BIOCHEMICALASPECTOFBLOOD
AbdulSalamM.SofroDept.ofBiochemistry,Fac.ofMedicine
YARSIUniversity
IntroducHon
AnaksayawaktudisunatdarahnyasusahberhenH,kenapayadok?
Tiapkalisayasikatgigikeluardarah,kenapayadok? Hidungadiksayaseringmimisen.Kenapayadok? Tekanandarahsayarendah.Apasayaharusmakansatekambingterusdok?
KadarHemoglobin(Hb)pamansayakurang.Katadokterharusditransfusi.Apakalaukurangdarahharusditransfusidok?
Anaksayakurangdarah,apaharusdiberilaukhaH&elaterusdok?
QsrelatedtoBlood
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
Venice, Italy Dec 2012
Theverybeginningofhumandevelopment
Fer$lizedovum
Zygote
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, 25th ed & other lecture sources
Coretopics
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
ComposiHonandmainfuncHonsofblood
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)
DeliveringOxygentotheHssues&helpinginthedisposalofcarbondioxide&protonsformedbyHssuemetabolism
MuchsimplerstructurethanmosthumancellsmembranesurroundingasoluHonofHb(about95%ofintracellularproteinoftheRBC)
Containcytoskeletalcomponentsimportantindeterminingtheirshape(Spectrin,ankyrin&otherperipheralmembraneprotein)
Red blood cells (cont.)
Possessmanybloodgroupsystems(eg.ABO,Rh&MNsystems)TheABOsystemiscrucialinbloodtransfusion
TheABOsubstancesareglycosphingolipids&glycoproteinssharingcommonoligosaccharidechains
Redbloodcells(cont.)
Lifespan:120days TheirproducHonisregulatedbyerythropoieHn(synthesizedinkidney&isreleasedtothebloodstreamandtravelstobonemarrowinresponsetohypoxia)
Redbloodcells(cont.)
About2millionRBCenterthecirculaHonpersecond
MetabolicallyacHve(butunique&relaHvelysimple)(facilitateddiusioninvolvingspecicprotein,i.e.glucosetransporter/permease,butnotinsulindependentlikeinmuscle&adiposecells)
Redbloodcells(cont.)
SOD,Catalase&GlutathioneprotectcellsfromoxidaHvestress&damagelinkedtoHexoseMonophosphateShunt(HMS=PentosePhosphatePathway)
Leukocyte(WBC)
Thereare3groups:granulocytes(polymorphonuclearleukocytes=PMNs):NeutrophilsBasophilseosinophils
monocyteslymphocytes
NeutrophilsphagocytosebacteriaandplayamajorroleinacuteinammaHon
Basophilsresemblemastcells,containhistamin&heparinandplayaroleinsometypesofimmunologichypersensiHvityreacHons
EosinophilsareinvolvedincertainallergicreacHons¶siHcinfecHons
Monocytesareprecursorsofmacrophageswhich,likeneutrophilsareinvolvedinphagocytosis
LymphocytesBlymphocytessynthesizeanHbodies,Tlymphocytesplaymajorrolesinvariouscellularimmunemechanisms,suchaskillingvirallyinfectedcells&somecancercells
Platelets(Thrombocytes)
cell-likeparHclessmallerthanRBCsandWBCs.
Helpwithcloingprocessbygatheringatbleedingsiteandclumpingtogethertoformaplugthathelpssealthebloodvessel.
Bloodgroupsystem
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&theirproductinABObloodgroupsystem
GeneH:fucosyltransferaseGeneA:N-acetylgalactosamineglycosyltransferaseGeneB:galactosyltransferaseGeneO:inacHveenzyme
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
RBCPrecursorStructure
Glucose
Galactose
N-acetylglucosamine
Galactose
Precursor Substance (stays the
same)
RBC
Source: cls.umc.edu/COURSES/.../ABOsystem.ppt
FormaHonoftheHanHgen
Glucose
Galactose
N-acetylglucosamine
Galactose
H antigen
RBC
Fucose cls.umc.edu/COURSES/.../ABOsystem.ppt
FormaHonoftheAanHgen
Glucose
Galactose
N-acetylglucosamine
Galactose
RBC
Fucose N-acetylgalactosamine
cls.umc.edu/COURSES/.../ABOsystem.ppt
FormaHonoftheBanHgen
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
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
HanHgen
Certain blood types possess more H antigen than others:
O>A2>B>A2B>A1>A1B Greatest
amount of H Least
amount of H
cls.umc.edu/COURSES/.../ABOsystem.ppt
Plasmaanditsproteins
Plasmaproteins
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
SomefuncHonsofplasmaproteins
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
Igclass Mol.Struct Carbohydr
IgG 2222 4%IgA 2222 10%IgM 2222 15%IgD 2222 18%IgE 2222 18%
IgfuncHonalgroups
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
Hemostasisandthrombosis
HemostasisisthecessaHonofbleedingfromacutorseveredvessel,whereasthrombosisoccurswhentheendotheliumliningbloodvesselsisdamagedorremoved(eg.uponruptureofanatheroscleroHcplaque)
Hemostasis&thrombosissharethreephases:FormaHonofaloose&temporaryplateletaggregateatthesiteofinjury
FormaHonofbrinmeshthatbindstotheplateletaggregate,formingamorestablehemostaHcplugorthrombus
ParHalorcompletedissoluHonofthehemostaHcplugorthrombusbyplasmin
Thrombi
Three types of thrombi: White thrombus Red thrombus Disseminated fibrin deposit in very
small blood vessels or capillaries
IntrinsicandExtrinsicpathwayofbloodcoagulaHon
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
Bloodcloingfactors
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
Bloodcloingfactors(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
Intrinsicpathway
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.
Intrinsicpathway(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
Intrinsicpathway(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
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
Extrinsicpathway
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
Extrinsicpathway(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
Finalcommonpathway
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
Finalcommonpathway(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
Somenotes
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)
Somenotes(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)
Somenotes(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)
Somenotes(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
TERIMAKASIHTHANKYOU
International Society of Blood Transfusion (ISBT) Meeting