DARAH

Olivia Herliani, dr., M.Si

 Aspek Biomedis
• Homeostasis
• Penelitian → perkembangan biokimia +
biokimia klinis
• ↑↓ protein plasma (enzim, Ig) → dx px ttn
Fungsi Darah
 Plasma Darah
• O/ plasma kecuali transport O2 + cell mediated
immunologic defense
• Plasma: air, elektrolit, metabolit, nutrien, protein,
hormon
• Komposisi air & elektrolit: plasma = cairan
ekstraseluler
• Konsentrasi total protein ± 7.0–7.5 g/dL → major
solids part of plasma.
• Protein → complex mixture: simple proteins +
conjugated proteins (glyco-, lipoproteins)
 Plasma Darah
• Proteomic techniques → isolasi + karakterisasi →
↑ plasma proteome
• Salting out: pemisahan protein dari kompleks
solven dan/atau elektrolit → sifat kelarutan →
fraksi → 3 kelompok besar: fibrinogen, albumin,
globulin
• Electrophoresis: supporting mediums (lab klinis:
selulose asetat) → stained strip (elektroforetogram)
→ albumin, α1, α2, β, γ globulin → densitometer
(kuantitas)
Protein plasma
Protein Plasma
 Starling Forces
• Arteriol: tekanan hidrostatik 37 mmHg –
tekanan interstitial/tissue 1 mmHg - tekanan
osmotik/oncotik protein plasma 25 mmHg =
outward force 11 mm Hg → cairan keluar ke
ruang interstitial
• Venul: tekanan hidrostatik 17 mmHg - tekanan
interstitial/tissue 1 mmHg - tekanan
osmotik/oncotik protein plasma 25 mmHg = 9
mmHg → menarik cairan kembali ke sirkulasi
 Edema
• Eg, malnutrition → protein def → plasma
protein concentration ↓↓ → fluid not attracted
back into intravasc → accumulate in extravasc
tissue space
 Protein Plasma
• Penelitian manusia & hewan → biosintesis,
turnover, struktur, fungsi, perubahan ∑ &
metabolisme pd px
• Gen → klon & struktur
• Antibodi protein plasma → presipitasi &
isolasi pure protein dari kompleks
• Isotop → jalur biosintesis & laju turnover
dalam plasma
 Sintesa Protein Plasma
• Pdu: liver, kec γ-globulin (sel plasma),
beberapa yang lain (tempat lain, misal sel
endothelial)
• Secretory route : rough endoplasmic membrane
→ smooth endoplasmic membrane → golgi
app → secretory vesicles → plasma
• Synthesized as preproteins + initially contain
amino terminal signal peptides → various
posttranslational modifications (proteolysis,
glycosylation, phosphorylation, etc)
 Sintesa Protein Plasma
• Transit times (hepatocyte → plasma) : 30 min -
several hours/> for individual proteins
• Most are glycoprotein → N- or O-linked
oligosaccharide chains, or both
• Ex alb → ≠ sugar residues
• Neuraminidase → removal terminal sialic acid
residues (ex ceruloplasmin) → ↓ ↓ T½ in
plasma
Sintesa Protein Plasma
 Polimorfism Protein Plasma
• Polymorphism = mendelian or monogenic trait
in population in at least two phenotypes
• Eg, ABO blood group substances, α1-AT, Hp,
Tf, ceruloplasmin, Ig → electrophoresis
isoelectric focusing
 T½ Protein Plasma
• Labeling isolated pure protein with 131I or
Cr51 → T½
• Alb → 20 d, Hp → 5 d
• Certain disease → altered T½
• Regional ileitis (Crohn disease) : inflamed
intestinal mucosa → >> plasma proteins (inc
alb) lost → protein-losing gastroenteropathy
→ T½ alb ↓ ≈ 1 d
 Acute Phase Proteins
• Acute-phase proteins ↑↑ (CRP/C-reactive
protein, α1-AT, Hp, α1-acid glycoprotein,
fibrinogen) in:
• Acute inflammatory state or certain tissue
damage
• Chronic inflammatory state
• Cancer
• Stimulator: cytokine & IL-1 + IL-6, NFkB
 Acute Phase Proteins
• CRP: stimulate classic complement pathway;
marker for tissue injury, infection &
inflammation, predictor in cardiovasc
• α1-AT: neutralize certain protease in
inflammation
Protein Plasma
Protein Plasma
 Albumin
• 69 kDa
• Major protein of plasma (3.4–4.7 g/dL)
• ± 60% of total plasma
• ± 40% in the plasma, 60% in extracellular
• Liver → 12 g/day → 25% of total hepatic
protein synthesis, 50% of total hepatic secreted
protein
 Albumin
• Preproprotein → signal peptide is removed
(cisternae of rough ER) → resulting
hexapeptide amino terminal is cleaved off
farther along the secretory pathway
 Albumin
• Mature albumin : 1 polypeptide chain → 585
aa + 17 disulfide bonds
• Proteases → subdivided → 3 domains + f/
• Ellipsoidal shape → ≠ ↑ plasma viscosity (≈
elongated molecule such as fibrinogen)
• Low molecular mass + high concentration →
responsible for 75–80% of osmotic pressure
 Albumin
• Liver disease → ↓ albumin-globulin ratio
• Protein malnutrition (eg kwashiorkor) → early
↓ albumin
• Alb prep → tx hemorrhagic shock, burns
 Haptoglobin
• Hp binds extracorpuscular Hb in a tight
noncovalent complex (Hb-Hp)
• ∑: 40 – 180 mg of hemoglobin-binding
capacity/deciliter
• ± 10% Hb degraded/day → circulation →
extracorpuscular; 90% in old/damaged rbc →
degraded by cells of the histiocytic system
• 3 polymorphic forms : Hp 1-1, Hp 2-1, Hp 2-2
 Haptoglobin
• Hb 65 kDa + simplest polymorphic form of Hp
(Hp 1-1) 90 kDa = Hb-Hp complex 155 kDa
• Free Hb → kidney glomerulus → tubules →
tends to precipitate therein
• Hb-Hp complex → ≠ glomerulus → prevent
loss of free Hb into the kidney → conserves Hb
iron
Transferrin
 Transferrin
• 76 kDa β1-globulin
• 20 polymorphic forms
• Transports iron (2 mol of Fe3+ per mole of Tf)
: gut → bone marrow
• 200 billion (± 20 mL) rbc → catabolized/day
→ 25 mg of iron
• Tf binds to receptor (TfR1 & TfR2) on surfaces
of many cells → internalized by receptor-
mediated endocytosis
 Transferrin
• Acid pH inside lysosome → iron dissociates
from protein → leaves endosome via DMT1 →
enter the cytoplasm
• ApoTf remains associated with its receptor →
returns to plasma membrane → dissociates
from its receptor → reenters the plasma →
picks up more iron → delivers needy cells
• N :Tf-iron turns over 10–20 x/day
 Transferrin
• Concentration ± 300 mg/dL → can bind 300
μg of iron/dl → TIBC
• However, N only ⅓ saturated with iron
• Iron deficiency anemia : <
• Storage of excess iron (eg,hemochromatosis): >
 Ceruloplasmin
• α2-globulin
• 160 kDa
• High copper content → blue color
• Carries 90% copper in plasma
• 1 ceruloplasmin binds 6 atoms of copper →
very tightly, copper ≠ exchangeable
• Albumin carries ~10% less tightly → donates
copper more readily → more important in
copper transport
 α1-antiproteinase
• 52 kDa
• Single-chain protein : 394 aa
• > 90% of α1 fraction plasma
• Synthesized by hepatocytes + macrophages
• Principal serine protease inhibitor (serpin, or
Pi) of plasma
• Inhibits trypsin, elastase, and certain other
proteases by forming complexes with them
 α1-antiproteinase
• 75 polymorphic forms
• Major genotype MM → phenotypic product
PiM
• Deficiency → ± 5% of emphysema in
genotype ZZ (PiZ) + in PiSZ heterozygotes →
secrete protein < PiMM
 α1-antiproteinase
• ∑ α1-at ↓ + polymorphonuclear wbc ↑ in the
lung (eg, pneumonia) → lacks a countercheck
to proteolytic damage of the lung by proteases
(elastase)
 α1-antiproteinase
• Methionine(residue 358) of α1-at ≈ binding to
proteases
• Smoking →oxidizes methionine → methionine
sulfoxide → inactivates it → α1-at no longer
neutralize proteases
• Devastating in (eg, PiZZ phenotype) who
already have low levels of α1-at
• Further diminution in α1-at by smoking → ↑
proteolytic destruction of lung tissue →
accelerating emphysema development
 α1-antiproteinase
• α1-at iv (augmentation tx) → emphysema due
to α1-at def
• α1-at def liver disease : molecules of ZZ
phenotypeaccumulate + aggregate cisternae ER
of hepatocytes → hepatitis with consequent
cirrhosis → tx: liver transplantation
 Immune system
• 3 : B lymphocytes, T lymphocytes, innate
immune system
• B lymphocytes : bone marrow cells →
synthesis of circulating humoral antibodies
(immunoglobulins)
• T lymphocytes : thymic origin → cell-mediated
immunologic processes (graft rejection,
hypersensitivity rx, malignant cells, viruses)
 Immune system
• Innate immune system : against infection in a
non-specific manner, not adaptive (unlike B &
T cells), variety of cells (phagocytes,
neutrophils, natural killer cells, others)
Immunoglobulins
Immunoglobulins
 Immunoglobulins
• Minimum 2 identical light (L) chains (23 kDa)
+ 2 identical heavy (H) chains (53–75 kDa) →
tetramer (L2H2) by disulfide bonds
• IgG : Y-shaped, binding Ag at both tips of Y
• Each chain → specific domains/regions ≈
structural and functional significance
• ½ L chain toward carboxyl terminal = constant
region (CL), ½ amino terminal = variable
region (VL)
 Immunoglobulins
• ¼ H chain at amino terminals = variable region
(VH), ¾ = constant regions (CH1, CH2, CH3)
• Binding of specific Ag @ amino terminal
portions of H & L chains (VH & VL) →
protein chains (2 sheets of antiparallel distinct
stretches of aa)
• Ig digestion by papain → 2 Ag-binding
fragments (Fab) + 1 crystallizable fragment
(Fc) → Ig functions other than Ag direct
binding
 Immunoglobulins
• 2 Fab regions → IgG bind 2 Ag molecules →
divalent
• Site on Ag to which an Ab binds = antigenic
determinant (epitope)
• Area in which papain cleaves Ig (between
CH1–CH2) = Hinge region → flexibility +
allows both Fab arms move independently →
helping them to bind to antigenic sites that
variable distances apart (eg, on bacterial
surfaces)
 Immunoglobulins
• Fc & Hinge regions differ in the different
classes of Ab, but overall Ab structure is
similar (Y)
 Immunoglobulins
• 2 general types of L chains : kappa (κ) &
lambda (λ) → distinguished on basis structural
differences of CL
• Always 2 κ or 2 λ L chains → never mixture of
κ&λ
• Humans : κ → more frequent
 Immunoglobulins
• 5 classes of H chain (differences in CH
regions) : λ, α, μ δ, and ε → μ and ε chains
have 4 CH domains
• Type of H chain → class of Ig (IgG, IgA, IgM,
IgD, and IgE) → effector function
Immunoglobulins
Immunoglobulins
Immunoglobulins
 Immunoglobulins
• Variable regions (VL & VH) → quite
heterogeneous → no 2 variable regions from
different humans have identical aa sequences
• Aa analyses : variable regions → relatively
invariable regions + other hypervariable
regions
• VL = 3 hypervariable regions; VH = 4
hypervariable regions
• Hypervariable regions (CDRs) → Ag binding
site ≈ specificity of Ab
 Immunoglobulins
• Constant regions, particularly CH2 & CH3 (&
CH4 of IgM and IgE) → Fc → class specific
effector functions of different Ig, eg,
complement fixation or transplacental passage
• IgG only the basic tetrameric structure, while
IgA and IgM can exist as higher order
polymers of 2, 3 (IgA), or 5 (IgM) tetrameric
units
 Immunoglobulins
• L & H chains synthesized as separate
molecules → assembled within B cell or
plasma cell → mature Ig
 Hemostasis & Thrombosis
• Medical emergencies
• Coronary & cerebral arteries → major cause of
death
• For rational tx
• Hemostasis: cut or severed vessel → cessation
of bleeding
• Thrombosis: damaged or removed blood vessel
endothelium lining (eg ruptur of atherosclerotic
plaque)
 Hemostasis & Thrombosis
• Hemostasis: intial vasoconstriction → ↓ blood
flow distal injury
• Hemostasis & thrombosis = 3 phases
• 1. formation of loose & temporary platelet
aggregate: platelets bind collagen →
thromboxane A2 + release ADP → activate
other platelet. Coagulation → thrombin →
further platelet activation. Platelets change
shape + fibrinogen → hemostatic
plug/thrombus
 Hemostasis & Thrombosis
• 2. platelet aggregate + fibrin mesh → more
stable hemostatic plug/thrombus
• 3. partial or complete dissolution hemostatic
plug/thrombus by plasmin
 Hemostasis & Thrombosis
• 3 types of thrombi/clots:
• White thrombus: platelets + fibrin + poor
erythrocytes; blood flow is rapid (arteries)
• Red thrombus: primarily red cells + fibrin;
morphologically = test tube, retarded blood
flow or stasis (eg, veins) with or without
vascular injury, or @ injury in conjunction with
an initiating platelet plug
• Fibrin deposit: very small blood vessels or
capillaries
Hemostasis & Thrombosis
Hemostasis & Thrombosis
 Hemostasis & Thrombosis
• Extrinsic & intrinsic pathway → fibrin clot
formation
• Tissue injury → extrinsic: initiation of fibrin
clot
• Negatively charged surfaces in vitro, eg, glass
→ intrinsic
• Both → prothrombin activation → thrombin →
fibrinogen cleavage → fibrin clot
Hemostasis & Thrombosis
Hemostasis & Thrombosis
 Hereditary Bleeding Disorders
• Hemophilia A: deficiency of f.VIII, X
chromosome-linked, history of royal families
of Europe
• Hemophilia B: deficiency of f.IX; clinical
features ≈ A, specific assays → distinguish
• Von Willebrand disease: 1% of population,
von Willebrand factor (endothelial cells →
large multimeric glycoprotein into plasma →
stabilizes f.VIII & promotes platelet adhesion)
deficiency or defect
Fibrinolysis
Fibrinolysis
 Fibrinolysis
• Injury: vascular endothelium → inactive tissue
plasminogen activator (t-PA) + fibrin →
active → cleaves plasminogen → plasmin →
digests fibrin → soluble degradation products
→ dissolves clot → released into fluid phase
→ inactivated
• Monocytes, macrophages, fibroblasts,
epithelial cells → urokinase → main action:
degradation of extracellular matrix
 Lab Tests
• Platelet count → number of platelet
• Bleeding time → overall test of platelet and
vessel wall function
• Platelet aggregation → responses to specific
aggregating agents
• Activated partial thromboplastin time (aPTT or
PTT) → intrinsic pathway; monitor heparin
therapy
 Lab Tests
• Prothrombin time (PT) → extrinsic pathway;
effectivenessof oral anticoagulants such as
warfarin
• Thrombin time (TT)
• Concentration of fibrinogen
• Fibrin clot stability
• Fibrin degradation products
 Stem Cells
• Stem cell: cell → unaltered daughter cells (ie,
self-renewal) & generate specialized cell types
(potency)
• Totipotent: all cells in organism
• Pluripotent: differentiate cells of 3 germ
layers
• Multipotent: only cells of closely related
family
• Unipotent: only 1 type of cell
 Stem Cells
• Stem cells: embryonic & adult
• Adult → more limited capabilities to
differentiate → overcomed by genetic
approaches
Hematopoietic Stem Cells
 Hematopoietic Stem Cells
• RBC & platelets → = diff until
megakaryocyte erythroid progenitors
• Lymphoid origin → multipotent progenitors
• WBC → common myeloid progenitors
• Regulator : stem cell factor, thrombopoietin,
various interleukins, erythropoietin, etc) +
transcription factors also involved
 Hematopoietic Stem Cells
• Stem cell factor → cytokine important role in
proliferation of hematopoietic stem cells &
progeny
• Thrombopoietin → glycoprotein important
regulating production of platelets by the bone
marrow
• Interleukins → cytokines produced by
leukocytes; regulate hematopoiesis & immune
system
 RBC
• F/: ↔ O2 & CO2, protons of tissue metabolism
• Membrane sorrounding Hb, no organelles
• ATP from glycolysis
• Biconcave shape + ion & water transport
• 120 d → ↓ ie hemolytic anemias
• Prod regulation by EPO
RBC
RBC
RBC
RBC
RBC
RBC
 Anemia
• WHO: Hb <130 g/L (♂) and <120g/L (♀)
Anemia
THANK YOU
 Take Home Exam
• Extrinsic & intrinsic pathways
• G6PD
• Spherocytosis & elliptocytosis