Abdul Salam M.

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

Q & A of Blood
Anak saya mudah mimisen (hidung berdarah,
kenapa ya dok?
Tiap kali sikat gigi keluar darah, kenapa ya dok?
Tekanan darah saya tinggi. Saya harus
bagaimana dok?
Tekanan darah saya rendah. Apa saya harus
makan sate kambing terus dok?
Bila saya kurang darah apa perlu transfusi
teratur dok?
Anak saya kurang darah, apa harus diberi lauk
hati & ampela terus dok?

Pendahuluan

Iqra bismi Rabbikalladzii kholaq

Kholaqal insaana min alaq .........
QS. AlAlaq
Venice, Italy
Dec 2012

cogito ergo sum =

saya berpikir maka saya ada
(Rene Descartes Filsuf Prancis)

Paris, France, Dec 2012

Cesky Crumlov, Cezk Dec 2013

Teaching aims
By

the 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

19-12

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

H&A

Tr-A
Ps
r u
e b
c s
u t
r a
s n
o c
r e

Antigen

Tr-B

Tr-H
O

H&B

hh
Precursor
substance

RBC Precursor Structure

RBC

Glucose

Precursor
Substance
(stays the
same)

Galactose
N-acetylglucosamine
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

Fucose

N-acetylgalactosamine

Formation of the B antigen

RBC

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

Glucose
Galactose
N-acetylglucosamine
Galactose

Fucose

Galactose

A
Group O

Many H
antigen sites

Group A

Fewer
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
amount of H

O>A2>B>A2B>A1>A1B

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

Least
amount of H

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

(antichymotrypsin, 1
antitrypsin, 2 macroglobulin,
antithrombin)
Blood clotting (various coagulation
factors, fibrinogen)
Hormones
Antiprotease

 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

IgA

22

22

10 %

IgM

22

22

15 %

IgD

22

22

18 %

IgE

22

22

18 %

4%

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

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

Involves

Blood clotting factors

F

I
F II
F III
F IV
FV
F

VII

: Fibrinogen
: Prothrombin
: Tissue factor
: Ca2+
: Proaccelerin, labile factor,
accelerator (Ac-) globulin
: Proconvertin, serum prothrombin
conversion accelerator (SPCA),
cothromboplastin

Blood clotting factors (cont.)

F

VIII

IX

X
F XI
F

XII
F XIII

: Antihemophilic factor A,
antihemophilic globulin (AHG)
: Antihemophilic factor B, Christmas
factor, plasma thromboplastin
component (PTC)
: Stuart Prower Factor
: Plasma thromboplastin antecedent
(PTA)
: Hageman factor
: 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.)

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
IX

VIII

Ca 2+

IXa
Ca 2+
PL

VIIIa

X
V

Xa
Va

Prothrombin

Ca 2+
PL
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

TERIMAKASIH
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