Gihan Gawish.Dr
Dr. Gihan Gawish
Coagulation
 Coagulation is a complex process by which
blood forms clots.
 Coagulation begins almost instantly after
an injury to the blood vessel has damaged
the endothelium (lining of the vessel).
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Hemostasis

Primary hemostasis: platelets
form a plug at the site of injury

immediately
Secondary
hemostasis:
occurs
simultaneously:
1. proteins in the blood plasma (clotting factors)
respond in a complex cascade
2. to form fibrin strands
3. which strengthen the platelet plug
Gihan Gawish.Dr
1. Primary hemostasis
Platelet activation
1. Damage to blood vessel walls exposes
sub endothelium proteins, most notably
collagen, present under the endothelium.
2. Circulating platelets bind collagen with
surface collagen-specific glycoprotein
Ia/IIa receptors.
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3. The adhesion is strengthened by circulating
proteins (vWF)
Binding intermediaries
von Willebrand factor
vWF
BLOOD
PLATELETS
ENDOTHELIAL CELL
vWF
vWF
vWF
EXPOSED
COLLAGEN
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4. This adhesion activates the platelets.
5. Activated platelets release the contents of stored
granules into the blood:
1.
2.
3.
4.
5.
6.
plasma ADP,
serotonin,
platelet activating factor (PAF),
von Willebrand factor (vWF) ,
platelet factor 4
thromboxane A2 (TXA2)
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6. The granules' contents activate:
Gq-linked protein receptor cascade
Increased calcium concentration in the platelets' cytosol
Activates protein kinase C
Activates phospholipase A2 (PLA2).
Modifies the integrin membrane glycoprotein
Increasing its affinity to bind fibrinogen.
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7. The activated platelets changed shape from
spherical to stellate
8. The fibrinogen cross-links with glycoprotein
aid in aggregation of adjacent platelets.
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2. Secondary hemostasis
The coagulation cascade

The coagulation cascade
hemostasis has two pathways:
of
secondary
1. The contact activation pathway (formerly known
as the intrinsic pathway)
2. The tissue factor pathway (formerly known as
the extrinsic pathway)
That lead to fibrin formation.
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The Clotting Cascade
Thrombin
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Fibrin
polymers
Fibrin
monomers
Fibrinogen
The biological of the coagulation
factors
 The coagulation factors are generally serine proteases
(enzymes).
 There are some exceptions. For example, FVIII and FV
are glycoproteins
 Factor XIII is a transglutaminase.
 Serine proteases act by cleaving other proteins at specific
sites.
 The coagulation factors circulate as inactive zymogens.
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The pathways are a series of
reactions
 zymogen + its glycoprotein co-factor are
activated to become active components that
then catalyze the next reaction in the
cascade, ultimately resulting in cross-linked
fibrin.
zymogen is inactive enzyme precursor of a
serine protease
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 Coagulation factors are generally indicated
by Roman numerals, with a lowercase a
appended to indicate an active form.
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Generation of Thrombin
 The prothrombin (Factor II) gene is located
on the eleventh chromosome (11p11-q12(
 Thrombin is produced by the enzymatic
cleavage of two sites on prothrombin by
activated Factor X (Xa).
 The activity of factor Xa is greatly enhanced
by binding to activated Factor V (Va), termed
the prothrombinase complex.
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 Prothrombin is produced in the liver and is
post-translationally modified in a vitamin Kdependent reaction that converts ten
glutamic acids on prothrombin to gammacarboxyglutamic acid (Gla).
 In the presence of calcium, the Gla residues
promote the binding of thrombin to
phospholipid bilayers
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 Deficiency of vitamin K or administration of
the anticoagulant warfarin inhibits the
production of gamma-carboxyglutamic acid
residues, slowing the activation of the
coagulation cascade.
 In human beings the level of prothrombin in
the blood stream increases after birth and
typically peaks on the 8th day after which
the prothrombin level lowers to normal
levels
Gihan Gawish.Dr
Action of Thrombin
 Thrombin converts fibrinogen to an active form
that assembles into fibrin.
 Thrombin also activates factor XI ,factor V ,and
factor VIII .This positive feedback accelerates the
production of thrombin.
 Factor XIII is also activated by thrombin. Factor
XIIIa is a transglutaminase that catalyzes the
formation of covalent bonds between lysine and
glutamine residues in fibrin. The covalent bonds
increase the stability of the fibrin clot.
Gihan Gawish.Dr
Action of Thrombin In platelets
 In addition to the thrombin activity in the
coagulation cascades, thrombin also
promotes platelet activation, via activation
of protease-activated
receptors on
the
platelet.
Gihan Gawish.Dr
Ca
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Electron Micrograph of Fibrin
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 A fibrin clot is formed by the interplay of the
intrinsic, extrinsic, and final common
pathways.
 The intrinsic pathway begins with the
activation of factor XII (Hageman factor) by
contact with abnormal surfaces produced by
injury.
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 The extrinsic pathway is triggered by trauma,
which activates factor VII and releases a
lipoprotein, called tissue factor, from blood
vessels. Inactive forms of clotting factors are
shown in red; their activated counterparts
(indicated by the subscript "a") are in yellow.
 Stimulatory proteins that are not themselves
enzymes are shown in blue.
 A striking feature of this process is that the
activated form of one clotting factor catalyzes the
activation of the next factor. I.
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Cofactors
Various substances are required for the
proper functioning of the coagulation
cascade:
1. Calcium and phospholipids (a platelet
membrane constituent)
 They are required for the tenase and
prothrombinase complexes to function.
Gihan Gawish.Dr

Calcium mediates the
binding of the complexes
via the terminal gammacarboxyl residues on
1. FXa
2. FIXa
(to
the
phospholipids
surfaces expressed by
platelets)
Prothrombin binds calcium ions with the modified
amino acid g-carboxyglutamate (red).
Gihan Gawish.Dr
The
CalciumBinding Region
of Prothrombin
2. Vitamin K
 It is an essential factor to a hepatic gammaglutamyl carboxylase that adds a carboxyl group
to glutamic acid residues on:
 Factor II,
 Factor VII,
 Factor IX
 Factor X,
 Protein S ,
 Protein C
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 In adding the gamma-carboxyl group to
glutamate residues on the immature clotting
factors Vitamin K is itself oxidized.
Gihan Gawish.Dr
 Another enzyme ,Vitamin K
epoxide reductase VKORC
reduces vitamin K back to its
active form.
 Vitamin K epoxide reductase
is pharmacologically important
as a target for anticoagulant
(antagonists) drugs warfarin
and related coumarins such
as acenocoumarol ,
phenprocoumon and
dicumarol .
Gihan Gawish.Dr
 These drugs create a deficiency of reduced
vitamin K by blocking VKORC, thereby inhibiting
maturation of clotting factors.
 Other deficiencies of vitamin K (e.g. in
malabsorption ,)or
disease( hepatocellular
carcinoma impairs the function of the enzyme and
leads to the formation of PIVKAs (proteins formed
in vitamin K absence) this causes partial or non
gamma carboxylation and affects the coagulation
factors ability to bind to expressed phospholipids
Gihan Gawish.Dr
The Clotting Process Must Be
Precisely Regulated
 There is a fine line between hemorrhage
and thrombosis. Clots must form rapidly
yet remain confined to the area of injury.
 Activated factors are short-lived because
they are diluted by blood flow, removed
by the liver, and degraded by proteases.
 For example, the stimulatory proteins
factors Va and VIIIa are digested by
protein C
Gihan Gawish.Dr
 Protein C is
switched on
thrombin.
a protease that is
by the action of
 Thus, thrombin has a dual function:
it catalyzes the formation of fibrin
and it initiates the deactivation of
the clotting cascade.
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Fibrinolysis
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 Fibrinolysis is the process wherein a fibrin
clot is broken down
 Plasmin cuts the fibrin mesh at various
places, leading to the production of
circulating fragments that are cleared by
other proteases or by the kidney and liver.
 Plasmin is produced in an inactive form,
plasminogen, in the liver.
Gihan Gawish.Dr
Coagulation factors and related
substances
 I fibrinogen Forms clot (fibrin (
 (II( prothrombin Its active form (IIa) activates I, V, VII, VIII,
XI, XIII, protein C, platelets
 Tissue factor Co-factor of VIIa (formerly known as factor III)
 Calcium Required for coagulation factors to bind to
phospholipid (formerly known as factor IV
 )V( proaccelerin, labile factor )Co-factor of X with which it
forms the prothrombinase complex
Gihan Gawish.Dr
 XII (Hageman factor) Activates factor XI and
prekallikrein
 XIII (fibrin-stabilizing factor) Cross links fibrin
 von Willebrand factor Binds to VIII, mediates
platelet adhesion
 Prekallikrein Activates
cleaves HMWK
XII
and
prekallikrein;
 high molecular weight kininogen (HMWK)
Supports reciprocal activation of XII, XI, and
prekallikrein
Gihan Gawish.Dr
 antithrombinIII Inhibits IIa, Xa, and other
proteases
 heparin cofactor II Inhibits IIa, cofactor for
heparin
 protein C Inactivates Va and VIIIa
 protein S Cofactor for activated protein C
Gihan Gawish.Dr
Specific inhibitors of
clotting factors
1.Antithrombin III is the most important one,
 It is a plasma protein that inactivates thrombin by
forming an irreversible complex with it.
 It resembles alpha 1-antitrypsin except that it
inhibits thrombin much more strongly than it
inhibits elastase.
 Also, it blocks other serine proteases in the
clotting cascade namely, factors XIIa, XIa, IXa,
and Xa.
Gihan Gawish.Dr
2.Heparin
 The inhibitory action
enhanced by heparin
of
antithrombin
III
is
 It is a negatively charged polysaccharide found in
mast cells near the walls of blood vessels and on
the surfaces of endothelial cells
 Heparin acts as an anticoagulant by increasing
the rate of formation of irreversible complexes
between antithrombin III and the serine protease
clotting factors.
 Antitrypsin and antithrombin are serpins, a family
of serine protease inhibitors.
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Electron Micrograph of a Mast Cell. Heparin
and other molecules in the dense granules are
released into the extracellular space when the
cell is triggered to secrete.
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3. Alpha 1-antitrypsin
 which normally inhibits elastase
 alpha 1-Antitrypsin activity normally increases
markedly after injury to counteract excess elastase
arising from stimulated neutrophils.
 The mutant a 1-antitrypsin caused the patient's
thrombin activity to drop to such a low level that
hemorrhage ensued.
Gihan Gawish.Dr
Disease and clinical significance
of thrombosis
1.
Hemophilias
are the
coagulation factor disorders.
best-known
The three main forms are:
hemophilia A
(factor VIII deficiency)
hemophilia B
(factor IX deficiency or
"Christmas disease")
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hemophilia C
(factor XI deficiency,
mild bleeding tendency).
 2. von Willebrand disease
 It is the most common hereditary bleeding disorder
and is characterized as being inherited autosomal
recessive or dominant.
 In this disease there is a defect in von Willebrand
factor (vWF) which mediates the binding of
glycoprotein Ib (GPIb) to collagen.
 This binding helps mediate the activation of
platelets and formation of primary hemostasis.
Gihan Gawish.Dr
3. Deficiency of Vitamin K
 It may also contribute to bleeding disorders
because clotting factor maturation depends
on Vitamin K.
4. Liver diseases:
 Some clotting factors; II, IX, VII, X are
synthesized in liver
 Liver diseases
deficiency of these
factors
bleeding disorders.
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Coagulation Tests
Gihan Gawish.Dr
Coagulation Cascade
PT
PTT
VIIIa
Heparin
Hirudin,
Argatroban
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Coagulation and Fibrinolysis
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Coagulation Tests
Screen test
Bleeding Time (Duke method,
Template method), Thrombin Time,
PT, PTT
Other coagulation inhibitor Study,
VIII, XI, XII
DIC profile
Fibrinogen, FDP, 3P Test, D-dimer
Antiphospholipid syndrome
Dilute Russell Viper Venom Time
(dRVVT), Anti-Cardiolipin Ab, ACA,
IgG, Anti-Phospholipid Ab, APA, IgG,
Anti-Cardiolipin Ab, ACA, IgM
Fibrinolysis
Euglobulin clot lysis time,
Plasminogen activator inhibitor,
Alpha2-antiplasmin
Coagulation factor
Factor I (Fibrinogen), II, V, VII, VWF,
VIII, IX, X, Urea solubility test,
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PLT function
Platelet aggregation
Thrombosis
APC Resistance, Protein S,
Antithrombin III, Protein C,
Plasminogen
1. Bleeding time
 Done with a template.
 Time taken for the blood to stop
 Normal range 2-10mts
 Prolonged in plt disorders, low plts, severe
anemia, Vwf, collagen vascular disease
 Great variability in results, unreliable,
invasive, insensitive
Gihan Gawish.Dr
2. Activated Partial
Thromboplastin Time (aPTT)
 It is a performance indicator measuring the
efficancy of both the "intrinsic" and the
common coagulation pathways.
 It is also used to monitor the treatment
effects with heparin.
 It is used in conjunction with the
prothrombin time (PT) which measures the
extrinsic pathway.
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Methodology (aPTT)
 Container: blue top (3.2% citrate) tube
 Collection: Deliver tubes immediately to the laboratory
 In order to activate the intrinsic pathway, phospholipid, an
activator (such as silica, celite, kaolin, ellagic acid), and
calcium (to reverse the anticoagulant effect of the citrate)
are mixed into the plasma sample .
 The time is measured until a thrombus (clot) forms.
 The test is termed "partial" due to the absence of tissue
factor from the reaction mixture.
Gihan Gawish.Dr
Performing APTT






At 37 ̊C
Plasma
Add kaolin/elgaic acid
Phospholipid source
Calcium
Time the appearance of clot
Gihan Gawish.Dr
Activated Partial Thromboplastin
Time (aPTT)
 Causes for Rejection: Specimen received
more than 4 hours after collection, tubes not
filled, clotted specimens, visible hemolysis
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Activated Partial Thromboplastin
Time (aPTT)
 Reference Interval: 20-25 to 32-39 seconds.
Prolong in newborns
 However, newborns and infants do not normally
experience bleeding, because a balance between
procoagulants and natural anticoagulants is
maintained.
 Critical Values: >100-150 seconds
Gihan Gawish.Dr
PTT in clinical states
 PTT prolonged in
 PTT shortened in
1. Congenital or acquired def of
intrinsic pathway factors
1. Pregnancy
2. Heparin
3. Lupus AC (antiphospholipid
antibody)
Gihan Gawish.Dr
2. In conditions causing
activation of factors
3. Prothrombin Time (PT)
 Clotting time from factor VII to fibrin clot
 PT↑: fibrinogen or factors II, V, VII, or X
deficiency,
therapeutic
anticoagulants
(heparin, hirudin, or argatroban)
 Container: Blue top (3.2% sodium citrate)
tube
Gihan Gawish.Dr
Prothrombin Time (PT)
 heparin prolongs the PT to a lesser extent
than PTT. Hirudin and argatroban prolong
the PT and PTT.
► Collection: directly from a peripheral vein
Causes for Rejection: Specimen received
more than 24 hours after collection, tube not
filled, clotted specimen, visible hemolysis
Gihan Gawish.Dr
Prothrombin Time (PT)
 Reference Interval: 10-12 to 12-14
seconds. Prolong in newborns. Up to 16
seconds at birth, and gradually shortens into
the adult normal range by the age of 6
months.
 Critical Values: >30 seconds
Gihan Gawish.Dr
Prothrombin Time (PT)
 Methodology: Reagent called thromboplastin
(phospholipid with tissue factor and calcium)
added, measure clot formation time.
 Vitamin K trial may be performed with an
unexplained PT prolongation. If vitamin K
deficiency, the PT becomes normal or significantly
shorter within 12-24 hours after vitamin K
administration.
Gihan Gawish.Dr
Prothrombin Time (PT)
 Monitoring warfarin: international normalized
ratio (INR), therapeutic goal is an INR of 2-3.
► INR = [patient PT/normal PT]ISI
► international sensitivity index (ISI), varies in
reagents
The ISI is usually between 1.0 and 1.4.
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Effects of Factor Deficiencies on
PT and PTT
 PTT Prolonged, PT Normal: Deficiencies of
factor VIII, IX, XI, and/or XII (intrinsic pathway)
 PT Prolonged, PTT Normal: Deficiency of factor
VII
(extrinsic
pathway),
mild-to-moderate
deficiencies of factor II, V, X, and/or fibrinogen
(common pathway)
 Both PT and PTT Prolonged: Deficiencies of
factor II, V, X, and/or fibrinogen (common
pathway), Multiple factor deficiencies
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4. Thrombin clotting time
 TT is a coagulation assay which is usually
performed in order to detect for the
therapeutic level of the anticoagulant
Heparin.
 It is also sensitive in detecting the presence
of a fibrinogen abnormality.
Gihan Gawish.Dr
Methodology of TT
 After liberating the plasma from the whole blood
by centrifugation ,bovine Thrombin is added to the
sample of plasma.
 The clot is formed and is detected optically or
mechanically by a coagulation instrument.
 The time between the addition of the thrombin
and the clot formation is recorded as the thrombin
clotting time
Gihan Gawish.Dr
Reference Interval TT
 The reference interval of the Thrombin
Clotting time is generally <21 seconds,
depending on the method and the endemic
patient population.
 Results outside of reference interval indicate
heparin
therapy,
Hypofibrinogenemia,
hyperfibrinogenemia fibrinogen abnormality,
or Lupus anticoagulant.
Gihan Gawish.Dr
5. D-Dimers and Fibrin
Degradation Products (FDP)
 Plasmin degrades fibrin clots
into
D-dimers
and
fibrin
degradation products (FDP).
 Limitations: elevate whenever
the coagulation and fibrinolytic
systems are activated.
 High rheumatoid factor (RF)
levels may cause false-positive
result.
Gihan Gawish.Dr
D-Dimers and Fibrin Degradation
Products (FDP)
 Methodology: semi quantitative or quantitative
immunoassays
 D-dimer is a specific FDP formed only by plasmin
degradation of fibrin, not of intact fibrinogen.
 D-Dimer and FDP(+): thrombosis, liver disease,
postoperatively, significant bleeding, hemodialysis,
eclampsia, sickle cell crisis, cancer, pregnancy
Gihan Gawish.Dr
6. Disseminated Intravascular
Coagulation (DIC) Screen
 D-dimer or fibrin degradation products
(FDP), prothrombin time (PT), activated
partial thromboplastin time (PTT), platelet
count, and fibrinogen. These tests are not
specific for DIC.
 Specimen: Plasma (and whole blood for
platelet count and peripheral blood smear)
Gihan Gawish.Dr
Disseminated Intravascular
Coagulation (DIC) Screen
 DIC is a common acquired coagulation
disorder resulting from excessive activation
of the coagulation system, usually due to
massive tissue injury, sepsis, or certain
pregnancy complications.
Gihan Gawish.Dr
Disseminated Intravascular
Coagulation (DIC) Screen
 disseminated micro vascular thrombi
→consumes platelets, coagulation factors,
and natural anticoagulants →PT, PTT
prolongations, bleeding
 Reference value: FDP< 5.0 ug/ml, DDimer<324 ug/L
Gihan Gawish.Dr