BIOCHEMICAL ASPECT OF

BLOOD
Abdul Salam M. Sofro
Dept.of Biochemistry, Fac. Of Medicine
YARSI University
R.C of Biotech. Gadjah Mada University

Teaching aims
By the end of the lecture, students would be able to
understand various biochemical aspects of blood


Reference:
Murray K et al. 2000. Harpers Biochemistry, 25
th
ed. &
various sources
Core topics
Introduction
Composition and main functions of blood
Plasma and its proteins
Hemoglobin
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)
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 (erythrocytes)
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)
Metabolically active (but unique & relatively
simple) glucose transporter/permease:
SOD, Catalase & Glutathione protect cells from
oxidative stress & damage linked to Hexose
Monophosphate Shunt (HMS =Pentose Phosphate
Pathway)
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

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
Precursor
substance
GALNAc
GAL
GNAc
GAL
FUC
A

B
A GalNAc B Gal
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 ammonium sulfate into three
major groups fibrinogen, albumin & globulins
by electrophoresis using cellulose acetate into
five bands albumin, o
1
, o
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
Each plasma protein has a characteristic half-
life in the circulation
The level of certain protein in plasma protein
increase during acute inflammatory states or
secondary to certain types of tissue damage
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)
Cont.
Transport or binding proteins
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

Cont.
Liproproteins (chylomicron, VLDL, LDL,
HDL)
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)
o
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)
o
1
-Antiproteinase (o
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
o
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
Ig structure
Tetramer :
* a pair of light chains (two identical
k=kappa or =lambda chains)
* a pair of heavy chains (two identical
o=alpha, =gamma, o=delta, c=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 (o, , o) or four (c, ) constant
regions
Immunoglobulin
(antibody)
molecule
Ig class Mol. Struct Carbohydr
IgG 2k2 22 4 %

IgA o2k2 o22 10 %

IgM 2k2 22 15 %

IgD o2k2 o22 18 %

IgE c2k2 c22 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
Schematic models of an antibody
molecule and a Fab fragment
Mudik yok!
Hemoglobin

General
Hemoglobin (four subunits) and its similar
molecule myoglobin (one subunit) are
iron-containing heme proteins consists
of apoprotein & non-protein heme
These heme proteins function in oxygen
binding, oxygen transport, electron
transport & photosynthesis carried
out by heme (a cyclic tetrapyrrole) & its
ferrous iron (at the center of the planar
ring)

Myoglobin :

MW 17,000 ; a monomer of protein with
153 AA residues
stores oxygen in red muscle tissue
will be released under condition of
oxygen deprivation (eg. Severe
aexercise) and used by muscle
mitochondria for ATP synthesis

75% of the AA residues are present
in 8 o-helix (helix A to H)
Histidin F8 and E7 perform unique
roles in oxygen binding
Oxygen-binding curve for myoglobin is
hyperbolic

Hemoglobin:

Transports oxygen, CO
2
& protons
Its allosteric properties results from its
quaternary structures
A tetramer composed of pairs of
different polypeptides/subunits (o, |, , o
etc. globin chains) a pair of globin chain
product of gene cluster in chromosome 11
& a pair of globin chain product of gene
cluster in chromosome 16
Globin genes
Chromosome 11
(|- cluster):
c-
G
-
A
- |-o-|
Chromosome 16
(o-cluster):

2
-
1
-o
2
-o
1
-o
2
-o
1
-u
2
2 2 2 2
2
2
2
1
1 1
Globin Genes :
Hb types :
Embryo
(Gower-I) (Portland) (Gower-II)
Chains Synthesized
5'
3'
Chromosome #16
2 2
2 2
2 2
2 2
Fetus
Adult
(Hb-F)
(Hb-A) (Hb-A)
2
3'
5'
G
G
G
A
A
A
Globin Genes :
Hb types :
Chains Synthesized
Chromosome #11
50
30
10
6 18 30 6 18 30 42
prenatal age (wks)
% of total
globin
synthesis
birth
postnatal age (wks)
Types of hemoglobin
Hb Gower 1 = ,2c2
Hb Portland = ,22
Hb Gower 2 = o2c2
Hb Fetal (HbF) = o22
Hb Adult (HbA) = o2|2
Hb Adult minor (HbA2) = o2o2
Oxygenation of Hb is accompanied by
conformational changes in the apoprotein
near the heme group (oxygenated Hb = R
form)
After releasing O
2
at the tissues
(deoxygenated Hb = T form), Hb
transports CO
2
& protons to the lungs
Hb can bind CO
2
directly when oxygen is
released (about 15% CO
2
carried in blood
is carried directly on the Hb molecule. CO
2

reacts with the amino terminal a-amino
groups of Hb, forming a carbamate &
releasing protons that contribute to the
Bohr effect).


CO
2
+ Hb NH
3
+
<=> 2 H
+
+ Hb N C O
-


conversion of the amino terminal from a
positive to a negative charge favours salt
bridge formation between the o & |
chains, a situation characteristic of the
deoxy state.


H
O
As CO
2
is absorbed in the blood, the
carbonic anhydrase (CA) in erythrocyte
catalyzes the formation of carbonic acid,
which in turn rapidly dissociate into
bicarbonate and a proton. To avoid
increasing the acidity of blood, a buffering
system must absorb these excess protons
this is carried out by Hb

CO
2
+ H
2
O H
2
CO
3
HCO
3
-
+ H
+

CA spontaneous
Hb binds 2 protons for every 4 oxygen
molecules released & thus contributes
significantly to buffering capacity of
blood increase in proton concentration
promotes oxygen release, while increase in
P
O2
promotes proton release.
At the lungs, oxygenation of Hb is
accompanied by expulsion and subsequent
expiration of CO
2
Bohrs effect (a
reversible phenomenon with that in the
peripheral tissues)


2,3-Bisphosphoglycerate (BPG) in Hb
Formed from glycolytic intermediate 1,3-
bisphosphoglycerate
One molecule of BPG is bound per Hb
tetramer in the central cavity the
space is wide enough when Hb is in the T
form (deoxygenated)
Binds more weakly to fetal Hb than to
adult Hb
Increase concentration of BPG lowers the
affinity of Hb for oxygen (decreases P
50
)
increasing the ability of Hb to release
oxygen at the tissues

Mutant human Hb
Causes hemoglobinopathy (when biologic
function is altered)
Due to mutations in the gene that code
for globin chains:
Structurally abnormal Hb (HbM, HbS,
HbE, HbC etc)
Reduced synthesis of Hb
(thalassemias)
Diagnosed by special method (e.g.
molecular diagnosis)
Batak
Melayu
Minang
Palembang
Bangka
Dayak
Banjar
Palu
Minahasa
Jawa
Tengger
Sumbawa
Bali
Sumba
Sasak
Alor
Toraja
Gambar 5.5. Pola distribusi dan prevalensi trait thalassemia-| dan hemoglobin-E
pada berbagai populasi di Indonesia. * adalah hemoglobin O
Ina
.
1,5 0
3,7
5,2
2,9
4,3
9,2 6,5
5,4 4,5
3,2 4,8
0 10,6
3,1 1,5
0 0
0 1,7
1,2 3,7
0 4*
1,2 6,1
2,9 4,3
2,5 36,6
5,1 6,8
0 0
= trait thalassemia-|
= trait hemoglobin-E
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:
(1) zymogens of serine dependent
proteases which become activated
during the process of coagulation
(2) cofactors
(3) fibrinogen
(4) a transglutaminase, which stabilizes
fibrin clot
(5) 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
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

http://liveonearth.livejournal.com/477946.html
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
http://liveonearth.livejournal.com/477946.html
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 (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