The current development of
(semi)-artificial platelet
substitutes
N. van Oorschot
N.van Oorschot
Catharijnesingel 131
3511 GZ Utrecht
0643975799
niek@vanoorschot.net
Studentnumber: 3257924
Supervisor: Dr. M. Roest
1
Index
Introduction _____________________________________________ 3
Platelet transfusion ________________________________________ 5
Fibrinogen substitutes _____________ Error! Bookmark not defined.
Coated erythrocytes ______________________________________ 11
Coated albumin microcapsules/microspheres__________________ 13
Coated liposomes ________________________________________ 17
Nanosheets _____________________________________________ 20
Discussion ______________________________________________ 22
References _____________________________________________ 25
2
Introduction
Blood platelets are small cell-fragments circulating in the blood. They originate from
megakaryocytes in the bone marrow. Because platelets are cell-fragments of the
megakaryocyte, they do not contain a nucleus. Platelets are about 1-2 µm in size and have a
lifespan of 7 – 10 days.
Platelets are essential in the primary haemostasis. Upon blood vessel damage, the collagen
fibers underlying the vessel will be exposed. Platelets in circulation will adhere to the
collagen fibers with the aid of von Willebrand factor (vWF). This reduces the speed of the
platelets in the circulation and activates them. Upon activation, other receptors on the
platelets plasma membrane will be able to bind their ligands. These receptors will anchor
the platelet strongly to the vessel wall, not able to be released by the circulating blood. This
involves several integrins (i.a. GPVI and GPIIb/IIIa). Then the platelet gets fully activated.
Upon activation, GPIIb/IIIa receptors open and they can bind other platelets. This assures the
forming of a platelet aggregate. This aggregate formed in the primary haemostasis is a
haemostatic plug which seals the vessel wall. Then the secondary haemostasis can take
place.
As stated, platelets play an essential role in the primary haemostasis. A lack of platelets can
therefore lead to higher risks of bleeding. A platelet count of <150͘͘͘͘͘͘ˑ10͘͘͘͘͘͘9 L-1 is diagnosed as
thrombocytopenia. A platelet count between 50͘͘͘͘͘͘ˑ10͘͘͘͘͘͘9 L-1 and 150͘͘͘͘͘͘ˑ10͘͘͘͘͘͘9 L-1 normally does not
lead to a bleeding tendency, and is mostly discovered accidently during an operation or
tooth extraction. The risks gets high at a platelet count of <10͘͘͘͘͘͘ˑ10͘͘͘͘͘͘9 L-1, leading to skin and
mucous membrane bleedings as purpura, petechiae or ecchymoses.
Thrombocytopenia can be caused by a decrease in platelet production, an increase in
platelet degradation or a disrupted distribution of platelets. Also blood suppletion with
erythrocyte concentrates and plasma after severe blood loss can lead to thrombocytopenia
due to dilution. In most patients the cause of thrombocytopenia is multi-factorial.
A decrease in platelet production can be caused by a decreased megakaryopoiësis i.e. as a
result of radiation or drugs, a decreased thrombopoiësis as a result of drugs or a lack of folic
acid and by hereditary factors. These hereditary factors include various syndromes such as
TAR-syndrome, Bernard-Soulier syndrome and gray-platelet syndrome.
Increased degradation is the most common cause of thrombocytopenia and can have
immunological causes as well as non-immunological causes. Drugs can cause an
immunological degradation of platelets. Some drugs can combine with platelet plasma
membrane proteins to form neo-epitopes. Auto-antibody’s against this neo-epitope will bind
and the platelets will be degraded. Some drugs (i.e. GP IIb/IIIa-antagonists) can cause a
3
conformational change, thereby exposing unusual parts of the receptor, which can be
recognized by antibodies, resulting in platelet degradation. Other immunological causes of
thrombocytopenia include auto-immunethrombocytopenia and alloimmunethrombocytopenia of neonates. Non-immunological thrombocytopenia causes
include thrombotic thrombocytopenic purpura (TTP), haemolytic-uraemic syndrome (HUS),
VWF disease type IIb and mass blood transfusion.
Disrupted distribution of platelets can be caused due to spleen enlargement, leading to
hypersplenism in which the spleen holds up a large number of platelets. However, the
platelet count can never fall to <20͘͘͘͘͘͘ˑ10͘͘͘͘͘͘9 L-1 as result of hypersplenism alone.
Thrombocytopenic patients can be treated by platelet transfusion. Platelet transfusions are
mainly given to patient with bone marrow diseases and patients with immunological caused
thrombocytopenia. Platelets collected from blood donors will be transfused into the
patient. Platelets can be obtained in several ways and with different preparation methods;
this is the subject of the next chapter.
The current methods of platelet transfusion have several disadvantage. Therefore, there is
an urgent need for alternative therapies. The most promising alternative for platelet
transfusion is the development of (semi)-artificial platelets; products which can overcome
several of the disadvantages of the current method of platelet transfusions. These (semi)artificial platelet contain thromboerythrocytes, albumin microspheres, liposomes and
nanosheets. This thesis will discuss the research of platelet substitutes taking the following
question in consideration. What is the current development of (semi)-artificial platelet
substitutes, and what are the advantages of these platelet substitutes over regular platelet
transfusion?
4
Platelet transfusion
Each year, 1,5 million platelet units are
transfused in the USA and 2,9 million
platelet units are transfused in Europe
(Maniatis, 2005). These large amount of
transfusions are therefore a great subject of
research. Currently, standard clinical
protocols for platelet transfusion are used,
but novel procedures are under research.
Platelet concentrates (PC) can be obtained
in two ways; either from anti-coagulated
whole blood or by plateletpheresis. In
European countries, both methods of PC
preparation are evenly used (Vasallo et al,
2006). Both methods have their own
advantages and disadvantages.
Whole-blood platelet production
Platelets are collected from anti-coagulated
whole blood in two ways, depending on the
type of centrifugation. This results either in
platelet-rich plasma (PRP) or buffy-coat
platelets (BCPs).
BCPs are most commonly used in Europe. In
the united states PRP accounts for 60% of
the platelets products transfused (Spiess,
2010). PRP is created by centrifugation of
anti-coagulated whole blood in two steps.
First a soft spin is applied, centrifugation
with a low g-force, thereby separating the
PRP from the red cells. The red cells are
stored for different use. Then a high g-force
Figure 1. Comparison of platelet production by the
platelet rich plasma (PRP)and buff y-coat (BC)
methods (Stroncek et al, 2006)
spin is applied, separating the platelets
from the plasma. Later on the platelets are
resuspended in 50-70 mL of donor plasma or
5
platelet-preserving solution. The end product contains about 65-70% of the donors platelets.
Most of the lost platelets are trapped in the red cell component, and lost after the first spin.
BCPs are produced by the exact opposite method of PRP. At first a hard spin is applied, and a
soft spin later on. After the hard spin, a layer of platelets (and white cells if they are not
filtrated before) is formed, this layer is called the buffy coat. The buffy coat is then
resuspended and a soft spin is applied, concentrating the platelets. During the hard spin,
about 25-30% of the red cells are destroyed, which could otherwise be used for other
applications.
BCP-PC’s and PRP-PC’s have an nearly equivalent platelet count. But BCP-PC’s have some
advantages. BCP-PC’s have 10% fewer contaminating leucocytes (Vasallo et el, 2006).
Furthermore, by using the BC preparation method, 30-75 mL more plasma can be recovered,
with 20 mL less erythrocytes as a consequence (regarding a standard 500 ml whole blood
donation)(Vasallo et al, 2006). PRP-PC’s must be separated from whole blood within 8 h of
collection. Whole blood, if rapidly cooled down to 22 °C, can be stored up to 24 hours before
processing by the BC-PC preparation method. Which makes the BC method more attractive,
in logistical perspective (Vasallo et al, 2006). The last advantage taken into account is more
theoretical; platelets isolated by the BC-method may be less activated during centrifugation.
This due to the fact that the platelets are centrifuged against a erythrocyte cushion rather
than being centrifuged against a non-physiological plastic container in the PRP-PC
production process (Vasallo et al, 2006)
Anyway, both production processes are limited in time, have the disadvantage of early
activation, can be contaminated (also after leucocyte reduction) and there is always a loss of
products (erythrocytes and plasma) that otherwise could be used for other clinical
applications.
Platelet apheresis
PC’s can be obtained from one single donor by platelet apheresis. This involves the use of
automated cell separation equipment. This apheresis machine harvests the platelets from
the donor’s whole blood. The remaining components are given back to the donor. This rises
the donors opportunity for donating blood more often.
PC’s produced by the whole-blood method are small doses. About four to six doses obtained
are pooled to make an adult therapeutic dose (Schrezenmeier et al, 2010) One adult
therapeutic dose contains approximately 3ˑ10͘͘͘͘͘͘11 platelets. The yield of platelets obtained
6
from a single donor with the apheresis method can vary depending on the donor, type of
machine, and procedure used but is equivalent to at least 3–13 random donor PCs obtained
with the whole blood method (Hardwick, 2008). Therefore, the PC obtained by apheresis can
be given as a single dose to one patient, or even sometimes split into 2 adult therapeutic
doses
Patients can become refractory to random donor platelets. This is due to alloimmunization
to HLA and/or platelet specific antigens. These patients need to be infused with HLA
matched platelets. 1 in 1500 patients are a HLA match, which makes it extremely difficult to
find a HLA match (SW medical center) Furthermore, HLA testing costs between $300 and
$500 in addition to the price of the platelets (SW medical center). This makes refractoriness
a problem in treatment of patients by platelet transfusion.
Infusing platelets of one single donor limits the risks alloimmunization to HLA antigens that
can be acquired due to exposure of a large number of donors. Also the risks of Transfusion
Transmitted Infections (TTI) are limited. Although limiting these risks by the use of a single
donor, does not eliminate them completely.
Leukocytes are reduced from platelet concentrates before storage, either by leukocytes
filtration or by apheresis protocols which uses size and density differences between platelets
and leukocytes to remove the leukocytes during the collecting of the platelets. Leukocytes
need to be removed because of the their unwanted complications in immune
compatibilities, including alloimunization to HLA and other leukocyte-associated antigens.
Furthermore, when the patient or the donor has antibodies against HLA -I or HLA-II of the
leukocytes, transfusion related acute lung injury can (TRALI) can occur. The mechanism of
TRALI is poorly understood, but it involves activation of neutrofilic granulocytes due to
antibody-antigen interaction. These granulocytes then bind to the lung endothelium. The
release of cytokines cause dilation and lead to lung edema. (van Stein and te Boekhorst,
2008). Other risk factors of transfusion of allogenic leukocytes include viral, bacterial, or
protozoal infections.
Leukocyte reduction is to be done pre-storage because of the release of biological active
substances during storage, for example interleukins which can cause fever in the patient.
Anyhow, some leukocytes might slip through and ends up in the platelet concentrate. This
problem, however, can have an advantage because of the fact that when bacteria are
harvested unwanted, leukocytes are likely to scavenge them.
Platelet concentrates obtained from donors need to be stored in blood banks, which can
lead to storage defects. When platelets are stored a complex series of changes may occur. At
7
first partial, graded activation takes place. This can be observed by the increase of GPIIb/IIIa
and GPIb expression on the surface of the membranes, as by decrease in the number or
concentration of their (mostly alpha-) granules. Activation during storage triggers loss of the
discoid shape, degranulation and vacuolization, thereby limiting their post-transfusional
recovery and survival in circulation (Maniatis, 2005). Also platelet and platelet-leukocyt
aggregates can be formed (Spiess, 2010)
Finally, the platelets may lose about 25-30% of their lipid membrane. The platelets are
budding off micro-particles of lipid membrane which are pro-thrombotic and therefore carry
risks (Spiess, 2010)
These signs of the platelets would seem to indicate that they are dying. However, the
lifespan of a normal platelet in circulation in vivo is about 10 days. After the platelets are
harvested and stored, their shelf life is 5 days. This means that a large number of platelets
present in the concentrate will die because they are already simply too old. Therefore Bruce
Spiess postulates the question in his 20͘͘͘͘͘͘10͘͘͘͘͘͘ article: “Does the harvest and storage of platelets
speed the normal ageing (dying) process? Or Are the changes part of a natural senesce
cycle?”(Spiess, 2010) Which is something to consider in future research
Taking into conclusion, all PC’s, whether they are obtained with apheresis or one of the
whole blood methods, have disadvantages. These disadvatages include alloimmunization,
transfusion transmitted infections or remaining leucocytes induced adverse effects such as
TRALI.
Then there is the platelet storage problem. The longer the platelets are stored, the less
usefull the product is. A large number of these disadvantages can be overcome by the use of
artificial platelets. Researchers have started with the development of semi-artificial
platelets, but cureently a lot of full-artificial platelet products exist. Ranging from liposomes
to albumin spheres and nanosheets, all fully synthetic and therefore crossing out a whole lot
of side effects and disadvantages. Furthermore, the most important factor; if all platelet
transfusion would be artificial there would not be any need for donors!
8
Fibrinogen substitutes
The glycoprotein(GP) IIb/IIIa complex is a heterodimeric receptor of the integrin family. It is
present on the membrane of platelets. Upon activation by an agonist (i.e. ADP or TRAP,) GP
IIb/IIIa receptors stored in internal pools move to the platelet surface (Matzdorf et al, 2006)
Furthermore a conformational change of the GPIIb/IIIa receptor (both the newly arrived as
the receptors already abundant) occurs, making the receptor accessible for interaction
(Becker, 1997). The GPIIb/IIIa receptor interacts with at least three adhesive proteins;
fibronectin, vWF and fibrinogen. These interactions are critical for platelet adhesion,
spreading, and aggregation (Andrieux, 1989).
Fibrinogen is an essential protein in thrombus formation and platelet aggregation. Platelets
bind to fibrinogen with their GPIIb/IIIa receptor, and thereby clustering of the platelets
occurs and aggregates are formed. The first (semi)-artificial platelets made were coated with
fibrinogen, so platelets were able to bind them and the substitutes can enhance platelet
aggregation. The use of the whole fibrinogen molecule to coat platelet substitutes has some
disadvantages. At first, the fibrinogen purification process is complicated because fibrinogen
from human blood is not stable, and its activity in solution is extremely low. It also rises the
potential for transmitting infectious diseases. (Takeoka, 2001)
There two classes of sequences in the fibrinogen protein that bind to the GPIIb/IIIa receptor;
the RGD-based sequences 95RGDF98 and 572RGDS575 in the Aα chains and
400HHLGGAKQAGDV411
, called H12, the fibrinogen γ-chain dodecapeptide, in the carboxyl-
terminal of the c-chain (Andrieux, 1989) These sequences will play a major role in future
experiments and investigation as you will see. The newer platelet substitutes are coated
with these sequences instead of the whole fibrinogen molecule.
At first the RGD sequences in fibrinogen, which are also implemented in fibronectin and
vWF, were discovered with a different goal at mind. By knowing these sequences, synthetic
peptides containing these sequences could be made. These synthetic peptides could inhibit
the interaction of each adhesive protein with platelets, by means of a strong competition.
Synthetic peptides could also be derived from the H12-sequences that is only present on
fibrinogen, with the same inhibition as a result. This inhibition could come in handy for
treating thrombosis, since it prevents the formation of blood clots. Later on these
developments were used to treat and investigate something other than thrombosis;
thrombocytopenia and the development of artificial platelet substitutes.
9
Okamura et al used the H12 fibrinogen sequence to coat their platelet substitutes, for the
first time. This decision derived from the general observations that the H12 sequence
interacts with the GPIIb/IIIa receptor specifically. The RGD sequence is the cell attachment
site of a large number of adhesive extracellular matrix, blood, and cell surface proteins, and
nearly half of the over 20 known integrins recognize this sequence in their adhesion protein
ligands [1]. As H12 binds specificially and only to GPIIb/IIIa, this sequence was preferred over
the RGD sequence.
Another possibility is to conjugate the artificial platelet substitutes with ligands which
interact with collagen or von Willebrand Factor (vWF), instead of the platelets. At a site of
vascular injury, collagen is exposed. vWF will bind to the collagen and facilitates the binding
of platelets to each other and to the collagen. If the (semi-) artificial platelet product has a
ligand that binds to either one of these molecules it will agglutinate specificially at sites of
vascular injury. The GPIa/IIa complex normally found on platelet membranes can bind to
collagen. Therefore, platelet substitutes can be coated with rGP1a/IIa. The advantage of this
conjugate is that they could also work in severe thrombocytopenic patients, because there is
no need for platelets to attach first to the sites of vascular injury. Platelet substitutes coated
with fibrinogen or fibrinogen sequences bind tot platelets, so their mechanism of action
relies on some platelets binding to the site of vascular injury first, which can be a limiting
step in severe thrombocytopenic patients. However, rGP1a/IIa conjugates cannot bind other
platelets. So platelet aggregates can not be made, and it does not enhance the primary
thrombosis.
Because of these advantages of the H12 en RGD peptides, the use of the rGPIa/IIa
conjugated artificial platelet products fell into decay and wasn’t investigated any further. All
later study’s used one of the fibrinogen sequences, to coat their platelet substitutes.
10
Coated erythrocytes
As soon as 1980, Coller et al discovered that inert
beads coated with fibrinogen cause platelets in rest to
agglutinate spontaneously (Coller, 1980) This
interaction however needed to be enhanced by the
addition of an agonist (i.e. ADP)(Coller,1980). In 1983
Agam and Livne found that formaldehyde fixed
platelets with fibrinogen covalently bound on their
surface, did the same (Agam and Livne, 1983) These
findings started a series of experiments in with a wide
range of platelets substitutes, starting with coated
erythrocytes (Agam and Livne, 1983,1984)
In 1992 Agam and Livne where the first to report
fibrinogen coated erythrocytes (Agam and Livne, 1992)
They covalently cross-linked fibrinogen molecules tot
the red cells membrane by incubation with
formaldehyde. These erythrocytes had the capability to
enhance agonist induced platelet aggregation in vitro.
The platelet-dependent aggregation, induced by ADP,
thrombin or ionophore, was dependent of the
fibrinogen concentration; the fibrinogen density on the
erythrocytes could vary between 58 to 1400 molecules
per cell. The tail bleeding time in thrombocytopenic
rats were shortened from 18 ± 1.5 minutes to 4.5±1.0
minutes 1 hour after fibrinogen-coated erythrocytes
injection. Interestingly the duration of the shortening
of bleeding time was longer for the erythrocytes
method than that seen after the infusion of fresh rat
platelets, which was the convential method for treating
thrombocytopenia.
Also in 1992 Coller et al did the same, apart from the
fact that they used only a part of the fibrinogen
molecule (Coller et al, 1992) They produced arginineglycine-asparatic acid (RGD) coated erythrocytes. These
platelet substitutes where called thromboerythrocytes
11
Figure 2. Microscopic image. control
erythrocytes: the dense lawn ofplatelets can be
seen with only a single adherent erythrocyte in
thefield. In sharp contrast, the
thromboerythrocytes bound extensively to the
adherent platelets. The binding of
thromboerythrocytes to the adherent platelets
was inhibited by the peptide RGDF (400
Mg/ml). (Adapted from Coller et al, 1992)
because of the fact that these are erythrocytes which gained a thrombocytic function. The
discovery that the RGD sequence on fibrinogen is recognized by the GPIIb/IIIa receptor led
Coller et al consider to covalently attaching an RGD-containing peptide instead of the whole
fibrinogen molecule. This would avoid the problems associated with purification of human
fibrinogen and the potential for transmission of infectious diseases. The
thromboerythrocytes were tested by binding them to platelets adhered to collagen. These
thromboerythrocyes formed a dense lawn over the platelets that were already adhered to
the collagen. Control erythrocytes however, did not. (Figure 2) Furthermore, adding of free
RGDF protein inhibited the binding of thromboerythrocytes by means of competition. This
means that the thromboerythrocytes bind the platelet with their conjugated RGDF
sequence.
Initial studies in vitro showed that thromboerythrocytes could interact with platelets
adhering to collagen under conditions of low shear rates of 50 to 100 per second, but not at
higher shear rates of 500 per second. An intitial study in which thromboerythrocytes were
infused in thrombocytopenic guinea pigs, showed a reduction of the standard ear bleeding
time (Cerasoli et al, 1994). However, the thromboerythrocytes did not significantly reduce
the prolonged bleeding time in thrombocytopenic primates (Alving et al, 1997)
Thromoberythrocytes are capable of selectively interact with activated platelets. The result
is the production of an aggregate containing platelets and erythrocytes. This is due to the
binding of the coated RGD sequences on the erythrocytes with the GPIIb/IIIa receptor on the
activated platelets.
There are several advantages for using thromboerythrocytes as an alternative for regular
platelet transfusions. At first, these cells can be made from the patient’s own blood. There is
no need for donor transfusions. The erythrocytes from the patient can be coated and
transformed into thromboerythrocytes in 1-2 hours. And since there are 20 times more
erythrocytes than platelets in the circulation of a normal individual, less blood is needed.
However, there are also disadvantages. It is possible that the thromboerythrocytes are
subject to prematural removal, due to their alteration. Furthermore, there was no reduction
of the prolonged bleeding time in vivo in primates. Lastly, the use of thromboerythrocytes
still comes with immunogenic issues, when the thromboerythrocytes have to be made from
donor blood depending on the situation of the thrombocytopenic patient. As
thromboerythrocytes are blood derived, alloimunization may occur. When non-blood
derived platelet susbtitutes could be made, the alloimmunization issue could be crossed out.
12
Coated albumin microcapsules/microspheres
The findings that inert beads and formaldehyde fixed platelets with bound fibrinogen, as
stated at the beginning of the previous chapter, also lead to the development of two kinds of
fibrinogen coated albumin particles (FAM). These were either microcapsules or
microspheres
In 1995, Yen et al. produces fibrinogen-coated albumin microspheres (FAM) which were
shown to be haemostatically active in thrombocytopenic rabbits. These were named
Thrombospheres (Yen et al, 1995) Aggregates formed in either perfusion chambers or seen
in ear bleeding time wound biopsies all contained a mixture FAMS, platelets and fibrin.
Administering of FAMS shortened the standard rabbit ear bleeding time from a mean of 21´7
min to 5´2 min at 15 min after infusion. Also the quantitative blood loss from a standard
surgical abdominal wall wound was decreased.
Later, in 1999, Levi et al. produced fibrinogen-coated albumin microcapsules called
synthocytes (Levi et al, 1999) The administration of synthocytes in rabbits made
thrombocytopenic, either by anti-platelet antibodies or by the means of chemotherapy,
resulted in a significant reduction of enhanced bleeding. The enhancement of primary
haemostatis seemed to be due to the facilitiation of adhesion of remaining platelets in
circulation by the synthocytes.
This mechanism of action was
discovered by a perfusion
experiment. The fibrinogen
coated microcapsules were added
to human whole blood and
perfused over a endothelial
matrix. Microscopic pictures
showed aggregates formed on the
endothelial matrix composed of
microcapsules, platelets and
connecting fibrin fibers. (Figure 3)
This was also confirmed by
microscopic analysis of biopsies
taken from the ear bleeding
Figure 3. Scanning electron microscope photograph showing the
interaction of the fibrinogen-coated albumin microcapsules and
platelet aggregates with connecting fibrin fibers (Levi et al, 1999)
wound of the rabbits.
Both products, FAMs and Synthocytes were not found to be thrombogenic. Both
experiments showed that the mechanism of their products effect relies on the facilitation of
13
adhesion of the remaining platelets to the endothelium. This makes the use of synthocytes a
new semi-artificial platelet product, that still relies on the presence of platelets.
Later on, in 2003, Takeoka et al started with a new novel platelet substitute. (Takeoka et al,
2003) These consisted of latex beads with a diameter of 1 µm. Human serum albumin was
adsorbed onto the surface of the latex beads and then either the H12 sequence or the RGD
peptide was conjugated on the surface via disulfide links. Bare latex beads, H12-latex beads
and RGD- latex beads were tested and compared by adding them to non-activated platelets,
centrifuging and measuring the percentage of agglutination by a flow cytometry. The
percentage agglutination did not increase for the bare latex beads and the H12-latex beads
but did increase for the RGD-latex beads to 2.9 ± 1.3%. (Takeoka et al, 2003)
They concluded that the H12 conjugated latex beads were shown to preferentially interact
with an activated platelet surface via GPIIb/IIIa receptors and to facilitate platelet
accumulation at sites of haemostasis. The adhesion of H12-latex beads was suppressed in
the presence of free H12 as an inhibitor of GPIIb/IIIa binding, showing that the adhesion was
specific. The RGD-coated beads on the other hand, seemed to cause agglutination with nonactivated platelets, which makes it a non-usable product. Agglutination of non-activated
platelets would lead to non-selective enhanced thrombosis in the human body.
With the conclusion that particles coated with the H12 sequence would be suitable
candidates for an alternative to human platelet concentrates transfused into
thrombocytopenic patients in mind, new platelet products were produced. In 2005,
Okamura et al. produced biocompatible and biodegradable particles by conjugating the H12
sequence to polymerized albumin particles (polyAlb) (Okamura et al, 2005). H12-poly-Alb
showed to specificially interact with an activated platelet surface via GPIIb/IIIa receptors and
to facilitate platelet accumulation at sites of haemostasis. The H12-polyAlb particles where
tested and compared with controls in vitro by the use of a flow experiment where imitation
thrombocytopenic blood flowed over a collagen-coated surface. The particles were also
tested in vivo with the standardized tail-bleeding time in thrombocytopenic rats. In both
tests the haemostatic ability was enhanced, therefore H12-polyAlb may be a suitable
candidate for an alternative to human platelet concentrates infused into thrombocytopenic
patients.
The problem with H12-PolyAlb is the fact that it’s half-life in blood is very short
(approximately 10 minutes) (Okamura et al, 2005) . By prolonging the blood residence time
in vivo, a more suitable product for further use can be obtained. Polyethylene glycol (PEG)
modification on the surface of carriers such as phospholipid vesicles or biocompatible
polymeric particles has been widely used to prolong the t1/2 or to stabilize their dispersion
14
states(Okamura et al, 2007) Therefore, Okamura et al constructed a H12-PEG-polyAlb
particles (Okamura et al, 2007). These particles maintained the specific binding ability to
activated platelets and the H12-PEG-polyAlb dose dependently shortened the tail bleeding
time of thrombocytopenic rats and the haemostatic effects lasted for at least 6 hours. H12PEG-polyAlb was also tested in thrombocytopenic rabbits, to check if the haemostatic
abilities also could be obtained in larger animals (Okamura et al, 2008). H12-PEG-polyAlb
particles significant shortened the template ear bleeding time in the rabbits. In the same
study the comparison between conventional platelet transfusion was calculated. The
haemostatic capacity of the H12-PEG-polyAlb was 31- or 65-fold greater than that of a
similar volume of platelets. This means that by the usage of this artificial platelet substitute,
which synthetically made and not blood-derived, less donor blood, which is always short in
supply, is needed. Okamura et al stated in their 2008 article that the next step in their H12PEG-polyAlb research is to treat animals with severe thrombocytopenia resulting from blood
loss during surgery. This subject of study, however, still to be done.
The mechanism of action of the H12-PEG-PolyAlb is as follows: At first, platelets still in the
circulation adhere to the collagen that is exposed at the site of the vascular injury. These
platelets are activated and bind to the H12-PEG-PolyAlb in the blood. Then, because the
H12-PEG-PolyAlb bond to the platelet, a lot of H12 sequences are available for binding other
platelets. H12-PEG-PolyAlb, hereby, promotes thrombus formation by accelerating and
enhancing the aggregating of the flowing platelets. H12-PEG-PolyAlb particles even
contribute to the thrombus by means of their own mass. Therefore, when administered at
higher doses, the H12-PEG-PolyAlb can also work in patients with severe thrombocytopenia.
In these patients, fewer platelets will adhere to the collagen. But because the H12-PEGPolyAlb particles also form a thrombus by the use of their own mass, it mechanism of action
still exists.
As stated before, it is also possible to use the rGPIa/IIa complex to conjugate as a ligand on
the albumin microspheres. Therefore, Teramura et al conjugated the rGPIa/IIa complex to
biocompatible albumin particles (poly-Alb) (Teramura et al, 2003) They concluded that
rGPIa/IIa-conjugated polyAlb reduced the tail bleeding time thrombocytopenic mice in vivo.
This reducement was significant but was only one-tenth of the platelets haemostatic
function. This is because platelets, and also fibrinogen (sequence) coated substitutes, can
bind other platelets to form an aggregate. This is an ability that rGP1a/IIa coated particles
lack, the only exerting haemostatic function is by means of their own mass, and they do not
recruit other platelets.
15
From all described studys can be concluded that albumin microparticles conjugated with the
H12-dodecapeptide are a promising artificial platelet substitute. Its ability to enhance
haemostasis has been shown in vivo and in vitro. Clinical research is still to be done, as also
more pre-clinical research is necessary. Unfortunately, coated albumin particles are no
subject of current research. Researchers and developers are focussing on liposome based
platelet substitutes; also a promising novel platelet substitute and therefore the subject of
the next chapter.
16
Coated liposomes
In 1988 came the idea of using liposomes as potential semi-artificial platelet substitute. Dr
Schwarz use several models to test the thrombogenicity of combinations of procoagluant
liposomes with activated factor X. This combination showed to be haemostatically active in
haemophilic dogs, but it was associated with an unacceptable toxicity (Blajchman, 2001)
In 1993, Rybak en Renzulli invented what they called plateletsomes. These consisted of
unilamellar lipid vesicles in which platelet membrane extracts were incorporated, including
the membrane receptors GPIb, GPIIb/IIa and GPIV. Plateletsomes did not show any effects
on platelet aggregation in vitro, but infusion of plateletsomes in thrombocytopenic rats
reduced the standard tail bleeding time (Rybak and Renzulli, 1993). Plateletsomes are a
semi-artificial platelet substitute and a blood derived product. Therefore, this product still
carries the risks of alloimmunization, as other transfusion side effects. A complete synthetic
platelet substitute would overcome these disadvantages.
Nishiya et al developed several liposome
based platelet substitutes, bearing
recombinant fragments of GPIb or
anticollagen antibodys. The first study
with significant haemostatic effects come
from Nishiya’s 2004 study in which
liposomes with H12 conjugated on their
surface were prepared. These liposomes
interacted with activated platetelets by
binding of the fibrinogen H12-seqeuence
to the GPIIb/IIIa receptor on the platelets.
Triggered by the interaction the liposome
released encapsulated material that was
within them. The rate of content release
was dose-dependent, dependent of the
surface density of H12. Furthermore,
liposome membranes are dynamic
structures. Ligands coupled to the surface
have the ability to move around the bilayer membrane, which allows them to
position themselves for substrate binding,
or even clustering together. This
Figure 4. Visualization of accumulation of H12(iopamidol)liposomes. (B) A cross-sectional CT image of the
side view of rat infused with H12-(iopamidol)liposomes. (C) a
3D CT image of B. Arrowheads indicate the accumulation
points of the H12-(iopamidol)liposomes. (D) A cross-sectional
CT image of the side view of rat infused with
(iopamidol)liposomes. (E) A 3D CT image of D. (F) A crosssectional CT image of the side view of rat infused with
iopamidol solution. (G) A 3D CT image of F. (Source: Okamura
et al, 2010)
17
clustering seemed to be involved in the encapsulated material release; the optimal substrate
interaction triggers release of the liposome contents. Also other interaction modes of
liposomes with platelets may occur: internalization of the liposomes by the platelets upon
interaction. When the liposomes are conjugated with octa-arginine instead of H12, the
liposomes are internalized, thereby releasing their contents inside of activated platelets; a
useful ability (Nishiya et al, 2004)
Using liposomes as artificial platelets has the advantage of strengthening the haemostatic
ability by installation of a drug delivery function; encapsulating potent platelet agonists in
the liposomes. Okamura et al used adenosine diphosphate (ADP) as a drug to be carried by
the liposomes (Okamura et al, 2008). ADP is normally stored in dense platelet granules and
released after cellular activation, it then functions to reinforce or maintain platelet
aggregation. This is what Okamura et al showed in their 2009 study on H12-(ADP)-liposomes.
They confirmed that H12-conjugated liposomes bind specifically to GPIIb/IIIa on
activated platelets and the standard tail bleeding time in rats and ear bleeding in rabbits
where significantly reduced. In 2010 Okamura et al also visualized the specific accumulation
of their liposomes at sites of vascular injury, by encapsulating the contrast dye iopamidol
into the liposomes creating H12-(iopamidol)-liposomes (Figure 4) (Okamura et al, 2010).
Hereby providing the first evidence that H12-conjugated liposomes accumulate specificially
in platelet aggregates at sites of vascular injury.
In their 2010 study , Okamura et al created different kinds of liposome vesciles, al bearing
the H12 sequence and containing ADP (H12-(ADP)-vesicles). These vesicles varied in
membrane flexibility and lamellarity, hereby regulating the release of the liposomes content.
They claimed to have obtained a recipe to regulate the haemostatic ability of their vesicles
by controlling the ADP release with the aid of different membrane properties (Okamura et
al, 2010) They indeed succeeded by correcting the bleeding time in severe
thrombocytopenic rabbits and rats, and even varying the haemostatic ability. The
mechanism of the ADP release and its regulation is as follows. Vesicles incorporated in the
platelet aggregates are strongly bound to neighboring platelets. Therefore, they are subject
to physical forces. This forces pull on the liposome and continuously change its form. This
deformability depends on the membrane flexibilities an lamellarities of the liposomes, which
is in turn dependent on the composition of the liposome. This deformability (including a
possible disruption) is correlated with the amount of ADP released from the liposome
vesicle. Less flexibility and higher lamellarity will release more ADP. (see figure 5).
18
Figure 5. Schematic view of ADP release from H12-(ADP)-liposomes incorporated into platelet aggregates.
(Okamura et al, 2010)
There is a major safety issue involved with this product, regarding the possibility of
enhanced platelet activation and aggregation in the circulation. The ADP released from 100%
of the liposomes contained by an effective dose injection would reach a concentration of 5
µmol L-1, a concentration that would induce acute aggregation in vitro [1]. However, when
studied in vitro, this wasn’t the case. By measuring the P-selectin expression, which is marker
of platelet activation, the platelet activation capability of ADP in the circulation can be
obtained. The p-selection expression did not differ between a control saline injection and an
injection of an effective dose (20 mg kg-1). Even an administration of a bolus infusion of
extremely high concentration of ADP did not induce P-selectin expression. The plasma ADP
catabolizing function, induced by nucleotidases present in the blood and the endothelium, is
a high functioning system and prevents systemic aggregation. Concluding that these
liposomes are safe for in vivo infusion. (Okamura et al, 2009)
Concluding, liposomes are a potential artificial platelet substitutes. Liposomes are studied
extensively in vitro and in vivo, with promising results. Liposome based substitutes are able
selectively bind platelets at sites of vascular injury, but not in circulating. Furthermore,
liposomes can encapsulated agonists and deliver them to sites of vascular injury with
enhanced haemostatic effects. An ability that albumin microparticles do have not. Preclinical studies on liposomes are promising but human clinical trials are still to be done.
19
Nanosheets
In 2009, Okamura et al published an article about a new artificial platelet substitute; diskshaped biodegradable nanosheets (Okamura et al, 2009) The nanosheets are made of
biodegradeable poly(D,L-lactide-co-glycolide) (PLGA). In this study the nanosheets were
coated with the H12 dodecapeptide mentioned before, and compared with spherical H12
coated PLGA microparticles with the same surface area and conjugation number of H12.
A flow experiment was conducted, in which thrombocytopenic blood flowed over a collagen
surface witch addition of either nanosheets or spherical carriers conjugated with a
fluorescent marker. This experiment showed that the nanosheets adhered to the collagen
surface at twice the rate of the spherical carriers (14.5 ±2.3 vs. 7.7±1.3 /mm2/s ). Concluding,
ellipsoidal nanosheets adhered more effectively than classical spherical carriers of the same
volume. Due to the larger contact area of the nanosheets more binding sites where
available, supporting the adhesive strength. This experimentconfirmed earlier findings;
Decuzzi et al showed that the adhesive strength was significantly increased by increasing the
aspect ratio of the minor and major axes of the particle from 1 (which is a sphere) tot 10.
(Decuzzi et al, 2006) So, the greatest advantage in the use of nanosheets is having a large
contact area for the targeting site, rather than the conventional small contact area of
spherical carriers.
Furthermore, by changing the aspect ratio the nanosheets tend to align in the more nearwall region of the blood vessel (Mortensen et al, 2006). Flowing near the vessel wall makes it
more likely for the nanosheets to bind to the site of injury. Okamura et al also produced
rectangular nanosheets with a very high aspect ratio of 60 that did not adhere to the
collagen surface. These nanosheets did not flow in the near wall region due to interference
Figure 6 (A) Graph of the total collagen surface coverage of
platelet thrombi in addition of H12-PLGA nanosheets (O), H12PLGA microparticles (∆), and PBS control (■) in the flow
experiment (B) Images of the collagen surface with platelet
thrombi after the flow experiment. (a) PBS, (b) H12-PLGA
nanosheets, and (c) H12-PLGA microparticles (Okamura et al,
2009)
20
with the erythrocytes also present in the blood. These experiments showed that the
adhesive rate of the carriers on a collagen surface can be controlled by the change of their
shape and that the best adherence is obtained with ellipsoidial nanosheets with a medium
aspect ratio as these will flow in the near wall
region of the vessel, seen in figure 6
Finally, the nanosheets induced two-dimensional
spreading of platelet thrombi , an ability that is
not assessed by using spherical microparticles .
The thrombi formed by the use of microparticles
did pile up dramatically, in contrast to the thrombi
formed by the use of the nanosheets (Okamura et
al, 2009) This is seen in Figure 7; the low contrast
in SEM image of the nanosheets shows the
spreading of the thrombus, as opposed to the
high contrast of the microspheres which shows
piling of the thrombus. The spreading of the
thombi might be due to the ultrathin structure of
the nanosheets, as platelets bind to either side of
the sheet, with a broad scaffold. Platelets binding
to spherical carriers may naturally lead to piled up
thrombi, as platelets bind in a 3-dimensional
manner. Piled up thrombi may lead to vessel
Figure 7. SEM images of platelet thrombi
involving H12-PLGA nanosheets and H12-PLGA
microparticles, respectively. Arrows in the SEM
images indicate nanosheets and microparticles
(Okamura et al, 2009)
occlusion and can have severe consequences.
Disk-shaped biodegradable nanosheets can be a
useful artificial platelet substitute. Their
adherence ability is twice that of spherical carriers. In vivo studies are however still to be
done. So nanosheets are a promising novel platelet substitute, which needs more extensive
study.
21
Discussion
Platelet transfusion is the current method of treating thrombocytopenic patients. Infusion of
platelet concentrates have several disadvantages. As stated these include alloimmunization,
transfusion transmitted infections or remaining leucocytes induced adverse effects such as
TRALI. Then there is the platelet storage problem. The longer the platelets are stored, the
less usefull the product is. These disadvantages translate to society in terms of money and
time. Platelet products are expensive, especially when they need to be HLA tested for
refractory patients. Mass production of synthetic artificial platelet substitutes have to be
much cheaper. Furthermore, curing procedures, including platelet infusion to
thrombocytopenic patients , must be as effective as possible. Especially when the
thrombocytopenia is a therapy induced side effect such as in patients with bone marrow
cancer. In general, therapists want to focus on the main problem of their therapy, and do
not want to worry about side effects. It is even more disastrous when curing the side effects
gives rise to other problems such as the problems current platelet transfusion can induce.
Availability of sufficient donor blood is a problem all over the world. As the production of
platelet transfusion products requires large volumes of whole blood, this problem is even
larger. The alternative of artificial platelet substitutes solves these problems, as these are
synthetic and can be made in laboratories.
As artificial platelet substitutes are so promising, several researchers focused on their
development An important step in the process of platelet substitute development was the
identifying of the various sequences in the fibrinogen protein which bind to the GPIIa/IIb
receptor complex on platelets. This raised opportunities for conjugating these sequences to
several artificial products.
The most promising products are liposomes and albumin microspheres. Liposomes do even
have an extra promising function; the delivery of encapsulated products such as adenosine
diphosphate. However, when the albumin microspheres can be modified in to different
shapes, such as the nanosheets described, the haemostatic ability will increase. Anyhow,
both products are tested extensively both in vivo and in vitro. The results were promising.
Both products reduces the standard ear bleeding time in rabbits significantly, and showed an
enhanced haemostatic effect in in vitro experiments under flow. As safety can be a major
concern with this product, the systemic aggregation in circulation was also studied. Both
products’ mechanism of action is at the site of vascular injury, and do not enhance
aggregation in circulation.
22
All studies performed on artificial platelet substitutes so far are pre-clinical. Clinical testing is
still necessary. Because the methods of platelet transfusion currently used are widely
accepted, despite of its problems, the needs for an alternative could be under-valued.
Despite that, there is a need for an alternative and clinical tests should take place in the near
future. Furthermore, it seems to be that research on artificial platelet substitutes has come
to a stop. Between 2000 and 2005 several platelet substitutes were developed and
researched. Since then, these platelet substitutes were subject of intensive research.
However, no major steps were taken. Maybe, clinical trials were executed, but the results
were not published because the outcome was not as expected. This is something we could
consider, but there is no denying in the fact that artificial platelet substitutes are a promising
product that are worth to be clinically tested.
Concluding, platelet substitutes are promising alternatives, having a lot of advantages over
regular platelet transfusions. There has been much progress so far on the development of
different products, but they still need to be finished, tested in clinical trials and produced.
23
TRAP induced platelet activation and the effects of EPO
Introduction
Human blood platelets can be activated by agonists such as adenosine diphosphate (ADP) or
thrombin receptor associated protein (TRAP). Upon activation P-selectin is expressed on the platelet
membrane. This receptor displayed on the membrane are therefore markers for platelet activation.
When the blood is incubated with fluorescencent antibodies against P-selectin, platelet activation
can be measured with the use of the FACS technique.
The use of the drug erythropoietin (EPO) is associated with elevated risks of thromboembolic events.
EPO can induce increasing platelet counts, but the higher risks of thrombosis in patients using EPO
cannot be accounted for by the increased platelet count alone. The true mechanism of the effect on
of EPO on platelets is still unclear (Testa, 2010). Here the effect of EPO on the activation of blood
platelets is measured.
Methods
The mastermix contains HBS buffer in addition of 1:25 α-P-selectin-RPE and either 1:25 α-GPIB-FITC
or 1:100 α-fibrinogen-FITC. Thrombin receptor associated protein (TRAP) is added to the mastermix
(1:80) and diluted 1:4 8 times resulting in a series of standards with decreasing TRAP concentration
range. Erythropoetin (EPO) is added to whole blood, either in a concentration of 1:100, 1:1000 or
1:10.000, and incubated for 30 minutes at 37 °C. This is added to 4 series of TRAP concentration
ranges, obtaining 3 ranges with different EPO concentration and one control range, and incubated
for 20 minutes. The incubation is stopped by addition of 0,2% formaline. The activation of the
platelets is measured by FACS analysis of the antibody fluorescence, which is a marker of activation.
Results & Discussion
P-Selectin expression
10000
10000
8000
8000
6000
Control 6000
EPO 1:100
EPO 1:1000
4000
EPO 1:10000
4000
2000
-2
2000
-1
1
-2000
Control
EPO 1:100
EPO 1:1000
EPO 1:10000
RFU
RFU
P-Selectin expression
[TRAP]
2
3-2
-1
0
1
2
3
[TRAP]
Graph 1 & 2. Both graphs show the P-selectin expression, which is a maker of platelet activation,
depending on the concentration of TRAP. Furthermore, EPO is added in different concentrations, and
compared with controls in absence of EPO. Graphs are the result of two of the same experiments,
24
with the same results
Graphs 1and 2 show that platelets incubated with EPO are less activated. The degree of activation of
platelets in absence of EPO is clearly higher then all of the platelets incubated with EPO. The
experiment was conducted 3 times (only two graphs shown), in which the platelets in the control
group in which no EPO was added consistently had more P-selectin expression. Note that the
concentration of EPO in the blood sample does not elicit any difference in activation. Furthermore, it
seems that in absence of EPO, P-selectin is expressed more at lower TRAP concentrations, resulting
in earlier activation.
EPO is associated with thrombotic events. The results of this experiment indicate the opposite;
addition of EPO decreases the P-selectin expression. As P-selectin is a marker of platelet activation,
EPO may decrease the platelet degree of activation. A also plausible explanation is that EPO has a
negative effect on P-selectin expression, without influencing the degree of activation. P-selectin is
known to bind neutrophils and monocytes, hereby forming bridges of platelets between these cells.
This results in mixed cellular aggregates which could be involved in vessel occlusion (Ryan and
Worthington, 2009). This marks EPO again as an anti-thrombotic molecule, instead of the prothrombotic risks mentioned earlier in the use of EPO as a drug.
In conclusion, EPO seems to have a negative effect on either P-selectin expression or platelet
activation. Further research is needed in which other activation markers have to be used, to
determine if EPO has an effect on P-selectin alone or on platelet activation as a whole. Either way,
the mechanism of action by which EPO acts on platelets or P-selectin, also has to subject of research.
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