BIOCHEMISTRY

Blood Chemistry
Dr. Igrobay
BLOOD INTRODUCTION Plasma Fraction
 Blood is one of the largest organ systems of the body  It has 2 constituents: diffusible and non-diffusible
 It is the liquid that flows in an enclosed circulation that is part of the  Diffusible constituents means it can spread in the body
circulatory or cardiovascular system  Non-diffusible constituents means it only focuses on a certain part
 It consists of blood cells suspended in the plasma of the body
 Plasma is the liquid portion of the unclotted blood  Diffusible constituents examples are:
 Blood constitutes around 5-7% of the total body weight o Electrolytes
 To know one’s blood volume, it is around 6% of your total weight in kg o Sodium
 Specific gravity of blood is 1.06 o Calcium
 pH of blood is 7.4 o Potassium
o Normal pH is 7.35-7.45 o Hormones
o Vitamins
o Average pH is 7.4
 Viscosity of blood is 1.7-2.0
 Non-diffusible constituents examples are:
 Freezing point of blood is -0.56oC
o Proteins
 Osmotic pressure of blood at 37oC is 7.6 atm pressure
o Polypeptides
MAJOR FUNCTIONS OF BLOOD
1. Respiration – transport oxygen from lungs to the tissues and of CO2 PLASMA
from the tissues to the lungs  Plasma is the liquid part of fluid blood
2. Nutrition – transport of absorbed food materials  Serum is the fluid remaining after blood coagulates
3. Excretion – transport of metabolic waste to the kidneys, lungs, skin,  Plasma proteins consist of water, electrolytes, metabolites,
and intestines for removal nutrients, proteins and hormone
 Water and electrolyte composition of plasma is practically the same
Capillaries are the main site of respiration, exchange as that of all extracellular fluids
nutrition, and waste products  Laboratory determination of levels of Na, K, Ca, Mg, HCO3 , PaCO2 and
of blood pH are important in clinical management
4. Maintenance of the normal acid-base balance in the body
5. Regulation of water balance through the effects of blood on the Plasma Proteins
exchange of water between the circulating fluid and the tissue fluid  Majority of the plasma proteins is made up of water
6. Regulation of body temperature by the distribution of body heat  It is around 7.0 – 7.5 g/dL
o Normal body temperature is 36.5-37.5oC  It is a complex mixture of glycoprotein and lipoproteins
o Average body temperature is 37oC  The plasma proteins can be separated into 3 major groups:
o >37.5oC = hyperthermia
1. Fibrinogen
o <36.5oC = hypothermia
2. Albumin
3. Globulins
7. Defense against infection by the white blood cells and circulating
Fibrinogen is involved in the coagulation cascade or clot
antibodies
formation. Fibrinogen will be converted to fibrin by the help of
8. Transport of hormones and regulation of metabolism
thrombin
9. Transport of metabolites and regulation of metabolism Albumin is a protein that serves as a transport vehicle that carries
10. Coagulation – clot formation also bile salts and fatty acids
Globulins are involved in the immune system (immunoglobulins)
COMPOSITION OF BLOOD
 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

 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
Picture above: inflammatory states or secondary to certain types of tissue damage
There are two fractions: one is liquid which is the plasma and the other one
 The diversity of their biochemical properties attributes to their
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 varying functions
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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 BIOCHEMISTRY
Blood Chemistry
Dr. Igrobay
 The normal total serum protein concentration is 6-8 g/dL (60-80 g/L) Acute hepatitis and Chronic Liver Disease
and is determined as a fraction containing albumin and the serum  If the liver is defective, plasma protein is
globulins affected
>8 g/dL = Hyperproteinemia
<6 g/dL = Hypoproteinemia 2. Genetic abnormalities
Familial or hereditary disease
 The fibrinogen was discarded in the clot that separated from the
plasma to form the serum specimen 3. Increased loss of albumin
 Increased concentration is termed as hyperproteinemia and this Nephrotic syndrome
could be due to water depletion, multiple myeloma,  Kidneys are defective = proteinuria
macroglobulinemia, and sarcoidosis Massive burns
 The higher the surface area of skin burned,
 Decreased concentration is termed hypoproteinemia and this could
the higher is the protein necessary to be
be secondary to any organ dysfunction such as congestive heart
repaired
failure, peptic ulcer, nephrosis, and cirrhosis
Protein losing enteropathy
 Protein losses at the GIT, goes in the stool
Functions of the Plasma Proteins
1. Antiproteases 4. Increased catabolism of albumin
2. Blood clotting factors Fast breaking own of albumin like in massive burns and
3. Enzyme hormones widespread malignancy (Stage 3 or 4)
4. Immune defense Cancer cells uses protein in order to survive
5. Inflammatory responses
6. Oncofetal 5. Multifactorial
7. Transport or binding proteins Cirrhosis of liver or heart
Congestive heart failure
MAJOR TYPES OF PLASMA PROTEINS Pregnancy
Albumin (69 kDa)
 The major plasma protein in humans Haptoglobin (Hp)
 3.4-4.7 g/dL  It is a plasma glycoprotein that binds extracorpuscular hemoglobin
 Makes up approximately 60% of the total plasma protein in a tight non-covalent complex [Hp-Hp (155kDa)], preventing the
 About 40% is present in the plasma and 60% in the extracellular space loss of free hemoglobin into the kidney thus conserving valuable iron
 It is initially synthesized as preprotein in the liver (12 g/day) present in hemoglobin
 The concentration of the serum albumin controls the passage of  The binding between haptoglobin and hemoglobin is highly specific
water through the cell membrane by osmosis and is responsible for  It exists in three polymorphic forms:
75-80% of the osmotic pressure of human plasma o Hp 1-1
 In addition, it serves as a protein stores for the body that can be o Hp 2-1
utilized when a deficit develops o Hp 2-2
 It serves as a solvent for fatty acids and bile salts  The simplest of which is Hp 1-1 (90kDa) and is found in humans
 It serves as a transport vehicle by loosely binding hormones, amino  In extensive hemolysis, haptoglobin in the serum is depleted
acids, drugs and metals  Thus, haptoglobin measurement is useful for the clinical assessment
Increased concentration (hyperalbuminemia) has no significant of hemolysis
correlation with diseases. The reduction of circulating albumin  Haptoglobin is an acute phase reactant and is frequently elevated in
concentration results in a shift of fluid from intravascular to medically stressful situations
extravascular spaces and is most commonly manifested as edema
and/or congestion Transferrin (76 kDa)
 The transport of iron in the plasma is accomplish by transferrin,
Decrease concentration (hypoalbuminemia) may be due to
which migrates in the ß-globulin region
several mechanisms but the most common one is a decrease in
the production of albumin in the liver  1 molecule of transferrin can bind 2 ions of iron
 It shuttles iron as Fe2+ (Ferrous) to sites where it is needed, either for
 Treatment for hypoalbuminemia is letting the patient drink egg storage or utilization
white In female who are part of the reproductive age group are prone
to iron deficiency anemia due to menstruation
 Causes of albuminemia:
1. Reduced synthesis of albumin
Treatment to this is iron supplements and Vitamin C
Malnutrition Iron supplements are in the Ferrous state (absorbable form)
 Protein calorie malnutrition such as in Vitamin C enhances the absorption of iron in the intestines
Kwashiorkor
Malabsorption syndrome
 Intestines cannot absorb properly
Chronic Inflammatory Disease
 Pulmonary tuberculosis

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 BIOCHEMISTRY
Blood Chemistry
Dr. Igrobay
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
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
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
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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
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:
 Stem cell is a cell with a unique capacity to produce unaltered
daughter cells (self-renewal) and to generate specialized cell
types (potency)
 Stem cells may be:
o Totipotent (capable of producing all the cells in an
organism)
o Pluripotent (able to differentiate into cells of any of
the three germ layers)
o Multipotent (produce only cells of a closely related
family)
o Unipotent (produce only one type of cell)
Different Progenitors of Hematopoietic Stem Cell
Cell-cell interaction and the release of the humoral factors (CSFs, e.g. GM-
CSF/G-CSF: deficiency of which may result to neutropenia) do control the
production and maturation of white cells
Lymphopoiesis
 This is the growth and maturation of lymphocytes
 Lymphocytes are conditioned by two main organ systems, the
thymus (T-lymphocytes) or the bone marrow (B-lymphocytes)
Megakaryopoiesis
 This is the process of platelet development from megakaryocytes
 Thrombopoietin is a hormone that controls proliferation and
maturation of megakaryocytes
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

RED BLOOD CELLS (ERYTHROCYTES)

Picture Above:  Non-nucleated, biconcave in shape and devoid of intracellular
The multipotential hematopoietic stem cell derived from the bone marrow organelles
can give rise to different progenitors. First it branches into two: Myeloid
 It functions mainly in the delivery of O2 and excretion of CO2
progenitor and Lymphoid progenitor.
In Myeloid progenitor it gives rise to:  It has a lifespan of 120 days (it goes to the spleen after it dies)
o Granulocytes – Basophils, Eosinophils, Neutrophils (BEN)  Approximately <1% of RBC is replaced daily at a rate of 2M/sec
o Erythrocytes  Has glucose transporter (GLUT 1) in its membrane and has powerful
o Monocytes – differentiates into macrophages or dendritic cells oxidants which are produced during the course of metabolism:
o Mast cells Superoxide, Hydrogen peroxide, Peroxyl and Hydroxyl radicals and
o Megakaryocytes – becomes thrombocytes (platelets) ROS
 Normal red cell count ranges from 4.2-6.2 x 10(6)
For the Lymphoid progenitor, it gives rise to:  Polycythemia literally means “many blood cells” but usually refers to
o B lymphocyte
increased red cell mass. A better term for this is erythrocytosis or
o T lymphocyte
erythremia
o Natural killer cell
 Secondary or reactive polycythemia is due to a recognizable
physiologic stimulus
Erythropoiesis
 Polycythemia vera or True polycythemia is spontaneous or
 Are derived from the committed erythroid precursor cells through a
unprovoked increased in the red blood cells
process of mitotic growth and maturation
 Anemia is defined as a decrease in the concentration of hemoglobin.
 Erythropoietin (EPO) is a hormone produced largely by the kidney
This is often the end result of red blood cell abnormalities
that stimulates CFU-E stem cells to speed up growth and enhance
maturation  The red blood cell has a unique and relatively simple metabolism

Granulopoiesis
 It is the replacement of leukocytes by cell division and production of
new white cells in the marrow
Continued next page…..
 A process of differentiation occurs, whereby immature white cells
gradually develop and exhibit characteristics of mature functional
leukocytes

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 BIOCHEMISTRY
Blood Chemistry
Dr. Igrobay
Red Blood Cell Membrane
 It consists of an integral layer of lipids, including phospholipids and
cholesterol, which contain an intimate association of proteins
 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
 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
Some of the Major Proteins of the RBC Membrane
3. Spectrin
o Major protein of the cytoskeleton and confers the
flexibility of the membrane of the RBC
o It has four binding sites: self association, Ankyrin, actin
and protein 4.1
o The disorder of which will lead to Hereditary
Spherocytosis and also Hereditary Elliptocytosis
Hereditary Spherocytosis
- Spherocytes (RBC’s without biconcave shape &
central pallor) having low surface-to-volume ratio
- Cause hemolytic anemia
- Causes splenomegaly due to plugging of easily-
ruptured 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
Hereditary Elliptocytosis
- Elliptical RBs
- Defective spectrin or Glycophorin C or protein 4.1
(points of attachment)

4. Ankyrin
o A pyramid-shaped protein that binds spectrin
o Also binds tightly to band 3, securing the attachment of
spectrin to the membrane
o 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
1. Anion exchange protein (Band 3) forming a protein 4.1 – spectrinactin ternary complex
o C terminal is external and N terminal is internal o Also binds to the integral proteins
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 Exists as a dimer in the membrane, in which it forms a

tunnel, permitting the exchange of chloride for
bicarbonate
o Cytosolic N terminal binds to Hb, protein 4.1 and 4.2,
Ankyrin and several glycolytic enzymes

2. Glycophorin A, B, and C
o Transmembrane glycoprotein which is of the single-pass
type Table above:
o C terminal bind spectrin to cell membrane via protein 4.1 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
o Contains binding sites for influenza virus and
Plasmodium falciparum (parasite that causes malaria)

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 BIOCHEMISTRY
Blood Chemistry
Dr. Igrobay
ABO BLOOD GROUP Immunodominant
Antigen Glycosyltransferase
Blood Group Sugar
 Defined system of RBC antigens (blood group substances controlled A α-3-N-acetylgalactosaminyltransferase
N-acetyl-D-
galactosamine
by a genetic locus having variable number of alleles
B α-3-D-galactosyltransferase D-Galactose
O α-2-L-fucosyltransferase L-Fucose
Blood Type
 It is the antigenic phenotype usually recognized by the use of
The product of A gene is the GalNac transferase that adds the terminal
appropriate antibodies
GalNac (immunodominant sugar of blood group A) to the O substance

ABO substances
 These are complex oligosaccharides present in most cells of the body
and in certain secretions
 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
 Also known as H antigen or O antigen or O substance
 The precursor of both A and B substances
 The blood group substance found in persons of type O
 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
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
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:
 Genotype – actual genetic information
o AA/AO , BB/BO, OO, AB
 Phenotype – observed inherited trait
o Blood types: A, B, AB, O

Bombay (Oh) Phenotype

 Phenotype: Oh
 Genotype: hh or H null
 Inheritance of two
recessive h genes
 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
WHITE BLOOD CELLS (LEUKOCYTES)  Tissue basophils, not blood basophils, have IgE receptors adherent
 Classified according to the presence of granules (Granulocytes) or to cell membranes which react with allergens and IgE to induce
absence of granules (Agranulocytes) and their staining release of vasoactive mediators
characteristics  Diapedesis - passage of blood cells through the intact walls of the
 They are formed in the bone marrow and lymphatic tissue with a life capillaries, typically accompanying inflammation
span of 13 to 20 days
 Half of the circulating white cells are granulocytes, the cytoplasm of Neutrophil
which contains readily visible granules of various chemical and  55-65% of WBC count
enzymatic compositions  Have several lobes in nucleus
 The three main types of granulocytes are the: neutrophils,  Present in acute bacterial infection
eosinophils, and basophils  Active Glycolysis
 The staining characteristics of granules define the cell type:  Active Pentose Phosphate Pathway
o Neutral staining – neutrophils  Moderate Oxidative phosphorylation
o Reddish – eosinophils  Rich in lysosomes and their degradative enzymes (used in killing
o Bluish – basophils bacteria and other phagocytosed substances)
 White cells are distinguished from circulating red cells by the  Unique enzymes: MPO (myeloperoxidase), NADPH oxidase
presence of a nucleus  Are recruited from the blood stream into the tissue to help eliminate
 Automated counting procedures enumerate all nucleated cells as the foreign invaders via chemotactic factors
white cells To reach the tissues, neutrophils must pass through the
 Normal white blood cell count is 4.3-10.8 x 10 (3 µl) capillaries by the migration along the blood vessel wall and
 ↑ in WBC count is Leukocytosis then adhere to the endothelial lining (Diapedesis)
 ↓ in WBC count is Leukopenia
Respiratory Burst – is when neutrophils and other
Lymphocytes phagocytic cells engulf bacteria, they exhibit a rapid
increase in oxygen consumption. This phenomenon
 Has large nuclei with small amount of cytoplasm
produces a large amount of reactive derivatives. Some of
 Smallest WBC (<10 µm in diameter)
these products are potent microbial agents
 25-35% of WBC
 Circulating blood lymphocytes constitute a tiny fraction (<5%) of the Neutrophil in Acute Inflammation:
total lymphocyte pool o Entry of activated neutrophils into the tissues
 Response in acute infection o Increased vascular permeability
 There are two primary subtypes: o Activation of platelets
o T-lymphocytes o Spontaneous resolution
o B-Lymphocytes
PLATELETS
Monocytes  Vary in size from to about 4 µ and circulate for approximately 8-12
 Have elongated, indented, or dark kidney-bean shaped nuclei days as disc-shaped, anucleate cells
 Highly phagocytic (cell-eating)  Regulation of platelet production is ascribed to thrombopoietin
 Largest WBC (16-20 µm)  Functions:
 With delicate nuclear chromatin 1. To protect the vascular integrity of the endothelium
 Constitute 5-8% of circulating blood leukocytes 2. Initiate repair when blood vessel walls are damaged
 Primary Homeostasis – platelet-vessel wall interaction
Eosinophils During homeostasis or thrombosis, they become activated & help
 Large, numerous granules form hemostatic plugs or thrombi which include 3 major steps:
 Nuclei with 2 lobes 1. Adhesion to exposed collagen in blood vessels
2. Release (exocytosis) of the contents of their storage
 2-5% WBC count
granules
 Found in lining of respiratory and digestive tracts
3. Aggregation
 Important functions involve protection against infections caused by
parasitic worms and involvement in allergic reaction  Normal platelet count is 150-350x10 (3) µl
 Secrete anti-inflammatory substances in allergic reactions Thrombocytopenia is defined as a platelet count < 100,000 µl and
causes may be due to production defects, sequestration due to
Basophils splenomegaly and accelerated destruction
 Least numerous (0.5-1% WBC count)
 Has large coarse cytoplasmic granules Thrombocytosis is platelet count >350,000 µl which could be
 Contains Histamine, Serotonin, Heparin primary or secondary. Primary thrombocytosis
 Histamine is responsible for allergic reactions (thrombocythemia) may be complicated by bleeding and/or
 Serotonin is responsible for making you feel sleepy thrombosis; Secondary rarely causes hemostatic problems.
Disorders of platelet function are due to defects in platelet
 Heparin is an anticoagulant
adhesion, aggregation or granule release

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 BIOCHEMISTRY
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)

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 EXTRINSIC PATHWAY (Factors III and VII)
3. Fibrin deposits – are common in capillaries  Initiation of fibrin clot formation in response to tissue injury (there is
damage to the blood vessels or to the endothelial cells)
Coagulation Cascade  This pathway is quicker than the intrinsic pathway
 Purpose is to generate active serine protease enzyme thrombin  Located in sub-endothelium and on activated monocyte
 In turn acts selectively on the soluble plasma protein fibrinogen,  Involves tissue factor, factors VII and X, and Ca2+ and results in factor Xa
converting it into soluble fibrin  Formation of complexes between tissue factor and factor VIIa is
 Fibrin is the visible end product of coagulation and acts as the cement considered the key process involved in initiation of blood coagulation
substance to stabilize the initial primary platelet plug in vivo
 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

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 BIOCHEMISTRY
Blood Chemistry
Dr. Igrobay

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-molecular-
weight 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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 BIOCHEMISTRY
Blood Chemistry
Dr. Igrobay
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
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

DISORDERS OF COAGULATION & THROMBOSIS  PT (Prothrombin Time)

 Disorders of blood coagulation may either be congenital or acquired o Measure of the Extrinsic Pathway and common
in origin pathways of coagulation
 Congenital Disorder: o Measures effectivity of anti-coagulants
o Hemophilia A – deficiency in factor VIII o Measures factors I, II, V, VII, X
o Hemophilia B- deficiency in factor IX o Normal prothrombin time is 12-15 seconds
o Von Willebrand’s disease – defect in factor VIII-related
antigen produced by platelets and endothelium result in Normal Values for Coagulation Studies
abnormal platelet function Clotting time, Lee-White 4-8 mins
 Acquired Coagulation Disorder: Activated coagulation (celite) < 100 sec
o Vitamin K deficiency Prothrombin time (PT) 11-13 sec or within 2 sec of control
o Liver disease Activated partial prothrombin time
30-40 sec or within 5 sec of control
o Rattlesnake bites (aPTT)
o Coagulation factor deficiencies due to autoimmune 10-15 sec or within 1.3 times as long
Thrombin clotting time (TCT)
as control
diseases and massive blood transfusion
Fibrinogen 150-450 mg/dl
 Thrombosis may occur either the venous or arterial system, and the
Clot dissolution (5-M urea) Clot intact at 1 hr, 24 hr
pathogenesis of each is somewhat different
Euglobulin lysis Lysis in 2-6 hrs
o Venous thrombosis involves venous stasis, vascular
Fibrinogen degradation products
damage and hypercoagulability Latex particles < 20 µg/ml
o Arterial thrombosis, it may involve platelet-platelet Tanned red cells < 5 µg/ml
interaction or platelet-vessel wall interaction and Antithrombin III
thrombin interaction Coagulation assay > 50% of normal pool
Spectrophotometric 85-125% of normal pool
Factors Favoring Thrombotic Tendency
Blood stasis
Local Factors
Vascular damage REFERENCES
Unimpeded activation of coagulation – DIC  Biochemistry Manual (2018)
(intravascular thrombin generation)
 Dr. Igrobay Recordings
Deficiency of natural anticoagulants
 Antithrombin III
Coagulation-fibrinolytic
 Protein C & S
imbalance
Defective fibrinolysis and abnormal plasmin
generation
 Quantitative depletion
 Qualitative abnormalities
Prosthetic valves
Increased platelet turnover
Valvular heart disease

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