Blood outgrowth endothelial cell - mediated coagulation

Summary

The risk of developing a bleeding or thrombosis is still difficult to determine
with the current understanding of the coagulation system. The aim of the
current fundamental research proposal is to improve the understanding the
coagulation system through a detailed characterization of endothelial
progenitor cells, the so called "blood outgrowth endothelial cells (BOECs),
which circulate in small quantities in the blood [1]. Improved information
about the specific role of endothelium-mediated mechanisms in the coagulation
system will help to identify new parameters that will allow for a better and
more accurate assessment of individual risk tendencies of developing a bleeding
diathesis or thrombosis.

The endothelial cell surface plays an important role in blood clotting.
However, in regular clotting assays the contribution of the endothelium is
excluded given that its function cannot be directly assessed in blood. In the
Einthoven Laboratory for Experimental Vascular Medicine, we have the expertise
to isolate and culture BOECs according to established procedures, resulting in
fully functional endothelial cells [2]. In this study, BOECs will be isolated
from the blood of healthy volunteers. In the subsequently generated endothelial
cell lines, we will characterize and quantify the biosynthesis and expression
of several endothelial proteins that play an important role in blood
coagulation. The results of this study will be incorporated into a mathematical
model of the coagulation system, with the aim of using this improved model to
indentify parameters that allow for assessing the risk of developing a bleeding
or thrombotic complication.


Background

Estimated risk of bleeding/thrombosis is complicated to assess
The coagulation system comprises a complex set of responses that protect the
body against bleeding and thrombosis. The latter is examplified by venous
thrombosis, which is the most frequent encountered complication during
hospitalization, and can be prevented provided the correct treatment protocol
is used [3]. Unfortunately, there are no clotting tests available that are able
to determine which patients are at increased risk of developing thrombosis.
Venous thrombosis is treated with so-called anticoagulants, of which the
effectiveness is tested using the International Normalized Ratio (INR).
Although this method usually prevents bleeding due to a high dosage of
anticoagulants [4], medication-related bleeding can still occur, even if the
measurements stay within the normal INR range. For example, one study
demonstrated that a severe (organ) bleeding occurred annually in 3% of patients
treated with oral anticoagulants regulated by the Dutch anticoagulation clinic
[5]. Clearly, there is a high need for new assays that determine the bleeding
and thrombosis risk in each individual. An improved mathematical model of the
coagulation system could serve as a tool to identify the parameters that can be
assessed in these assays.

Role of endothelial cells in blood
Since endothelial cells line the vessel wall and contain many membrane-bound
proteins, the contribution of these cells to coagulation is not directly
measurable in blood and/or plasma. However, these cells and their components
play an important role in the coagulation process and their actions should
therefore be taken into account in order to make reliable predictions about
coagulation. Endothelial cells are involved in coagulation through various
mechanisms, such as through the production of von Willebrand factor (VWF). When
the endothelium is activated, VWF is released into the bloodstream, where it
contributes to the adhesion and aggregation of platelets. VWF also functions as
a carrier protein for coagulation factor VIII; both of these functions are
important for the procoagulant effect of VWF [6]. Other endothelial-specific
coagulation proteins take part in the protein C system [7]. Activation of this
system occurs upon the interaction of thrombin with thrombomodulin (TM), a
membrane protein found on endothelial cells. The so formed complex converts
protein C to activated protein C (APC) that, together with its cofactor protein
S, inactivates the clotting factors Va and VIIIa, thereby downregulating
coagulation. APC can also bind to the endothelial protein C receptor (EPCR),
another membrane protein that is anchored to the endothelium. This complex is
capable of activating protease activated receptor 1 (PAR-1), an endothelial
transmembrane protein, thereby initiating a cytoprotective effect on the
endothelium [8]. Recently, it was discovered that the enzyme protein disulfide
isomerase (PDI) is released when the endothelium adopts a prothrombotic
phenotype. PDI converts encrypted tissue factor to activated tissue factor and
as such contributes to blood coagulation [9].

The above mentioned mechanisms indicate that the endothelium plays an important
role in the clotting cascade through various mechanisms. However, a detailed
characterization of their direct contribution to clotting is lacking thus far.
This study aims to characterize these endothelial-mediated mechanisms and their
relationship to the coagulation system by characterizing and quantifying the
biosynthesis and expression of various proteins in BOECs. This will be
performed under normal conditions and under conditions that may cause
thrombosis in vivo (such as low oxygen, low nutrients, low pH, and in the
presence of inflammatory mediators). The knowledge gained will provide unique
insight into the contribution of endothelial cell-specific components to normal
coagulation and thrombosis.

Mathematical model
All information obtained in this study will be imported in a new mathematical
model of coagulation. By including the endothelial cell-mediated mechanisms
into this model we anticipate to be able to better describe and predict the
coagulation response. This simulation model will provide the basis for
developing an optimal combination of coagulation assays to determine the
hemostatic balance and the risk of developing a bleeding diathesis or
thrombosis in individual patients.


Referenties (1)
1. Lin Y, Chang L, Solovey A, Healey JF, Lollar P, Hebbel RP. Use of blood
outgrowth endothelial cells for gene therapy for hemophilia A. Blood. 2002 Jan
15;99(2):457-62
2. van den Biggelaar M, Bouwens EA, Kootstra NA, Hebbel RP, Voorberg J, Mertens
K. Storage and regulated secretion of factor VIII in blood outgrowth
endothelial cells. Haematologica. 2009 May;94(5):670-8
3. Cohen AT, Tapson VF, Bergmann JF, Goldhaber SZ, Kakkar AK, Deslandes B,
Huang W, Zayaruzny M, Emery L, Anderson FA Jr; ENDORSE Investigators. Venous
thromboembolism risk and prophylaxis in the acute hospital care setting
(ENDORSE study): a multinational cross-sectional study. Lancet. 2008 Feb
2;371(9610):387-94
4. Cannegieter SC, Rosendaal FR, Wintzen AR, van der Meer FJ, Vandenbroucke JP,
Briët E. Optimal oral anticoagulant therapy in patients with mechanical heart
valves. N Engl J Med. 1995 Jul 6;333(1):11-7
5. Veeger NJ, Piersma-Wichers M, Tijssen JG, Hillege HL, van der Meer J.
Individual time within target range in patients treated with vitamin K
antagonists: main determinant of quality of anticoagulation and predictor of
clinical outcome. A retrospective study of 2300 consecutive patients with
venous thromboembolism. Br J Haematol. 2005 Feb;128(4):513-9
6. de Wit TR, van Mourik JA. Biosynthesis, processing and secretion of von
Willebrand factor: biological implications. Best Pract Res Clin Haematol. 2001
Jun;14(2):241-55
7. Dahlbäck B, Villoutreix BO. Regulation of blood coagulation by the protein C
anticoagulant pathway: novel insights into structure-function relationships and
molecular recognition. Arterioscler Thromb Vasc Biol. 2005 Jul;25(7):1311-20
8. Ruf W, Dorfleutner A, Riewald M. Specificity of coagulation factor
signaling. J Thromb Haemost. 2003 Jul;1(7):1495-503
9. Ahamed J, Versteeg HH, Kerver M, Chen VM, Mueller BM, Hogg PJ, Ruf W.
Disulfide isomerization switches tissue factor from coagulation to cell
signaling. Proc Natl Acad Sci U S A. 2006 Sep 19;103(38):13932-7

The aim of this research is to improve the understanding of the coagulation
system through a detailed characterization and quantification of biosynthesis
and expression of several coagulation proteins in BOECs. The data obtained in
the study will be included in a mathematical model describing the entire
coagulation system, with the ultimate goal of identifying parameters that
predict the risk of developing a bleeding or thrombotic tendency in individual
patients.

The specific research objectives are:
1. The quantitative and qualitative determination of the transcription and
expression of endothelial specific blood components, including VWF, TM, EPCR,
PAR-1, PDI, under various conditions:
a. under normal physiological conditions,
b. under conditions that in vivo may cause thrombosis (such as low oxygen, low
nutrients, low pH and in the presence of inflammatory mediators).
2. To determine the immediate effect of endothelial factors on blood
coagulation by including BOECs in existing clotting assays such the thrombin
generation assay [10.11].


Referenties (2)
10. Hemker HC, Al Dieri R, De Smedt E, Béguin S. Thrombin generation, a
function test of the haemostatic-thrombotic system. Thromb Haemost. 2006
Nov;96(5):553-61
11. Bos MH, Meijerman DW, van der Zwaan C, Mertens K. Does activated protein
C-resistant factor V contribute to thrombin generation in hemophilic plasma? J
Thromb Haemost. 2005 Mar;3(3):522-30

Selecting subjects
For this study blood, will be collected from 50-100 healthy adults. Prospective
subjects will be informed about this study by advertisement. The study will
result in fundamental biochemical findings that have no direct clinical
significance. Given the nature of the experiments we do not anticipate to make
discoveries that directly relate to the study subjects. Therefore, no feedback
on the data obtained will be communicated to the volunteers. Since the
isolation of BOECs from blood is not always successful, the subjects will be
asked whether they agree upon blood collection at later time points as well (up
to three times total).

Implementation and blood collection
The volunteers will be informed about the research through advertisements. If
interested in participating, the subjects will be informed about the study both
in writing and by phone. Subsequently, they will be requested to come to the
LUMC. After giving a written consent to participate in the study, 50-100 ml
citrated blood will be sampled.

Coding of the blood samples
The personal data of the subjects will only be available to the responsible
researcher. The blood samples will be encrypted, and the code to link the
samples to the subjects is only available to the responible researcher, not to
other employees.


Processing of blood samples
The BOECs will be isolated and cultured in the Einthoven Laboratory for
Experimental Vascular Medicine. Subsequently, the generated endothelial cell
lines will be stored in liquid nitrogen for future experiments. The cells will
be preserved for ten years. Furthermore, DNA will be isolated from the cells to
confirm the identity of some endothelial components. The mRNA levels of some
proteins will be determined, as well as their expression levels. The BOECs will
be further characterized under normal and prothrombotic conditions and
included in existing coagulation assays.


Ethical aspects

Burden for the subjects
The subjects have no direct interest in the research. The study will provide
more insight into the role of endothelial cells in blood, as this information
will be used to create a new mathematical model of coagulation. The burden on
the subjects is minimal, basically just a single blood sample, although in some
cases a second or third blood sample might be requested. Subjects receive a
reimbursement for travel costs and a ¤ 25 gift certificate as thanks for
participating. The risk for the participants is negligible because it involves
a single blood withdrawal only.

Informed consent
Participants in the research will be informed about the study in advance, both
verbally and in writing. Subjects will be enrolled after they have given their
permission. All participants can contact Dr. W.M. Lijfering, Tel: 071 526 5639,
to obtain further information. For independent advice, participants can contact
Dr. F.J.M. van der Meer, Tel: 071 526 3901.

Privacy
The blood samples will be coded as previously described and only the
responsible researcher, Dr. M.H.A. Bos, has access to the key linking the code
to personal data. The subject will not receive feedback of the research
findings.

The burden on the subjects is minimal: basically just a single blood sample,
although in some cases a second or third blood sample might be requested. The
risk for the participants is negligible because it involves blood sampling
only. A bruise may occur due to blood withdrawal, which can be prevented by
firmly pressuring the puncture.