Plasma Proteins

INT’
• Blood
• Total volume is about 4.5 to 5 liters in adult human being
• If mixed with an anticoagulant and centrifuged, the cell components (RBC and WBC) are precipitated.
• The supernatant is called plasma
• Plasma
• About 55–60% of blood
• Clear creamy/yellowish cell free fluid of blood
• Total protein content of normal plasma is 6 to 8 g/100 mL.
• Serum
• defibrinated plasma
• If blood is withdrawn without anticoagulant and allowed to clot
• after about 2 hours liquid portion is separated from the clot
• lacks coagulation factors including prothrombin and fibrinogen
Plasma
• Intravascular – extracellular fluid
• In contact with the other body fluids
• Extracellular fluid
• CSF
• Urine
• An open medium of transport
• Aware system that reacts, responds to and reflects the metabolic and
pathological changes in the body
• Consist of
• 0.9% inorganic and 0.8% organic molecules
• About 7 gm/dl protein
Plasma Proteins
• Plasma Proteins
• Almost all, except immunoglobulins are synthesized in liver
• generally synthesized on membrane-bound polyribosomes
• Most are glycoproteins
• The plasma proteins consist of
• Hormonal
• Catalytic – enzymes
• Transport – various binding proteins
• Defense – immunoglobulins and complement
• Blood clotting, anticoagulants and fibrinolytic proteins
Fractionation of plasma proteins
• Plasma proteins are separated by
• Precipitation
• Gel filtration
• Chromatography
• Electrophoresis
Precipitation
• Plasm proteins sequentially precipitated by two methods
• Cohn’s fractionation
• Alcohol as a precipitating agent
• Used for commercial preparation of human serum albumin
• For clinical laboratories and research are Cohn’s fraction V
• Salt precipitation
• By Increasing salt concentration – called ‘salting out’
• Water is extracted from proteins
• Makes them insoluble resulting in their concentration
• Salt in the solution is represented as percent saturation
• Increased percent of saturation causes different proteins to be precipitated
• ‘Salting in’ dissolving by lowering salt concentration
• Commonly used salts – sodium sulphate and ammonium sulphate
Electrophoresis
• Electrophoresis refers
• The movement of charged particles through an electrolyte when subjected
to an electric field
• In clinical laboratory, electrophoresis is employed regularly for
separation of serum proteins
• Protein bands are visualized/quantitated by
• Exposing them to dyes – Coomassie blue/Amido black
• Dissolving in NaOH sol’n and concentration measured colorimetrically
• Normal and abnormal electrophoretic patterns can be compared
Cont
Normal patterns of electrophoresis
• In agar gel electrophoresis
• Normal serum is separated into 5 bands.
• Their relative concentrations are given below:
• Albumin : 55–65%
• Alpha-1 globulin : 2–4%
• Alpha-2 globulin : 6–12%
• Beta globulin : 8–12%
• Gamma globulin : 12–22%
• Pre albumin and transthyretin at front and fibrinogen at the back are
not seen as a separate bands
Cont
Cont
• Albumin has the maximum and gamma globulin has the minimum
mobility in the electrical field
• Most of the alpha-1 fraction is made up of
• alpha-1 antitrypsin
• Alpha-2 band is mainly made up by
• alpha-2 macroglobulin
• Beta fraction contains
• low density lipoproteins
• Gamma globulins contain the
• antibodies (immunoglobulins)
Abnormal Patterns in Clinical Diseases

Various abnormalities can be identified in the electrophoretic pattern

Infection, trauma or surgery
• Stimulate the release of cytokines and stress hormones
• Acute phase protein conc. increases
• α2 globulin peak is increased
• Albumin concentration decrease
• Negative acute phase reactant protein
• Peak is lowered
Chronic infections
• Gamma globulins are increased
• Smooth and wide based
Cont
Multiple Myeloma
• Proliferation of a single clone of plasma cells – mono clonal
• Gives rise to huge quantities of a single type of immunoglobulin
• Immunoglobulins appear as a sharp discrete band – known as M-band
Protein losing conditions
• Severe lose of proteins through kidneys
• In nephritic syndrome
• Due to damage to the glomerular basement membrane
• Small proteins are lost very fast – plasma albumin conc decreased too much
• Lose of gamma globulins
• α2 peak is raised - α2 macroglobulin being a very large molecule is retained in plasma
Cont
• Plasma proteins lost through GIT
• GIT ulcers or protein losing enteropathies
• A lot of proteins lost in burn patients
• Secretion from burnt body surfaces
• These conditions also show similar electrophoretic pattern as in nephrotic
syndrome
Hepatic disease
• Most plasma proteins synthesized in hepatocytes
• Any kind of liver disease would result decreased plasma proteins
• The synthesis of α2 globulins is stimulated in cirrhosis
• Lowered peaks for albumin, α1 globulin and β globulin
Cont
Severe malnutrition
• Plasma proteins serve as reservoirs of proteins
• May be used as energy source
• Lowering of albumin peak
Leukemia and primary immune deficiency
• The gamma globulin decreased in plasma
Cont
• 

• Plasma protein electrophoresis in various disease states. A, Normal. ALB, Albumin. B, Immediate response pattern. C, Delayed response

pattern. D, Liver cirrhosis. E,Protein-losing conditions (nephrotic syndrome, protein-losing enteropathy). F, Monoclonal gammopathy
(“paraprotein”).
Transporter Proteins
• Blood is a watery medium
• lipids and lipid soluble substances will not easily dissolve in the aqueous medium
• Hence carried by specific carrier proteins like
• Albumin - carries bilirubin, free fatty acids, calcium and drugs
• Pre albumin and transthyretin - carries thyroid hormones, thyroxin (T4) and tri-iodo thyronine (T3)
• Retinol binding protein RBP – carries Vit-A
• Thyroxin binding globulin TBG - specific carrier molecule for thyroxine and tri-iodo thyronine
• Transcortin - transport protein for cortisol and corticosterone
• Haptoglobin - for hemoglobin
• Hemopexin - for heme and
• Transferrin - for iron are important to prevent loss of iron from body
Albumin
• Name derived from the white precipitate of boiled egg
• Latin, albus = white
• Constitutes the major part of plasma proteins
• has one polypeptide chain
• 585 amino acids
• Elliptical in shape
• Synthesized by hepatocytes; therefore, estimation of albumin is a liver
function test
• Albumin is synthesized as a precursor, and the signal peptide is removed
as it passes through endoplasmic reticulum
Cont
• Half-life of albumin is about 20 days.
• Liver produces about 12 g of albumin per day, representing about 25%
of total hepatic protein synthesis
• Due to small size
• Can seep out of the blood vessels
• Is a component of most extravascular fluids
• CSF
• Interstitial fluid
• Urine
• Amniotic fluid
Functions of Albumin

• Colloid Osmotic Pressure of Plasma

• The total osmolality of serum is 278–305 mosmol/kg (about 5000 mm of Hg)
• produced mainly by salts, which can pass easily from intravascular to extravascular space
• electrolytes inside and outside the vascular compartments will cancel each other
• Effective osmotic pressure by proteins
• proteins cannot easily escape out of blood vessels
• It is about 25 mm Hg, and 80% of it is contributed by albumin
• The maintenance of blood volume is dependent on this
• According to Starling's hypothesis, at the capillary end, the blood pressure (BP) or
hydrostatic pressure expels water out, and effective osmotic pressure (EOP) takes
water into the vascular compartment
• Edema is seen in conditions where albumin level in blood is less than 2 g/dL
Cont
• Transport Function
• Carries many charges on its surface
• provide anchoring sites
• Albumin is the carrier of various hydrophobic substances in the blood.
• Bilirubin and non-esterified fatty acids
• Drugs (sulfa, aspirin, salicylate, dicoumarol, phenytoin)
• Only the unbound fraction of drugs is biologically active
• Hormones: Steroid hormones, thyroxine.
• Metals: Albumin transports copper.
• Calcium and heavy metals are non-specifically carried by albumin
Cont
• Buffering Action
• All proteins have buffering capacity
• albumin has maximum capacity
• Because of its high concentration in blood
• Albumin has a total of 16 histidine residues which contribute to this buffering
action.
• Nutritional Function
• All tissue cells can take up albumin by pinocytosis
• So albumin may be considered as the transport form of essential amino
acids from liver to extrahepatic cells
Cont
• Drug Interactions
• When two drugs having high affinity to albumin
• There may be competition for the available sites
• Consequent displacement of one drug.
• e.g. phenytoin-dicoumarol interaction.
• Protein-bound Calcium
• Calcium level in blood is lowered in hypoalbuminemia
• Thus, even though total calcium level in blood is lowered, ionized calcium
level may be normal, and so tetany may not occur
• Calcium is lowered by 0.8 mg/dL for a fall of 1 g/dL of Albumin.
Cont
• Therapeutic Use
• Human albumin is therapeutically useful to treat burns, hemorrhage
and shock.
• Edema
• Hypoalbuminemia will result in tissue edema (Starling's law).
• Malnutrition, where albumin synthesis is depressed (generalized edema)
• Nephrotic syndrome, where albumin is lost through urine (facial edema)
Pre-albumin or Transthyretin
• Faster mobility in electrophoresis than albumin – so pre albumin
• Small proteins that circulate in plasma as 1:1 dimer with RBP
• Migrate ahead of albumin in electrophoresis
• Homotetramer – half life about 48 hours
• Functions
• Transport of thyroid hormones
• Triiodothyroonine and thyroxine
• Has two binding sites
• About 10% the total plasma thyroid hormones
Retinol binding proteins RBP
• Monomeric protein
• Synthesized in liver
• Rate of secretion from liver influenced by
• binding of retinol to RBP
• Functions
• Transport of all trans retinol
• Symbiotic interaction with transthyretin as hetero dimer
• Provides protection to transthyretin
• Against being excreted in urine due to its small size
• Transthyretin enhances the binding kinetics of retinol with RBP
• Once retinol is delivered the dimer dissociates
• Zinc is required for the synthesis of RBP
• RBP as well as Vit A deficiency is associated with zinc deficiency
Proteins in Hb and iron metabolism
• The proteins involved are
• Haptoglobin Hp – for hemoglobin
• Hemopexin – for heme
• Ceruloplasmin Cp – as ferroxidase
• Transferrin TRF – for iron
Haptoglobin - Hp
• α2-glycoprotein
• Synthesized in hepatocytes
• Composed of 4 polypeptide chains
• Two types - Assuming (αβ)2 configuration
• α-chain – 83 amino acids and β-chain 245 amino acids
• Plasma half life 5.4 days
• Functions
• Binds to Hb and prevents the loss of heme iron
• Prevents renal tubules from damage by excreted Hb molecules
• Controlling inflammation – stimulated by inflammatory processes
• Has peroxisase enzyme activity – prevent lipid peroxidation
• Binds lymphocytes, monocytes and granulocytes controls their proliferation
• Removed by reticulo-endothelial cells
Hemopexin
• β- globulin
• By hepatic parenchyma
• Functions
• Binds ferro-protoporphyrin – released from brake down of Hb
• Has affinity for other porphyrins also from
• Myoglobin
• Catalase
• One hemopexin binds one porphyrin
• Removed from circulation by hepatocytes
Ceruloplasmin - Cp
• α2 globulin
• Single polypeptide – 1046 aa with 3 polysaccharide chains
• Contain about 95% total serum copper ions
• Each with 6 to 8 atoms
• Synthesis
• In liver mostly
• Small proportions by macrophages and lymphocytes
• Half life – 4-5 days
• Functions
• As ferroxidase to regulate the ionic state of iron
• Facilitate its incorporation into transferrin by oxidizing ferrous to ferric
• Cannot be copper transporting protein B/c of slow release of copper from Cp
Transferrin - TRF
• Iron transporter single polypeptide chain
• Synthesized in liver and small extent in reticuloendothelial cells
• Half life – 7 days
• About 50% in the extravascular compartment – CSF and lymph
• Affinity for binding iron Fe3+ but also for others Cu, Zn, Co and Ca
• Functions
• Binds iron absorbed from intestine or released from Hb in ferric form
• Most body cells have receptors for transferrin
• Iron in to the cells diffuses and incorporated into ferritin and hemosiderin
• Used for the synthesis of Hb, myoglobin and cytochrome
Iron conservation by haemopexin and haptoglobin
Acute phase reactants
• Concentration significantly increases during inflammatory processes
• The main acute phase reactants are
• α1-Acid glycoprotein – Orosomucoid
• C-reactive protein
• α1-Antitrypsin (AAT)
• α2-Macroglobulin (AMG)
α1-Acid glycoprotein – AAG Orosomucoid

• Synthesized primarily in liver

• Massively glycated – 45% CHO and 12% sialic acid
• Plasma half life 3 days
• Functions
• One of the lipocalins – bind with a number of lipophilic substances
• Implicated in down regulation of immune response
• Inhibits mitosis, might control replication of viruses/parasities
• Inhibits platelet aggregation and phagocytosis by neutrophils
• Could supply CHO for healing process
C-reactive protein - CRP

• Called CRP due to its ability to bind with C-polysaccharide on the cell wall of
streptococcus pneumoni
• Cyclic homo-pentamer
• Synthesized in liver
• Binds polysaccharides in bacteria, fungi, protozoa
• Functions
• After binding it activates the classical complement pathway to initiate
• Opsonization
• Phagocytosis
• Lysis of the antigens
• Detoxification of endogenous toxins from damaged tissue by
• binding with them
• Leading to their clearance
α1-Antitrypsin (AAT)

• Potent broad spectrum inhibitor of

• serine proteinases
• Eg Trypsin, plasmin, thrombin, chymotrypsin, elastase, and cathepsin.
• Single polypeptide
• 394 amino acids
• Half life 6 to 7 days
• Primarily synthesized in liver also locally by monocytes and macrophages
• Very small molecule and can diffuse into interstitial fluid
• Functions
• Protects tissue against protease digestion
• Inhibitors of leukocyte elastase from polymorphonuclear cells
• Uninhibited elastase causes emphysema
• Inhibitor of neutral protases, kallikrein, rennin , urokinase, plasmin and thrombin other from clotting cascade
α2-Macroglobulin (AMG)

• Protease inhibitor of plasma

• Tetramer
• Synthesized by liver parenchyma cells
• Monocytes and astrocytes in small amount
• Has large size
• Primarily intravascular protein
• Found in CSF or urine only under pathological conditions
• Meningitis and renal failure respectively
• 8-10% of plasma protein
• Functions
• Inhibits enzymes of kinin, complement, coagulation and fibrinolytic pathways
• Doesn’t inactivate enzymes – binds them at a site away and immobilize them
• Transport of cytokines and growth factors to the targeted tissue
Blood clotting factors
• Control of bleeding from cut skin or blood vessels – hemostasis
• Hemostasis
• Constriction of blood vessels
• Platelet aggregation
• Formation of protein meshwork to plug the injury site
• Coagulation
• Process of blood clotting
• A series of plasma proteins participate in the process
• Formation of fibrin clot through sequential activation of mediator proteins
• Rapid as each activation step multiplies the signal through a cascade
Cont
• The coagulation factors
• present in circulation as inactive zymogen
• converted to their active forms only when the clotting process is initiated
• prevent unnecessary intravascular coagulation.
• Several of these factors require calcium for their activation
• The calcium ions are chelated by the gamma carboxyl group of glutamic acid
residues of the factors, prothrombin, VII, IX, X, XI and XII.
• The gamma carboxylation of glutamic acid residues is dependent on
vitamin K and occurs after synthesis of the protein (post-translational
modification).
Pathways for the formation of fibrin clot

• Intrinsic pathway
• Begins when negatively charged surface exposed
• Triggers the activation of factor XII and factor XI
• Von willibrand factor binds factor XII to convert it to active XIIa
• Active factor XIIa acts on factor XI
• Leads to the assembly of factors VIIIa, IXa
• Factor X is converted to Xa which acts on prothrombin to convert it to
thrombin
• Factor VIII is activated by thrombin and is a cofactor that serves as receptor
for factor IXa on the platelets
Cont
• Extrinsic pathway
• Starts
• When the tissue factor is exposed
• on the surface of endothelial cells
• At the site of tissue injure
• The tissue factor activates factor VII to VIIa
• The tissue factor complex (tissue factor + factor VIIa)
• Activate factor IX of intrinsic pathway
• Thus providing a point of a cross talk
Prothrombin
• α2 globulin
• Single polypeptide
• At the time of tissue injury
• Prothrombin is activated to thrombin
• By activated factor Xa by the cleavage of an N-terminal peptide
• Thrombin is a serine protease
• Thrombin gets attached through negative charges on the surface platelets
• Calcium helps the interaction
• Thrombin acts on circulating fibrinogen around the platelets
• Fibrin dimers are formed
Fibrinogen to fibrin
• Fibrinogen is dimeric molecule
• Composed of two sets of three polypeptide chains
• Platelet associated thrombin cleaves 2 peptides from fibrinogen converting it to
fibrin
• by cleaving of Arg-Gly peptide bonds of fibrinogen.
• The fibrin molecules interact with each other
• Form fibrin dimer
• Dimers set align side to side to form fibrils
• Fibrils converge in parallel arrays to form
• bilateral network branches and
• thick fiber cables
Plasmin and fibrinolysis
• After the injury site has healed
• Fibrin clot must be retracted or dissolved
• Dissolution is tightly regulated
• To prevent thrombosis
• Dislodged clots can get attached and trapped in blood arteries
• Dangerous and can put the person at risk of coronary artery disease or stroke
• Plasmin
• The enzyme responsible for the dissolution
• Exist as zymogen plasminogen
• Converted to active plasmin by plasminogen activator
• Brakes the fibrin molecule into five pieces
Defense Proteins
• Plasma contains two fractions as per its resistance to heat
• The heat labile fraction
• The heat sable fraction
• Ehrlich named the heat labile fraction as complement and the heat stable
fraction as amboceptor
• The complement system
• At least 20 proteins
• Participate in two pathways – cascades
• Classical pathways and Alternate pathway
• Activation leads to production of active molecules – effectors
• Initiate inflammation and neutralization of the invading antigen/bacteria
• By lysis or phagocytosis
Cont
• Complement C3
• Primarily synthesized in hepatocytes
• Bacterial endotoxins can stimulate its synthesis from
monocytes/fibroblasts
• Synthesized as large propolypeptide that is cleaved into two chains
• α and β
• A thio-ester bond is formed between glu and cys residues, which is also
found in the structurally related C4,C5,C6
• Cleavage of this bond is essential for the interaction of these proteins
with cell membranes and other structures
Cont
• C3 provides functional link between the two complement activation
pathways
• Its activation through either of the pathways results in its cleavage in
to C3b and C3a
• Once released into circulation, the C3b components can have the
following effects
• Release of histamine from mast cells
• Stimulation of smooth muscle contraction
• Increase in vascular permeability
• Solubilization of antigens
Cont
• Complement C4
• Second component in the activation of classical pathway cascade
• Essential for this pathway
• Synthesized in hepatocytes as a single polypeptide which is cleaved
into three chains which are held together by disulfide bonds
• Just like C3 the a chain contains the thiol-ester bond
• C4 is activated by C1s and upon activation is cleaved into C4a and C4b
• The active C4b fragment binds with C2 leading to it cleavage again by
C1s
Cont
• The resultant C4b2b complex proceeds with the activation of the rest of
the cascade
• C4b2b complex binds to the immune complex and promotes their
clearance from the blood
• As C5 convertase activates C5 and C5b with C6, C6,C8 and C9 forms
membrane attack complexes which drills the bacterial membrane
• Drugs like procainamide interfere with the C4 mediated clearance of
immune complexes and could form the basis of drug induced lupus
syndromes
• C4a fragment is a mild anaphylatoxin
Cont
• Immunoglobulins
• The active component in Ehrlich’s heat stable protective fraction of plasm
immunoglobulin IG
• The gamma globulin on electrophoresis of serum is rich in Ig
• Some Igs have β or even α mobility
• Ig are large molecules that are synthesized in the plasma cells upon
stimulation by a foreign substance in plasma
• Called antibody since they have a property to bind with the specific
antigens against which they have been produced
• Five types of Ig classified as IgG, IgA, IgM, IgD and IgE