HbS binding to GP1bα Activates Platelets in Sickle Cell disease

Running Head: SICKLE CELL DISEASE 0
HbS binding to GP1bα Activates Platelets in Sickle Cell disease
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SICKLE CELL DISEASE 1
Table of Contents
Introduction....................................................................................................................................1
Structure of Sickle haemoglobin...................................................................................................3
Epidemiology of sickle cells disease.............................................................................................5
Genetic inheritances.......................................................................................................................7
Pathophysiology..........................................................................................................................10
Vaso Occlusion........................................................................................................................10
Hemolytic Anemia...................................................................................................................11
Platelet activation and aggregation..............................................................................................12
References....................................................................................................................................15

SICKLE CELL DISEASE 2
Introduction
Sickle cells disease is not a new concept. It has been affecting people for many years. It was
discovered in 1910 for the first time in the United States in a patient from West Indies by a
cardiologist named Herrick. In 1951 a Nobel Prize winner chemist Pauling with his team found
that the oxygen-transporting proteins named hemoglobin had an altered structure in a patient
with SCD. Ingram and Hunt in 1956 discovered the replacement of glutamic acid by valine on
6th position. In 1970 the relation between the abnormal structure of hemoglobin and RBCs has
been discovered. The cure of this disease has been reported by bone marrow transplantation
which was applied on a child. The preventive treatment for this disease is proposed in 1995 and
to stop the complications of SCD, Hydroxyurea was found to be a proven drug (Information
Centre for Sickle Cell and Thalassemia Disorder, 2002). Some of the studies suggested that the
history of sickle cell disease is thousands of years old in Africa and known by various different
names in different languages (Serjeant, 2013).
Sickle cells disease is a genetic disorder inherited from parents to the children’s. It is associated
with defect or abnormality in red blood cells (World Health Organisation, 2006). The normal
red blood cells are disc-shaped but the changes occurred in hemoglobin causes the sickle shape
of erythrocytes (Brent Sickle Cell & Thalassemia Centre, 2015). These cells with altered shape
are rigid and may be burst when transported via blood vessels. This may become the reason for
the decreased amount of red blood cells inside the body and causes anemia. When
abnormalities or inheritance occur in oxygen-carrying proteins hemoglobin S also called sickle
hemoglobin, it leads to the sickle cell anemia (a sickle cell disease). Inheritance word itself
indicated that the disease is this disease is transfer to the next generation by passing abnormal
haemoglobin genes. Some of the diseases associated to sickle cell disease are: haemoglobin SC
disease, sickle cell anaemia and haemoglobin Sβ thalassemia, haemoglobin SS disease,

SICKLE CELL DISEASE 3
haemoglobin SC, haemoglobin SB 0 or β0 thalassemia, haemoglobin SD, SE and SO
(Healthline, 2017). This thesis also involves the discussion of hemoglobin and glycoprotein role
in platelet activation and aggregation. Other molecules and processes associated with sickle cell
disease also will be discussed. Hemoglobin is the molecule that maintains the highly conserved
state in different species within erythrocytes. The main role of hemoglobin is to carry oxygen to
various tissues form lungs. Persons with sickle cell disease Hb changes to stiff rods inside the
RBCs. Hb the depletion of nitric acid by Hb produces hydroxyl radicals which trigger
peroxidation of lipid membrane which results in cellular damage. Increased level of Hb in
plasma associated with vascular and organ abnormality like hemolysis. Release of Hb during
hemolysis produces ROS, which results in platelet activation (Helms et al. 2013). Various
studies suggested that when these Hb binds to GP1bα, the platelet activation has been triggered.
This interaction of hemoglobin to GP1bα stimulates some events like change in platelet shape,
secretion of granules and signalling process which leads to activate the function of integrin’s.
These pathways involved in activation of platelets mediated by GP1bα. This glycoprotein is a
surface membrane of platelet is consists of heterodimers where one alpha chain and one beta
chain linked to each other via a disulfide bond. This glycoprotein works as a receptor for VWF
(Madabhushi, Zhang, Kelkar, Dayananda, and Neelamegham, 2014). It is also initiated events
of intracellular signaling like the increased level of calcium in the cytoplasm, protein kinase
pathway activation which leads to activation of platelets. The initiation of GP1b alpha by Hb is
responsible for platelet apoptosis (Annarapu, Singhal, Sheetal, Ojha and Guchhait, 2016).
Some methods like measurement of platelet activation markers by using flow cytometry,
extracellular hemoglobin measurement by using ELISA, Coagulation essay, VWF: RCO assay
and platelet activation will be discussed in this dissertation to understand the activation of
platelets by binding of Hb to GP1bα. The epidemiological aspects, genetical aspects involved in
this study will also be discussed to provide a better understanding of this topic.

SICKLE CELL DISEASE 4
Structure of Sickle haemoglobin
The Normal haemoglobin is composed of total four protein chains and about four short non
protein molecules called heme. There are two alpha globin chains and two beta globin chains in
single haemoglobin. Each globin chains associated with heme prosthetic group, it is the site for
binding and release of oxygen. During binding and release of an oxygen compound a αβ dimer
moves (Weatherall and Clegg 2001). Haemoglobin S which is the altered form of normal
haemoglobin is a tetramer contains four subunits, where two subunits are of α and other two are
beta globin. The structure of two alpha subunits is same as in normal haemoglobin but beta
subunits are different. These subunits are the site for valine residues substitution to glutamic
acid. Alpha subunit contains 141 amino acids and on the other hand primary structure of beta
subunit has 146 amino acid (Marengo-Rowe, 2006). There are single heme binding sites for all
four subunits; these sites are responsible for oxygen transport. The secondary structure of
haemoglobin s contains complete alpha helicase which is separated and joint by random coils.
In secondary structure there is no beta sheet. Both alpha and beta subunits made consists of
eight alpha helicase. The binding between these four subunits is strongly bounded with salt
bridges and hydrogen bonds (Mackey, no date). The tertiary structure is similar as secondary. In
quaternary structure there are two confirmations called T and R protein states are same as wild-
type hemoglobin A R confirmation which is open and in the mobile state responsible for
keeping home binding pocket open. These pockets also unhindered by salt bridges and
hydrogen bonds. At the distal end of an alpha chain between heme binding pockets the T state
has been constricted and stabilize by salt bridges. In a normal haemoglobin A, this T
confirmation accounts for decreasing oxygen affinity. It is also responsible for promoting
polymerisation of haemoglobin S in haemoglobin S both the subunits are divided as valine
acceptors and donors. The polymerisation occurred in Hb S is a primary pathogenic event that
occurs in SCD (Steinberg, 2018).

SICKLE CELL DISEASE 5
In a hydrophobic binding pocket the donating subunit contains beta val- 6 and accepting beta
subunit receives beta val-6. There are two residues in acceptor beta chains: BetaPhe-85 and
BetaLeu-88. There is a seven double-stranded structure has been found in hemoglobins tetramer
after betaGlu-6 to betaVal-6 mutation. These strands are twisted toward one another with the
helical pitch of 2,900 Å. The basic fibre of haemoglobin is 210 Å thick. The building blocks of
this fibre is called Wishner-love double strand which contains single helical twist (Roufberg,
and Ferrone, 2000).
Figure 1 the chain with colored squares shows eight amino acids in a beta chain which has a
glutamic acid at sixth position and sickle beta chain contains valine. (Sickle cell and
thalassemia disorder, 2002)