Group No 2:

Members: Hasnain Riaz,Hassnat Yaqoob,Muhammad

Shakeel Nawaz,Saifullah,Muhammad Huzaifa
Presentation: Hematology(Practical)
Course Instructor: Doctor Hafiz Ifthkhar Hussain
Topic: Anticoagulants:Types,Mode of
action and Preparation of
anticoagulant bottles.
Introduction:
Anticoagulants, commonly known as blood thinners, are chemical
substance that prevent or reduce coagulation of blood prolonging
the coagulation time. Also used to dissolve blood clots form in the
blood vessels(emboli and thrombi).Thrombus is a clot that adhere
to walls of blood vessels and emboli is a clot that keeps moving.
Some of them occur naturally in blood eating animals such
as leeches and mosquitoes, where they help keep the bite area
unclotted.
long enough for the animal to obtain some blood and some
are synthesized in laboratory. As a class of medications,
anticoagulants are used in treatment of thrombotic
disorders. Oral anticoagulants are taken by many people
in pill or tablet form, and
various intravenous anticoagulant dosage forms are used in
hospital. Some anticoagulants are used in medical equipment
such
as sample tubes, blood transfusion bags, heart-lung machines
and dialysis equipment.
One of the first anticoagulants, warfarin , was initially approved as
a rodenticide.
Examples of anticoagulants include:
1)Apixaban (Eliquis)
2)Dabigatran (Pradaxa)
3)Edoxaban (Savaysa)
4)Enoxaparin (Lovenox)
5)Heparin
6)Rivaroxaban (Xarelto)
7)Warfarin

Anticoagulation:
The clotting mechanism of the blood to prevent or treat thrombosis and
embolism.
The anticoagulant drugs either inhibit the action of the coagulation factors(the
thrombin inhibitors, such as heparin and heparin-related agents)
Or interfere with the synthesis of the coagulation factors (the vitamin K
antagonists, such as warfarin) .The fibrinolytic system dissolves intravascular clots
as a result of the action of plasmin, an enzyme that digests fibrin. Plasminogen,
an inactive precursor, is converted to,
plasmin by cleavage of a single peptide bond. Plasmin is a relatively
nonspecific protease, it digests fibrin clots and other plasma
proteins, including several coagulation factors. Therapy with
thrombolytic drugs can dissolve both pathological thrombi and fibrin
deposits at sites of vascular injury, thus, such drugs also may
promote hemorrhage.

Usage of Anticoagulation Therapy:

* Treatment and Prevention of Deep Venous Thrombosis
* Pulmonary Emboli
* Prevention of stroke in patients with atrial fibrillation, artificial
heart
valves, cardiac thrombus.
* Ischemic heart disease
*During procedures such as cardiac catheterisation and apheresis

Types of anticoagulants:
Vitamin K antagonists
Direct Oral Anticoagulants(DOAS)
Low molecular weight heparins
Naturally occurring anticoagulants
Vitamin K antagonist:
Vitamin K “antagonists” like warfarin stop your liver from processing
vitamin K into “factors” that normally help clot your
blood. This curbs blood clotting.Cumarins are group of oral
anticoagulants that acts as vitamin K antagonist. Most common
example is warfarin. It works by reducing the effects of vitamin K,
which is a vitamin your body uses in the process of blood-clotting.
Warfarin is used to prevent unwanted clots from forming if you have
a condition that puts you at risk of conditions such as
atrial fibrillation. It is also used to prevent any clots that may have
already . formed in the blood vessels of your legs, lungs or heart
from becoming larger and causing problems. Warfarin is currently the
most prescribed anticoagulant although newer
anticoagulants are increasingly being prescribed.

Direct Oral anticoagulants:

DOACs work more quickly than vitamin K antagonists, which have been
around longer. DOACs can also be more predictable. These drugs tend to
work for shorter periods, so you may need to take them twice a day,
compared to once daily for other anticoagulants. DOACs include:

Direct thrombin inhibitors: These drugs interfere with your

body’s use of thrombin, a key enzyme that helps clot your
blood. Though usually injected under the skin, you can take it in
pill form as dabigatran (Pradaxa).
Direct factor Xa inhibitors: This type of anticoagulant stops
the Xa factor in the clotting process from working as it should.
These medications, which come in pill form, include apixaban
(Eliquis), betrixaban (Bevyxxa), edoxaban (Lixiana, Savaysa), and
rivaroxaban (Xarelto).

Low molecular weight heparins:

Low-molecular-weight heparins (4500 Da, or 15 monosaccharide
units) are isolated from standard heparin by gel filtration
chromatography, precipitation
with ethanol, or partial depolymerization with nitrous acid and other
chemical or enzymatic reagents. Low-molecular-weight heparins differ from
standard heparin and from each other in their pharmacokinetic properties
and mechanism of action.

Naturally occurring anticoagulants:

The most important natural anticoagulants are protein C, protein S,
and antithrombin (which used to be called antithrombin III until its
name was changed to antithrombin.
Heparin is a naturally occurring anticoagulant that
prevents the formation and extension of blood clots. Heparin does
not break down clots that
Have already formed (unlike tissue plasminogen activator) but
allows fibrinolysis to work normally to break down clots. Heparin is
commonly extracted from porcine intestinal mucosa or bovine
lung. Despite the heterogeneity in composition among different
commercial preparations of heparin, their biological activities are
similar (150 USP units/mg). The USP unit is the quantity of heparin
that prevents 1 mL of citrated sheep plasma from clotting for 1
hour after the addition of 0.2 mL of 1% CaCl2.
Mode of Action:
Naturally occurring anticoagulants:
Thrombosis is prevented by several regulatory mechanisms
that require a normal vascular endothelium.
▪ Prostacyclin (prostaglandin I2; PGI2) is synthesized by
endothelial cells and inhibits platelet aggregation and
secretion
Heparin sulfate proteoglycans synthesized by endothelial cells
stimulate the activity of antithrombin.
▪ Protein C is a plasma zymogen that is homologous to factors II,
VII, IX, and X; its activity depends on the binding of Ca2+ to g
carboxyglutamate (Gla) residues within its amino terminal domain.
▪ Activated protein C, in combination with its nonenzymatic Gla-
containing cofactor (protein S), degrades cofactors Va and
VIIIa and thereby greatly diminishes the activation of
prothrombin and factor Protein C is activated by thrombin
only in the presence of thrombomodulin, an integral
membrane protein of endothelial cells.
. Like antithrombin, protein C exerts an anticoagulant effect
in the vicinity of intact endothelial cells. Tissue factor
pathway inhibitor (TFPI) is found in the lipoprotein fraction
of plasma. When bound to factor Xa, TFPI inhibits factor Xa
and the factor VIIa –tissue factor complex. By this mechanism,
factor Xa may regulate its own production.
Heparin catalyzes the inhibition of several coagulation proteases
by antithrombin, a glycosylated, single chain polypeptide.
Antithrombin is synthesized in the liver and circulates in plasma,
inhibition occurs when the protease attacks a specific Arg- Ser
peptide bond in the reactive site of antithrombin and becomes
trapped as a stable 1:1 complex.
Heparin increases the rate of the thrombin antithrombin reaction
at least 1000-fold by
serving as a catalytic template to which both the inhibitor and the protease
bind. Binding of heparin also induces a conformational change in antithrombin
that makes the reactive site more accessible to the protease. Once thrombin
has become bound to antithrombin, the heparin molecule is released from the
complex.

Low molecular weight heparin:

LMWH binds to anti-thrombin, a serine protease inhibitor, and creates
a conformational change. This change accelerates its inhibition of
activated factor X in conversion of prothrombin to thrombin. Thus,
thrombin cannot convert fibrinogen to fibrin strands and clot
formation. Once this change occurs LMWH is freed
and can bind to another anti-thrombin molecule .LMWH also
directly inhibits thrombin as it is a heterogeneous mixture of
molecules, some containing enough polysaccharide sequence, but
this effect is much less than that of unfractionated heparin.
Direct thrombin inhibitor:
Bivalirudin: As a bivalent DTI, bivalirudin is a short synthetic peptide that
consists of the N-terminal and C-terminal active peptide domains of hirudin
joined by a linker5. It blocks thrombin activity by binding the active site (N-
terminus of bivalirudin) and
the fibrinogen-binding
exosite (C-terminus of bivalirudin)
of circulating
and clot-bound thrombin.
The majority of bivalirudin is
enzymatically eliminated
.
and considered safest to use in the presence of both hepatic and
renal dysfunction.

Lepirudin: Approved for anticoagulation in patients with HIT by the

Food and Drug Administration (FDA) in 1998, lepirudin is a desulfated
recombinant hirudin. Not only can it inhibit free and clot-bound thrombin,
6 ,it is not activated by platelet factor 4, making it more effective in the
presence of platelet-rich thrombi 7, In addition, lepirudin does not
interact with HIT antibodies. Because lepirudin is excreted mainly through
the kidneys, dose reductions are recommended in patients with renal
dysfunction.
Argatroban:
Argatroban is an L-arginine derivative that binds to the active site of free
fibrin and clot-associated thrombin, thereby preventing fibrin formation,
Factor V, VIII, and XIII activation, protein C activation, and platelet
aggregation.
This DTI is cleared by the liver
and, therefore, not recommended
for patients with
hepatic dysfunction. In 2000, the FDA
approved argatroban for the
treatment of thrombosis in
patients with HIT, and in 2001 approved its use during
percutaneous coronary interventions(PCI) in patients with or at
risk of HIT.

Direct factor Xa inhibitor:

These new agents exert their anticoagulant effect via direct
inhibition of a single Factor within the coagulation cascade (such
as Factor Xa or thrombin). ... Unlike indirect Factor Xa inhibitors,
rivaroxaban inhibits both free and clot-bound Factor Xa, as well as
prothrombinase activity, thereby prolonging clotting times.
Vitamin K antagonist:
The vitamin K antagonist warfarin sodium inhibits γ-carboxylation
activation of coagulation factors II, VII, IX, and X and proteins C
and S. The full anticoagulant effect of warfarin requires about 5
days of therapy. Warfarin is a vitamin K antagonist and has been
the most common choice of oral anticoagulant worldwide since the
1950s. It acts through inhibition of the enzyme vitamin K epoxide
reductase, which catalyzes the γ-carboxylation of the so-called
vitamin K–dependent coagulation factors (II, VII, IX, and X).
Inhibition of this enzyme results in the production of functionally
deficient factors thereby leading to impaired hemostasis.
Preparation of anticoagulant Bottles:
Purple top bottle:
Purple blood bottles contain EDTA (ethylenediaminetetraacetic acid),
which acts as a potent anticoagulant by binding to calcium in the blood.
Potassium EDTA(K2 or K3 ) is more preferred, rather than Sodium EDTA,
because Sodium EDTA is less soluble in water. 10% solution of
potassium EDTA (w/v) in distilled water is prepared as stock
anticoagulant for hematological studies. To collect 1ml blood, 10 ul of
this solution is added to the collection tube. Commonly used for
complete count,erythrocyte sedimentation rate and blood film for
abnormal cells and parasites.
Blue top bottles:
Contain buffered sodium citrate, which acts as a reversible
anticoagulant by binding to calcium ions in the blood and
subsequently disrupting the clotting cascade. Sodium citrate is also
added to blood products for transfusion and acts as a preservative
by stopping them from clotting in the bag. Commonly used for
coagulation screen: including bleeding time for platelet function
Green top bottles:
Heparin tubes are coated on the inside wall with spray-dried
lithium, ammonium or sodium heparin and are used to determine
analytes in clinical chemistry. The additive acts as an
anticoagulant, and blocks the clotting cascade.
Black top tube:
This tube contain sodium citrate as anticoagulant and is mainly
used for erythrocyte sedimentation rate.
Pink top tube:
This tube also contain the anticoagulant EDTA.And is specifically
used for whole blood samples. Common test done are group and
save ,crossmatch and direct combs test.
Grey top tube:
This tube contains two agents first one is sodium fluoride that
ensure no glucose breakdown occurs and potassium oxalate which
acts as anticoagulant. It is used for biochemistry tests and for
whole blood analysis.
Light green top bottle:
It contains lithium heparin which acts as anticoagulant and plasma
separator gel which separates plasma layer. Most commonly used
for biochemistry tests.
Yellow top tube:
This tube contain citrate dextrose as an anticoagulant. This tube is
used for collection of whole blood for special studies.
Why blood clot outside the body:
Many factors contribute blood clotting outside the body
1) Change in pressure
2) Change in fluidity of blood
3) Air lead to activation of clotting proteins like fibrinogen
and prothrombin , calcium a clotting factor that cause blood
clotting
4) Change in temperature
.
5) Heparin an anticoagulant produced by liver prevent blood
clotting inside body .Similarly blood in heparin added tube
may not undergo clotting cascade but heparin mixed blood in
watch glass clot due to air.
YouTube links:
https://youtu.be/_yQD0U3ZtCs
https://youtu.be/VxEIsPOdOLw
https://youtu.be/21VV9VKN_bY

Reference:
Anticoagulation drugs by Mina Kelleni
The Coumadin ( warfarin ) Help Book
Anticoagulation therapy: A clinical practice Guide Second
Addition