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).

Gihan Gawish.Dr
 Hemostasis
Primary hemostasis: platelets immediately
form a plug at the site of injury

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
 Primary hemostasis . 1
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.

Gihan Gawish.Dr
 3. The adhesion is strengthened by
circulating proteins (vWF) Binding
intermediaries
vWF
von Willebrand factor

BLOOD PLATELETS

vWF ENDOTHELIAL CELL

vWF

vWF
EXPOSED COLLAGEN
Gihan Gawish.Dr
4. This adhesion activates the platelets.

5. Activated platelets release the contents of stored

granules into the blood:

1. plasma ADP,
2. serotonin,
3. platelet activating factor (PAF),
4. von Willebrand factor (vWF) ,
5. platelet factor 4
6. thromboxane A2 (TXA2)
Gihan Gawish.Dr
 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.

Gihan Gawish.Dr
7. The activated platelets changed shape from
spherical to stellate

8. The fibrinogen cross-links with glycoprotein

aid in aggregation of adjacent platelets.

Gihan Gawish.Dr
 Secondary hemostasis. 2
The coagulation cascade
The coagulation cascade of secondary
hemostasis has two pathways:

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.

Gihan Gawish.Dr
 The Clotting Cascade

Thrombin

Fibrin Fibrin Fibrinogen

Gihan Gawish.Dr polymers monomers
 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.

Gihan Gawish.Dr
 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

Gihan Gawish.Dr
 Coagulation factors are generally indicated
by Roman numerals, with a lowercase a
appended to indicate an active form.

Gihan Gawish.Dr
 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.
Gihan Gawish.Dr
 Prothrombin is produced in the liver and is
post-translationally modified in a vitamin K-
dependent reaction that converts ten
glutamic acids on prothrombin to
gamma-carboxyglutamic acid (Gla).

In the presence of calcium, the Gla residues

promote the binding of thrombin to
phospholipid bilayers

Gihan Gawish.Dr
 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
Gihan Gawish.Dr
increase the stability of the fibrin clot.
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

Gihan Gawish.Dr
Gihan Gawish.Dr
 Electron Micrograph of Fibrin

Gihan Gawish.Dr
 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.

Gihan Gawish.Dr
 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.
Gihan Gawish.Dr
 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 gamma-
carboxyl residues on

1. FXa
2. FIXa
(to the phospholipids
surfaces expressed by
platelets)
The Calcium-
Binding Region
Prothrombin binds calcium ions with the modified
of Prothrombin
amino acid g-carboxyglutamate (red).
Gihan Gawish.Dr
 2. Vitamin K
It is an essential factor to a hepatic gamma-
glutamyl carboxylase that adds a carboxyl group
to glutamic acid residues on:
Factor II,
Factor VII,
Factor IX
Factor X,
Protein S,
Protein C

Gihan Gawish.Dr
 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 a protease that is
switched on by the action of
thrombin.

Thus, thrombin has a dual function:

it catalyzes the formation of fibrin
and it initiates the deactivation of
the clotting cascade.

Gihan Gawish.Dr
 Fibrinolysis

Gihan Gawish.Dr
 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 XII and prekallikrein;

cleaves HMWK

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 of antithrombin III is
enhanced by heparin

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.
Gihan Gawish.Dr
 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.
Gihan Gawish.Dr
 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 best-known
coagulation factor disorders.
The three main forms are:

hemophilia A hemophilia C
)factor VIII deficiency( , factor XI deficiency(
hemophilia B ).mild bleeding tendency
(factor IX deficiency or
"Christmas disease")

Gihan Gawish.Dr
 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.

Gihan Gawish.Dr
 Coagulation
Tests

Gihan Gawish.Dr
 Coagulation Cascade

PT

VIIIa
PTT
Heparin

Hirudin,
Argatroban

Gihan Gawish.Dr
 Coagulation and Fibrinolysis

Gihan Gawish.Dr
 Coagulation Tests
Screen test Other coagulation inhibitor Study,
Bleeding Time (Duke method, VIII, XI, XII
Template method), Thrombin Time, DIC profile
PT, PTT Fibrinogen, FDP, 3P Test, D-dimer

Antiphospholipid syndrome Fibrinolysis

Dilute Russell Viper Venom Time Euglobulin clot lysis time,
(dRVVT), Anti-Cardiolipin Ab, ACA, Plasminogen activator inhibitor,
IgG, Anti-Phospholipid Ab, APA, IgG, Alpha2-antiplasmin
Anti-Cardiolipin Ab, ACA, IgM
PLT function
Platelet aggregation
Coagulation factor
Factor I (Fibrinogen), II, V, VII, VWF,
VIII, IX, X, Urea solubility test, Thrombosis
APC Resistance, Protein S,
Antithrombin III, Protein C,
Gihan Gawish.Dr
Plasminogen
 Bleeding time. 1
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
 Activated Partial. 2
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.

Gihan Gawish.Dr
 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

Gihan Gawish.Dr
 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 1. Pregnancy

intrinsic pathway factors
2. In conditions causing
2. Heparin activation of factors

3. Lupus AC (antiphospholipid
antibody)

Gihan Gawish.Dr
 Prothrombin Time (PT). 3
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.

Gihan Gawish.Dr
 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
Gihan Gawish.Dr
 Thrombin clotting time. 4
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
 D-Dimers and Fibrin. 5
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
 Disseminated Intravascular. 6
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, D-

Dimer<324 ug/L

Gihan Gawish.Dr