BIOCHEM A

Blood Chemistry – Dra. Gabaldon

BLOOD Three Generalizations:

 Viscous fluid with a cellular and fluid portion
o Cellular – RBCs, WBCs, platelets Most plasma proteins are synthesized in the liver
o Fluid – plasma, serum  Roughly 70% to 80% of all plasma proteins are synthesized in the
liver and they are membrane bound
o These include albumin, fibrinogen, transferrin, and most
components of the complement and blood coagulation
cascade
o Exception of von Willebrand factor, which is synthesized in
the vascular endothelium
o Exception is the γ-globulins, which are synthesized in the
lymphocytes.
 Genes for plasma proteins code for an aminoterminal signal
sequence that targets them to the endoplasmic reticulum. As this
leader sequence emerges from the ribosome, it binds to a
transmembrane protein complex in the endoplasmic reticulum
called the signal recognition particle. The emerging polypeptide
chain is pulled through the signal recognition particle into the
lumen of the endoplasmic reticulum, during which process the
leader sequence is cleaved off by an associated signal peptidase.
The newly synthesized proteins then traverse the major secretory
route in the cell prior to entering the plasma, during which
process they are subject to various posttranslational
PLASMA
modifications. Transit times through the hepatocyte from the site
 Composition
of synthesis to the plasma vary from 30 minutes to several hours
o Water, electrolytes, metabolites, nutrients, proteins and
for individual proteins.
hormones
 Most plasma proteins are glycoproteins covalently modified by
o Almost the same as the ECF
the addition of either N- or O-linked oligosaccharide chains
o Albumin is the major exception
PLASMA PROTEINS
 Total protein: 7-7.5 mg/dl
 Comprises total solids of plasma
Exhibit polymorphism
 Simple proteins, glycoproteins, lipoproteins
 A polymorphism is a Mendelian or monogenic trait that exists in
 Three major groups:
the population in at least two phenotypes, neither of which is rare
o Fibrinogen
 The ABO blood group substances are the best known examples of
o Albumin
human polymorphisms.
o Globulin
 Other human plasma proteins that exhibit polymorphism include
α1-antitrypsin, haptoglobin, transferrin, ceruloplasmin, and
Plasma Proteins in Distribution of Fluid:
immunoglobulins
 In the arterioles:
o The osmotic pressure (oncotic pressure) exerted by the
Have a half in the circulation
plasma proteins is approximately 25 mm Hg
 The half-life of a plasma protein is the time required for 50% of
o Since the hydrostatic pressure in the arterioles is
the molecules present at any given moment to be degraded or
approximately 37 mm Hg, with an interstitial (tissue)
otherwise cleared from the blood.
pressure of 1 mm Hg opposing it, a net outward force of
 Under normal circumstances, the resulting turnover, or
about 11 mm Hg drives fluid out into the interstitial spaces.
replacement, of older protein molecules with newly synthesized
 In venules:
ones occurs without any change in their total concentration.
o The hydrostatic pressure is about 17 mm Hg, with the
oncotic and interstitial pressures as described above; thus, a
net force of about 9 mm Hg attracts water back into the
circulation
 This is referred to as the Starling forces
 PATHOLOGIC: If the concentration of plasma proteins is markedly
diminished (eg, due to severe protein malnutrition), fluid will no
longer flow back into the intravascular compartment. The
resulting accumulation of fluid in the extravascular tissue spaces
results in a condition known as edema.

Page 1 of 7
  Because of its relatively low molecular mass (about 69 kDa) and
high concentration, albumin is thought to be responsible for 75%
to 80% of the osmotic pressure of human plasma.
 One of the major roles of albumin is to bind to and thereby
facilitate the transport of numerous ligands
o Free fatty acids (FFA), calcium, certain steroid hormones,
bilirubin, copper, and some of the plasma tryptophan
o A variety of drugs, including sulfonamides, penicillin G,
dicumarol, and aspirin

Haptoglobin
 A plasma glycoprotein that binds extra-corpuscular hemoglobin
(Hb) to form a tight noncovalent complex (Hb-Hp).
o Extracorpuscular Hgb – hemoglobin that is degraded and
released into the circulation
o About 10% is released and the other 90% is resent in old,
damaged red blood cells which will be degraded by the
histiocytic complex
 Amount of haptoglobin ranges in 40-180mg of Hgb-binding
capacity per dL
 Functions:
o Prevents loss of free hemoglobin into the kidney
o Conserves the valuable iron
o Protects the kidney
 Patients suffering from hemolytic anemias exhibit low levels of
haptoglobin. This is because, while the half-life of haptoglobin is
approximately 5 days, the Hb-Hp complex is removed rapidly by
the hepatocytes (half-life 90 minutes). Thus, when haptoglobin is
bound to hemoglobin, it is cleared from the plasma about 80
times faster than normally

Hemopexin
 Is a β1-globulin that binds free heme
 Albumin will bind some metheme (ferric heme) to form
methemalbumin, which then transfers the metheme to
Albumin
hemopexin
 Major protein of human plasma (3.4-4.7 g/dL)
 Makes up approximately 60% of the total plasma protein and 40%
Iron
is present in the plasma
 Is a key constituent of many human proteins, including
 The liver produces about 12g of albumin per day and would
hemoglobin, myoglobin and cytochrome P450 group of enzymes
represent 25% of the total hepatic protein synthesis
 A healthy adult loses only about 1.0 to 1.5 mg (<0.05%)
 Initially synthesized as preproalbumin (preproprotein according to
 However, an adult premenopausal female can experience iron
Harper’s). Its signal peptide is removed as it passes into the
deficiency due to blood loss during menstruation
cisternae of the rough endoplasmic reticulum by the signal
peptidase producing now the proalbumin. Proalbumin will bind
Iron Absorption
with furin and the hexapeptide at the resulting amino terminal is  Absorption of nonheme iron:
subsequently cleaved off farther along the secretory pathway
o Done by enterocytes of the proximal duodenum and is a
 Certain humans suffer from genetic mutations that impair their highly regulated process.
ability to synthesize albumin. Individuals whose plasma is
o Inorganic dietary iron in the ferric state (Fe3+) is reduced to
completely devoid of albumin are said to exhibit analbuminemia.
its ferrous form (Fe2+) by a brush border membrane-bound
o Although albumin is normally the major determinant of
ferrireductase, duodenal cytochrome b (Dcytb)
plasma osmotic pressure, persons suffering from
o Vitamin C, gastric acid, and a number of other reducing
analbuminemia show only moderate edema
agents present in food may also favor reduction of ferric to
 The plasma of patients with liver disease often shows a decrease
ferrous iron
in the ratio of albumin to globulins (decreased albumin-globulin o The transfer of iron across the apical membrane of the
ratio). The synthesis of albumin decreases relatively early in
enterocytes is accomplished via the divalent metal
conditions of protein malnutrition, such as kwashiorkor transporter 1 (DMT1 or SLC11A2)
 Mature human albumin consists of a single polypeptide chain 585
o Once inside the enterocytes, iron can either be stored
amino acids in length that is organized into three functional
bound to the iron storage protein ferritin but should be in
domains.
the ferric form
 Its ellipsoidal conformation is stabilized by a total of 17 intrachain
o The ferrous form can also be transferred across the
disulfide bonds
basolateral membrane into the circulation by the iron

Page 2 of 7
 exporter protein, ferroportin or iron-regulated protein 1
(IREG1 or SLC40A1)
o Hephaestin, a copper-containing ferroxidase homologous to
ceruloplasmin, oxidizes Fe2+ to Fe3+ prior to export. Iron is
transported in plasma in the Fe3+ form by the transport
protein, transferrin.
 Dietary iron that is ingested as heme is taken up by a distinct
mechanism. Following absorption by the enterocytes, iron is
released from heme by the enzymatic action of heme oxygenase.
Once released, the iron is either stored in association with ferritin
or transported into circulation by ferroportin.
Role of Transferrin
 The extreme toxicity of free iron is largely a consequence of its
ability to catalyze the formation of damaging reactive oxygen
 Transferrin (Tf) is a glycoprotein synthesized by the liver. This β1-
globulin has a molecular mass of approximately 76 kDa and
contains two high-affinity binding sites for Fe3
 The form of the protein in which both sites are occupied is called
holotransferrin (Tf- Fe)
 The concentration of Tf in plasma is approximately 300 mg/dL,
sufficient to carry a total of approximately 300 μg of iron per
deciliter of plasma.
 The binding sites in transferrin are not normally fully occupied, or
saturated. Typically, about 30% of the iron binding sites in
transferrin are occupied
 Saturation can decrease to less than 16% during severe iron
deficiency and may increase to more than 45% in iron overload
conditions
Transferrin Cycle
 For the delivery of transported iron, the recipient cell must bind
circulating transferrin via a cell surface transferrin receptor 1
(TfR1).
 The receptor-transferrin complex is then internalized by receptor-
mediated endocytosis as your early endosome. Once it matures
to a late endosome, the internal pH will now decrease or acidify.
The low internal pH will cause dissociation of your ferric iron from
the transferrin receptor complex
 The ferric iron needs to go out of the cell via DMT1, but DMT1
only carries ferrous form of iron
 Ferric iron is converted to its ferrous form by the ferrireductase,
Steap 3, and is then transported to the cytosol via DMT1
 The TfR1-apoTf complex is recycled back to the cell surface
 At the cell surface, apoTf is released from TFR1. TfR1 then binds
to new Tf-Fe
Ceruloplasmin
 Is an a2 globulin
 Carries 90% of copper present in the plasma, but although it
carries 90% it is not the major source of copper in the tissues
o The major source of copper in the tissues is albumin
 Synthesized in the liver and has a ferrioxidase activity
 One molecule of ceruloplasmin can bind 6 copper atoms
Iron in senescent RBCs is recycled by the macrophages
 Senescent RBCs are phagocytosed by the macrophages
 Hgb dissociated into globin and heme
 Heme is degraded by your heme oxygenase, releasing now the
ferrous form of iron and carbon monoxide
 The ferrous form is transported through the natural resistant
associated macrophage (NRAM-1) which will now bring the iron
out of the cell via ferroportin
 The ferrous has to be converted to ferric form via the
ceruloplasmin which has a ferroxidase capacity, in order to be
carried by the transferrin
*ferroportin – has role in both absorption and iron release
Page 3 of 7
Iron Hemostasis
Ferritin
 Human body can typically store up to 1 g of iron, the vast majority
of which is bound to ferritin.
 Ferritin (MW 440 kDa) is composed of 24 identical subunits, which
surround as many as 3000 to 4500 ferric atoms
 The subunits may be of the H (heavy) or the L (light) type
 The H-subunit possesses ferroxidase activity, which is required for
iron-loading of ferritin
 Plasma ferritin levels considered to be an indicator of body iron
stores.
 Hemosiderin, a partly degraded form of ferritin that contains iron,
can be detected in tissues by histological stains (eg, Prussian
blue), under conditions of iron overload (hemosiderosis)
Synthesis of TfR1 and Ferritin are Reciprocally Regulated
 The rates of synthesis of TfR1 and ferritin are reciprocally linked
to intracellular iron levels. When iron is low, TfR1 synthesis
increases and that of ferritin declines.
 Control is exerted through the binding of iron regulatory proteins
(IRPS) to hairpin loops structures called iron response elements
(IREs)
o Located in the 5′ untranslated region for ferritin
o Located in the 3’ untranslated region for TfR1
 IRPs bind to the IREs only when intracellular iron levels are low
o For transferrin: Binding at the 3′ UTR of mRNA for TfR1
increases TfR1 synthesis
o For ferritin: Binding of an IRP to the IRE located at the 5′
UTR of ferritin mRNA blocks translation
 Similarly, when iron levels are high, the IRBs dissociate and
doesn’t bind with IRE
o Under these circumstances translation of ferritin mRNA is
facilitated and TfR1 mRNA is rapidly degraded.
Hepcidin
 Is the chief regulator of systemic iron homeostasis
 Hepcidin binds to the cellular iron exporter, ferroportin,
triggering its internalization and degradation
 The consequent decrease in ferroportin results in decreased iron
absorption in the intestine and depressed iron recycling by
macrophages
 Together, these result in a reduction in circulating iron levels
(hypoferremia) as well as reduced placental iron transfer during
pregnancy
 When plasma iron levels are high, hepatic synthesis of hepcidin
increases, thus reducing iron absorption and macrophage iron
recycling
HEMOSTASIS AND THROMBOSIS
 Hemostasis is the cessation of bleeding from a cut or severed
vessel
 Thrombosis occurs when the endothelium lining blood vessels is
damaged or removed
 In hemostasis, there is initial vasoconstriction of the injured
vessel, causing diminished blood flow distal to the injury
Then, hemostasis and thrombosis share three phases:
 Formation of a loose and temporary platelet aggregate at the site
of injury. Platelets bind to collagen at the site of vessel wall injury,
form thromboxane A2, and release ADP, which activate other
platelets flowing by the vicinity of the injury
o Thrombin, formed during coagulation at the same site,
causes further platelet activation. Upon activation, platelets
change shape and, in the presence of fibrinogen and/or von
Willebrand factor, aggregate to form the hemostatic plug (in
hemostasis) or thrombus (in thrombosis).
 Formation of a 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.
Two pathways lead to fibrin clot formation
 The extrinsic and the intrinsic pathways
 Initiation of fibrin clot formation in response to tissue injury is
carried out by the extrinsic pathway.
 The intrinsic pathway can be activated by negatively charged
surfaces in vitro, for example glass
 Both pathways lead to the proteolytic conversion of prothrombin
to thrombin. Thrombin catalyzes cleavage of fibrinogen to initiate
fibrin clot formation
Page 4 of 7

EXTRINSIC PATHWAY
 Involves tissue factor, factors VII and X, and Ca2+
 The extrinsic pathway is initiated at the site of tissue injury with
the exposure of tissue factor (TF), located in the subendothelium
and on activated monocytes
 TF interacts with and activates factor VII
o TF is a zymogen containing vitamin K–dependent γ-
carboxyglutamate [Gla]
o Synthesized in the liver
o High-affinity binding sites for Ca2+
 TF acts as a cofactor for factor VIIa, enhancing its enzymatic
activity to activate factor X
 The reaction by which factor X is activated requires the assembly
of the extrinsic tenase complex (Ca2+-TF-factor VIIa)
 Factor VIIa cleaves an Arg-Ile bond in factor X to produce the two-
chain serine protease, factor Xa
 TF and factor VIIa also activate factor IX in the intrinsic pathway.
 Tissue factor pathway inhibitor (TFPI) is a major physiologic
inhibitor of coagulation. TFPI is a protein that circulates in the
blood where it directly inhibits factor Xa by binding to the enzyme
near its active site. This factor Xa-TFPI complex then inhibits the
factor VIIa-TF complex
INTRINSIC PATHWAY
 The formation of factor Xa is the major site where the intrinsic
and extrinsic pathways converge
 Involves factors XII, XI, IX, VIII, and X, as well as prekallikrein, high-
molecular-weight (HMW) kininogen, Ca2+, and cell-surface
exposed phosphatidylserine.
 This pathway results in the production of factor Xa by the intrinsic
tenase complex, in which factor IXa serves as the serine protease
and factor VIIIa as the cofactor.
 The intrinsic pathway can be initiated by “contact” in which
prekallikrein, HMW kininogen, factor XII, and factor XI are
exposed to a negatively charged activating surface.
 Factor XII is activated to factor XIIa upon proteolysis by kallikrein.
This factor XIIa, generated by kallikrein, attacks prekallikrein to
generate more kallikrein, setting up a positive feedback activation
loop.
 Factor XIIa, once formed, activates factor XI to XIa and also
releases bradykinin (a peptide with potent vasodilator action)
from HMW kininogen
o Thrombin can also activate factor XI, as well as factor VIII, V
and XIII
 In the presence of Ca2+, factor XIa activates factor IX
 Factor IXa cleaves an Arg-Ile bond in factor X to produce factor Xa
 The intrinsic tenase complex, composed of Ca2+-factor VIIIa-
factor X, which forms on the membrane surface of platelets
activated to expose procoagulant phosphatidylserine.
 Factor VIII (330 kDa), a circulating glycoprotein, is not a protease
precursor but a cofactor that serves as a receptor on the platelet
surface for factors IXa and X.
o Factor VIII is activated by minute quantities of thrombin to
form factor VIIIa
Page 5 of 7
COMMON PATHWAY  Thrombin also activates factor XIII to factor XIIIa
 Factor Xa, produced by either the extrinsic or the intrinsic  Factor XIIIa is a highly specific transglutaminase that covalently
pathway, activates prothrombin (factor II) to thrombin (factor IIa) cross-links γ–chains and, more slowly, α-chains of fibrin molecules
 The activation of prothrombin requires the assembly of a by forming peptide bonds between the amide groups of
prothrombinase complex, consisting of Ca2+, factor Va, and glutamine and the ε-amino groups of lysine residues
factor Xa  Such crosslinking yields a more stable fibrin clot with increased
 Factor V (330 kDa) is synthesized in the liver, spleen, and kidney resistance to proteolysis. This fibrin mesh serves to stabilize the
and is found in platelets as well as in plasma. hemostatic plug or thrombus
o Functions as a cofactor in a manner similar to that of factor
VIII
 Factor V is activated by thrombin and inactivated by activated
protein C
 Activated protein C limits the activation of prothrombin to
thrombin thereby controlling the formation of clot
 Prothrombin is a single-chain glycoprotein synthesized in the
liver. The amino terminal region of prothrombin contains 10 Gla
residues, and the serine-dependent active protease site close to
CONTROL OF THROMBIN ACTIVITY
the carboxy terminal region of the molecule.
 Once active thrombin is formed in the course of hemostasis or
 Upon binding to the complex of factors Va and Xa on the platelet
thrombosis, its concentration must be carefully controlled to
membrane, prothrombin is cleaved by factor Xa at two sites to
prevent further fibrin formation or platelet activation.
generate the active, two-chain thrombin molecule, which is then
 This is achieved in two ways
released from the platelet surface
o Feedback mechanisms
o Inactivation of any thrombin formed by circulating
FIBRINOGEN TO FIBRIN
inhibitors, the most important of which is antithrombin
 Thrombin converts fibrinogen to fibrin
Thrombin Inhibitors
Fibrinogen
 Antithrombin
 Is an abundant (3 mg/mL) soluble plasma glycoprotein that
o The most important
consists of a dimer of three polypeptide chains, (Aα, Bβ, γ), that is
o Contributes approximately 75% of the antithrombin activity.
covalently linked by disulfide bonds
o The endogenous activity of antithrombin is greatly
 The Bβ and γ chains contain asparagine-linked complex
potentiated by the presence of sulfated glycosaminoglycans
oligosaccharides
(heparins)
 The amino terminal regions of the six chains are held in close
 Heparin
proximity by a number of disulfide bond while the carboxyl
o Bind to a specific cationic site of antithrombin, which
terminal regions are spread apart.
induces a conformational change that promotes binding of
 The N-terminal A and B portions of the Aα and Bβ chains are
antithrombin to thrombin, as well as to its other substrates.
termed fibrinopeptide A (FPA) and fibrinopeptide B (FPB),
o The anticoagulant effects of heparin can be antagonized by
respectively; these domains are highly negatively charged as a
strongly cationic polypeptides such as protamine sulfate,
result of an abundance of aspartate and glutamate residues
which bind strongly to heparin, thus inhibiting heparin
 The negative charges contribute to the solubility of fibrinogen in
binding to antithrombin
plasma and importantly also serve to prevent aggregation by
 Thrombin is involved in an additional regulatory mechanism that
causing electrostatic repulsion between fibrinogen molecules.
operates in coagulation. It combines with thrombomodulin, a
glycoprotein present on the surfaces of endothelial cells. The
complex activates protein C on the endothelial protein C receptor
o In combination with protein S, activated protein C (APC)
degrades factors Va and VIIIa, thereby limiting their actions
in coagulation.

Thrombin
 Serine protease
 Hydrolyzes the four Arg-Gly bonds between the N-terminal
fibrinopeptides and the α and β portions of the Aα and Bβ chains
of fibrinogen releasing the FPA or FPA
 The release of FPA and FPB will now promote exposure of the
binding site and subsequent aggregation fibrin monomer in
staggered array
o Fibrin monomers spontaneously polymerize in a half-
staggered pattern to form long strands (protofibrils)
 Although insoluble, this initial fibrin clot is unstable, held together
only by the noncovalent association of fibrin monomers
Coumarin Drugs
 The coumarin drugs, which are used as anticoagulants, inhibit the
vitamin K–dependent carboxylation of Glu to Gla residues in the
amino terminal regions of factors II, VII, IX, and X and also
proteins C and S
 Coumarins inhibit vitamin K epoxide reductase thereby inhibiting
the reduction of the quinone derivatives of vitamin K to the active
hydroquinone forms
 Administration of vitamin K will bypass the coumarin-induced
inhibition and allow the posttranslational modification of
carboxylation to occur

Page 6 of 7
FIBRIN CLOT DEGRADATION
Plasmin
 Serine protease mainly responsible for degrading fibrin and
fibrinogen, circulates in the form of its inactive zymogen,
plasminogen (90 kDa)
 Rapidly inactivated by the fast-acting plasmin inhibitor, α2-
antiplasmin
 Activators of plasminogen of various types are found in most
body tissues, and all cleave the same Arg-Val bond in plasminogen
to produce the disulfide bridgelinked two-chain serine protease,
plasmin (Figure 55–6).
 Plasmin(ogen) specifically binds lysine residues on fibrin and so is
increasingly incorporated into the fibrin mesh as it cleaves it.
 TAFIa (activated thrombin activatable fibrinolysis inhibitor)
removes terminal lysines from fibrin, is able to inhibit fibrinolysis.
o Thrombin activates TAFI to TAFIa, thereby inhibiting
fibrinolysis during clot formation
 Tissue plasminogen activator (t-PA) is a serine protease that is
released into the circulation from vascular endothelium under
conditions of injury or stress and is catalytically inactive unless
bound to fibrin
o Upon binding to fibrin, t-PA cleaves plasminogen within the
clot to generate plasmin, which in turn digests the fibrin to
form soluble degradation products and thus dissolves the
clot
PLATELET ACTIVATION
 Platelets normally circulate in an unstimulated disk-shaped form.
Three major steps are involved:
 Platelets adhere to collagen via specific receptors on the platelet
surface via von Willebrand
o Together with thrombin, vWF will produce a conformational
change leading to pseudopod formation
 These adherent platelets release the contents of their storage
granules (the dense granules and the alpha granules)
o Granule release is also stimulated by thrombin.
o The interaction of thrombin with its G protein–coupled
receptors PAR-1 and PAR-4 stimulates the activity of an
intracellular phospholipase Ca (PLCa). This enzyme
hydrolyzes the membrane phospholipid
phosphatidylinositol 4,5-bisphosphate (PIP2, a
polyphosphoinositide) to form the two internal effector
molecules (1,2-diacylglycerol (DAG) and 1,4,5-inositol
trisphosphate (IP3)
o DAG stimulates protein kinase C whichresults in aggregation
and release of the contents of the storage granules
o IP3 causes release of Ca2+ which in turn will lead to
activation of a platelet cytosolic phospholipase A2 leading
to the formation of thromboxane A2
o Thromboxane A2, in turn, by binding to its protein–coupled
receptor, can further activate phospholipase C
 Aggregation
Page 7 of 7