Investigating the Impact of HbS Binding to GP1bα on Patients with Sickle Cell Disease

This research proposal aims to investigate the role of platelet activation induced by binding of HbS to GP1bα in intravascular clot formation among Sickle cell disease patients. The study will involve collection of blood samples from patients suffering with sickle cell disease and normal healthy individuals across three hospitals in the district. The methodology will include the purchase of several antibodies such as p-Akt (S473), (p)-Lyn, p-ERK, Akt, PI3K and ERK.

Running head- DISSERTATION PROPOSAL
Dissertation Proposal
Name of the Student
Name of the University
Author Note

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Executive summary
Sickle cell disease encompasses a group of blood related disorders that affect the hemoglobin.
Hemoglobin is the respiratory pigment responsible for delivery of oxygen to all cells and tissues
throughout the body. People suffering from this disorder have abnormal hemoglobin molecule,
called HbS, which distorts red blood cells and converts them to a crescent or sickle shape.
The most common signs and symptoms of this disease include reduction in the count of red
blood cells, recurrent infections, and episodes of pain. It often results in frequent
hospitalizations. Breakdown of these red blood cells result in manifestation of anemia that leds to
fatigue, shortness of breath, and delayed growth among children. It affects millions of people all
over the world and is one of the most common inherited disorders. Research studies have
established links between the disease and platelet cell activation. The report is based on a
research proposal that will investigate impact of HbS binding to GP1bα on patients suffering
from sickle cell disease.

2DISSERTATION PROPOSAL
Table of Contents
Introduction......................................................................................................................................3
Background......................................................................................................................................3
Research objective...........................................................................................................................5
Research aim....................................................................................................................................6
Research hypothesis.........................................................................................................................6
Literature review..............................................................................................................................6
Methodology....................................................................................................................................8
Data analysis....................................................................................................................................9
Ethical considerations....................................................................................................................10
Timeline.........................................................................................................................................11
References......................................................................................................................................12
Appendix........................................................................................................................................16

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Introduction
Sickle cell disease refers to a group of blood stream related disorders that are typically
inherited from the parents. The most common type of sickle cell disease is sickle cell anaemia
which leads to the formation of abnormality in hemoglobin, the oxygen carrying pigment present
in red blood cells. This abnormality results in a rigid sickle like shape of the red blood cells.
Inheritance of two abnormal copies of the gene that produces hemoglobin, one from each of the
parent, results in this disease (Ware et al. 2017). Certain factors that increase susceptibility of a
person to get affected with sickle cell disease are namely, stress, high attitude, temperature
changes, and dehydration. This disease is a major global health concern, owing to the fact that
more than 4.4 million people were found to be affected with it in the year 2015. Approximately
43 million people were found to be carriers of the sickle cell trait in the same here. In addition,
80% prevalence of the disease is found in sub Saharan African regions. It also contributed to
more than 114,800 deaths globally in 2015 (Piel et al. 2013). Previous studies have suggested
that inactivation of blood platelets place a significant role in reducing risk of sickle cell disease,
nearby lowering rates of associated mortality and morbidity (Yawn et al. 2015). This research
proposal will therefore investigate effects of platelet activation on sickle cell disease severity.
Background
Evidence-based nursing refers to the approach that encompasses the process of making
quality decisions in order to provide optimal nursing care to patients. It often combined clinical
expertise, with personal demands and preferences of the patients, and most relevant research
materials available on the related topic (Melnyk et al. 2012). Implementing updated methods of
providing care to patients are accompanied with a thorough critical appraisal of high quality

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studies that have significant findings. Thus, the primary goal of evidence-based nursing is to
bring about an improvement in the overall health well being and safety of the patient, while
providing them health care services in a cost effective manner. This in turn improves patient
satisfaction as well as health outcomes. Therefore, collection interpretation and appraisal of
relevant literature and integration of their findings in clinical scenario forms the foundation of
evidence-based nursing. The research proposal will utilize the concept of evidence-based nursing
to identify the findings that have been reported by several articles on the impacts of binding of
GP1bα with hemoglobin of a sickle-celled patient, for formulating effective interventions. On the
other hand, critical appraisal of a literature refers to the process that involves a systematic and
careful examination of the research in order to judge its value, trustworthiness and relevance in
context of the health scenario (Cottrell 2017). Critical appraisal is an essential component of
evidence-based practice as it helps in asking answerable questions, searches for evidences, and
evaluates relevance of the findings, which is followed by deciding what action can be taken from
the findings.
According to research studies, mutations in the Hbb gene are responsible for causing
sickle cell disease (Ribeil et al. 2017). The gene is principally made up of four subunits of
proteins, namely, 2 subunits of alpha globin and 2 of beta globin, respectively (Mehari et al.
2012). This gene provides instructions for translation of beta globin protein. A particular
mutation in the gene is found to produce an abnormal version of the aforementioned protein,
which results in the formation of haemoglobin S or HbS. Similar findings have also been
reported by other studies that suggested replacement of one beta globin subunit among people
suffering from Sickle cell disease (Nath and Hebbel 2015). Abnormal versions of this beta globin
protein are found to create distortions in red blood cell that results in a sickle shape (Hoban,

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Orkin and Bauer 2016). These sickle shaped cells often have a premature death and result in
anaemia. These variations in the beta globin gene often contribute to increase in mortality and
morbidity.
Furthermore, research studies also indicate that presence of Sickle cell trait in one parent
increases the likelihood of a child of getting affected with the disease by 50%. Moreover, a child
with both parents showing sickle cell trait has a 25% chance of acquiring the disease. Several
researches have been carried out to investigate the geographical prevalence of sickle cell
anaemia. According to the studies, sickle cell gene is commonly found in regions that have high
prevalence of malaria (Sun and Zhao 2014). It is found to frequently occur in several parts of
sub-saharan Africa, in addition to the Arabian Peninsula and among people of African origin.
Therefore, epidemiological studies have established highest frequency of the disease in tropical
regions and the Middle East. Substantial amount of migration of people originating from these
high prevalence area two countries like the Europe and America have also created a significant
increase in incidence of the disease (Serjeant 2013). Furthermore, according to research
evidences, presence of a single cell early or trade in regions where malaria is prevalent offers a
selective advantage to the people. Findings suggest that heterozygous condition for sickle cell
anaemia is advantageous, and helps in conference resistance to malaria among the infected
person (Steinberg and Sebastiani 2012).
Research objective
Owing to the fact that sickle cell disease is the most commonly inherited blood related
disorder in major countries of the world, there is a need to formulate effective intervention
strategies. The objectives include the following:

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 Identifying the underlying factors that contribute to sickle cell disease
 Discovering the role of platelet activation in the disorder
 Identifying the importance of GP1bα in clot formation
Research aim
The aim of this research is to investigate the role of platelet activation induced by binding
of HbS to GP1bα in intravascular clot formation among Sickle cell disease patients.
Research hypothesis
H1- GP1bα binding to HbS will lead to platelet activation among sickle cell diseased
patients
H0- GP1bα binding to HbS will have no effect on platelets among sickle cell diseased
patients
Literature review
Studies have recognised presence of increased platelet activation among people suffering
from Sickle cell disease. Increase platelet activation has been determined as an essential
component of hemolytic action among patients suffering from sickle cell disease. An activation
of the increased percentage in platelet count has also been found during the steady-state among
Sickle cell patients. Furthermore, according to research findings, activation of platelets has been
established to play an important role, in development of complications related to chronic
vascular disorders. Increased platelet count has been found to promote hyperplasia by the
secretion of vasoactive and mitogenic substances (Manwani and Frenette 2013).

7DISSERTATION PROPOSAL
Research studies also established correlation between activation of pathologic platelet
count and onset of acute thrombosis. Similar findings have also been reported by other studies
that examined sickle cell patients for evidence of platelet activation. An increase in the
expression of activation dependent cells present on platelets, among patients suffering from
sickle cell disease compared to their African-American and Caucasian controls suggested that
sickle cell disease patients manifest increased amount of platelet microparticles (Zhang et al.
2016). Consistency was also found with other research findings that presented a significant
increase in the number of platelet and micro aggregate formation among sickle cell patients,
upon comparison with healthy controls. This also suggested that platelet hyperactivity plays an
important role in sickle cell disease.
Further, research findings of suggested that platelet activation occurs after events related
to attachment and adhesion. Such activation is also observed after triggering activation
mechanisms of thromboxane A2, thrombin or platelet activating factors that has been released
from endothelial cells. According to research findings, upon encountering a break in epithelium,
platelets come in contact with molecules, such as, collagen that trigger activation. In addition
molecules, like ADP, thromboxane A2 and thrombin being also trigger similar activation. This
results in exocytosis of alpha granules sentence granules, followed by activation of the enzyme
phospholipase A2 (Annarapu et al. 2016). It also results in a change in shape to an amorphous
form and the platelets adhere to each other, thereby resulting in the formation of a platelet plug.
Endothelial platelet activation has also been associated with sickle cell anaemia due to adhesion
to vascular endothelium. Research studies have contributed formation of vaso-occlusion to
increased platelet activation (Belcher et al. 2014). Studies have provided evidences which
indicate circulation of platelets in the activated state in blood stream of people suffering from

8DISSERTATION PROPOSAL
sickle cell disease (Tantawy et al. 2013). This becomes capable of stimulating endothelial cell
activation and appears to be mediated by adhesion or direct contact (Sparkenbaugh and
Pawlinski 2013).
Furthermore, according to several studies, GP1bα has been identified as a surface
membrane protein present on platelets that plays an essential role in formation of platelet plug
(Potoka and Gladwin 2015). It is bound to bind to the A1 domain of Von willebrand factor,
bound to the sub-endothelium. Similar findings have been reported by other studies, which state
that the risk of thrombosis and hypercoagulation increases among people suffering from
hemolytic disorders, upon the onset of intravascular hemolysis. The study has also correlated
platelet activation two binding of hemoglobin to GP1bα. Results of a recent study has also
shown that adult HbA induces activation of platelets on binding with GP1bα (Wun et al. 2013).
Moreover, platelet activation has also been directly correlated with increased levels of free
hemoglobin in plasma (Singhal et al. 2015). The research proposal will therefore aim to
investigate the effect of sickle cell haemoglobin binding with GP1bα, on platelet activation.
Methodology
The methodology will involve collection of blood samples from patients suffering with
sickle cell disease and normal healthy individuals across three hospitals in the district. 100
patients will form the sample group. Similar number of patients will form the control. They will
be age and gender matched. This will help in producing statistically significant results at the end
of the study and prevent selection bias. All participants will be recruited after taking their
informed consent in a written format. 5 to 10 ml of blood sample will be collected from each
participant (both sample and control) in acid citrate dextrose or sodium citrate anticoagulant.

9DISSERTATION PROPOSAL
Inclusion criteria will focus on enlisting patients who have been diagnosed homozygous for
sickle cell disease, following and HPLC fractionation method.
Exclusion criteria will include patients suffering from cognitive disability, as they might
not understand implication of the research and the reason for recruiting them. Infants will not be
recruited in either of the experimental groups. The participants will principally be recruited
across hematology departments of the three selected hospitals. Before conducting the research, a
pilot study will be conducted on a smaller scale among 10 patients. This will reduce likelihood of
errors in the result and will also determine feasibility of the formulated protocol.
The study would require purchase of several antibodies such as, p-Akt (S473), (p)-Lyn,
p-ERK, Akt, PI3K and ERK. Furthermore, goat anti-rabbit/mouse IgG that is conjugated with
peroxidase enzyme will also be required. In addition, anti-hemoglobin α, fluorescent antibody,
and synthetic peptides will have to be purchased. Haemoglobin S that has been isolated with the
use of Sephadex G-25 column, followed by an ion exchange chromatography will also be
purchased. ATP colorimetric assay kit will be used for the methodology.
Data analysis
Platelet rich plasma obtained from the anticoagulant blood will be resuspended in a
buffer of calcium free thyroid. Human platelets will be used to purify glyocalicin. Binding of
haemoglobin to glyocalicin will be measured using the BIAcore-T200 system. An Elisa test will
also be carried out to evaluate the binding. Different concentrations of HbS will be incubated
with glyocalicin, immobilized on the ELISA plate for an hour, which will be detected by goat
anti-Hbα antibody and HRP tagged donkey anti-goat IgG antibody. The washed platelets will be

10DISSERTATION PROPOSAL
incubated with different concentrations of HbS, followed by processing in SDS PAGE and
immunoblotting.
Flow cytometry will be performed for determining activation markers, present on the
surface of platelets. Confocal fluorescent microscopy will also be performed. The amount of
hemoglobin in blood plasma will be measured by performing sandwich Elisa test. This will be
followed by a statistical analysis where the experimental values will be presented in the form of
standard deviation or mean ± standard error of mean (SEM). One way anova, student's T test and
Pearson analysis of correlation will be used for data analysis. Value less than 0.05 will be
considered as statistically significant. The results will show whether HbS binding activated the
platelet. The Anova test will identify differences between means of unrelated sample and control
group (González-Rodríguez, Colubi and Gil 2012). On the other hand, the Pearson correlation
test will measure the linear association between the two variables (Eisinga, Te Grotenhuis and
Pelzer 2013).
Ethical considerations
Since the study involves collection of blood samples from patients suffering from sickle
cell disease, as well as healthy individuals, ethical considerations include obtaining approval
from the human research ethics committee, before taking informed consent from the participants.
Timeline
The study will begin from March 15 and will be completed within June 28, 2018. A gantt
chart is given in the appendix.

11DISSERTATION PROPOSAL
References
Annarapu, G.K., Singhal, R., Peng, Y. and Guchhait, P., 2016. Inhibition of Hb binding to
GP1bα abrogates Hb-mediated thrombus formation on immobilized VWF and collagen under
physiological shear stress. PloS one, 11(4), p.e0154276.
Belcher, J.D., Chen, C., Nguyen, J., Milbauer, L., Abdulla, F., Alayash, A.I., Smith, A., Nath,
K.A., Hebbel, R.P. and Vercellotti, G.M., 2014. Heme triggers TLR4 signaling leading to
endothelial cell activation and vaso-occlusion in murine sickle cell disease. Blood, 123(3),
pp.377-390.
Cottrell, S., 2017. Critical thinking skills. Macmillan Education, pp.23-75.
Eisinga, R., Te Grotenhuis, M. and Pelzer, B., 2013. The reliability of a two-item scale: Pearson,
Cronbach, or Spearman-Brown?. International journal of public health, 58(4), pp.637-642.
González-Rodríguez, G., Colubi, A. and Gil, M.Á., 2012. Fuzzy data treated as functional data:
A one-way ANOVA test approach. Computational Statistics & Data Analysis, 56(4), pp.943-
955.
Helms, C.C., Marvel, M., Zhao, W., Stahle, M., Vest, R., Kato, G.J., Lee, J.S., Christ, G.,
Gladwin, M.T., Hantgan, R.R. and Kim‐Shapiro, D.B., 2013. Mechanisms of hemolysis‐
associated platelet activation. Journal of Thrombosis and Haemostasis, 11(12), pp.2148-2154.
Hoban, M.D., Orkin, S.H. and Bauer, D.E., 2016. Genetic treatment of a molecular disorder:
gene therapy approaches to sickle cell disease. Blood, 127(7), pp.839-848.

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Manwani, D. and Frenette, P.S., 2013. Vaso-occlusion in sickle cell disease: pathophysiology
and novel targeted therapies. Blood, 122(24), pp.3892-3898.
Mehari, A., Gladwin, M.T., Tian, X., Machado, R.F. and Kato, G.J., 2012. Mortality in adults
with sickle cell disease and pulmonary hypertension. Jama, 307(12), pp.1254-1256.
Melnyk, B.M., Fineout-Overholt, E., Gallagher-Ford, L. and Kaplan, L., 2012. The state of
evidence-based practice in US nurses: critical implications for nurse leaders and
educators. Journal of Nursing Administration, 42(9), pp.410-417.
Nath, K.A. and Hebbel, R.P., 2015. Sickle cell disease: renal manifestations and
mechanisms. Nature reviews Nephrology, 11(3), p.161.
Piel, F.B., Hay, S.I., Gupta, S., Weatherall, D.J. and Williams, T.N., 2013. Global burden of
sickle cell anaemia in children under five, 2010–2050: modelling based on demographics, excess
mortality, and interventions. PLoS medicine, 10(7), p.e1001484.
Potoka, K.P. and Gladwin, M.T., 2015. Vasculopathy and pulmonary hypertension in sickle cell
disease. American Journal of Physiology-Lung Cellular and Molecular Physiology, 308(4),
pp.L314-L324.
Ribeil, J.A., Hacein-Bey-Abina, S., Payen, E., Magnani, A., Semeraro, M., Magrin, E.,
Caccavelli, L., Neven, B., Bourget, P., El Nemer, W. and Bartolucci, P., 2017. Gene therapy in a
patient with sickle cell disease. New England Journal of Medicine, 376(9), pp.848-855.
Serjeant, G.R., 2013. The natural history of sickle cell disease. Cold Spring Harbor perspectives
in medicine, 3(10), p.a011783.

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Singhal, R., Annarapu, G.K., Pandey, A., Chawla, S., Ojha, A., Gupta, A., Cruz, M.A., Seth, T.
and Guchhait, P., 2015. Hemoglobin interaction with GP1bα induces platelet activation and
apoptosis: a novel mechanism associated with intravascular hemolysis. Haematologica, 100(12),
pp.1526-1533.
Sparkenbaugh, E. and Pawlinski, R., 2013. Interplay between coagulation and vascular
inflammation in sickle cell disease. British journal of haematology, 162(1), pp.3-14.
Steinberg, M.H. and Sebastiani, P., 2012. Genetic modifiers of sickle cell disease. American
journal of hematology, 87(8), pp.795-803.
Sun, N. and Zhao, H., 2014. Seamless correction of the sickle cell disease mutation of the HBB
gene in human induced pluripotent stem cells using TALENs. Biotechnology and
bioengineering, 111(5), pp.1048-1053.
Tantawy, A.A.G., Adly, A.A.M., Ismail, E.A.R., Habeeb, N.M. and Farouk, A., 2013.
Circulating platelet and erythrocyte microparticles in young children and adolescents with sickle
cell disease: Relation to cardiovascular complications. Platelets, 24(8), pp.605-614.
Ware, R.E., de Montalembert, M., Tshilolo, L. and Abboud, M.R., 2017. Sickle cell disease. The
Lancet, 390(10091), pp.311-323.
Wun, T., Soulieres, D., Frelinger, A.L., Krishnamurti, L., Novelli, E.M., Kutlar, A., Ataga, K.I.,
Knupp, C.L., McMahon, L.E., Strouse, J.J. and Zhou, C., 2013. A double-blind, randomized,
multicenter phase 2 study of prasugrel versus placebo in adult patients with sickle cell
disease. Journal of hematology & oncology, 6(1), p.17.

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Yawn, B.P., Buchanan, G.R., Afenyi-Annan, A.N., Ballas, S.K., Hassell, K.L., James, A.H.,
Jordan, L., Lanzkron, S.M., Lottenberg, R., Savage, W.J. and Tanabe, P.J., 2014. Management of
sickle cell disease: summary of the 2014 evidence-based report by expert panel
members. Jama, 312(10), pp.1033-1048.
Zhang, D., Xu, C., Manwani, D. and Frenette, P.S., 2016. Neutrophils, platelets, and
inflammatory pathways at the nexus of sickle cell disease pathophysiology. Blood, 127(7),
pp.801-809.

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Appendix