Abdul Salam M.

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

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?
Q & A of Blood

Iqra bismi Rabbikalladzii kholaq
Kholaqal insaana min alaq .........
Venice, Italy
Dec 2012
QS. AlAlaq
Pendahuluan
cogito ergo sum =
saya berpikir maka saya ada
(Rene Descartes Filsuf Prancis)
Cesky Crumlov, Cezk Dec 2013
Paris, France, Dec 2012
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, 25
th
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)
19-12
Composition of Blood
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
H & B
H
H
P s
r u
e b
c s
u t
r a
s n
o c
r e
Tr-A
Tr-B
O
Precursor
substance
Tr-H
hh
RBC Precursor Structure
Glucose
Galactose
N-acetylglucosamine
Galactose
Precursor
Substance
(stays the
same)
RBC
Source: cls.umc.edu/COURSES/.../ABOsystem.ppt
Formation of the H antigen
Glucose
Galactose
N-acetylglucosamine
Galactose
H antigen
RBC
Fucose
cls.umc.edu/COURSES/.../ABOsystem.ppt
Formation of the A antigen
Glucose
Galactose
N-acetylglucosamine
Galactose
RBC
Fucose
N-acetylgalactosamine
cls.umc.edu/COURSES/.../ABOsystem.ppt
Formation of the B antigen
Glucose
Galactose
N-acetylglucosamine
Galactose
RBC
Fucose
Galactose
cls.umc.edu/COURSES/.../ABOsystem.ppt

Group O Group A
Many H
antigen sites
Fewer
H antigen
sites
A
A
A
A
A
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:


O>A
2
>B>A
2
B>A
1
>A
1
B
Greatest
amount of H
Least
amount of H
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, 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
10
6
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 (V
L
) &
one constant region (C
L
)
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 (V
L
/V
H
& C
L

/C
H
1) => antigen binding fragment
C terminal of L chain (C
L
) => interchain
disulphide bond
C terminal of H chain (C
H
) 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+
F V : 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 Ca
2+
activates F
IX. This in turn cleaves an Arg-Ile bond in
F X to produce F Xa

Intrinsic pathway
PK
HK
XII XIIa
IX IXa
X Xa
Prothrombin Thrombin
XI XIa
HK
X
VIIa/Tissue factor
Extrinsic pathway
VII
Ca
2+
Ca
2+
Ca
2+
PL
Ca
2+
PL
VIII VIIIa
V Va
Prothrombin Thrombin
Fibrinogen
Fibrin monomer
Fibrin polymer
Cross-linked
Fibrin polymer
XIII
XIIIa
Extrinsic pathway
Also leads to activation of F X but by
different mechanism.
Involves tissue factor, F VII, F X & Ca
2+

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
TERIMAKASIH
THANK YOU
International Society of Blood Transfusion (ISBT) Meeting