Anti-Clotting Mechanisms

Dr Sujanthi Wickramage
Dept. of Physiology
FMAS
06/12/2017
 What keeps the blood fluid?

• Smooth intact vascular endothelium

– Inhibits intrinsic pathway of blood coagulation

• Glycocalyx attached to the endothelium

– A mucopolysaccharide
– Repels clotting factors and platelets
What keeps the blood fluid? cont.
• Synthesis of prostacyclin (PGI2) and nitric
oxide (NO) by the vascular endothelium
– Cause vasodilatation
– Inhibit platelet aggregation

• Thrombomodulin expression
– A protein bound to endothelium
– Binds thrombin
What keeps the blood fluid? cont.
• Protein C
– A plasma protein
– Inactivates activated clotting factors V and VIII
– Inactivates inhibitor of t-PA

• Antithrombin III
– Binds and inactivates thrombin and other clotting
factors
What keeps the blood fluid? cont.

• Heparin
– Inactivates Thrombin and other clotting factors

• Plasminogen Activator
– Forms Plasmin
Thrombomodulin

• A protein bound to the endothelium

• Binds with Thrombin in the blood and forms
Thrombomodulin – Thrombin complexes
• Activates Protein C
– Activated Protein C (APC), along with co-factor
Protein S, inactivates clotting factors V and VIII
 Thrombumodulin – Thrombin complex

Activate

Remove Thrombin Protein C

from circulation
Protein S
Inactivate
Factor Va and VIIIa

Prevent
clotting!
• When endothelium is damaged:
– Endothelial smoothness lost
– Glycocalyx – Thrombomodulin layer lost
– Activates Factor XII and platelets
– Sets off intrinsic pathway of clotting
Antithrombin III
• The most important anticoagulants in the
blood are the ones that remove thrombin
i.e.
– Fibrin fibers
– Antithrombin III (Antithrombin – Heparin cofactor)
When a clot is forming:
A) 85 – 90% of the Thrombin formed gets
absorbed into fibrin fibers.
This prevents the excessive spread of the
Thrombin, and therefore the clot.

B) The Thrombin that is left (10 – 15%) combines

with Antithrombin III.
This would inactivate this Thrombin in 12 – 20
minutes.
Heparin

• Powerful anticoagulant
• Found in low concentrations in blood
• Produced by mast cells and basophils in
pericapillary connective tissues
– In lungs, liver and many other tissues
• Highly negatively charged polysaccharide
Heparin cont.
• By itself – has little/no anticoagulant
properties
• However, when combined with Antithrombin
III – increases the effectiveness of
Antithrombin III by 100 fold to 1000 fold!
• Heparin – Antithrombin III complex removes:
– Thrombin
– Activated clotting factors IX, X, XI, XII
 Lysis of a clot
• When a clot is being formed at the site of an
injury, many plasma proteins get trapped in
the clot
• A plasma protein called Plasminogen also gets
trapped in the clot in large amounts
• This plasminogen is in the inactive state, and
therefore cannot lyse the clot
• Plasminogen receptors are located on
endothelial cells
Lysis of a clot cont.
• A few days after the injury, after the bleeding
has completely ceased, a substance called
Tissue Plasminogen Activator (t-PA) slowly
start getting released from
– Injured tissues
– Vascular endothelial cells
• t-PA can activate Plasminogen and form
Plasmin
Plasmin
• Resembles Trypsin (Most important proteolytic
digestive enzyme of pancreatic secretion)
• Digests:
– Fibrin fibers
– Fibrinogen
– Prothrombin
– Clotting factors V, VIII, XII
• Causes lysis of the clot
 Plasminogen t-PA Plasmin
(Profibrinolysin) (Fibrinolysin)

Digests clotting factors

& Fibrin fibers

Removes remaining
unnecessary blood clot
i.e. Removes minute clots from
peripheral vessels & re-opens vessels
• Action of plasmins – opposed by antiplasmins
produced in the liver
Plasminogen system:
• Also called Fibrinolytic system
• Specially designed to dissolve any fibrin
formed
• Lysis of fibrin and fibrinogen will produce
fibrin degradation products (FDP)
• FDP inhibits Thrombin
Fibrinogen, Fibrin

Plasmin

FDP

(-)

Thrombin
Plasminogen can be activated into Plasmin
by:

• t-PA
• u-PA (Urokinase-type Plasminogen activator)
• Thrombin
• Activated clotting factor XII
• Other activators formed from endothelial and
other cells
– Kallikrein
 Commercially available t-PA
• Clinically used to dissolve thrombi (clots)
formed inside pathologically damaged blood
vessels (ex: Myocardial infarction).
i.e.
– Urokinase
– Streptokinase (Bacterial enzyme, Fibrinolytic)
– t-PA (Human t-PA now produced by recombinant
DNA techniques)
 Anticoagulants
1. Heparin
– Naturally occurring anticoagulant
– Clinically given in larger doses for anticoagulant
properties
– Facilitates the action of antithrombin III

2. Aspirin
– Inhibits platelet aggregation
Anticoagulants cont.
3. Calcium ions
– For in vitro prevention of clotting in collected blood
– By adding agents that bind Ca2+
i.e.
• Sodium citrate
• Potassium oxalate
• ethylene diamine tetraacetate (EDTA)
– These cannot be used in vivo as calcium is essential
for other body functions
Anticoagulants cont.

4. Coumarin derivatives
– Inhibit the action of Vitamin K
– Used in vivo
i.e.
• Warfarin
• Dicumarol
Vitamin K
• A fat soluble vitamin
• Need fats in the diet for absorption
• Also produced by colonic bacteria
• Is a cofactor for the enzyme that catalyzes the
conversion of glutamic acid residues to γ-
carboxyglutamic acid residues.
Vitamin K cont.
• Six of the proteins involved in clotting require
conversion of a number of glutamic acid
residues to γ-carboxyglutamic acid residues
before being released into the circulation.
• Therefore, all six are vitamin K-dependent.
• These proteins are factors II (prothrombin),
VII, IX, and X, protein C, and protein S
Laboratory Investigations
 Laboratory Investigations
1. Blood film and platelet count
– Show the number and morphology of platelets
– Normal range for the platelet count:
150 – 400 x 109 /L

Abnormalities:
– Thrombocytopaenia (Low platelet count)
– Abnormal platelet morphology seen in leukemia
and lymphoma
Lab investigations cont.
2. Bleeding time (BT)
– Tests the efficiency of vasoconstriction and
platelet plug formation
– A sharp prick is made on the earlobe and time
taken for the bleeding to stop is noted.
– Normal range: 2 – 9 minutes

Prolonged in:
– Thrombocytopaenia
– Thrombasthenia (platelet function defect)
Lab investigations cont.
3. Clotting time (CT)
– The time required for the blood to clot in a glass
tube
– Tests the intrinsic pathway
– Factor XII gets activated by contact with glass
– Normal: 5 – 10 minutes

Prolonged in:
– Liver disease
– If the patient is on warfarin
Lab investigations cont.
4. Prothrombin time (PT)
– Tests the extrinsic pathway
– Oxalated plasma (calcium ions removed) is treated
with calcium ions and tissue factor
– Time taken for clotting is noted
– Normal: 11 – 16 seconds
Prolonged in:
– Abnormalities of factors VII, X, V, II or I
– Liver disease
– If the patient is on warfarin
Lab investigations cont.

5. Activated partial thromboplastin time (APTT)

– Tests the intrinsic pathway
– Normal: 25 – 39 secs

Prolonged in:
– Liver disease
– If the patient is on warfarin
 Lab investigations cont.
6. Thrombin time (TT)
– Performed by adding thrombin to a sample of the
patient's plasma
– Time taken for a clot to form is noted
– Normal: 10 – 15 seconds
Prolonged in:
– Fibrinogen deficiency
– dysfibrinogenaemia (abnormal function of fibrinogen)
– Inhibitors such as heparin or FDPs
Bleeding Disorders
 Inherited coagulation disorders
1. Haemoplilia A
– Also called ‘Classic haemophilia’
– Genetic disorder, occurs almost exclusively in males
– Sex linked recessive (carried through X chromosomes)
– Caused by an abnormality or deficiency of Factor VIII
– 85% of those with haemophilia
– Bleeding into joints (Haemarthroses)
– Treated with Factor VIII concentrates
Inherited coagulation disorders cont.

2. Haemoplilia B
– Also called ‘Christmas disease’
– Caused by an abnormality or deficiency of Factor
IX
– 15% of those with haemophilia
– The inheritance and clinical features are identical
to haemophilia A
– Treated with factor IX concentrates
Inherited coagulation disorders cont.

3. vWF deficiency
– Due to a deficiency or abnormality of vWF
– Problems with platelet adhesion to damaged
subendothelium as well as stabilizing Factor VIII in
plasma
– The result is defective platelet function and
Factor VIII deficiency
 Acquired coagulation disorders
1. Vitamin K deficiency
– Clotting factors II, VII, IX and X and proteins C and
S are vitamin K dependent
– Without it, these factors cannot bind calcium
Vitamin K deficiency maybe seen in:
– Malabsorption of fats
– Use of oral anticaogulant drugs (i.e. Warfarin)
– New borns
– Cholestasis
Acquired coagulation disorders cont.

2. Liver disease
May give rise to a number of defects in haemostasis:
– Vitamin K deficiency (Due to intrahepatic and
extrahepatic cholestasis)
– Reduced synthesis of clotting factors (Due to
extensive hepatocellular damage)
Acquired coagulation disorders cont.

3. Disseminated intravascular coagulation (DIC)

– Results from the presence of large amounts of
traumatized or dying tissue in the body
– This would release great quantities of tissue factor
into the blood
– Widespread generation of fibrin & clot formation
– Block vessels, blood supply to tissues reduced
– At the same time – person may bleed due to
clotting factor depletion!
Acquired coagulation disorders cont.

• DIC maybe seen in:

– Widespread trauma
– Burns
– Septicaemia (Bacterial endotoxins activating
clotting mechanisms)
– Haemolytic transfusion reactions
– Malignant disease
– Obstetric causes
– Snake bite
– Surgery
Changes in Lab Investigations…
PT APTT BT Plt
count
Thrombocytopaenia
Haemophilia
vW disease
Vit K deficiency/
Warfarin
Aspirin
DIC
The End