Biochemical Aspect of Blood

8/3/2019 Biochemical Aspect of Blood

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BIOCHEMICAL ASPECT OFBLOOD

Abdul Salam M. Sofro

Dept.of Biochemistry, Fac. Of MedicineYARSI UniversityR.C of Biotech. Gadjah Mada University


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Teaching aims

By the end of the lecture, students would be able tounderstand various biochemical aspects of blood

Reference:Murray K et al. 2000. Harpers Biochemistry, 25th ed. &

various sources


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Core topics

Introduction

Composition and main functions of blood Plasma and its proteins

Hemoglobin

Hemostasis and thrombosis


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Introduction

Blood is a liquid tissue circulates in what

is virtually a closed system of bloodvessels

Blood consists of solid elements (RBC,WBC & platelets) suspended in a liquidmedium called plasma critical for the

maintenance of health


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Composition and main

functions of blood


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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


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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 ofall extracellular fluids Once the blood has clotted

(coagulated), the remaining liquid phase(called serum) lacks of the clotting

factors (including fibrinogen)


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Red blood cells (erythrocytes)

Delivering Oxygen to the tissues & helpingin the disposal of carbon dioxide & protonsformed by tissue metabolism

Much simpler structure than most humancells membrane surrounding a solution ofHb (about 95% of intracellular protein ofthe RBC)

Contain cytoskeletal components importantin determining their shape (Spectrin,ankyrin & other peripheral membrane

protein)


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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


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Red blood cells (cont.)

Life span : 120 days Their production is regulated by erythropoietin

(synthesized in kidney & is released to theblood stream and travels to bone marrow inresponse to hypoxia)

Metabolically active (but unique & relativelysimple) glucose transporter/permease:

SOD, Catalase & Glutathione protect cells fromoxidative stress & damage linked to HexoseMonophosphate Shunt (HMS =Pentose PhosphatePathway)


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About 2 million RBC enter the circulationper second

Metabolically active (but unique &relatively simple) (facilitateddiffusion involving specific protein, i.e.

glucose transporter/permease, but notinsulin dependent like in muscle & adiposecells)


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SOD, Catalase & Glutathione

protect cells from oxidative stress& damage linked to HexoseMonophosphate Shunt (HMS=Pentose Phosphate Pathway)


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Leukocyte (WBC)

There are 3 groups :

granulocytes (polymorphonuclearleukocytes = PMNs):

Neutrophils

Basophils

eosinophils monocytes

lymphocytes


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Neutrophils phagocytose bacteria andplay a major role in acute inflammation

Basophils resemble mast cells, contain

histamin & heparin and play a role in sometypes of immunologic hypersensitivityreactions

Eosinophils are involved in certain allergicreactions & parasitic infections


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Monocytes are precursors ofmacrophages which, like neutrophils areinvolved in phagocytosis

Lymphocytes B lymphocytessynthesize antibodies, T lymphocytesplay major roles in various cellular

immune mechanisms, such as killingvirally infected cells & some cancer cells


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Genes & their product in ABO

blood group system

Gene H : fucosyltransferase

Gene A : N-acetylgalactosamine

glycosyltransferase

Gene B : galactosyltransferase

Gene O : inactive enzyme


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Gene product Antigen Gene product Antigen

H & A

H & B

H

HP s

r ue bc su tr a

s no cr e

Tr-A

Tr-B

O

Precursorsubstance

Tr-H

hh


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Precursorsubstance

GALNAc

GALGNAc

GAL

FUC

A

B

A GalNAc B Gal


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Plasma and its proteins


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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 ofantibodies

Can be separated

by sodium or ammonium sulfate into threemajor groups fibrinogen, albumin & globulins by electrophoresis using cellulose acetate into

five bands albumin, 1, 2, & globulin


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Cont. Concentration of plasma protein is important

in determining the distribution of fluidbetween 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 attractswater back into circulation


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Cont.

The above pressures are often referredto as the Starling forces.

If plasma protein concentration ismarkedly diminished (eg. due to severeprotein malnutrition fluid is notattracted back into the intravascular

compartment and accumulates inextravascular tissue spaces oedema


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Cont.

Most plasma proteins are synthesized in theliver

Plasma proteins are generally synthesized onmembrane-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 orsecondary to certain types of tissue damage


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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)


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Cont.

Transport or binding proteins albumin for bilirubin, FFA, ions, metals,metheme, steroids, other hormones,variety of drugs

Ceruloplasmin contains Cu but albumin ismore important in physiological transportof Cu

Corticosteroid-binding globulin(transcortin)

Haptoglobin

binds extracorpuscular Hb


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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)


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Detail functions of some plasmaprotein

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, particularlythose 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


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Cont.

Haptoglobin:

A plasma glycoprotein that bindsextracorpuscular Hb in a tightnoncovalent Hb-Hp complex

Prevent loss of free Hb into kidney

Its plasma levels are of somediagnostic use low level in hemolyticanemias


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Cont.

Transferrin: a 1-globulin, a glycoprotein,

synthesized in liver

Plays an important role in the bodysmetabolism of iron (two mol of Fe3+ permole of transferrin) diminishes

potential toxicity of free iron Plasma concentration is approx. 300mg/dL can bind 300 g of iron per dL(Total Iron Binding Capacity of plasma)


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Ceruloplasmin (Cp)

2-globulin

Binds copper (Cu)

Exhibits a copper-dependent oxidaseactivity

Low levels of Cp are associated withWilson disease

Tissue levels of Cu & certain other metalsare regulated in part by metallomethionins(small protein found in the cytosol ofcells particularly liver, kidney & intestine)


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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 incertain cases (approx. 5%) ofemphysema


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2-Macroglobulin

A large plasma glycoprotein Comprises 8-10% of the total plasmaprotein in human

Synthesized by a variety of cell types,including monocytes, hepatocytes &astrocytes.

Binds many proteinases (an importantpanproteinase inhibitor)

Binds many cytokines


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Immunoglobulin

Play a major role in the bodysdefence mechanism

Synthesized by B lymphocytes


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Immunoglobulin (Ig)

A group of proteins involved inmediating immune response in higherorganisms

In gamma globulin fraction of serum Very heterogeneous Similar in different species

106 different antibodies may beproduced in human adult


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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 (, ) constantregions


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Immunoglobulin(antibody)molecule


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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 %


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Ig functional groups

N terminal of H & L chains (VL/VH & CL /CH1)=> antigen binding fragment

C terminal of L chain (CL) => interchaindisulphide 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 specificeffector function => complement fixation orplacental transfer, cell surface binding etc


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Schematic models of an antibodymolecule and a Fab fragment


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Mudik yok!


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Hemoglobin


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General

Hemoglobin (four subunits) and its similarmolecule myoglobin (one subunit) areiron-containing heme proteins consists

of apoprotein & non-protein heme These heme proteins function in oxygenbinding, oxygen transport, electrontransport & photosynthesis carriedout by heme (a cyclic tetrapyrrole) & itsferrous iron (at the center of the planarring)


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Myoglobin :

MW 17,000 ; a monomer of protein with153 AA residues

stores oxygen in red muscle tissue will be released under condition ofoxygen deprivation (eg. Severe

aexercise) and used by musclemitochondria for ATP synthesis


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75% of the AA residues are presentin 8 -helix (helix A to H)

Histidin F8 and E7 perform uniqueroles in oxygen binding

Oxygen-binding curve for myoglobin ishyperbolic


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Hemoglobin:

Transports oxygen, CO2 & protons

Its allosteric properties results from its

quaternary structures A tetramer composed of pairs ofdifferent polypeptides/subunits (, , , etc. globin chains) a pair of globin chainproduct of gene cluster in chromosome 11& a pair of globin chain product of genecluster in chromosome 16


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Globin genes

Chromosome 11

(- cluster):

-G-A- -- Chromosome 16

(-cluster):

2-1-2-1-2-1-


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2

2 22 22

2

2

1 1 1

Globin Genes :

Hb types :

Embryo

(Gower-I) (Portland) (Gower-II)

Chains Synthesized

5' 3'

Chromosome #16


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2 2 222 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


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50

30

10

6 18 30 6 18 30 42prenatal age (wks)

% of totalglobinsynthesis

birth

postnatal age (wks)


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Types of hemoglobin

Hb Gower 1 = 22

Hb Portland = 22

Hb Gower 2 = 22

Hb Fetal (HbF) = 22

Hb Adult (HbA) = 22 Hb Adult minor (HbA2) = 22

O ti f Hb i i d b


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Oxygenation of Hb is accompanied byconformational changes in the apoproteinnear the heme group (oxygenated Hb = Rform)

After releasing O2 at the tissues(deoxygenated Hb = T form), Hb

transports CO2 & protons to the lungs Hb can bind CO2 directly when oxygen isreleased (about 15% CO2 carried in bloodis carried directly on the Hb molecule. CO2reacts with the amino terminal a-aminogroups of Hb, forming a carbamate &releasing protons that contribute to the

Bohr effect).


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CO2 + Hb NH3+ 2 H+ + Hb N C O-

conversion of the amino terminal from apositive to a negative charge favours saltbridge formation between the &

chains, a situation characteristic of thedeoxy state.

H O


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As CO2 is absorbed in the blood, the

carbonic anhydrase (CA) in erythrocytecatalyzes the formation of carbonic acid,which in turn rapidly dissociate intobicarbonate and a proton. To avoid

increasing the acidity of blood, a bufferingsystem must absorb these excess protons this is carried out by Hb

CO2 + H2O H2CO3HCO3- + H+

CA spontaneous

Hb bi d 2 t f 4


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Hb binds 2 protons for every 4 oxygenmolecules released & thus contributes

significantly to buffering capacity ofblood increase in proton concentrationpromotes oxygen release, while increase inP

O2promotes proton release.

At the lungs, oxygenation of Hb isaccompanied by expulsion and subsequentexpiration of CO2Bohrs effect (areversible phenomenon with that in theperipheral tissues)

2 3 Bi h h l (BPG) i Hb


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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 thespace is wide enough when Hb is in the Tform (deoxygenated)

Binds more weakly to fetal Hb than toadult Hb

Increase concentration of BPG lowers theaffinity of Hb for oxygen (decreases P50) increasing the ability of Hb to release

oxygen at the tissues


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Mutant human Hb

Causes hemoglobinopathy (when biologicfunction is altered)

Due to mutations in the gene that codefor globin chains:

Structurally abnormal Hb (HbM, HbS,HbE, HbC etc)

Reduced synthesis of Hb(thalassemias)

Diagnosed by special method (e.g.molecular diagnosis)


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Batak

Melayu

Minang

Palembang

Bangka

Dayak

Banjar

Palu

Minahasa

Jawa

Tengger

SumbawaBali

Sumba

Sasak

Alor

Toraja

Gambar 5.5. Pola distribusi dan prevalensi trait thalassemia- dan hemoglobin-Epada berbagai populasi di Indonesia. * adalah hemoglobin OIna.

1,5 0

3,7

5,2

2,9

4,3

9,2 6,5

5,44,5

3,2 4,8

0 10,6

3,1 1,5

0 0

0 1,7

1,2 3,7

0 4*

1,26,1

2,9 4,3 2,536,6

5,1 6,80 0

= trait thalassemia-

= trait hemoglobin-E


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Hemostasis and

thrombosis

H i i h i f bl di f


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Hemostasis is the cessation of bleeding from acut or severed vessel, whereas thrombosisoccurs when the endothelium lining bloodvessels is damaged or removed (eg. uponrupture of an atherosclerotic plaque)

Hemostasis & thrombosis share threephases: Formation of a loose & temporary platelet

aggregate at the site of injury Formation of fibrin mesh that binds to

the platelet aggregate, forming a morestable hemostatic plug or thrombus Partial or complete dissolution of the

hemostatic plug or thrombus by plasmin

Thr mbi


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Thrombi

Three types of thrombi: White thrombus

Red thrombus

Disseminated fibrin deposit in very smallblood vessels or capillaries

Intrinsic and Extrinsic pathway of


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Intrinsic and Extrinsic pathway ofblood coagulation

Two pathways lead to fibrin clotformation

These pathways are not independent

Initiation of fibrin clot in response totissue injury is carried out by extrinsicpathway, but how intrinsic pathway isactivated in vivo is unclear (but itinvolves a negatively charged surface)

Intrinsic & extrinsic pathways convergein a final common pathway


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Involves many different proteins canbe classified into 5 types:

(1) zymogens of serine dependentproteases which become activatedduring the process of coagulation

(2) cofactors

(3) fibrinogen

(4) a transglutaminase, which stabilizesfibrin clot

(5) regulatory & other proteins


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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, serumprothrombin conversion accelerator(SPCA), cothromboplastin


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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


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Intrinsic pathway

Involves factors XII, XI, IX, VIII, & Xas 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 negativelycharged activating surface.


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Intrinsic pathway (cont.)

When the components of the contactphase assemble on the activating surface,F XII is activated to F XIIa upon

proteolysis by kallikrein. This F XIIaattacks prekallikrein to generate morekallikrein, setting up a reciprocalactivation

F XIIa once formed, activates F XI to FXIa and also release bradykinin fromHMW kininogen


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Intrinsic pathway (cont.)

F XIa in the presence of Ca2+ activates FIX. This in turn cleaves an Arg-Ile bond inF X to produce F Xa


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

Intrinsic pathway


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PKHK

XII XIIa

IX IXa

X Xa

Prothrombin Thrombin

XI XIa

HK

X

VIIa/Tissue factor

Extrinsic pathwayVII

Ca 2+

Ca 2+

Ca 2+

PL

Ca 2+

PLVIII VIIIa

V Va


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Prothrombin Thrombin

Fibrinogen

Fibrin monomer

Fibrin polymer

Cross-linkedFibrin polymer

XIII

XIIIa


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


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Extrinsic pathway

Also leads to activation of F X but bydifferent 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 injurywith the expression of tissue factor onendothelial cells

Extrinsic pathway (cont )


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Extrinsic pathway (cont.)

Tissue factor interacts with & activatesF VII. Tissue factor acts as a cofactorfor F VIIa, enhancing its enzymatic

activity to activate F X Activation of F X provides an important

link between those two pathways


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Final common pathway

Involves activation of prothrombin tothrombin 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 theassembly of a prothrombinase complex,consisting of platelet anionic phospholipid,Ca2+, F Va, F Xa, & prothrombin

l h ( )


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Final common pathway (cont.)

Conversion of fibrinogen to fibrin iscatalyzed by thrombin (thrombin alsoconverts F XIII to F XIIIa, a factor

highly specific transglutaminase thatcovalently cross-links fibrin molecules byforming peptide bonds between theamide groups of glutamine & the e-amino

groups of lysine recidues, yielding a morestable fibrin clot with increasedresistance to proteolysis

Some notes


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Some notes

Levels of circulating thrombin must becarefully controlled achieved in 2 ways:

Feedback mechanism through a cascade

of enzymatic reactions for theconversion of prothrombin to thrombin

Inactivation of any thrombin formed by

circulating inhibitors (the mostimportant of which is antithrombin III)

Some notes(cont )


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Some notes(cont.)

Endogenous activity of antithrombinIII is greatly potentiated by thepresence of heparin

Coumarin anticoagulants (eg.Warfarin) inhibit vit.K-dependentcarboxylation of F II, VII. IX & X

Fibrin clots are dissolved by plasmin(circulates in plasma in the form ofits inactive zymogen, plasminogen)

Some notes(cont )


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Some notes(cont.)

Activators of plasminogen are foundin most body tissues e.g. tissue plasminogen activator (alteplse, t-

PA) is a serine protease that isreleased into circulation from vascularendothelium under condition of injury orstress & is catalytically inactive unless

bound to fibrin (recombinant t-PA isused therapeutically as a fibrinolyticagent as is Streptokinase

Urokinase (precursor: prourokinase)

S ( )


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Some notes(cont.)

Hemophilia A is due to deficiency of FVIII

Hemophilia B is due to deficiency of F IX

Endothelial cells synthesize prostacyclin(potent inhibitor of plateletaggregation)& other compounds thataffect clotting & thrombosis

Aspirin is an effective antiplatelet drug Some laboratory tests measure

coagulation & thrombolysis


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Biochemical Aspect of Blood

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