Abdul Salam M.

Sofro
Dept.of Biochemistry, Fac. of Medicine
YARSI University
Teaching aims

 Bythe end of the lecture, students would

be able to understand & describe various
biochemical aspects of blood

 Reference:
Murray K et al. 2000. Harper’s
Biochemistry, 25th ed & other lecture
sources
Core topics

 Introduction
 Composition and main functions of blood
 Plasma and its proteins
 Hemostasis and thrombosis
Introduction

 Blood is a “liquid” tissue circulates in what

is virtually a closed system of blood
vessels
 Blood consists of solid elements (RBC,
WBC & platelets) suspended in a liquid
medium called plasma  critical for the
maintenance of health
Composition and main functions of
blood
Functions

 Respiration
 Nutrition
 Excretion
 Maintenance of normal acid-base balance
 Regulation of water balance
 Regulation of body temperature
 Defense against infection by WBC &
circulating antibodies
 Transport of hormones & regulation of
metabolism
 Transport of metabolites
 Coagulation
 Composition
 Solid elements : RBC, WBC, Platelets
 Liquid medium : plasma consisting of water,
electrolytes, metabolites, nutrients, proteins,
hormones, etc.
 Water & electrolyte composition of
plasma is practically the same as that of all
extracellular fluids
 Once the blood has clotted (coagulated),
the remaining liquid phase (called serum)
lacks of the clotting factors (including
fibrinogen)
Composition of Blood

19-9
Red blood cells (erythrocytes)

 Delivering Oxygen to the tissues & helping

in the disposal of carbon dioxide & protons
formed by tissue metabolism
 Much simpler structure than most human
cells  membrane surrounding a solution
of Hb (about 95% of intracellular protein of
the RBC)
 Contain cytoskeletal components important
in determining their shape (Spectrin,
ankyrin & other peripheral membrane
protein)
Red blood cells (cont.)

 Possess many blood group systems (eg.

ABO, Rh & MN systems)
 The ABO system is crucial in blood
transfusion
 The ABO substances are
glycosphingolipids & glycoproteins
sharing common oligosaccharide chains
 Red blood cells (cont.)

 Lifespan : 120 days

 Their production is regulated by
erythropoietin (synthesized in kidney & is
released to the blood stream and travels to
bone marrow in response to hypoxia)
Red blood cells (cont.)

 About 2 million RBC enter the circulation

per second
 Metabolically active (but unique & relatively
simple)  (facilitated diffusion involving
specific protein, i.e. glucose
transporter/permease, but not insulin
dependent like in muscle & adipose cells)
Red blood cells (cont.)

 SOD, Catalase & Glutathione protect

cells from oxidative stress & damage
 linked to Hexose Monophosphate
Shunt (HMS =Pentose Phosphate
Pathway)
Leukocyte (WBC)
 There are 3 groups :
 granulocytes (polymorphonuclear
leukocytes = PMNs):
○ Neutrophils
○ Basophils
○ eosinophils
 monocytes
 lymphocytes
 Neutrophils phagocytose bacteria and
play a major role in acute inflammation
 Basophils resemble mast cells, contain
histamin & heparin and play a role in
some types of immunologic
hypersensitivity reactions
 Eosinophils are involved in certain allergic
reactions & parasitic infections
 Monocytes are precursors of
macrophages which, like neutrophils are
involved in phagocytosis
 Lymphocytes  B lymphocytes
synthesize antibodies, T lymphocytes
play major roles in various cellular
immune mechanisms, such as killing
virally infected cells & some cancer cells
Platelets (Thrombocytes)

 cell-like particles smaller than RBCs

and WBCs.
 Help with clotting process by
gathering at bleeding site and
clumping together to form a plug that
helps seal the blood vessel.
Blood group system

 Very important in blood transfusion

 Determined by antigens in blood cell
membrane and antibody in plasma
 ABO blood group system:
 Blood group A : antigen A, antibody Anti B
 Blood group B : antigen B, antibody Anti A
 Blood group AB : antigen A & B, antibody non
 Blood group O: antigen non, antibody anti A &
anti B
A GalNAc GALNAc
GAL
GNAc

A GAL

B
Precursor
B Gal substance
FUC
Genes & their product in ABO blood
group system

 Gene H : fucosyltransferase
 Gene A : N-acetylgalactosamine
glycosyltransferase
 Gene B : galactosyltransferase
 Gene O : inactive enzyme
 Gene product Antigen Gene product Antigen

H&A
Tr-A
Ps H
r u Tr-B
e b Tr-H H&B
c s O
u t
r a hh H
s n
o c Precursor
r e substance
RBC Precursor Structure

RBC

Glucose

Galactose
Precursor
Substance
(stays the N-acetylglucosamine
same)
Galactose

Source: cls.umc.edu/COURSES/.../ABOsystem.ppt
Formation of the H antigen

RBC

Glucose

H antigen Galactose

N-acetylglucosamine

Galactose

Fucose cls.umc.edu/COURSES/.../ABOsystem.ppt
 Formation of the A antigen

RBC

cls.umc.edu/COURSES/.../ABOsystem.ppt

Glucose

Galactose

N-acetylglucosamine

Galactose

N-acetylgalactosamine
Fucose
 Formation of the B antigen

RBC

cls.umc.edu/COURSES/.../ABOsystem.ppt

Glucose

Galactose

N-acetylglucosamine

Galactose

Galactose
Fucose
Genetics

 The H antigen is found on the RBC with

the Hh or HH genotype, but NOT from
the hh genotype
 The A antigen is found on the RBC with
the Hh, HH, and A/A, A/O, or A/B
genotypes
 The B antigen is found on the RBC with
the Hh, HH, and B/B, B/O, or A/B
genotypes
 H antigen

 Certain blood types possess more H

antigen than others:

Greatest Least
amount of H amount of H
O>A2>B>A2B>A1>A1B

cls.umc.edu/COURSES/.../ABOsystem.ppt
 A A

A A
Group O Group A

Many H Fewer A
antigen sites H antigen
sites

Most of the H antigen sites in a

Group A individual have been
converted to the A antigen

cls.umc.edu/COURSES/.../ABOsystem.ppt
Plasma and its proteins
Plasma proteins

 Total plasma protein approx. 7.0-7.5 g/dl

 A complex mixture of simple & conjugated
proteins such as glycoproteins & various
types of lipoproteins, thousands of antibodies
 Can be separated by:
 sodium or amm. sulfate into three major
groups  fibrinogen, albumin & globulins
 electrophoresis using cellulose acetate into
five bands  albumin, 1, 2,  &  globulin
 Cont.
 Concentration of plasma protein is important in
determining the distribution of fluid between
blood & tissues
 Osmotic pressure (oncotic pressure) exerted
by plasma protein is approx. 25 mm Hg.
 Hydrostatic pressure in arterioles is approx.
37 mm Hg  a net outward force of about 11
mm Hg drives fluid out into interstitial spaces.
 Hydrostatic pressure in venules is approx. 17
mm Hg  a net force of about 9 mm Hg
attracts water back into circulation
Cont.

 The above pressures are often referred to

as the Starling forces.
 If plasma protein concentration is markedly
diminished (eg. due to severe protein
malnutrition  fluid is not attracted back
into the intravascular compartment and
accumulates in extravascular tissue
spaces  oedema
 Cont.

 Most plasma proteins are synthesized in

the liver
 Plasma proteins are generally synthesized
on membrane-bound polyribosomes
 Almost all plasma proteins are
glycoproteins
 Many plasma proteins exhibit
polymorphism
Some functions of plasma proteins

 Antiprotease (antichymotrypsin, a1 –
antitrypsin, a2 macroglobulin,
antithrombin)
 Blood clotting (various coagulation
factors, fibrinogen)
 Hormones
 Immune defence (Ig, complement
proteins, b2-microgloblin)
 Involvement in inflammatory
responses (acute phase response
protein eg. C-reactive protein, a1-
acid glycoprotein
 Oncofetal (a1-fetoprotein = AFP)
 Transport or binding proteins such
as:
Cont.
 albumin for bilirubin, FFA, ions, metals,
metheme, steroids, other hormones, variety
of drugs
 Ceruloplasmin contains Cu but albumin is
more important in physiological transport of
Cu
 Corticosteroid-binding globulin (transcortin)
 Haptoglobin  binds extracorpuscular Hb
 Liproproteins (chylomicron, VLDL, LDL,
HDL)
Cont.
 Hemopexin
 Retinol-binding protein
 Sex hormone-binding globulin
 Thyroid-binding
 Transferrin
 Transthyretin (formerly pre albumin, binds
T4 & forms a complex with Retinol-binding
protein)
Detail functions of some plasma protein

 Albumin:
 Major protein of human plasma (3.4-4.7 g/dL)
 Some 40% in plasma, 60% in extracellular space
 Synthesized in liver as preproprotein, depressed in
a variety of diseases, particularly those of liver
(decreases albumin/globulin ratio)
 Responsible for 75-80% of osmotic pressure of
human plasma
 Ability to bind various ligands (include FFA, Ca,
certain steroid hormones, bilirubin etc.
 Play an important role in transport of Cu, drugs
Cont.
 Haptoglobin:
 A plasma glycoprotein that binds
extracorpuscular Hb in a tight
noncovalent Hb-Hp complex
 Prevent loss of free Hb into kidney
 Its plasma levels are of some
diagnostic use  low level in
hemolytic anemias
Cont.
 Transferrin:
 a 1-globulin, a glycoprotein, synthesized
in liver
 Plays an important role in the body’s
metabolism of iron (two mol of Fe3+ per
mole of transferrin)  diminishes potential
toxicity of free iron
 Plasma concentration is approx. 300
mg/dL  can bind 300 µg of iron per dL
(Total Iron Binding Capacity of plasma)
 Ceruloplasmin (Cp)
 2-globulin
 Binds copper (Cu)
 Exhibits a copper-dependent oxidase activity
 Low levels of Cp are associated with Wilson
disease
 Tissue levels of Cu & certain other metals
are regulated in part by metallomethionins
(small protein found in the cytosol of cells
particularly liver, kidney & intestine)
 1-Antiproteinase (1-antitrypsin)
 Synthesized by hepatocytes &
macrophages
 Principal serine protease inhibitor of
human plasma  inhibits trypsin,
elastase & certain other proteases
 Deficiency of this protein has a role in
certain cases (approx. 5%) of
emphysema
 2-Macroglobulin
 A large plasma glycoprotein
 Comprises 8-10% of the total plasma
protein in human
 Synthesized by a variety of cell types,
including monocytes, hepatocytes &
astrocytes.
 Binds many proteinases (an important
panproteinase inhibitor)
 Binds many cytokines
 Immunoglobulin
 Play a major role in the body’s
defence mechanism
 Synthesized by B lymphocytes
Immunoglobulin (Ig)

 A group of proteins involved in mediating

immune response in higher organisms
 In gamma globulin fraction of serum
 Very heterogeneous
 Similar in different species
 106 different antibodies may be
produced in human adult
Source:
http://pathmicro.med.sc.edu/mayer/IgStruct2000.htm
 Ig structure
 Tetramer :
* a pair of light chains (two identical
=kappa or =lambda chains)
* a pair of heavy chains (two identical
=alpha, =gamma, =delta, =epsilon or
=mu chains)
 Light chain has one variable region (VL) &
one constant region (CL)
 Heavy chain has one variable region (VH)
and three (, , ) or four (, ) constant
regions
Ig class Mol. Struct Carbohydr

IgG 22 2 2 4%

IgA 22 2  2 10 %

IgM 22 22 15 %

IgD 22 22 18 %

IgE 22 22 18 %

Ig functional groups

N terminal of H & L chains (VL/VH & CL

/CH1) => antigen binding fragment
 C terminal of L chain (CL) => interchain
disulphide bond
 C terminal of H chain (CH) particularly C 2
& C 3 * and C 4 of IgM & IgE) constitute
the Fc fragment responsible for class
specific effector function => complement
fixation or placental transfer, cell surface
binding etc
Hemostasis and thrombosis
 Hemostasis is the cessation of bleeding
from a cut or severed vessel, whereas
thrombosis occurs when the endothelium
lining blood vessels is damaged or
removed (eg. upon rupture of an
atherosclerotic plaque)
 Hemostasis & thrombosis share three
phases:
 Formation of a loose & temporary platelet
aggregate at the site of injury
 Formation of fibrin mesh that binds to the
platelet aggregate, forming a more stable
hemostatic plug or thrombus
 Partial or complete dissolution of the
hemostatic plug or thrombus by plasmin
Thrombi

 Three types of thrombi:

 White thrombus
 Red thrombus
 Disseminated fibrin deposit in very
small blood vessels or capillaries
Intrinsic and Extrinsic pathway of blood
coagulation
 Two pathways lead to fibrin clot formation
 These pathways are not independent
 Initiation of fibrin clot in response to tissue
injury is carried out by extrinsic pathway,
but how intrinsic pathway is activated in
vivo is unclear (but it involves a negatively
charged surface)
 Intrinsic & extrinsic pathways converge in a
final common pathway
 Involves many different proteins  can
be classified into 5 types:
 zymogens of serine dependent
proteases which become activated
during the process of coagulation
 cofactors
 fibrinogen
 a transglutaminase, which stabilizes
fibrin clot
 regulatory & other proteins
 Blood clotting factors

F I : Fibrinogen
 F II : Prothrombin
 F III : Tissue factor
 F IV : Ca2+
FV : Proaccelerin, labile factor,
accelerator (Ac-) globulin
F VII : Proconvertin, serum prothrombin
conversion accelerator (SPCA),
cothromboplastin
 Blood clotting factors (cont.)
F VIII : Antihemophilic factor A,
antihemophilic globulin (AHG)
F IX : Antihemophilic factor B, Christmas
factor, plasma thromboplastin
component (PTC)
F X : Stuart Prower Factor
 F XI : Plasma thromboplastin antecedent
(PTA)
F XII : Hageman factor
 F XIII : Fibrin stabilizing factor (FSF),
fibrinoligase
Intrinsic pathway

 Involves factors XII, XI, IX, VIII, & X as

well as prekallikrein, HMW kininogen,
Ca2+ & platelet phospholipids  results
in the production of factor Xa.
 Commences with the “contact phase” in
which prekallikrein, HMW kininogen, F
XII & F XI are exposed to a negatively
charged activating surface.
 Intrinsic pathway (cont.)

 When the components of the contact

phase assemble on the activating surface,
F XII is activated to F XIIa upon
proteolysis by kallikrein. This F XIIa
attacks prekallikrein to generate more
kallikrein, setting up a reciprocal activation
 F XIIa once formed, activates F XI to F
XIa and also release bradykinin from
HMW kininogen
Intrinsic pathway (cont.)

F XIa in the presence of Ca2+ activates F

IX. This in turn cleaves an Arg-Ile bond in
F X to produce F Xa
 Intrinsic pathway
PK
HK

XII XIIa
HK Extrinsic pathway
VII
Ca 2+
XI XIa

VIIa/Tissue factor
Ca 2+
IX IXa
VIII VIIIa Ca 2+
PL

X Xa X

V Va Ca 2+
PL

Prothrombin Thrombin
 Prothrombin Thrombin

XIII
Fibrinogen

XIIIa

Fibrin monomer

Fibrin polymer

Cross-linked
Fibrin polymer
 Extrinsic pathway

 Also leads to activation of F X but by

different mechanism.
 Involves tissue factor, F VII, F X & Ca2+
and results in the production of F Xa
 It is initiated at the site of tissue injury with
the expression of tissue factor on
endothelial cells
Extrinsic pathway (cont.)

 Tissue factor interacts with &

activates F VII. Tissue factor acts as a
cofactor for F VIIa, enhancing its
enzymatic activity to activate F X
 Activation of F X provides an
important link between those two
pathways
 Final common pathway
 Involves activation of prothrombin to
thrombin
 F Xa produced by either intrinsic or
extrinsic pathway, activates prothrombin (F
II) to thrombin (F IIa)
 Activation of prothrombin, like that of factor
X, occur on the surface of activated
platelets & requires the assembly of a
prothrombinase complex, consisting of
platelet anionic phospholipid, Ca2+, F Va,
F Xa, & prothrombin
 Final common pathway (cont.)
 Conversion of fibrinogen to fibrin is
catalyzed by thrombin (thrombin also
converts F XIII to F XIIIa, a factor highly
specific transglutaminase that covalently
cross-links fibrin molecules by forming
peptide bonds between the amide groups
of glutamine & the e-amino groups of
lysine recidues, yielding a more stable
fibrin clot with increased resistance to
proteolysis
 Some notes

 Levels of circulating thrombin must be

carefully controlled  achieved in 2 ways:
 Feedback mechanism through a
cascade of enzymatic reactions for the
conversion of prothrombin to thrombin
 Inactivation of any thrombin formed by
circulating inhibitors (the most important
of which is antithrombin III)
 Some notes(cont.)

 Endogenous activity of antithrombin III is

greatly potentiated by the presence of
heparin
 Coumarin anticoagulants (eg. Warfarin)
inhibit vit.K-dependent carboxylation of F II,
VII. IX & X
 Fibrin clots are dissolved by plasmin
(circulates in plasma in the form of its
inactive zymogen, plasminogen)
 Some notes(cont.)
 Activators of plasminogen are found in
most body tissues e.g.
 tissue plasminogen activator (alteplse, t-
PA)  is a serine protease that is
released into circulation from vascular
endothelium under condition of injury or
stress & is catalytically inactive unless
bound to fibrin (recombinant t-PA is used
therapeutically as a fibrinolytic agent as
is Streptokinase
 Urokinase (precursor: prourokinase)
Some notes (cont.)

 Hemophilia A is due to deficiency of F VIII

 Hemophilia B is due to deficiency of F IX
 Endothelial cells synthesize prostacyclin
(potent inhibitor of platelet aggregation)&
other compounds that affect clotting &
thrombosis
 Aspirin is an effective antiplatelet drug
 Some laboratory tests measure coagulation
& thrombolysis