Far Eastern University-Dr.

Nicanor Reyes Medical Foundation

Plasma Proteins and Hemostasis and Thrombosis
Harper’s-Chapter 50 and 51
Dr. May Gabaldon 2014

Functions of Blood HAPTOGLOBIN(Hp)

Plasma
 Total protein: 7-7.5 mg/dl
o Comprises total solids of plasma
 Methods of separation of CHONs:
o Solvents
o Electrolytes
 Based on solubility characteristics
o “Salting out” method
 Sodium ammonium sulfate
o Electophoresis- most common medthod of
analyzing plasma CHONs
 Cellulos acetate –most widely
used medium
*Plasma proteins exerts oncotic pressure
*Starling forces
*Decrease in plasma protein concentration = edema
 Characteristics of plasma proteins:
o Synthesized in the liver
o Most are glycoproteins
 Except albumin
o Exhibit polymorphism
 Mendelian or monogenic trait that
exists in the population in at least
two phenotypes, neither of which
is rare.
o Have a half-life in the circulation
o Acute phase reactans
 C-reactive protein (CRP)
 Fibrinogen
 Nuclear factor kappa-B(NFkB)
 Involved in stimulation of
synthesis of certain of
acute phase reactants
 Bids extracorpuscular haemoglobin in a tight non-
covalent complex (Hb-Hp)
 It prevents loss of free haemoglobin to the kidney.
 This conserves the valuable iron present in
haemoglobin
 Hemolytic anemia
o ↑Hgb due to hemolysis - ↓Haptoglobin
levels
 Why? Half life of haptoglobin is
approx.5 days. Hb-Hb complex
reduces at about 90 minutes
which is rapidly being removed by
the liver.
 Thus when haptoglobin is bound
to haemoglobin, it is cleared from
the circulation 80 times faster than
normal.
 Haptoglobin-related protein
o Homologue of haptoglobin
o Inc. in cancers
o Mechanism not understood.
 Hemopexin
o Binds to free heme
o B1-globulin
 Albumin binds to metheme (Ferric heme) to for
methalbumin which then transfers the metheme to
hemopexin.
TRANSFERRIN- involved in iron transport
Non-Heme Iron Transport
1

ALBUMIN
 Major protein found in plasma
 Not a glycoprotein
 60% of total plasma protein
 Synthesized in the liver 1. Ferric iron is reduced to the ferrous form by a
 Synthesized as pre-proteinSignal peptide is luminal ferrireductase, duodenal cytochrome b
removed as it passes into the cisternae of RER (Dcytb).
 Decreased in hepatic diseases, protein malnutrition  Vitamin C, gastric acid may also reduce
and kwashiorkor. 2. Ferrous iron is transported into theenterocyte via
divalent metal transporter-1 (DMT-1).
 Responsible for 75-80% of osmotic pressure.
 DMT is non specific and may transport any
 Analbuminemia-lack of albumin
divalent cations such as Mg, Co, Zn, Cu,
o Mild edema
Pb
o Other plasma proteins compensate
 Major transport protein in the plasma

1| Honeylyn Bumanlag
Far Eastern University-Dr. Nicanor Reyes Medical Foundation
Plasma Proteins and Hemostasis and Thrombosis
Harper’s-Chapter 50 and 51
Dr. May Gabaldon 2014
3. Within the enterocyte, iron is either stored as ferritin, 5. Apotransferrin (apo-Tf) remains bound to TfR1.
or transported out of the cell, across the baslolateral Ferric iron is converted to its ferrous form by the
membrane, by ferroportin (Fp). ferrireductase, Steap 3.
 Ferroportin of iron regulated protein 1 6. Ferrous iron is then transported into the cytosol via
(IREG1/ SLC40A1) DMT1.
4. Ferrous iron is oxidized to its ferric form by the 7. The TfR1-apo-Tf complex is recycled back to the
ferroxidase, hephaestin. cell surface.
 Hephaestin is a copper-containing 8. At the cell surface, apo-Tf is released from TFR1.
ferroxidase homologous to ceruloplasmin 9. TfR1 then binds to new Tf-Fe.
which oxidizes ferrous to ferric 10. This completes the transferrin cycle.
5. Ferric iron is then bound by transferrin in blood
which transports it to various sites in the body. Iron in senecent erythrocytes is recycled by
 Excess iron that is stored in the macrophages
enterocytes as ferritin is lost when
enterocytes are slough off into the gut
lumen.
 Free iron is toxic due to formation of free radicals
 Fenton Reaction:

 In the plasma iron is tightly bound to transferrin

 Transferrin is a B1-globulin
 Holotransferrin (Tf-Fe)- Transferrin bound to two
irons.

TRANSFERRIN CYCLE
1. Senescent erythrocytes are phagocytosed by
macrophages.
2. Hemoglobin is degraded and iron is released from
heme by the action of the enzymeheme oxygenase.
3. Iron, in the ferrous form, is then transported out of
the macrophage via ferroportin (Fp).
4. In the plasma, it is oxidized to the ferric form by
ceruloplasmin before binding to transferrin (Tf).
5. Iron circulates in blood tightly bound to Tf.

*Plasma ferritin level is an indicator of body iron stores.

*Hemosiderin is an ill-defined molecule and appears to be
partly degraded form of ferritin that contains iron.

Intracellular Iron Hemostasis

1. Holotransferrin (Tf-Fe) binds to transferrin receptor 1

(TfR1) present in clathrin-coated pits on the cell
surface.
2. The TfR1–Tf–Fe complex is endocytosed
andendocytic vesicles fuse to form early
endosomes.
3. The early endosomes mature to late endosomes,
which have an acidic pH inside.
4. The low pH causes release of iron from its binding
sites on transferrin.

2| Honeylyn Bumanlag
Far Eastern University-Dr. Nicanor Reyes Medical Foundation
Plasma Proteins and Hemostasis and Thrombosis
Harper’s-Chapter 50 and 51
Dr. May Gabaldon 2014
1. The mRNA for ferritin is represented on the left, and
that for TfR1 on the right of the diagram. HEMOSTASIS AND THROMBOSIS
2. At high concentrations of iron, the iron bound to the
IRP prevents that protein from binding the IREs on  Hemostasis- cessation of bleeding from a cut or
either type of mRNA. severed vessels.
3. The mRNA for ferritin is able to be translated under  Thrombosis occurs when the endothelium lining of
these circumstances, and ferritin is synthesized. blood vessels is damaged or removed.
4. On the other hand, when the IRP is not able to bind
to the IRE on the mRNA for TfR1, that mRNA is CLOTTING FACTORS:
degraded.
5. In contrast, at low concentrations of iron, the IRP is Zymogens of Serine
able to bind to the IREs on both types of mRNA. proteases
6. In the case of the ferritin mRNA, this prevents it Factor XII Binds to negatively charged surface,
frombeing translated. eg, kaolin, glass; activated by high-
molecular-weight kininogen and
7. Hence ferritin is not synthesized. In the case of the kallikrein
mRNA for TfR1, binding of the IRP prevents that Factor XI Activated by factor XIIa
mRNA from being degraded, it is translated, and Factor IX Activated by factor XIa and factor
TfR1 is synthesized. VIIa
8. IRP, iron regulatory protein; IRE, iron response Factor VII Activated by factor VIIa, factor Xa,
and thrombin
element. Activated on the surface of activated
Factor X
platelets by tenase complex (Ca2+,
Hepcidin is a chief regulator of Systemic Iron factors VIIIa and IXa) and by factor
Hemostasis VIIa in the presence of tissue factor
and Ca2+
 It is synthesized in the liver as prohepcidiin Factor II Activated on the surface of activated
 Cleaved to generate bioactive hepcidin platelets by prothrombinase complex
Functions: (Ca2+, factors Va and Xa) [Factors
II, VII, IX, and X are Gla-containing
1. Decreases absorption of iron in the intestines zymogens] (Gla
2. Prevents recycling of iron from macrophages = -carboxyglutamate)
3. Reduce placental transfer of iron
Cofactors
Factor VII Activated by thrombin; factor VIIIa is
a cofactor in the activation of factor X
by factor IXa

Factor V Activated by thrombin; factor Va is a

cofactor in the activation of
prothrombin by factor Xa
Tissue factor (factor A glycoprotein located in the
subendothelium and expressed on
III) activated monocytes to act as a
cofactor for factor VIIa

Fibrinogen Factor I Cleaved by thrombin to form

fibrin clot
Thiol-Dependent Factor XIII Activated by thrombin;
stabilizes fibrin clot by covalent cross-
Transglutaminase linking

Regulatory and Other

Proteins
1. Hepcidin binds to and triggers the internalization Activated to activated protein C
Protein C
and degradation of ferroportin expressed on the (APC) by thrombin bound to
surface of enterocytes and macrophages. thrombomodulin; then degrades
2. This decreases iron absorption from the intestine factors VIIIa and Va
and inhibits iron release from macrophages, leading Acts as a cofactor of protein C; both
Protein S
to hypoferremia. proteins contain Gla ( -
carboxyglutamate) residues

Thrombomodulin Protein on the surface of endothelial

--end-- cells; binds thrombin, which then
activates protein C

3| Honeylyn Bumanlag
Far Eastern University-Dr. Nicanor Reyes Medical Foundation
Plasma Proteins and Hemostasis and Thrombosis
Harper’s-Chapter 50 and 51
Dr. May Gabaldon 2014

Intrinsic:
Activated by glass or
negatively charged surfaces

Extrinsic Pathway

Arg-Ile
Arg-Ile

4| Honeylyn Bumanlag
Far Eastern University-Dr. Nicanor Reyes Medical Foundation
Plasma Proteins and Hemostasis and Thrombosis
Harper’s-Chapter 50 and 51
Dr. May Gabaldon 2014
Extrinsic Pathway
1. Initiated with exposure of tissue factor
2. Tissue factor interacts with VII  VIIa
3. VII is a Gla-containing zymogen which has a high
affinity for Calcium
4. Tissue factor acts as a cofactor for factor VIIa,
enhancing it’s enzymatic activity to activate Factor X
(EXTRINSIC TENASE COMPLEX: Calcium, tissue
factor, VIIa, X)
5. Factor VIIa cleaves ARG-ILE bond in Factor X to
produce the two-chain serine protease Xa
6. Factor VIIa can also activate IXa in the intrinsic
pathway
Tissue factor pathway inhibitor (TFPI) is a major
physiologic inhibitor of coagulation. It is a protein that
circulates in the blood associated with lipoproteins. TFPI
directly inhibits factor Xa by binding to the enzyme near its
active site.
***This factor Xa–TFPI complex then inhibits the factor VIIa–
tissue factor complex.
Intrinsic Pathway
1. Initiated with the “contact phase”
2. Prekallikrein, HMWK, Factor XII and Factor XI are
exposed to a negatively charged activating surface
Ex. Kaolin in in vitro tests
3. Factor XII Factor XIIa by kallekrein
4. XIIa attacks prekallikrein to generate mor kallikrein
5. XII activates Factor XIXia
6. XIa in presence of Calcium activates IXIXa
7. IXa cleaves ARG-ILE bond in Factor X to produce
factor Xa
8. (INTRINSIC TENASE COMPLEX: Calcium, VIIIa,
IXa, X)
Common Pathway
1. Xa activates Prothrombin Factor IIIIa
2. Assembly of prothrombinase complex: Calcium,
Factor Va, factor Xa, Prothrombin which takes place
on platelets with exposed phospholipids
3. Thrombin converts Fibrin to Fibrinogen (FIBRIN
MONOMER; unstable clot)
FIBRINOGEN
FPA
FPB
 Amino terminal end bears excess negative charges
due to the presence of Aspartate and Glutamate, as
well as Tyrosine O-sulfate in FPB
 Negative charges contribute to fibrinogen in plasma
 It also serves to prevent aggregation by causing
electrostatic repulsion between fibrinogen
molecules.
5|
THROMBIN
 Serine protease formed by prothrombinase complex
 Hydrolyzes the four ARG-GLY bonds between
fibrino eptides and α and β portions of the Aα and
Bβ chains of fibrinogen
 The release of fibrinopeptides by thrombin
generates fibrin monomer.
 The removal of fibrinopeptides exposes binding
sites that allow the molecules of fibrin to aggregate
spontaneously in a regular staggered array forming
an insoluble fibrin clot.
 The initial form is rather weak, held together only by
the non-covalent association of fibrin monomers.
 In addition to converting Fibrinogen to fibrin, it also
converts Factor XIII to XIIIa
 XIIIa is a highly specific transglutaminase that
covalently cross-links fibrin molecules by forming
peptide bonds between amide groups of
glutamine and ε-amino groups of lysine
 Yielding a more stable firbin clot that is resistible to
proteolysis. It stabilizes the hemostatic plug or
thrombus.
Thrombin levels are fully controlled
 Thrombin level increases with intermediate clotting
factors increasing in concentration as one proceeds
down the cascade
 Inactivated by circulating inhibitors:
o Antithrombin- 75% of antithrombin activity
of the blood.
 Also inactivates factors IXa, Xa,
Xia, XIIa, and VIIa complexed with
tissue factor.
o α2-Macroglobulin- remainder of
antithrombin activity.
o Heparin co factor II
o α1-antitrypsin
 Heparin- potentiates antithrombin
 Protamine- binds strongly to heparin thus inhibiting
it’s binding to antithrombin (antidote).
 Thrombomodulin- a glycoprotein
Honeylyn Bumanlag
Far Eastern University-Dr. Nicanor Reyes Medical Foundation
Plasma Proteins and Hemostasis and Thrombosis
Harper’s-Chapter 50 and 51
Dr. May Gabaldon 2014
o Binds with thrombin to activate Protein C
on endothelial protein C receptor.
o In combination with Protein S, activated
protein C degrades factor Va and VIIIa

COUMARIN DRUGS
 Ex. Warfarin
 Inhibit Vitamin K-dependent carboxylation of Glu to
Gla residues in the amino terminal of factors II, VII,
IX, X, Protein C and S.
o These proteins all of which are synthesized
in the liver are dependent on Ca-binding
properties of Gla residues.
 Act by Inhibit Vitamin-K epoxide reductase
 thereby inhibiting the reduction of quinone
derivatives of Vitamin K to active hydroquinone PLATELET ACTIVATION
forms. 1. Adhesion to exposed collagen in blood vessels
 Antidote: VITAMIN K  Platelets adhere to collagen via:
o GPIa-IIa
Fibrin clots are dissolved by Plasmin o GPIb-IX-V- mediated by von
 Plasmin- degrades fibrin and fibrinogen Williebrand factor
 Plasminogen- inactive form of Plasmin, is activated o GPVI
through cleavage of its ARG-VAL bonds to form  Important in platelet adherence to
plasmin. subendothelium.
2. Release (exocytosis) of contents of their storage
granules
 Dense and alpha granules

 α2-antiplasmin- inhibitor of plasmin

 Activated thrombin activatable fibronolysis
inhibitor (TAFIa)
o Thrombin activates TAFI  TAFIa
o Acts by removing terminal lysine
residues from fibrin to inhibit
fibrinolysis
 Tissue plasminogen activator (tPa)
o Serine protease
o Activated when bound to fibrin
o Upon binding to fibrin, tPa cleaves
plasminogen to plasmin
o Plasmin in return dissolves the clot.
 Urokinase
o Prokinase Urokinase
o Isolated from urine
o Also activates plasminogen to plasmin
a. Thrombin initiates activation by interacting
with its receptor PAR-1, PAR-4, and GPIb-
IX-V on platelet plasma membrane
b. It’s reaction in return activates intracellular
Phospholipase Cβ.
c. Phospholipase Cβ hydrolyzes
phosphatidylinositol 4,5-biphosphate (PIP2)
d. PIP activates Diacylglycerol (DAG) which
stimulates protein kinase c (PKC) which
phosphorylates the protein pleckstrin
causing platelet aggregation.
e. PIP also activcates 1,4,5-Inositol
Triphosphate (IP3) which causes release of

6| Honeylyn Bumanlag
Far Eastern University-Dr. Nicanor Reyes Medical Foundation
Plasma Proteins and Hemostasis and Thrombosis
Harper’s-Chapter 50 and 51
Dr. May Gabaldon 2014
Ca2+ in the cytosol mainly from the dense
granules.
2+
f. Increase in Ca causes activation of platelet
cytosolic phospholipase A2 results in
liberation of arachidonic acid leading to
formation of Thromboxane A2
g. Thromboxane A2 binds to its protein-
coupled TP receptor which further activates
Phospholipase C.
3. Platelet aggregaton

ANTIPLATELET DRUGS
 ASPIRIN
o Antiplatelet drug
o Irreversibly acetylates cyclooxygenase system
involved in formation of thromboxane A2,
o It is a potent aggregator of platelets and also a
vasoconstrictor.
 CLOPIDOGREL

7| Honeylyn Bumanlag