BIOCHEMISTRY
Blood Chemistry
Dr. Igrobay
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BLOOD INTRODUCTION
Blood is one of the largest organ systems of the body
It is the liquid that flows in an enclosed circulation that is part of the
circulatory or cardiovascular system
It consists of blood cells suspended in the plasma
Plasma is the liquid portion of the unclotted blood
Blood constitutes around 5-7% of the total body weight
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To know one’s blood volume, it is around 6% of your total weight in kg
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Specific gravity of blood is 1.06
pH of blood is 7.4
o
Normal pH is 7.35-7.45
o
Average pH is 7.4
Viscosity of blood is 1.7-2.0
Freezing point of blood is -0.56oC
Osmotic pressure of blood at 37oC is 7.6 atm pressure
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MAJOR FUNCTIONS OF BLOOD
Respiration – transport oxygen from lungs to the tissues and of CO2
from the tissues to the lungs
Nutrition – transport of absorbed food materials
Excretion – transport of metabolic waste to the kidneys, lungs, skin,
and intestines for removal
Capillaries are the main site of respiration, exchange
nutrition, and waste products
4.
5.
6.
Maintenance of the normal acid-base balance in the body
Regulation of water balance through the effects of blood on the
exchange of water between the circulating fluid and the tissue fluid
Regulation of body temperature by the distribution of body heat
o Normal body temperature is 36.5-37.5oC
o Average body temperature is 37oC
o >37.5oC = hyperthermia
o <36.5oC = hypothermia
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7.
Defense against infection by the white blood cells and circulating
antibodies
8. Transport of hormones and regulation of metabolism
9. Transport of metabolites and regulation of metabolism
10. Coagulation – clot formation
Plasma Fraction
It has 2 constituents: diffusible and non-diffusible
Diffusible constituents means it can spread in the body
Non-diffusible constituents means it only focuses on a certain part
of the body
Diffusible constituents examples are:
o
Electrolytes
o
Sodium
o
Calcium
o
Potassium
o
Hormones
o
Vitamins
Non-diffusible constituents examples are:
o
Proteins
o
Polypeptides
PLASMA
Plasma is the liquid part of fluid blood
Serum is the fluid remaining after blood coagulates
Plasma proteins consist of water, electrolytes, metabolites,
nutrients, proteins and hormone
Water and electrolyte composition of plasma is practically the same
as that of all extracellular fluids
Laboratory determination of levels of Na, K, Ca, Mg, HCO3 , PaCO2 and
of blood pH are important in clinical management
Plasma Proteins
Majority of the plasma proteins is made up of water
It is around 7.0 – 7.5 g/dL
It is a complex mixture of glycoprotein and lipoproteins
The plasma proteins can be separated into 3 major groups:
1. Fibrinogen
2. Albumin
3. Globulins
Fibrinogen is involved in the coagulation cascade or clot
formation. Fibrinogen will be converted to fibrin by the help of
thrombin
Albumin is a protein that serves as a transport vehicle that carries
also bile salts and fatty acids
Globulins are involved in the immune system (immunoglobulins)
COMPOSITION OF BLOOD
Picture above:
There are two fractions: one is liquid which is the plasma and the other one
is solid which is cellular (buffy coat + RBC). The plasma has the highest
percentage compared to the cellular part. For the cellular fraction it is
exemplified by the RBC which is the most abundant followed by the buffy
coat. The buffy coat is composed of the white blood cells and the platelets
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The concentration of proteins in the plasma is important in
determining the hydrostatic pressure and osmotic pressure in the
body
Edema can happen when either of the hydrostatic or oncotic
pressure malfunctions
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Most plasma proteins are synthesized in the liver
They are generally synthesized on membrane-bound polyribosomes
Most of which are synthesized as preproteins
Many of them exhibit polymorphism
But each plasma protein has a characteristic half-life in the
circulation
The levels of certain proteins in plasma increase during acute
inflammatory states or secondary to certain types of tissue damage
The diversity of their biochemical properties attributes to their
varying functions
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BIOCHEMISTRY
Blood Chemistry
Dr. Igrobay
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The normal total serum protein concentration is 6-8 g/dL (60-80 g/L)
and is determined as a fraction containing albumin and the serum
globulins
>8 g/dL = Hyperproteinemia
<6 g/dL = Hypoproteinemia
The fibrinogen was discarded in the clot that separated from the
plasma to form the serum specimen
Increased concentration is termed as hyperproteinemia and this
could be due to water depletion, multiple myeloma,
macroglobulinemia, and sarcoidosis
Decreased concentration is termed hypoproteinemia and this could
be secondary to any organ dysfunction such as congestive heart
failure, peptic ulcer, nephrosis, and cirrhosis
Functions of the Plasma Proteins
Antiproteases
Blood clotting factors
Enzyme hormones
Immune defense
Inflammatory responses
Oncofetal
Transport or binding proteins
MAJOR TYPES OF PLASMA PROTEINS
Albumin (69 kDa)
The major plasma protein in humans
3.4-4.7 g/dL
Makes up approximately 60% of the total plasma protein
About 40% is present in the plasma and 60% in the extracellular space
It is initially synthesized as preprotein in the liver (12 g/day)
The concentration of the serum albumin controls the passage of
water through the cell membrane by osmosis and is responsible for
75-80% of the osmotic pressure of human plasma
In addition, it serves as a protein stores for the body that can be
utilized when a deficit develops
It serves as a solvent for fatty acids and bile salts
It serves as a transport vehicle by loosely binding hormones, amino
acids, drugs and metals
Increased concentration (hyperalbuminemia) has no significant
correlation with diseases. The reduction of circulating albumin
concentration results in a shift of fluid from intravascular to
extravascular spaces and is most commonly manifested as edema
and/or congestion
Decrease concentration (hypoalbuminemia) may be due to
several mechanisms but the most common one is a decrease in
the production of albumin in the liver
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Acute hepatitis and Chronic Liver Disease
 If the liver is defective, plasma protein is
affected
Treatment for hypoalbuminemia is letting the patient drink egg
white
Causes of albuminemia:
1. Reduced synthesis of albumin
Malnutrition
 Protein calorie malnutrition such as in
Kwashiorkor
Malabsorption syndrome
 Intestines cannot absorb properly
Chronic Inflammatory Disease
 Pulmonary tuberculosis
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2.
Genetic abnormalities
Familial or hereditary disease
3.
Increased loss of albumin
Nephrotic syndrome
 Kidneys are defective = proteinuria
Massive burns
 The higher the surface area of skin burned,
the higher is the protein necessary to be
repaired
Protein losing enteropathy
 Protein losses at the GIT, goes in the stool
4.
Increased catabolism of albumin
Fast breaking own of albumin like in massive burns and
widespread malignancy (Stage 3 or 4)
Cancer cells uses protein in order to survive
5.
Multifactorial
Cirrhosis of liver or heart
Congestive heart failure
Pregnancy
Haptoglobin (Hp)
It is a plasma glycoprotein that binds extracorpuscular hemoglobin
in a tight non-covalent complex [Hp-Hp (155kDa)], preventing the
loss of free hemoglobin into the kidney thus conserving valuable iron
present in hemoglobin
The binding between haptoglobin and hemoglobin is highly specific
It exists in three polymorphic forms:
o
Hp 1-1
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Hp 2-1
o
Hp 2-2
The simplest of which is Hp 1-1 (90kDa) and is found in humans
In extensive hemolysis, haptoglobin in the serum is depleted
Thus, haptoglobin measurement is useful for the clinical assessment
of hemolysis
Haptoglobin is an acute phase reactant and is frequently elevated in
medically stressful situations
Transferrin (76 kDa)
The transport of iron in the plasma is accomplish by transferrin,
which migrates in the ß-globulin region
1 molecule of transferrin can bind 2 ions of iron
It shuttles iron as Fe2+ (Ferrous) to sites where it is needed, either for
storage or utilization
In female who are part of the reproductive age group are prone
to iron deficiency anemia due to menstruation
Treatment to this is iron supplements and Vitamin C
Iron supplements are in the Ferrous state (absorbable form)
Vitamin C enhances the absorption of iron in the intestines
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BIOCHEMISTRY
Blood Chemistry
Dr. Igrobay
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Ceruloplasmin (160 kDa)
It is an α2-macroglobulin with a blue color because of its high copper
content
It carries 90% of the copper present in plasma
Each molecule of ceruloplasmin binds six atoms of copper very
tightly, so that the copper is not readily exchangeable
Albumin carries the 10% of plasma copper and bind to it less tightly,
donating it more readily to tissues than ceruloplasmin
Ceruloplasmin exhibits a copper-dependent oxidase activity but
physiologic significance has not been clarified
Copper Metabolic Diseases:
Menke’s diseases
 Also known as kinky or steely hair disease
 X-linked disorder due to mutation in the gene
encoding a Copper-binding P-Type ATPase
Willson’s disease
 Genetic disease wherein copper fails to be excreted in
the bild
 Results to copper toxicosis
 Kayser-Fleischer ring - green or golden pigment ring
around the cornea due to deposition of copper in
Descemet’s membrane
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α1 – Antiproteinase (α1-Antitrypsin) (52 kDa)
It is a major component of the α1 fraction (>90%) of human plasma
It is synthesized by hepatocytes and macrophages
Function of α1-antitypsin is to inhibit the production of elastase
Elastase is secreted by bacterial neutrophils
Elastase functions to recoil the lungs
If α1-antitrypsin is affected, there is no recoil = barreled chest
It is the principal serine protease inhibitor (serpin, or Pi) and has at
least 75 polymorphic forms
Congenital deficiency of α1 – antiproteinase can result in premature
emphysema
Volatile irritants found in cigarette smoking stimulate the release of
proteases in the lungs
Without α1-antiproteinase to inhibit these enzymes, it would cause
considerable destruction to the lung parenchyma leading to severe
often fatal emphysema
A second clinical syndrome associated with congenital deficiency of
α1-antiproteinase in children is cirrhosis of liver
This disorder may require liver transplantation in severe cases
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α2 – Macroglobulin (720 kDa)
A large plasma glycoprotein which transports approximately 10% of
Zinc in plasma
It is synthesized by variety of cell types including monocytes,
hepatocytes, and astrocytes
It is a major member of a group of plasma proteins that include
complement proteins C3 and C4
These proteins contain a unique internal cyclic thiol ester bond and
for this reason have been designated as the thiol ester plasma
protein family
This bond is highly reactive and is involved in some of the biologic
actions of α2-macroglobulin
It is an important panproteinase inhibitor, binding to many
proteinases
Panproteinase inhibitor means that α2-macroglobulin inhibits the
enzyme responsible for degrading or hydrolyzing the whole
polypeptide chain
In deficiency of α1-proteinase, α2-macroglobulin probably is the
primary protease inhibitor
In nephrotic syndrome, α2-macroglobulin is allowed to retain in the
circulation because of its large molecular weight, even in profound
proteinuria
In severe cases, the concentration of α2-macroglobulin in serum may
approach or even exceed that of albumin
Fibrinogen (340 kDa)
A soluble plasma glycoprotein which consists of three non-identical
pairs of polypeptide chains covalently linked by disulfide bonds
All three chains are synthesized in the liver
Half-life: 3.5 – 4 days
It is a clotting factor 1: involve in the clot formation
Causes of increase:
Homeostatic stress (bleeding or hemorrhage)
Nonspecific stressor of inflammation
Pregnancy
Autoimmune disorders
All are pathologic except pregnancy
Amyloidosis
Amyloidosis is a disorder due to accumulation of various insoluble
fibrillary proteins between cells of tissues to an extent that affects
function
It is generally due to either increased production of certain proteins
or accumulation of mutated forms of other proteins
DIFFERENTIATION OF RBC and OTHER BLOOD CELLS
Hematopoiesis
It refers to the formation and development of all types of blood cells
from their parental precursors
The marrow is a special environment for hematopoietic growth and
development
When the hematopoietic marrow cells are mature and ready to
circulate to the peripheral blood, the cells leave the marrow
parenchyma and emerge into venous sinuses
The parental precursors are hematopoietic stem cells. Stem cells are
pluripotential cells which means that they have the capability to become
different kind of cells
Stem cells can be found in the bone marrow or umbilical cord
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BIOCHEMISTRY
Blood Chemistry
Dr. Igrobay
Clinical Importance of Hematopoietic Stem Cells:
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Stem cell is a cell with a unique capacity to produce unaltered
daughter cells (self-renewal) and to generate specialized cell
types (potency)
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Stem cells may be:
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Totipotent (capable of producing all the cells in an
organism)
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Pluripotent (able to differentiate into cells of any of
the three germ layers)
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Multipotent (produce only cells of a closely related
family)
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Unipotent (produce only one type of cell)
Cell-cell interaction and the release of the humoral factors (CSFs, e.g. GMCSF/G-CSF: deficiency of which may result to neutropenia) do control the
production and maturation of white cells
Lymphopoiesis
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This is the growth and maturation of lymphocytes
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Lymphocytes are conditioned by two main organ systems, the
thymus (T-lymphocytes) or the bone marrow (B-lymphocytes)
Megakaryopoiesis
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This is the process of platelet development from megakaryocytes
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Thrombopoietin is a hormone that controls proliferation and
maturation of megakaryocytes
Different Progenitors of Hematopoietic Stem Cell
Platelets are formed by the development of demarcation membranes within
the cytoplasm, and individual platelets are extruded into the venous sinuses
The RBC and platelets share a common pathway of differentiation. Each
pathway is regulated by various factors (e.g. Stem cell factor (SCF),
thrombopoietin (TPO), Interleukins (IL), erythropoietin (EPO), various colony
stimulating factors (CSFs)
Stem cell factor is a cytokine that plays an important role in the proliferation
of hematopoietic stem cells and some of their progeny
Thrombopoietin is a glycoprotein that is important in regulating the
production of platelets by the bone marrow
Interleukins are cytokines produced by leukocytes; they regulate various
aspects of hematopoiesis and of the immune system
Picture Above:
The multipotential hematopoietic stem cell derived from the bone marrow
can give rise to different progenitors. First it branches into two: Myeloid
progenitor and Lymphoid progenitor.
In Myeloid progenitor it gives rise to:
o
Granulocytes – Basophils, Eosinophils, Neutrophils (BEN)
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Erythrocytes
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Monocytes – differentiates into macrophages or dendritic cells
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Mast cells
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Megakaryocytes – becomes thrombocytes (platelets)
For the Lymphoid progenitor, it gives rise to:
o
B lymphocyte
o
T lymphocyte
o
Natural killer cell
Erythropoiesis
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Are derived from the committed erythroid precursor cells through a
process of mitotic growth and maturation
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Erythropoietin (EPO) is a hormone produced largely by the kidney
that stimulates CFU-E stem cells to speed up growth and enhance
maturation
Granulopoiesis
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It is the replacement of leukocytes by cell division and production of
new white cells in the marrow
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A process of differentiation occurs, whereby immature white cells
gradually develop and exhibit characteristics of mature functional
leukocytes
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RED BLOOD CELLS (ERYTHROCYTES)
Non-nucleated, biconcave in shape and devoid of intracellular
organelles
It functions mainly in the delivery of O2 and excretion of CO2
It has a lifespan of 120 days (it goes to the spleen after it dies)
Approximately <1% of RBC is replaced daily at a rate of 2M/sec
Has glucose transporter (GLUT 1) in its membrane and has powerful
oxidants which are produced during the course of metabolism:
Superoxide, Hydrogen peroxide, Peroxyl and Hydroxyl radicals and
ROS
Normal red cell count ranges from 4.2-6.2 x 10(6)
Polycythemia literally means “many blood cells” but usually refers to
increased red cell mass. A better term for this is erythrocytosis or
erythremia
Secondary or reactive polycythemia is due to a recognizable
physiologic stimulus
Polycythemia vera or True polycythemia is spontaneous or
unprovoked increased in the red blood cells
Anemia is defined as a decrease in the concentration of hemoglobin.
This is often the end result of red blood cell abnormalities
The red blood cell has a unique and relatively simple metabolism
Continued next page…..
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BIOCHEMISTRY
Blood Chemistry
Dr. Igrobay
Red Blood Cell Membrane
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It consists of an integral layer of lipids, including phospholipids and
cholesterol, which contain an intimate association of proteins
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These proteins may be integral or peripheral proteins
Peripheral membrane proteins – associated with its surface,
generally via protein-protein interactions, determining shape
and flexibility
Integral membrane proteins – glycosylated and span the
membrane
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3.
Spectrin
o
o
o
Major protein of the cytoskeleton and confers the
flexibility of the membrane of the RBC
It has four binding sites: self association, Ankyrin, actin
and protein 4.1
The disorder of which will lead to Hereditary
Spherocytosis and also Hereditary Elliptocytosis
Hereditary Spherocytosis
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The protein-lipid composition is important in maintaining the
integrity of the red cell membrane
The membrane also resists an uncontrolled influx of sodium ions,
which are present in higher concentrations in plasma and an efflux
of potassium ions, which are present in higher concentrations
within the red cell
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Spherocytes (RBC’s without biconcave shape &
central pallor) having low surface-to-volume ratio
Cause hemolytic anemia
Causes splenomegaly due to plugging of easilyruptured spherocytes in sinusoids of the spleen
Susceptible to osmotic lysis
Abnormal spectrin makes it unable to react with
membrane proteins → weakened cell membrane
and spherocytic shape
Some of the Major Proteins of the RBC Membrane
Hereditary Elliptocytosis
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4.
Ankyrin
o
o
o
1.
Anion exchange protein (Band 3)
o
C terminal is external and N terminal is internal
o
A multipass membrane protein, extending across the
bilayer
o
Responsible for the reaction wherein from the tissues,
CO2 is obtained
CO2 from tissues enter the RBC as HCO3- → exchanged
for Cl- in the lungs (chloride shift)
Maintain electroneutrality within the cell
o
o
2.
Elliptical RBs
Defective spectrin or Glycophorin C or protein 4.1
(points of attachment)
A pyramid-shaped protein that binds spectrin
Also binds tightly to band 3, securing the attachment of
spectrin to the membrane
Sensitive to proteolysis
5.
Actin (Band 5)
o
Exists in RBC as short, double-helical filaments of F-actin
o
The tail end of spectrin dimers bind to actin
o
Also binds to protein 4.1
6.
Protein 4.1
o
A globular protein that binds tightly to the spectrin
forming a protein 4.1 – spectrinactin ternary complex
o
Also binds to the integral proteins
Exists as a dimer in the membrane, in which it forms a
tunnel, permitting the exchange of chloride for
bicarbonate
Cytosolic N terminal binds to Hb, protein 4.1 and 4.2,
Ankyrin and several glycolytic enzymes
Glycophorin A, B, and C
o
Transmembrane glycoprotein which is of the single-pass
type
o
C terminal bind spectrin to cell membrane via protein 4.1
o
Contains binding sites for influenza virus and
Plasmodium falciparum (parasite that causes malaria)
Table above:
Just take not that most of the major proteins are peripheral proteins. The
only integral proteins are anion exchange protein and glycophorin A, B, C
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BIOCHEMISTRY
Blood Chemistry
Dr. Igrobay
ABO BLOOD GROUP
Blood Group
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Defined system of RBC antigens (blood group substances controlled
by a genetic locus having variable number of alleles
Blood Type
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It is the antigenic phenotype usually recognized by the use of
appropriate antibodies
ABO substances
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These are complex oligosaccharides present in most cells of the body
and in certain secretions
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On membranes of the red blood cell, oligosaccharides that determine
the specific natures of the ABO substances appear to be mostly
present in glycosphingolipids¸ whereas in secretions are present in
glycoproteins
H substance
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Also known as H antigen or O antigen or O substance
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The precursor of both A and B substances
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The blood group substance found in persons of type O
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Formed by the action of fucosyltransferase (product of the H gene)
which catalyzes the addition of the fucose to the terminal Gal residue
of the precursor substance resulting to H substance
Antigen
Glycosyltransferase
Immunodominant
Sugar
A
α-3-N-acetylgalactosaminyltransferase
N-acetyl-Dgalactosamine
B
O
α-3-D-galactosyltransferase
α-2-L-fucosyltransferase
D-Galactose
L-Fucose
The product of A gene is the GalNac transferase that adds the terminal
GalNac (immunodominant sugar of blood group A) to the O substance
The product of B gene is the Gal transferase that adds the terminal Gal
(immunodomonant sugar of blood group B) to the O substance
Individuals with type AB possess both enzymes (GalNac and Gal transferase)
and thus have two oligosaccharide chains
Individuals of type O apparently synthesize an inactive protein, thus H
substance in their ABO blood group substance
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ABO BLOOD GROUP SYSTEM
System was first discovered by Landsteiner in 1900 when
investigating the basis of compatible and incompatible transfusion in
humans
The genes responsible for the production of the ABO substances are
present on the long arm of chromosome 9
Three alleles, two of which codominant (A & B) and the third (O)
recessive; ultimately determine the four phenotypic produces are:
A, B, AB, and O substances
Picture Above:
The one in the blue box is called the oligosaccharide chain or precursor
substance. It is composed of 2 galactose and 1 N-acetylglucosamine linked
together by ß1→4 and ß1→3 linkage. With the action of fucosyltransferase,
the sugar “fucose” will then be attached to the terminal Gal residue with a
α1→2 linkage creating the H substance
OLIGOSACCHARIDE SEQUENCE IN RBC MEMBRANE
Definition of Terms:
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Genotype – actual genetic information
o
AA/AO , BB/BO, OO, AB
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Phenotype – observed inherited trait
o
Blood types: A, B, AB, O
Bombay (Oh) Phenotype
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Phenotype: Oh
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Genotype: hh or H null
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Inheritance
of
two
recessive h genes
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Lack of L-fucose which is
needed
for
the
attachment of A and B
sugars
Take note of the structure of each antigens. O antigen has only fucose at the
terminal Gal. A antigen has GalNac attached to the terminal Gal with α-1,3
linkage. B antigen has Gal attached to the terminal Gal with α-1,3 linkage
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BIOCHEMISTRY
Blood Chemistry
Dr. Igrobay
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WHITE BLOOD CELLS (LEUKOCYTES)
Classified according to the presence of granules (Granulocytes) or
absence of granules (Agranulocytes) and their staining
characteristics
They are formed in the bone marrow and lymphatic tissue with a life
span of 13 to 20 days
Half of the circulating white cells are granulocytes, the cytoplasm of
which contains readily visible granules of various chemical and
enzymatic compositions
The three main types of granulocytes are the: neutrophils,
eosinophils, and basophils
The staining characteristics of granules define the cell type:
o
Neutral staining – neutrophils
o
Reddish – eosinophils
o
Bluish – basophils
White cells are distinguished from circulating red cells by the
presence of a nucleus
Automated counting procedures enumerate all nucleated cells as
white cells
Normal white blood cell count is 4.3-10.8 x 10 (3 µl)
↑ in WBC count is Leukocytosis
↓ in WBC count is Leukopenia
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Monocytes
Have elongated, indented, or dark kidney-bean shaped nuclei
Highly phagocytic (cell-eating)
Largest WBC (16-20 µm)
With delicate nuclear chromatin
Constitute 5-8% of circulating blood leukocytes
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Basophils
Least numerous (0.5-1% WBC count)
Has large coarse cytoplasmic granules
Contains Histamine, Serotonin, Heparin
Histamine is responsible for allergic reactions
Serotonin is responsible for making you feel sleepy
Heparin is an anticoagulant
Tissue basophils, not blood basophils, have IgE receptors adherent
to cell membranes which react with allergens and IgE to induce
release of vasoactive mediators
Diapedesis - passage of blood cells through the intact walls of the
capillaries, typically accompanying inflammation
Neutrophil
55-65% of WBC count
Have several lobes in nucleus
Present in acute bacterial infection
Active Glycolysis
Active Pentose Phosphate Pathway
Moderate Oxidative phosphorylation
Rich in lysosomes and their degradative enzymes (used in killing
bacteria and other phagocytosed substances)
Unique enzymes: MPO (myeloperoxidase), NADPH oxidase
Are recruited from the blood stream into the tissue to help eliminate
the foreign invaders via chemotactic factors
To reach the tissues, neutrophils must pass through the
capillaries by the migration along the blood vessel wall and
then adhere to the endothelial lining (Diapedesis)
Respiratory Burst – is when neutrophils and other
phagocytic cells engulf bacteria, they exhibit a rapid
increase in oxygen consumption. This phenomenon
produces a large amount of reactive derivatives. Some of
these products are potent microbial agents
Lymphocytes
Has large nuclei with small amount of cytoplasm
Smallest WBC (<10 µm in diameter)
25-35% of WBC
Circulating blood lymphocytes constitute a tiny fraction (<5%) of the
total lymphocyte pool
Response in acute infection
There are two primary subtypes:
o
T-lymphocytes
o
B-Lymphocytes
Eosinophils
Large, numerous granules
Nuclei with 2 lobes
2-5% WBC count
Found in lining of respiratory and digestive tracts
Important functions involve protection against infections caused by
parasitic worms and involvement in allergic reaction
Secrete anti-inflammatory substances in allergic reactions
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Neutrophil in Acute Inflammation:
o
Entry of activated neutrophils into the tissues
o
Increased vascular permeability
o
Activation of platelets
o
Spontaneous resolution
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PLATELETS
Vary in size from to about 4 µ and circulate for approximately 8-12
days as disc-shaped, anucleate cells
Regulation of platelet production is ascribed to thrombopoietin
Functions:
1. To protect the vascular integrity of the endothelium
2. Initiate repair when blood vessel walls are damaged
Primary Homeostasis – platelet-vessel wall interaction
During homeostasis or thrombosis, they become activated & help
form hemostatic plugs or thrombi which include 3 major steps:
1. Adhesion to exposed collagen in blood vessels
2. Release (exocytosis) of the contents of their storage
granules
3. Aggregation
Normal platelet count is 150-350x10 (3) µl
Thrombocytopenia is defined as a platelet count < 100,000 µl and
causes may be due to production defects, sequestration due to
splenomegaly and accelerated destruction
Thrombocytosis is platelet count >350,000 µl which could be
primary
or
secondary.
Primary
thrombocytosis
(thrombocythemia) may be complicated by bleeding and/or
thrombosis; Secondary rarely causes hemostatic problems.
Disorders of platelet function are due to defects in platelet
adhesion, aggregation or granule release
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Blood Chemistry
Dr. Igrobay
Certain Drugs that Inhibit Platelet Responses:

Aspirin
o
Irreversibly acetylates and inhibits the platelet COX-1
involved in the formation of thromboxane A2, a potent
platelet aggregator and vasoconstrictor

Clopidogrel
o
Inhibits ADP receptor

Abciximab
o
Interfere with fibrinogen and vWF binding (Von
Willenberg Factor binding)
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HOMEOSTASIS
Cessation of bleeding from a cut or severed vessel
Process by which body simultaneously stops bleeding from an injured
site, yet maintains blood in the fluid state
Failure of homeostasis leads to hemorrhage
Failure to maintain fluidity leads to thrombosis
Homeostatic mechanisms comprise 4 main systems:
1. Vascular system
2. Platelets
3. Coagulation system
4. Fibrinolytic system

3 Phases of Homeostasis and Thrombosis:
1. Formation of loose and temporary platelet aggregate
2. Formation of fibrin mesh
3. Partial or complete dissolution of platelet plug

3 Types of Thrombi:
1. White thrombus – consists of platelets and fibrin, forms
in the arteries
2. Red thrombus – consists of red cells and fibrin, forms in
veins
3. Fibrin deposits – are common in capillaries
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Coagulation Cascade
Purpose is to generate active serine protease enzyme thrombin
In turn acts selectively on the soluble plasma protein fibrinogen,
converting it into soluble fibrin
Fibrin is the visible end product of coagulation and acts as the cement
substance to stabilize the initial primary platelet plug
Both Intrinsic and Extrinsic Pathways results in the formation of
fibrin
Coagulation pathways are complex and involved in many different
proteins
Proteins can be classified as:
o
Zymogens of serine proteases
o
Cofactors
o
Fibrinogen
o
Transglutaminase
o
Regulatory proteins
EXTRINSIC PATHWAY (Factors III and VII)

Initiation of fibrin clot formation in response to tissue injury (there is
damage to the blood vessels or to the endothelial cells)

This pathway is quicker than the intrinsic pathway

Located in sub-endothelium and on activated monocyte

Involves tissue factor, factors VII and X, and Ca2+ and results in factor Xa

Formation of complexes between tissue factor and factor VIIa is
considered the key process involved in initiation of blood coagulation
in vivo
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INTRINSIC PATHWAY (Factors XII, XI, IX, VIII)

Activated by negatively charged surface in vitro

Involves factors XII, XI, IX, VIII as well as prekallikrein, high-molecularweight kininogen, Ca2+ , and phospholipid

It results in the production of factor Xa that is cleaved by the tenase
complex

With factor IXa as the serine protease and factor VIIIa as the cofactor
of the intrinsic pathway

Can be initiated with the “contact phase” in which prekallikrein,
HMW kininogen, factor XII, and factor XI are exposed to a negatively
charged activating surface
FIBRINOLYTIC PATHWAY

Fibrinolysis is one of the natural anticoagulant defense mechanism
which is a multi-component enzyme system that results in the
generation of an active enzyme plasmin

Plasmin is a serine protease mainly responsible for degrading fibrin
to fibrinogen, circulates in the form of inactive zymogen,
plasminogen, and any small amounts of plasmin that are formed in
the fluid phase under physiologic conditions are rapidly inactivated
by plasmin inhibitor α2-antiplasmin

Plasmin that is bound to fibrin is protected from α2-antiplasmin

Other plasmin inhibitors are the plasminogen activator inhibitor and
TAFIa

Several activators of plasmin are: streptokinase, urokinase, and t-PA
COMMON PATHWAY (Factors X, V, II, I, XIII)

Both the extrinsic and the intrinsic pathways converge to form the
common pathway (via the activation of factor X to Xa), which will
ultimately activate the plasma protein prothrombin (factor II) into
thrombin (factor IIa). This require the assembly of prothrombinase
complex (ionized calcium, prothrombin, factor Va, factor Xa)

Thrombin converts fibrinogen to fibrin

It also converts factor XIII to factor XIIIa, a highly specific
transglutaminase that covalently cross-links molecules, yielding a
more stable fibrin clot

This fibrin mesh serves to stabilize the hemostatic plug or thrombus
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Dr. Igrobay
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Regulation of Thrombosis:
Outside the vascular system, blood can be kept fluid by either
removing fibrinogen or by adding anticoagulants, most of which
prevent coagulation by chelating or removing calcium ions
Citrate, Oxalate, and EDTA are anticoagulants of the chelating
category
Heparin prevents coagulation by directly inhibiting thrombin;
prevents conversion of fibrinogen to fibrin by augmenting a natural
anticoagulant molecule, anti-thrombin III to neutralize thrombin
Protein C, like vitamin K-dependent coagulation factors, is also a
vitamin K-dependent polypeptide manufactured by the liver and
circulates in its inactive form. This process is also modulated by an
additional substance produced by the blood vessel termed
thrombomodulin, which may help focus neutralization at the site of
vascular injury. Protein C as an anticoagulant is due to its rapid
neutralizing activity of factors VIIIa and Va
Protein S accelerates the inactivation of Va and VIIIa by protein C
DISORDERS OF COAGULATION & THROMBOSIS
Disorders of blood coagulation may either be congenital or acquired
in origin
Congenital Disorder:
o
Hemophilia A – deficiency in factor VIII
o
Hemophilia B- deficiency in factor IX
o
Von Willebrand’s disease – defect in factor VIII-related
antigen produced by platelets and endothelium result in
abnormal platelet function
Acquired Coagulation Disorder:
o
Vitamin K deficiency
o
Liver disease
o
Rattlesnake bites
o
Coagulation factor deficiencies due to autoimmune
diseases and massive blood transfusion
Thrombosis may occur either the venous or arterial system, and the
pathogenesis of each is somewhat different
o
Venous thrombosis involves venous stasis, vascular
damage and hypercoagulability
o
Arterial thrombosis, it may involve platelet-platelet
interaction or platelet-vessel wall interaction and
thrombin interaction
Factors Favoring Thrombotic Tendency
Blood stasis
Local Factors
Vascular damage
Unimpeded activation of coagulation – DIC
(intravascular thrombin generation)
Deficiency of natural anticoagulants

Antithrombin III
Coagulation-fibrinolytic

Protein C & S
imbalance
Defective fibrinolysis and abnormal plasmin
generation
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Quantitative depletion
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Qualitative abnormalities
Prosthetic valves
Increased platelet turnover
Valvular heart disease
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LABORATORY TESTS
Closure time/Clotting time
o
Reflects the time required for the generation of thrombin
o
If the plasma concentration of prothrombin or of some of
the other factors are low (or if the factor is absent, or
functionally inactive), clotting time will be prolonged
o
The expected range for clotting time is 3-6 minutes using
slide and capillary method
o
Looks at overall platelet & vessel wall function

aPTT (Activated Partial Prothrombin Time)
o
Measure of the Intrinsic Pathway and common pathways
of coagulation
o
Monitors heparin toxin
o
Measures factor I, II, V, VIII, IX, X, XI, XII
o
Normal activated partial prothrombin time is 30-35
seconds

PT (Prothrombin Time)
o
Measure of the Extrinsic Pathway and common
pathways of coagulation
o
Measures effectivity of anti-coagulants
o
Measures factors I, II, V, VII, X
o
Normal prothrombin time is 12-15 seconds
Normal Values for Coagulation Studies
Clotting time, Lee-White
4-8 mins
Activated coagulation (celite)
< 100 sec
Prothrombin time (PT)
11-13 sec or within 2 sec of control
Activated partial prothrombin time
30-40 sec or within 5 sec of control
(aPTT)
10-15 sec or within 1.3 times as long
Thrombin clotting time (TCT)
as control
Fibrinogen
150-450 mg/dl
Clot dissolution (5-M urea)
Clot intact at 1 hr, 24 hr
Euglobulin lysis
Lysis in 2-6 hrs
Fibrinogen degradation products
Latex particles
< 20 µg/ml
Tanned red cells
< 5 µg/ml
Antithrombin III
Coagulation assay
> 50% of normal pool
Spectrophotometric
85-125% of normal pool
REFERENCES
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Biochemistry Manual (2018)
Dr. Igrobay Recordings
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