Abdul Salam M.

Sofro
Dept.of Biochemistry, Fac. of Medicine
YARSI University
Introduction
 Qs related to Blood
Anak saya waktu disunat darahnya susah berhenti,
kenapa ya dok?
Tiap kali saya sikat gigi keluar darah, kenapa ya dok?
Hidung adik saya sering mimisen. Kenapa ya dok?
Tekanan darah saya rendah. Apa saya harus makan
sate kambing terus dok?
Kadar Hemoglobin (Hb) paman saya kurang. Kata
dokter harus ditransfusi. Apa kalau kurang darah
harus ditransfusi dok?
Anak saya kurang darah, apa harus diberi lauk hati &
ampela terus dok?
 Iqra bismi Rabbikalladzii
Fertilized
ovum
kholaq (Bacalah, dengan
nama Rabb/Tuhan mu yang
telah menciptakan)
Kholaqal insaana min alaq
........ (Yang menciptakan
manusia dari alaq -
segumpal darah, sesuatu
yang beginning of human- QS. AlAlaq
menempel)
The very
development
Venice, Italy
Dec 2012 Zygote
cogito ergo sum =
saya berpikir maka saya ada
(Rene Descartes Filsuf Prancis)

Paris, France, Dec 2012

Cesky Crumlov, Cezk Dec 2013

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. Harpers
Biochemistry, 25th ed & other lecture
sources
Core topics

Introduction
Composition and main functions of blood
Plasma and its proteins
Hemostasis and thrombosis
Hemoglobin synthesis and degradation
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

19-13
 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.)

Life span : 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
 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
 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
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
Agglutination
 Antigens

b a
b
a

b b a a

b a
a b
b b
a
a
Reaksi Agglutinasi
Identifikasi Golongan darah ABO
Keterkaitan golongan darah
dengan individu tertentu
Contoh Gol. Darah di USA

Blood Type & Rh How Many Have It Frequency

O Rh Positive 1 person in 3 37.4%

O Rh Negative 1 person in 15 6.6%
A Rh Positive 1 person in 3 35.7%
A Rh Negative 1 person in 16 6.3%
B Rh Positive 1 person in 12 8.5%
B Rh Negative 1 person in 67 1.5%
AB Rh Positive 1 person in 29 3.4%
AB Rh Negative 1 person in 167 .6%
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, 1
antitrypsin, 2 macroglobulin,
antithrombin)
Blood clotting (various coagulation
factors, fibrinogen)
Hormones
 Immune defence (Ig, complement
proteins, 2-microgloblin)
Involvement in inflammatory
responses (acute phase response
protein eg. C-reactive protein, 1-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 bodys
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 g 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 bodys
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 22 22 4%

IgA 22 22 10 %

IgM 22 22 15 %

IgD 22 22 18 %

IgE 22 22 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 (alteplase,
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
International Society of Blood Transfusion (ISBT) Meeting

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