) Schematic illustration of the vessel, b) platelet
adhesion to collagen in the subendothelial matrix, c) platelet aggregation and activation; forming a
platelet plug that initially stops bleeding, d) fibrin network formation (coagulation) stabilizes the platelet
plug [15].
After an injury has occurred, a cascade of reactions takes place. The first one is the
primary haemostasis which includes vasoconstriction, where contraction by muscles in
the cell wall narrows the blood vessel leading to a reduced blood flow, platelet adhesion
and aggregation (Figure 2b and 2c) and formation of a platelet plug (Figure 2c).
Thereafter the secondary haemostasis takes place which includes activation of
coagulation factors and finally fibrin formation (Figure 2d) [14].
6
2.2.1 Plasma
Plasma is the light yellow fluid that remains when red blood cells, white blood cells and
platelets are separated from whole blood [16]. Plasma contains mostly water (~90 %)
which functions as a solvent for carrying other substances. Except for water, plasma
contains proteins like albumin, immunoglobulins (also called antibodies) and
fibrinogen. Additional components are salts (Ca, K, Mg, and Na), nutrients, enzymes,
hormones and nitrogenous waste products [17]. One critical group in plasma is the
coagulation and fibrinolysis proteins and their inhibitors. This group of proteins helps to
facilitate normal coagulation and dissolves clots once they are formed [16]. The
difference between plasma and serum is that serum does not contain fibrinogen [18].
2.2.2 Platelets
Blood contains three major types of cells; red blood cells, white blood cells and platelets
[19]. Platelets are not complete cells but more correctly cell fragments, produced in the
bone marrow from megakaryocytes, and they do not contain any nucleus. Platelets
circulate in the blood stream for about ten days and are then finally cleared by
macrophages in the spleen and liver. During this time, most platelets are never involved
in the haemostatic response caused by damage to the blood vessels [20]. Platelets
contain proteins that let them stick to the each other and to the blood vessel wall. They
also contain proteins that allow them to change shape when they adhere [19].
Platelets have three primary functions; adhering to an injured blood vessel (platelet
adherence), attaching to platelets to enlarge the forming plug (platelet aggregation) and
providing support for the processes of the coagulation cascade. Platelets are part of
several processes like haemostasis, thrombosis and inflammation [21]. They are the first
ones to react when an injury or cut occurs in a blood vessel. The subendothelial matrix
that surround the vessel wall facilitate platelet adhesion and upon adherence stimulate
them to change shape and aggregate to stop the bleeding [19].
7
The generation of thrombin from the coagulation process activates and recruits more
platelets into the growing thrombus by exposing and activating receptors on the platelet
surface. This allows platelets to adhere to each other and this process is called
aggregation [20]. ADP plays a vital role in normal haemostasis and thrombosis [22].
Platelets are activated by ADP, which induce the platelet shape change and make them
aggregate. Platelets have three known receptors for ADP; P2Y1, P2Y12 and P2X1,
which can be blocked by different drugs [23]. The P2Y1 and P2Y12 receptors are quite
similar and important for aggregation, shape change etc. P2X1 is an ion channel which
causes shape change and aids in the activation process [22].
2.2.3 The coagulation cascade
The complex coagulation cascade includes about 30 interacting proteins. In case of a
vessel cut or other injury, the tissue factor, (TF), gets exposed to the blood stream. It
binds the coagulation factor VII which leads to activation of the coagulation cascade
[24]. When TF activates the coagulation, it eventually results in an induction of the
coagulation protein thrombin. Thrombin has the ability to cleave fibrinogen which
generates fibrin [25]. The main function for the coagulation cascade is to prevent big
blood loss in case of an injury. This is accomplished by forming stable haemostatic clots
which consists of a mesh of fibrin reinforcing the previously aggregated platelets [26].
The coagulation cascade can be schematically separated into three areas; the intrinsic
pathway which is activated by contact with surfaces, the extrinsic pathway which is
activated by vascular injury and the common pathway which is initiated by the
activation from the intrinsic and/or extrinsic pathway [27].
8
Figure 3. Overview of the coagulation cascade. Modifications have been done on the original picture
[28].
In case of damage to the vascular wall, TF is exposed and the extrinsic pathway is
initiated [29]. Factor VII is a circulating coagulation factor which forms a complex with
tissue factor in presence of calcium. This complex quickly converts the proenzyme
factor X to the enzyme form factor Xa (Figure 3) [30].
The intrinsic pathway, also called the contact activation pathway, is the other branch of
the coagulation cascade. When circulating factor XII comes in contact with and is
bound to a negatively charged surface it spontaneously activates, and the intrinsic
pathway is thus activated. The activated factor XII enzyme is then able to activate factor
XI into the active enzyme factor XIa. In presence of calcium, factor XIa converts factor
IX to its active form, factor IXa. Factor X can then be activated to factor Xa by factor
IXa in the presence of factor VIII, calcium and platelet phospholipids (Figure 3) [31].
The end of both the extrinsic and the intrinsic pathway meet in the activation of factor X
to factor Xa and from here, the common pathway continues. [32].
9
Activated factor Xa and factor V acts as catalysts when prothrombin (factor II) is
converted to thrombin (factor IIa). This leads in turn to the conversion of fibrinogen
(factor I) to fibrin [33].
2.2.4 Fibrinolysis
Fibrinolysis is the process where the fibrin clot is dissolved. These degrading
mechanisms are performed by enzymes and regulated by inhibitors. There is a fine
balance in the fibrinolytic system and an infraction might lead to bleeding or thrombosis
[34].
Figure 4. Overview of the process of fibrinolysis. Modifications have been done on the original picture
[35].
The fibrinolytic process is initiated by the activation of plasminogen to plasmin.
Plasmin splits the fibrin clot into fibrin degradation products (Figure 4). The role of the
inhibitors is to balance the activity [34].
Primary fibrinolysis takes place naturally when the body clears clots that no longer are
needed because the damaged tissue is healed. Secondary fibrinolysis can be induced
with medications or occur as the result of stress or disease [36].
10
2.3 Fibrinogen
The soluble protein fibrinogen has a complicated molecular structure which consists of
two copies of three different polypeptide chains; α, β and γ. The chains are twisted and
linked together with disulfide bonds [37].
Figure 5. Schematic drawing of the fibrinogen molecule. The structure consists of three pairs of
polypeptide chains: α, β and γ. FPA = fibrinopeptide A, FPB = fibrinopeptide B, * = carbohydrate
cluster, † = disulfide rings [38].
Fibrinogen is located in blood plasma and can, via thrombin, be converted into fibrin.
Fibrin is an insoluble network of fibers which is the main component of the formation
of a blood clot during the haemostatic response to tissue injury [38].
One of the initial haemostatic events that stop bleeding from a vascular injury is platelet
aggregation. Aggregation is mediated by the binding of fibrinogen to platelet surface
receptors [39].
Fibrinogen is an acute-phase protein and a strong independent cardiovascular risk factor
[40]. The acute-phase proteins are a result of the initiation of inflammatory processes in
the body. They are triggered by the innate immune response. The proteins are important
during inflammatory processes and their plasma concentrations can be used as markers
of inflammation. If there is an inflammation, the amount of fibrinogen is rapidly
increasing [41].
11
2.4 Drugs
2.4.1 Bisphosphonates
Bone is constantly remodeling by a balance between osteoblasts, cells that build up
bone, and osteoclasts, cells that breaks down bone [42]. When the osteoclasts work
faster than the osteoblasts, diseases like osteoporosis, Paget’s disease and hypercalcemi
occurs. BP’s (Figure 6) is a class of drugs that is able to prevent bone resorption and
increase in bone mass [43]. This is not only important in connection with different
diseases but also when an implant has been inserted into the body.
Figure 6. Basic structure of Bisphosphonate [44].
BP’s adhere firmly to the bone surface and inactivates the osteoclasts. They are released
from the bone mineral by the action of the osteoclast itself, and become internalized in
the osteoclast as it starts to resorb the bone [42].
Two common types of BP’s are alendronate and zoledronate. Both have a hydroxyl
group as side chain R2. The difference between the two BP’s is the side chain R1 which
is an amino group for alendronate and an imidazole group for zoledronate (Figure 7).
Figure 7. Alendronate to the left and zoledronate to the right [45].
12
2.4.2 Heparin
Heparin is a polysaccharide and acts mainly by inhibiting thrombin and factor Xa [46].
Figure 8. The chemical formula for heparin [47]
This hinders the activated coagulation factors from clotting the blood [48]. Heparin
releases TFPI, tissue factor pathway inhibitor, from the vessel wall endothelium. TFPI
is an important physiological regulator of the coagulation factors that start the blood
coagulation and contributes to the anticoagulant effect of heparin. Heparin is commonly
used as an anticoagulant to prevent thrombosis [49].
2.4.3 Hirudin
Hirudin is a polypeptide with 65 amino acids which originally is produced by the
medicinal leech. [50].
Figure 9. The chemical formula for hirudin [51].
Hirudin is a direct thrombin inhibitor and the function of it is to bind to the thrombin
molecule and by that block the platelet activating effect [52]. The drug is licensed for
treatment of arterial or venous thrombosis and is an alternative to heparin [53].
2.4.4 Melagatran
Another anticoagulant, melagatran, is a dipeptide derivative.
13
Figure 10. The chemical formula for melagatran [54].
It works as a direct inhibitor of α-thrombin and thereby it resists forming of thrombin
and development of thrombosis [55, 56, 57]. The molecule has two stereoisomeric
centers, where the form (1R, 2S) is used clinically [58].
2.4.5 Cangrelor
Cangrelor is a potent inhibitor of the P2Y12 receptor.
Figure 11. The chemical formula for cangrelor [59].
It is fast able to achieve almost complete inhibition of ADP-induced platelet
aggregation [60]. It is an antagonist that has been primarily described as rapid-onset,
competitive, reversible inhibitor of P2T receptors. The P2T receptor is one of the three
known subtypes of ADP receptor on platelets together with P2X1 and P2Y1.
Chemically cangrelor is an ATP analogue [61].
2.4.6 Apyrase
Apyrase is anti-platelet aggregation protein [62]. It has the ability to hydrolyse ADP
which is a key component of platelet aggregation [63].
Figure 12. The protein structure of apyrase [64].
Apyrase is able to prevent platelet aggregation by directly decrease the ADP
concentration through ATP/ADP-hydrolysis [65].
14
2.5 Protein adsorption
Protein adsorption is a spontaneously driven process that occurs almost immediately on
clean surfaces in contact with a protein solution. This happens for example on a foreign
surface when biomaterials are implanted into humans or animals. When the surface of
the implant comes in contact with blood, a thin layer of plasma proteins will cover it.
The adsorbed proteins will affect the healing process, coagulation etc since the cells on
the artificial surface most likely interact with the adsorbed protein layer itself rather
than with the material. Hence, the initial protein adsorption onto a biomaterial surface
plays a major role in how the body reacts to on implanted biomaterial. A lot of studies
have been done on the influence of protein adsorption on the inflammatory response
because in case of an injury, inflammation is the first response from the body [66]. At
constant pressure and temperature, protein adsorption can only take place if Gibbs
energy, G, of the system decreases.
ΔadsG = ΔadsH – TΔadsS < 0
Formula 1. Gibbs energy, G, for a system, where Δads = the change in the thermodynamic functions of
state resulting from the adsorption process, H = enthalpy, T = temperature and S =entropy The
adsorption process is an outcome of the net interactions within the system [67].
The key processes that lead to protein adsorption are structural changes in the protein
molecule, electrostatic attraction between the surface and the protein, and dehydration
of the surface and the protein. All interactions and forces, like ones between molecules,
atoms, proteins, the surface and the solvent can affect the adsorption process.
The structure of protein changes when they are close to hydrophobic surfaces. Proteins
can partly lose their quaternary and on occasion their tertiary structure so the inner
hydrophobic parts of the protein are able to interact with the hydrophobic surface.
In terms of Gibbs energy, the structural loss increases the conformational entropy of the
protein and the hydrophobic interaction with the surface increases the entropy by
increasing the disorder of water when the surface is dehydrated. This results in a
decrease in Gibbs energy. The structural change of the protein means that the adsorption
is irreversible [67].
15
2.6 Testing methods
2.6.1 Ellipsometry
Ellipsometry is a sensitive optical technique which is used to determine various material
characteristics like layer thickness, optical constants, surface roughness and
composition [68]. The technique uses the polarization of light. Monochromatic light is
plane polarized and directed to the surface. The parallel (s-polarized) and the
perpendicular (p-polarized) components of the incident light are not reflected in the
same way. That is the reason why the reflected light is elliptically polarized [69].
Figure 13. Polarized light can be divided in two components; light perpendicular to the plane of
incidence, Es, and light parallel to the plane of incidence, E p Elliptically polarized light is a result when
the phase difference ≠ 0° or 180° [70].
Ellipsometry measures the change in polarization state of light reflected from the
surface of a sample. The measured values are expressed as Δ and Ψ. Δ represents the
phase change and Ψ is the relative amplitude change [71]. The changes obtained by Δ
and Ψ can be converted to an equivalent layer thickness by using the McCrackin
algorithm [72]. Advantages with ellipsometry are that the method is highly accurate and
reproducible and it is non-destructive to the sample. It is fast and easy to use.
Measurements can be made both in air and in a liquid cell. There is no need of reference
samples. The dynamic range of the ellipsometer is 1-1000 Å and it is so sensitive that it
easily detects a thickness change of only a few Angstroms. Requirements of the method
are that the sample that is to be measured must be a flat surface, the film has no light
adsorption and the refractive index has to be known [68, 70, 71].
16
2.6.2 Imaging of coagulation
Imaging of coagulation is a visual method where the coagulation process can be
followed. The surface is put in a cuvette in a way that the viewer can see what happens
on the surfaces from the side. The cuvette is then filled with citrate plasma. The addition
of citrate in the plasma sets the coagulation on hold. To start the experiment, Ca2+ is
added to the cyvettes which allows the coagulation to begin. In front of the four
cyvettes, a camera is set up to be able to take pictures of the process with a preset
amount of pictures during a certain time with a constant time interval.
Before the experiment starts, the plasma is very light yellow and transparent. When the
coagulation starts, the plasma is getting more opaque and the colour turns to almost
white. All the pictures for one cuvette are put together to a video sequence by a
computer program. When analyzing the process the scattered light, which differs
between coagulated and non-coagulated plasma, is measured and converted into
coagulation times for the cyvettes. Both coagulation times on the surface and in the bulk
solution can be measured [15].
2.6.3 Fluorescence microscopy
Fluorescence microscopy is a commonly used microscopy technique. Two of its
principal advantages are that light microscopy allows the observation of structures
inside a live sample in real time and specific cellular components can be observed
through molecule-specific labeling. One limitation of the method is though the
relatively low spatial resolution because of the diffraction of light [73]. When a photon
is absorbed by a fluorophore, an electron is raised to an excited state. Thereafter it
relaxes to the electronic ground state and emits a lower energy photon. This is when
fluorescence occurs [74].
Figure 14. Fluorescence [75]
17
2.6.4 Cone-and-plate setup
To be able to study how cells behave on different surfaces under similar circumstances
as in the intravascular space, the method cone-and-plate can be used. An artificial flow
over a surface is created. The cone-and-plate setup consists of a rotatable cone which is
run by a motor, an O-ring to put the sample on and a plate connected to a vacuum
pump. The surface is put on the O-ring which in turn is put on the plate and everything
is held in place by vacuum. A drop of blood is applied to the surface and the surface is
raised up until the cone is in close proximity to the surface and then the cone is set in a
rotational motion by the motor. The blood is subjected to a shear force over the surface
for a certain amount of time. This experiment can be done to make stability tests of
surface coatings by measuring the thickness of a protein matrix on a surface before and
after the cone-and-plate experiment to see how stable the matrix is. Another option for
experiment is to study adhered platelets in fluorescence microscope after exposure to
high shear. The shear rates can be adjusted to simulate a range of conditions found in
blood vessels. 100 rpm correspond to a shear rate at approximately 100 s -1 and simulate
blood flowing in the veins and 1200 rpm correspond to a shear rate at approximately
1200 s-1 and simulate blood flowing in the arteries [15].
18
3. Materials and methods
For all experiments, silicon wafers with 2000 Å evaporated titanium on one side was
used. The fibrinogen comes from CSL Behrings (USA). For the experiments with BP’s,
zoledronate from LKT Laboratories (USA) was used.
3.1 Preparations of titanium surfaces
The wafer was cut to smaller pieces suitable for the different experiments (0.5x1 cm for
fluorescence microscopy, 0.5x2 cm for imaging of coagulation and 1x1 cm for coneand-plate). The surfaces were put in an UVO-cleaner model 42 (Jelight Company Inc.
USA) for four minutes. In this step, contamination is removed by short-wavelength UV
radiation [76]. After that the surfaces were rinsed in MilliQ and dried flowing N2.
To get reference values for further measurements, three surfaces were randomly picked
out and measured with ellipsometry (AutoEL III null ellipsometer Rudolph Research,
USA). The mean values of the measurement points were calculated for the variables Δ
and Ψ, which was used in the McCrackin algorithm [72].
3.2 FibMat2.0
FibMat2.0 contains of a matrix of fibrinogen on the surface. The fibrinogen solution
was prepared according to a protocol made by AddBIO AB.
When making the protein multilayer, the surface was put in an eppendorf tube and
fibrinogen solution was added. Also the production of the multilayer was made due to a
protocol made by AddBIO AB.
19
3.3 FibMat2.0 with bisphosphonates
The difference between FibMat2.0 and FibMat2.0/Z is that the latter has BP’s
incorporated in the matrix. BP’s are very small and have no problem reaching into the
matrix and staying there. The integration of the bisphosphonates into the fibrinogen
matrix was made according to a protocol made by AddBIO AB.
3.4 FibMat2.0 with antithrombotic drugs
FibMat2.0/X is a further development of the FibMat2.0 where the X represents an
antithrombotic drug. Here, three different anticoagulants and two platelet inhibitors
have been tested due to how well they are integrated into the matrix and what effect
they have in the matrix in presence of blood. For the experiments with the addition of
different drugs, all the steps for the fibrinogen matrix was to be done at first. TF,
heparin, hirudin and melagatran were used for the method imaging of coagulation. TF
was added to the plasma shortly before the experiment was started. The reason for usage
of TF was to accelerate the coagulation process and by doing that, be able to get results
in a reasonable amount of time. For the three anticoagulants, solutions with as high
concentration as possible were prepared for each drug (heparin: 5000IE/ml, hirudin:
6500 IE/ml, melagatran (180 µM). The FibMat surfaces were then incubated in the
solution on a rotator. For the experiments with fluorescence microscopy; ADP (13
µM)), apyrase (1000 U/ml)) and cangrelor (100 nM) were used. Different ways of
incubations were done as seen in Table 2 and Table 3 but one thing was common for all
surfaces; first the fibrinogen matrix was produced and then the drugs were added
afterwards.
20
3.5 Platelet and coagulation experiments
3.5.1 Blood collection and plasma preparation
For all experiments, day fresh blood from healthy volunteers was drawn using a 0.8 mm
Venoject® needle form Terumo (Leuven, Belgium). For the experiments made with the
imaging of coagulation method, the blood was drawn into a 7.5 ml S-Monovette® tube
from Sarstedt (Nümbrecht, Germany), which was filled with 1/10 of 130 mM sodium
citrate.
After leaving the tube to cool down in room temperature for 10 minutes it was
centrifuged for 15 minutes at 2500 x g. The blood became separated and the plateletpoor plasma (PPP) ended up on top in the tube. The plasma was then filtered through a
0.20 µm pore size disposable Minisart® filter from Sartorius (Göttingen, Germany) to
platelet-free plasma (PFP).
Figure 15. A tube with blood that has been separated into its components [77].
Whole blood was used for the experiments including fluorescence microscopy and the
blood was drawn into tubes containing the anticoagulant heparin.
21
3.5.2 Imaging of coagulation
The surfaces (0.5x2 cm) were put in disposable PMMA spectrophotometry cuvettes
from Kartell (Noviglio, Italy) with the edge facing the digital camera from Canon
(Tokyo, Japan). For all experiments, four cuvettes were photographed at each time.
When everything was set up, 0.5 M CaCl2 was added to the plasma (36 µl CaCl2
solution to 1000 µl plasma). This reversed the anticoagulant effect of citrate. Then, 450
µl plasma was added to each cuvette and the coagulation was able to start. The program
connected to the camera was turned on and preset to take a picture of the cuvettes every
15 second and the total number of photos was set to 800.
With a program written in Matlab® (v. 7.2) from the Mathworks Inc. (Natick, USA), all
the pictures for each cuvette was converted into a film sequence. Another Matlab®
program then processed the sequences and, depending on light intensity, calculated a
coagulation time directly at the surface and a coagulation time in the bulk (in the
interval 0.5-1 mm from the surface). With these data, it was possible to compare the
coagulation process for different coatings on the surface.
3.5.3 Platelet adhesion
Low shear model: The surfaces (0.5x1 cm) were put in 0.5 ml eppendorf tubes which
were filled with 0.5 ml whole blood. The tubes were then put on a rotator for 20 minutes
at room temperature and during that time the blood was slowly flowing over the
surfaces. The surfaces were picked up, carefully rinsed with PBS and put in a Petri dish.
To fix the platelets and white blood cells, 3.7 % PFA in PBS was dropped on the
surfaces and then incubated 10 minutes. The surfaces were then rinsed in PBS twice.
The next step was incubation with Triton-X (0.1 % in PBS with an addition of 0.1
mg/ml BSA) for 3 minutes. Triton-X works as a detergent and permeabilized the
adhered cells. Again the surfaces were rinsed with PBS two times. The surfaces were
then incubated with Alexa Fluor® 546 phalloidin, diluted in PBS (1:100), for 20
minutes. In this step the cells were stained so they can be seen in a fluorescent
microscope. The surfaces covered with tin foil during the incubation. After this last
incubation, the surfaces were rinsed first with PBS, and then, de-ionized water, and
finally dried with compressed air. On a slide for microscopy (24x60 mm) drops of
Prolong Gold Antifade with DAPI were placed out and the tested surfaces were put
upside down on the drops. DAPI makes it possible to see the nuclei of the white blood
cells.
22
It was then possible to look at the surfaces in the fluorescent microscope and study the
adhesion and aggregation of platelets and the presence of white blood cells for different
surfaces with different preparations. Microscopy of the cells was done with the
microscope Zeiss AxioObserver Z1 and the software Zeiss AxioVision 4.6 (Carl Zeiss
MicroImaging GmbH, Germany)
High shear model: To see how the platelets behaved at a higher shear the method coneand-plate was used. The surface (1x1 cm) was centered on the O-ring and the vacuum
pump was turned on. On the surface, a small drop of blood, with or without addition of
drugs, was dispensed and the table was brought up so the cone was as close to the
surface as possible. The motor from IKA (Staufen, Germany) was started and the
rotational speed was set to 1200 rpm, which corresponds to a shear rate of
approximately 1200 s-1. After the motor and vacuum pump was turned off, the surface
was rinsed in PBS and fixed in 3.7 % PFA. After that, the staining procedure was the
same as described for the low shear model.
3.6 Statistics
The data from the experiments were expressed as mean ± standard deviation. For
measurements of the thickness of the protein layer, five adjacent points were measured
with ellipsometry for a number of surfaces. With the McCrackin algorithm, [72] the
thickness in Å is calculated from Δ and Ψ. A mean value and standard deviation for all
thicknesses was then calculated.
From the experiments with the fluorescence microscope, three pictures were taken from
each surface and mean values and standard deviations were calculated from the data
given by the Matlab® program.
23
4. Results
The results are presented, starting with the thickness measurements of FibMat2.0 made
with ellipsometry, then the results connected to FibMat2.0/Z and at last, the
FibMat2.0/X results. The results from FibMat2.0/X experiments are divided under the
titles “Coagulation”, “Low shear platelet adhesion” and “High shear platelet adhesion”
and do not appear in chronological order. All platelet adhesion experiments were
analyzed with fluorescent microscopy.
4.1 Measurement of protein film thickness with ellipsometry
To estimate the thickness of the multilayer, the surface was measured with ellipsometry
(AutoEL III null ellipsometer Rudolph Research, USA). The refractive index for
proteins was set to nf=1.465. Five measurement points were taken from each surface
and the value for Δ and Ψ was noted and calculated with help of the McCrackin
algorithm [72]. The mean value and standard deviation of the thicknesses of the surfaces
were calculated for each size of the fibrinogen surfaces. Different sizes of surfaces have
been used depending of which method that have been applied; 0.5x1 cm for low shear
platelet adhesion experiments, 0.5x2 cm for imaging of coagulation experiments and
1x1 cm for experiments made with the cone-and-plate setup (high shear platelet
adhesion). It is not the size of the surface that causes the variations in thickness of the
fibrinogen layer but rather the volume of the fibrinogen solution relative the surface
area.
24
Figure 16. Three different sizes; 0.5x1 cm, 0.5x2 cm and 1x1 cm, of titanium surfaces were incubated in
fibrinogen and the thickness of the protein layer was calculated with the McCrackin algorithm. (n = 3-6)
4.2 FibMat2.0 with bisphosphonates
The aim for the first experimental part was to compare surfaces with clean titanium with
titanium surfaces coated with fibrinogen and fibrinogen with BP’s. The three types of
surfaces were tested in different experiments in presence of blood. The purpose was to
find out how they differ regard to coagulation and platelet adhesion.
4.2.1 Coagulation
The imaging of coagulation experiment was made the same way six times with blood
from six different donors. The blood was centrifuged and filtered to PFP. Four cuvettes
were used with the following content:
Table 1. Content of cuvettes for imaging of coagulation, FibMat2.0/Z.
Cuvette 1
Cuvette 2
Cuvette 3
Cuvette 4
Clean surface of Titanium
Titanium surface with fibrinogen
Titanium surface with fibrinogen + BP
Reference; cuvette with no surface
25
As seen in Figure 17, the cuvette with only PFP and an addition of CaCl2, have the
longest coagulation time. The coagulation times for the surfaces with fibrinogen and
fibrinogen + BP respectively, do not differ significantly. Another observation for those
two cuvettes is that the coagulation time in the bulk is much longer than the one near the
surface. The coagulation time for clean titanium is much shorter than the coated
surfaces.
Figure 17. Surface coagulation times of PFP on titanium, fibrinogen, and fibrinogen + BP surfaces. The
reference corresponds to a cuvette without a titanium surface (n=6). The surfaces with fibrinogen and
fibrinogen + BP activated the coagulation similarly.
26
4.2.2 Low shear platelet adhesion
In conformity with the method imaging of coagulation, six experiments with blood from
six different donors were made and studied with fluorescence microscopy. For each
experiment, three surfaces were incubated in whole blood and left on a rotator for 20
minutes. One surface was of clean titanium, one was covered with fibrinogen and the
third was covered with fibrinogen + BP. The platelet coverage did not differ
significantly between the three surfaces.
Figure 18. Platelet adhesion on titanium, fibrinogen and fibrinogen + BP surfaces (n=6) after exposure
of whole blood on a rotator for 20 minutes. Platelets were stained with Alexa Fluor® 546 phallodin.
27
4.3 FibMat2.0 with antithrombotic drugs
In the second part of the project, other drugs were used in combination with the
fibrinogen matrix instead of zoledronate.
4.3.1 Coagulation
In the first experiment, it was investigated how hard the drugs had bound inside the
fibrinogen matrix. This was studied in two identical tests with blood from two donors.
At first, the surfaces were incubated with heparin (5000 IE/ml), hirudin (6500 IE/ml)
and melagatran (180 µM) respectively. The FibMat surfaces were put in 1.5 ml tubes
with the highly concentrated solution of one drug in each tube. The tubes were put on a
rotator and the incubation time was 30 minutes. A surface with only the fibrinogen
matrix served as a reference. PFP was used with an addition of 0.5 M CaCl2 and TF
(1:2000).
Coagulation times were measured directly after the incubation. For the stability tests,
the procedure was done with eight new FibMat surfaces. The difference was that after
the drug incubation, the surfaces were incubated in PBS. Four surfaces were incubated
in PBS for 15 minutes on a rotator and after that the coagulation times were measured.
The last four surfaces had a 30 minutes incubation time on a rotator before the
coagulation times were measured.
For the surfaces not incubated in PBS, time = 0 min shown in the charts in Figure 19,
the coagulation times were much longer for heparin and hirudin. There was also a big
difference between coagulation time on the surface and in the bulk; compare the charts
above for the coagulation times on the surface with the charts below for the coagulation
times in the bulk. The longer time for the surface shows good effect from the drugs.
After incubation in PBS for 15 minutes, the effect had been washed away for the
surfaces covered with heparin and hirudin and the coagulation times were similar to the
reference surface. For melagatran the coagulation times differed a lot between the two
donors, which can be seen in Figure 19.
When the rinsing time with PBS was doubled, the coagulation times were much shorter
and quite similar for all surfaces as seen in Figure 19.
28
29
Figure 19. Coagulation times of PFP on fibrinogen (reference) and fibrinogen + heparin (5000 IE/ml),
hirudin (6500 IE/ml) and melagatran (180 µM) surfaces (n=2). The drug incubation time for the surfaces
was 30 minutes.
During these first experiments, heparin was the anticoagulant that showed the longest
coagulation times. That is why heparin and not hirudin or melagatran was used for
further tests.
30
In the second coagulation experiment, heparin and cangrelor were mixed in the
fibrinogen solution in an attempt to integrate the drugs into the matrix. Here, platelet
rich plasma, PRP, was used with an addition of 0.5 M CaCl2 and TF (1:2000). As seen
in Figure 20, the coagulation times were short. The surface incubated in heparin, for
example, had a coagulation time of less than seven minutes in Figure 20, which can be
compared to the time for the same drug of over 40 minutes on the surface in Figure 19
(time = 0 min). Another thing that can be observed is that the coagulation starts on the
surface in all four cases. The experiment was made twice with blood from two different
donors.
Figure 20. Surface coagulation times of PRP on fibrinogen (reference) and fibrinogen in combination
with heparin (5000 IE/ml)), cangrelor (100 nM) and heparin + cangrelor (1 part cangrelor to 4 parts
heparin) surfaces (n=2). The drugs were mixed in the fibrinogen solution in connection with the
production of the surfaces
.
31
In the last coagulation experiment, three FibMat surfaces were incubated in heparin for
20 hours. One of them was then incubated in PBS for 30 minutes, one for 15 minutes
and the third was not incubated in PBS at all. A surface with only the fibrinogen matrix
served as a reference surface. PFP was used with an addition of 0.5 M CaCl2 and TF
(1:2000). Two identical experiments were done with blood from two different donors.
As seen in Figure 21, heparin only had effect on the surface that has not been incubated
in PBS, the other two acted similar to the reference surface.
Figure 21. Surface coagulation times of PFP on fibrinogen (reference) and fibrinogen + heparin (5000
IE/ml) surfaces (n=2). The drug incubation time for the surfaces was 20 hours. One heparin surface was
incubated in PBS for 30 minutes, one for 15 minutes and the third heparin surface was not incubated in
PBS at all. For the surfaces with heparin, PBS 15 min, heparin, PBS 30 min and reference; the
coagulation times was the same for both surface and bulk.
32
4.3.2 Low shear platelet adhesion
For FibMat2.0/X, two experiments were made using a rotator and a fluorescence
microscope.
In the first experiment, seven different surfaces were tested in two experiments using
whole blood from two different donors. The first surface was used as a reference and it
was first incubated in saline solution and then in whole blood. Surface 2 and 3 was also
incubated in two steps; first in cangrelor and then in whole blood. The difference
between surface 2 and 3 was an addition of ADP in the blood for surface 3. For the last
four surfaces, only one incubation was made; surface 4 was incubated in whole blood,
surface 5 in whole blood with an addition of cangrelor, surface 6 in whole blood with
ADP and the last surface in whole blood and a combination of cangrelor and ADP. The
time for incubation 1 was 10 minutes and incubation 2 was made on a rotator for 20
minutes. Table 2 shows the details about incubations and concentrations of the drugs.
Table 2. The different types of surfaces used for the experiment with fluorescence microscopy, FibMat2.0.
Surface
1 (Reference)
2
3
4
5
6
7
Incubation 1
Saline solution
Cangrelor (100 nM)
Cangrelor (100 nM)
Incubation 2
500 µl whole blood
500 µl whole blood
500 µl whole blood + 5 µl ADP (13 µM)
500 µl whole blood
500 µl whole blood + 5 µl cangrelor (100 nM)
500 µl whole blood + 5 µl ADP (13 µM)
500 µl whole blood + 5 µl cangrelor (100 nM)+
5 µl ADP (13 µM)
33
Figure 22a. Platelet adhesion for different combinations including saline solution, cangrelor (100 nM)
and ADP (13 µM) on FibMat2.0 surfaces (n=2) in presence of blood after exposure of whole blood on a
rotator for 20 minutes. Platelets were stained with Alexa Fluor® 546 phallodin.
As seen in Figure 22a, the results differ a lot between donor 1 and donor 2. There are
also big variations for donor 2 regarding the surface incubated with an addition of ADP
in whole blood.
To see what the surfaces look like, one photo representing each surface from donor 2 is
shown in Figure 22b. Surfaces 1-3 were incubated in saline solution (surface 1) and
cangrelor (surface 2 and 3) before the blood incubation. It seems though to be an
increase in platelets when the surfaces are incubated in the platelet inhibitor.
Regarding surface 4-7 in Figure 22b, the drugs have been added in the blood and there
has only been done one incubation. The surface incubated in just blood (surface 4) was
quite similar to the ones incubated in cangrelor (surface 5) and the combination of
cangrelor and ADP (surface 7). Surface 6, the surface with ADP, had a clear increase in
platelets.
34
1
2
4
5
6
7
3
Figure 22b. Surface 1-3 are incubated in two steps. 1: first saline solution and then whole blood. 2: first
in cangrelor (100 nM) and then whole blood. 3: first in cangrelor (100 nM) and then whole blood + ADP
(13 µM). Surface 4-7 are incubated in one step in whole blood with or without an addition of some drug.
4: only whole blood. 5: addition of cangrelor (100 nM). 6: addition of ADP (13 µM). 7: addition of a
combination of cangrelor (100 nM) and ADP (13 µM).
All photos in this thesis are taken from pictures from a fluorescence microscope.
Everything that is white or gray is platelets, aggregates of platelets or, in later
experiments, fibrin network. Everything that is black corresponds to the background.
35
The second experiment was repeated three times with blood from three different donors.
It was conducted in the low shear model, two antiplatelet agents (cangrelor and apyrase)
and platelet activator ADP. For all surfaces, the drugs were mixed in the blood and
incubated in one step. Surface 1 served as a reference and was incubated in whole blood
without any additions. Surface 2-6 was incubated in whole blood with an addition
drug/drugs; surface 2 with apyrase, 3 with cangrelor, 4 with ADP, 5 with a combination
of apyrase and ADP and surface 6 with a combination of cangrelor and ADP. Table 3
shows details about the six surfaces.
Table 3. The different types of surfaces used for the experiment with fluorescence microscopy, FibMat2.0.
Surface
1 (Reference)
2
3
4
5
6
Incubation
500 µl whole blood
500 µl whole blood + 0.5 µl apyrase (1000 u/ml)
500 µl whole blood + 5 µl cangrelor (100 nM)
500 µl whole blood + 5 µl ADP (13 µM)
500 µl whole blood + 0.5 µl apyrase (1000
u/ml)+ 5 µl ADP (13 µM)
500 µl whole blood + 5 µl cangrelor (100 nM) +
5 µl ADP (13 µM)
Figure 23a. Platelet adhesion for different combinations including apyrase (1000 u/ml)), cangrelor(100
nM) and ADP (13 µM) on FibMat2.0 surfaces (n=3) in presence of blood after exposure of whole blood
on a rotator for 20 minutes. Platelets were stained with Alexa Fluor® 546 phallodin.
36
In this experiment there were big variations between the results, which can be seen in
Figure 23a and Figure 23b. The biggest variations is seen on surface 2 and 5, which are
the ones including apyrase. In Figure 23a, for example, the bar for surface 2 shows that
the platelet coverage differs from less than 20 % to over 80 % between the three donors.
Donor 1
Donor 2
Donor 3
Reference
Apyrase
Cangrelor
ADP
Apyrase
+ ADP
Cangrelor
+ ADP
Figure 23b. Photos of the different surfaces for the three donors from the experiment described in Table 3
and shown in Figure 23a.
37
The fact that apyrase behaves very diverse for different donors can also be seen in the
photos (Figure 23b) where the pictures from the three donors indicates totally different
adhesion patterns.
Because of the difficulties in interpreting the results, it could be concluded that it is not
easy to do these kinds of tests on a rotator, which creates only a low shear. For the
remaining experiments the method cone-and-plate, which creates a high shear, was used
in an attempt to get more consistent results.
4.3.3 High shear platelet adhesion
The first cone-and-plate experiment was performed five times with blood from five
different donors. The rotation was set to 1200 rpm and the experiment went on for four
minutes for each surface. Surface 5 was incubated in whole blood + apyrase and surface
6 was incubated in whole blood + cangrelor for 10 minutes before the cone-and-plate
experiment started. Those surfaces had an addition of ADP and CaCl2 during the
experiment. Surface 1 worked as a reference surface and had whole blood with an
addition of CaCl2 dropped on it. For surface 2, 3 and 4; the whole blood that was put on
the surface had an addition of apyrase, cangrelor and ADP respectively. For each
surface, the amount of blood with or without additions was 40 µl and the amount of
CaCl2 for each surface was 1.5 µl. Table 4 shows details about the six surfaces.
Table 4. The different types of surfaces used for the Cone-and-plate experiment, FibMat2.0.
Surface
1 (Reference)
Incubation
On the surface
Fibrinogen surface with whole blood +
CaCl2
Whole blood + apyrase (1000 u/ml)+
CaCl2
Whole blood + cangrelor (100 nM)+
CaCl2
Whole blood + ADP (13
+ CaCl2
Addition of ADP (13 µM)+ CaCl2
2
3
4
5
6
10 minutes in whole blood +
apyrase (1000 u/ml)
10 minutes in whole blood +
cangrelor (100 nM)
Addition of ADP (13 µM)+ CaCl2
38
Figure 24a. Platelet adhesion for different combinations including apyrase (1000 u/ml)), cangrelor (100
nM) and ADP (13 µM) on FibMat2.0 surfaces (n=5) in presence of blood. The rate was 1200 rpm and the
experiment went on for four minutes for each surface. Platelets were stained with Alexa Fluor® 546
phallodin.
The trend for the surfaces Figure 24a was that cangrelor seemed to be the drug that
worked best in this model. The results were more consistent in the bars for surface 3,
representing cangrelor, than the ones for surface 2, which represented the experiments
including apyrase. From these results, cangrelor was chosen to be used in further
experiments and apyrase was considered not suitable for this kind of model.
39
Donor 1
Donor 2
Donor 3
Reference
Apyrase
Cangrelor
ADP
Apyrase
+ ADP
Cangrelor
+ ADP
Figure 24b. Photos taken from surfaces used in the experiment described in Table 4 and shown as a
diagram in Figure 24a.
40
Some observations can be done about the surfaces by looking at the appearance of the
platelets. By looking at photos from donor 2 it can be said that the reference surface has
a characteristic appearance for a surface in a cone-and-plate experiment. The platelets
form in strings and there are also some aggregations. On the surface covered with
cangrelor, one can see a significant decrease in number of platelets. They also have a
different shape; they are round and do not likely form aggregates. For the surface
covered with ADP, a clear increase in platelets has occurred and there are aggregates.
The surface covered with a combination of cangrelor and ADP has quite many, round
platelets and hardly any aggregations. There are more platelets than the surface with
only cangrelor but less platelet than the surface incubated with only ADP.
The last experiment was made three times with blood from three different donors. The
rate was 1200 rpm and it went on for five minutes. The first surface worked as a
reference surface and it was incubated in saline solution, the second one was incubated
in heparin, the third one in cangrelor and the last surface was incubated in a
combination of heparin and cangrelor. For all surfaces the incubation time was 90
minutes. In the cone-and-plate experiment; whole blood, TF (1:400) and CaCl2 was
used. Table 5 shows details about the four surfaces.
Table 5. The different types of surfaces used for the Cone-and-plate experiment, FibMat2.0/X.
Surface
1 (Reference)
2
3
4
Incubation
Fibrinogen surface in saline solution
for 90 minutes
Fibrinogen surface in heparin (5000
IE/ml) for 90 minutes
Fibrinogen surface in cangrelor (100
nM) for 90 minutes
Fibrinogen surface in heparin (5000
IE/ml) + cangrelor (100 nM) (1 part
cangrelor in 4 parts heparin) for 90
minutes
41
On the surface
Whole blood + TF 1:400 + CaCl2
Whole blood + TF 1:400 + CaCl2
Whole blood + TF 1:400 + CaCl2
Whole blood + TF 1:400 + CaCl2
In this experiment, both platelets and fibrin network were studied. Platelets were stained
with Alexa Fluor® 546 phallodin and the fibrin network with anti-fibrinogen [FITC]. As
seen in the left chart in Figure 25a it is difficult to conclude the results because of the
variations. It is strange that the platelet inhibitor cangrelor results in a higher percentage
of platelets than the reference surface. By comparing the reference surfaces in Figure
24a and the left chart in Figure 25a, it can be seen that the latter ones have a significant
lower percentage of platelet coverage.
In the same experiment, studying the coverage of the fibrin network instead of platelets,
the results were much more consistent. In a comparison with the reference surface and
the surface incubated in cangrelor against the surfaces incubated in heparin and heparin
+ cangrelor respectively, The right chart in Figure 25a shows that heparin had an effect
in preventing the fibrin network to be formed.
Figure 25a. Platelet adhesion (the left picture) and fibrin network (the right picture) for different
combinations including heparin (5000 IE/ml), cangrelor (100 nM) and a combination of heparin and
cangrelor (1 part cangrelor in 4 parts heparin) on FibMat2.0 surfaces (n=3) in presence of blood. The
rate was 1200 rpm and the experiment went on for five minutes for each surface. Platelets were stained
with Alexa Fluor® 546 phallodin and the fibrin was stained with anti-fibrinogen [FITC].
42
Platelets
Fibrin network
Reference
Heparin
Cangrelor
Heparin +
Cangrelor
Figure 25b. Photos of four different surfaces showing platelets to the left and fibrin network to the right.
Information about the experiment can be found in Table 5 and Figure 25a.
The pictures to the left in Figure 25b show the platelets. It can be seen that there are not
that big differences between the four surfaces. There is not much platelet adhesion on
neither of the surfaces.
The pictures to the right in Figure 25b show the fibrin network for the four different
surfaces. Nice fibrin networks were formed on the reference surface and the surface
with cangrelor. For the surface with heparin, the drug has shown a big impact and only
fragments of the network have been created. For the combination of heparin and
cangrelor, the surface was covered with dots and aggregates but no fibrin network.
43
5. Discussion
5.1 FibMat2.0 with bisphosphonates
According to the results in Figure 17 and Figure 18, it can be concluded that an
integration of BP’s do not make the fibrinogen matrix behave differently in presence of
blood. It can be seen that a surface coated with FibMat2.0 with or without BP’s act
similar to a clean titanium surface regard to platelet adhesion. When it comes to
coagulation; surfaces with a fibrinogen matrix act similar to a fibrinogen coated surface
with integrated BP’s. Those surfaces have longer coagulation times compared to a clean
titanium surface but shorter compared to the coagulation that occurs in the cuvette with
no titanium surface.
The BP’s are quickly released from the fibrinogen matrix to the surrounding bone and
will have a long retention time at the same location in the bone according to Weiss et al
[78]. In case of cell apoptosis, the BP’s are released and can adhere to bone mineral
again. The BP’s are therefore able to be recycled which means that they have a longterm residence in the same part of the bone [79] According to other studies with BP’s,
one dental by Abtahi et al and one regarding knee prosthesis in human by Hilding et al,
it seems that treatment before implantation is safe and with no visible complications
[80, 81]. Another study, made by Moroni et al, on osteoporotic patients with a hip
fracture has shown that bisphosphonates is effective in improving screw fixation in the
cancellous bone [82].
5.1.1 Coagulation
As seen in Figure 17, the coagulation times for the surfaces with fibrinogen and
fibrinogen + BP respectively, are similar. The coagulation time for the surface with
clean titanium is significantly shorter. One explanation for that is that titanium is more
negatively charged than fibrinogen in its native form which leads to an activation of
factor XII and that implies a faster coagulation [83]. The reference surface corresponds
to the surface of the cuvette wall. It is made of PMMA, polymethyl methacrylate, which
is an inert material in terms of contact activation [15]. The reason for the fact that the
fibrinogen surfaces, with or without BP’s, have a shorter coagulation time than the
reference can be that the PMMA cuvette is more inert than the more negatively charged
fibrinogen surfaces and thereby have longer coagulation time.
44
There is also a big difference between the coagulation times near the surface and in the
bulk for the clean titanium surface as well as for the titanium surfaces coated with
fibrinogen, with or without BP’s. That shows that the coagulation starts on the surface
and then spreads out in the bulk.
For the reference surface, the fully coagulation could not be seen during the 200
minutes experiment. It might not happen at all in the cuvette made of PMMA. For the
other three cuvettes, the coagulation process started at the surface and then spread out in
the bulk.
In comparison with results from [84] according to the clean titanium surface and the
surface with fibrinogen in combination with frozen plasma, the coagulation times agree
with the ones for the experiments in this thesis.
5.1.2 Low shear platelet adhesion
The results are quite similar between the three surfaces. It can be seen in Figure 18 that
there is a slightly lower percentage of platelets on the surface with fibrinogen + BP, but
on the other hand, there are large variations for all three surfaces. The blood used in this
experiment came from six different donors, which naturally lead to great variations.
There are many factors affecting the blood and even if the blood would come from the
same donor, but from two different days, the results might differ. This phenomenon was
also seen seen in Figure 22 [84], where there are photos of two titanium surfaces with
adhered platelets. The surfaces were treated in the same way but the appearance of the
platelets differed a lot.
In this project, the platelet coverage is about 30 % each for all three surfaces. When
studying the photo in Figure 23 B in [84] with an experiment done in the same way, the
coverage seems to be far more than 30 %. That shows the large variations that occur
because of differences in blood.
45
5.2 FibMat2.0 with antithrombotic drugs
5.2.1 Coagulation
For these experiments, blood from only two donors each was used. It should be noted
that with this number of repetition the conclusions are somewhat limited. Some trends
though, could be seen. In the first experiment it was investigated how well the drugs
were retained inside the fibrinogen matrix. Two identical tests with blood from two
donors were performed and the surfaces were incubated with heparin, hirudin and
melagatran respectively. A surface with the fibrinogen matrix alone served as a
reference. Figure 19, shows that coagulation occurred first in the bulk for all types of
surfaces. That might indicate that the anticoagulants have been successfully integrated
into the fibrinogen matrix because some kind of effects has been seen due to preventing
the coagulation to occur directly on the coated surface.
By looking at the charts regarding donor 2 in Figure 19 it seems like the drugs are
loosely bound to the matrix because there is hardly any effect left after just 15 minutes
incubation in PBS. For donor 1 in Figure 19 the coagulation times for the surface
incubated in melagatran are prolonged after the 15 minutes incubation in PBS compared
to the surface with no PBS incubation. It would be interesting to redo this experiment to
find out if melagatran behave this way or if it was a coincidence.
During this first experiment, heparin was the anticoagulant that showed the longest
coagulation times. That is the reason why heparin and not hirudin or melagatran was
used for further tests.
In the second coagulation experiment, heparin and cangrelor were mixed in the
fibrinogen solution in an attempt to integrate the drugs into the matrix as during its
formation. As seen in Figure 20 the coagulation times were very short. The surface
incubated in heparin, for example, had a coagulation time of under seven minutes,
which can be compared to the time for the same drug of over 40 minutes on the surface
in Figure 19 (time = 0 min) The drugs bound into the fibrinogen matrix but the
coagulation was speeded. This might depend on incubation times or temperature that are
optimal for the production of the matrix, but not suitable for the drug loading process.
46
Regarding where the coagulation starts, there is a difference between the experiment
shown in Figure 19, where the coagulation starts in the bulk, and the experiment shown
in Figure 20, where the coagulation starts on the surface. This observation is also an
indication of a successful integration of drugs when the incubation occurs after the
fibrinogen matrix is produced.
When it comes to the question of long term effects of the drug loaded matrix, Figure 19,
shows that as early as after 15 minutes in PBS, there were not much effect left from the
drugs. As seen in Figure 21, even though the incubation time in heparin was prolonged
to 20 hours, there was no effect of the drug after the 15 minutes incubation time in PBS.
The coagulation experiments show a fast drug release from the fibrinogen matrix. If a
slower drug release is wanted, one possible way could be to actively bind drugs to the
surface (coulombic interaction or covalent binding).
5.2.2 Low shear platelet adhesion
As seen in Figure 22a, there were big differences between the results from the different
donors. This shows that the haemostatic properties of blood from two different donors
can vary a lot. Another observation is that for Donor 2 in Figure 22a, there was a higher
percentage of platelets on the surface incubated in cangrelor before the incubation in
blood (bar 2) compared to the reference surface (bar 1). Furthermore, the percentage is
lower when adding cangrelor directly in the blood (bar 5). One thing to have in mind
regarding the experiments is the low shear flow conditions. According to Jackson [85]
there are differences between which mechanisms that initiate platelet aggregation
depending on shear rates. At a low shear rate, < 1000 s -1, platelet aggregation is
predominately mediated by the interaction of fibrinogen with integrin α IIbβ3. In Nilsson
et al [86] it is shown in different diagrams that the platelet coverage is lower under a
low shear (150 s-1) than under a higher shear (1500 s -1). Reasons why platelet adhere
and aggregate differently under high and low shear can be due to tissue factor
conformation and that receptors and ligands behave differently.
According to the results seen in Figure 23a, the surfaces with blood (bar 1), blood +
cangrelor (bar 3), blood + ADP (bar 4) and blood + cangrelor + ADP (bar 6) have more
consistent results than the ones including apyrase. Apyrase seems to have the opposite
effect compared to cangrelor when used at a low shear rate.
47
The question is why apyrase is unable to inhibit platelet adhesion when integrated into
the fibrinogen matrix. In [87], apyrase was connected to a polystyrene surface and
succeeded in platelet inhibition.
Regarding ADP, it can be seen in both Figure 22a and Figure 23a that is has large
variations in the results. One explanation is that ADP likely forms aggregates. It can
both be big aggregates that gets stuck on the surface, or too big aggregates that not is
able to get attached to the surface.
Another observation from the two experiments regarding platelet adhesion under a low
shear is that neither a loaded matrix nor a non-loaded matrix was suitable in this kind of
shear model. By looking at Figure 22a and Figure 23a it can be seen that there are large
variations between the results for the different donors. The fact that for example the
platelet inhibitor apyrase results in a higher percentage of platelets compared to the
reference surface gives a signal that the experiment should be made in another way.
That is why the nextcoming experiments were done in a high shear model instead.
5.2.3 High shear platelet adhesion
As seen in Figure 24a, the results varied a lot between different donors. The most
consistent results came from the reference (bar 1) and cangrelor (bar 3). The platelet
coverage was calculated from differences between objects on the background and says
nothing about how the platelets covered the surface. In the photos shown in Figure 24b,
it is possible to see what the platelets look like, what shape they have and if they form
aggregates or not. Cangrelor seem to be more suitable for this model because of more
consistent results and the fact that it actually inhibit platelet adhesion. Figure 24a shows
a reduction in platelets for the surface incubated in cangrelor. For apyrase on the other
hand, the platelet coverage is higher than for the reference surface for some of the
donors. Apyrase has shown more unpredictable results and will not be used in more
experiments. The platelet inhibitor that will be used in further experiments is cangrelor.
In the left chart in Figure 25a, the results for the reference and cangrelor alone are most
consistent. For the experiments including heparin, there are large variations. Overall, the
percentage is much lower compared to previous experiments. That fact is also clear
when studying the photos of platelets in Figure 25b.
48
When looking at the right chart in Figure 25a, it can be seen that the results were quite
consistent regarding the fibrin network from the same experiment. There seemed to be a
lot of fibrin network for the reference surface and the surface incubated in cangrelor
which is reasonable because the platelet inhibitor cangrelor should not have any direct
impact on fibrin network formation. For the surfaces incubated with heparin and the
combination of heparin and cangrelor the fibrin network were clearly not formed.
Another study regarding vascular stents shows that a combination of heparin and a
platelet inhibitor significantly reduces the coagulation compared to one of the two
components alone [88].
The pictures to the right in Figure 25b show photos of four surfaces that are
representative for the whole experiment. On the surface incubated with heparin, there
were just small fragments of fibrin network. This shows that heparin has prevented the
fibrin network to be formed. On the surface incubated with a combination of heparin
and cangrelor the surface looked the same with the addition of some kind of aggregates.
49
6. Conclusions
The aim of this thesis was to investigate how different drugs could be integrated in the
fibrinogen matrix and what effect they might show. Haemostatic studies have then been
done on the coated surfaces to see what effect the drugs have, when integrated into the
matrix.
6.1 FibMat2.0 with bisphosphonates
The results confirm the initial thesis that surfaces incubated with fibrinogen with or
without integrated BP’s act similar to clean titanium surfaces in regard platelet
adhesion. In the coagulation experiments, surfaces coated with fibrinogen, with or
without BP’s, showed longer coagulation times than clean titanium and shorter times
compared to the reference, which consisted of a PMMA.
6.2 FibMat2.0 with antithrombotic drugs
6.2.1 Anticoagulants integrated in the fibrinogen matrix
From the results, it was found that heparin and hirudin showed the best anticoagulant
effects. In a comparison between adding the anticoagulants already in the fibrinogen
solution and incubation with anticoagulants afterwards, the latter method is preferable.
The drug release was not surprisingly very fast in all coagulation experiments.
6.2.2 Platelet inhibitors integrated in the fibrinogen matrix
The results show that cangrelor seemed to have better effect when added in blood and
incubated compared to incubation with the platelet inhibitor on the surface before
incubation in blood. This was shown in experiments performed at a low shear rate.
According to my results, cangrelor inhibit platelet adhesion more effectively than
apyrase both under high and low shear rate.
50
6.2.3 Anticoagulant and platelet inhibitor in the fibrinogen matrix
For the last experiment, one anticoagulant and one platelet inhibitor was chosen in an
attempt to combine the two inhibitory mechanisms. Heparin and cangrelor was picked
because they had shown most effect and the most consistent results previously. Very
clear differences in regard to formation of fibrin network could be seen when comparing
the four surfaces. The surface incubated with both heparin and cangrelor showed the
least formation of fibrin network.
51
7. Future aspects
Control; try to control the drug release by finding a connection between drug
concentration and effect.
Try to actively bind drugs to surfaces (coulombic interaction or covalent
binding) for a slower drug release.
Detection; find a way to detect how much of the drug or drugs that has been
integrated into the fibrinogen matrix. That can be done with liquid scintillation
or by binding radioisotope-labeled compounds to the surface.
Try other drugs and other combinations of drugs into the fibrinogen matrix, for
example heparin + polyamines.
Make the experiments with many more donors due to the big variations between
donors.
52
8. Acknowledgements
First of all I would like to thank Ph.D. Trine Vikinge, CEO of AddBIO AB, for giving
me the opportunity to work with this project and always being interesting in discussing
my lab results and contributing with valuable comments.
My sincere thanks go to my supervisors; MSc. Henrik Aronsson for all assistance in the
IFM lab and Ph.D. Lars Faxälv for all guidance at the lab at Klinisk Kemi. I have really
enjoyed your company and you both have helped me but also taught me to work
independently, which I appreciate.
I will also thank Prof. Pentti Tengvall for accepting being my examiner, although we
have not met yet.
A big thank you to Emma Gundersen and Frida Olsson for brighten up every Monday
morning at the meetings at AddBIO AB, Maja Richter for great company while writing
the report and drinking coffee, the staff at the department of Klinisk Kemi for good
times at the lab, especially Kerstin Gustafsson for her excellent skills with the needle
and everyone who has helped out and donated blood for my experiments!
My greatest thanks to Friends, Family and of course, my better half Robin.
53
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