BIOCHEMICAL

ASPECT OF BLOOD

Abdul Salam M. Sofro

Dept.of Biochemistry, Fac. of Medicine
YARSI University

IntroducHon

Qs related to Blood
Anak saya waktu disunat darahnya susah berhenH,
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 haH &
ampela terus dok?

Fer$lized
ovum

Iqra bismi Rabbikalladzii kholaq

(Bacalah, dengan nama Rabb/Tuhan
mu yang telah menciptakan)
Kholaqal insaana min alaq ........ (Yang
menciptakan manusia dari alaq segumpal darah, sesuatu yang
menempel) - QS. AlAlaq
The very beginning of human
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
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

IntroducHon
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

 ComposiHon and main funcHons of blood

FuncHons

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 Hssues & helping in
the disposal of carbon dioxide & protons
formed by Hssue metabolism
Much simpler structure than most human cells
membrane surrounding a soluHon 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 producHon is regulated by erythropoieHn
(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 circulaHon per
second
Metabolically acHve (but unique & relaHvely
simple) (facilitated diusion involving
specic 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 oxidaHve 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 inammaHon
Basophils resemble mast cells, contain
histamin & heparin and play a role in some
types of immunologic hypersensiHvity
reacHons
Eosinophils are involved in certain allergic
reacHons & parasiHc infecHons

 Monocytes are precursors of macrophages

which, like neutrophils are involved in
phagocytosis
Lymphocytes B lymphocytes synthesize
anHbodies, T lymphocytes play major roles
in various cellular immune mechanisms,
such as killing virally infected cells & some
cancer cells

Platelets (Thrombocytes)
cell-like parHcles smaller than RBCs and
WBCs.
Help with cloing 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 : inacHve 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

H
Tr-H

Antigen

Tr-B
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

FormaHon of the H anHgen

RBC

Glucose

H antigen

Galactose
N-acetylglucosamine
Galactose

Fucose

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

FormaHon of the A anHgen

RBC

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

Glucose
Galactose
N-acetylglucosamine
Galactose

Fucose

N-acetylgalactosamine

FormaHon of the B anHgen

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

GeneHcs
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 anHgen
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 funcHons 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

Basic structure of immunoglobulins

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

IgE

22 22

18 %

22 22

18 %

 Ig funcHonal 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 cessaHon 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 atheroscleroHc plaque)
Hemostasis & thrombosis share three phases:
FormaHon of a loose & temporary platelet
aggregate at the site of injury
FormaHon of brin mesh that binds to the
platelet aggregate, forming a more stable
hemostaHc plug or thrombus
ParHal or complete dissoluHon of the
hemostaHc 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

coagulaHon
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 cloing factors

F I : Fibrinogen
F II : Prothrombin
F III : Tissue factor
F IV : Ca2+
F V : Proaccelerin, labile factor,
accelerator (Ac-) globulin
F VII : Proconvertin, serum prothrombin
conversion accelerator (SPCA),
cothromboplastin

Blood cloing 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
XI

Ca 2+

Extrinsic pathway
VII
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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