Platelets

• Platelets or thrombocytes are disc-shaped,

anucleated “cell fragments.”
• Normal count- 1.5- 4 lac cells / cumm
 Platelet Count in Neubauer Chamber
Rees Ecker fluid – Sodium citrate, Formalin , Brilliant cresyl blue
 Thrombopoiesis
Pleuri Potent Haemopoietic Cell ( PPHSC)

Megakaryoblast (Progenitor cell )

Megakaryocyte

Platelets

• 1 Megakaryocyte forms 2000 - 4000 platelets.

• Regulated by feedback mechanism ( Thrombopoietin. )

 Life span & Fate of platelets

• 8 -12 days, average 10 days. Destroyed by tissue

macrophage system in Spleen .

• So spleenomegaly decreases platelet count.

• So spleenectomy increases platelet count.

 Dense granules

α granules

PDGF

Factor XIII

Contractile proteins - Actin , Myosin (Thrombosthenin) cause

platelet to contract & help in clot retraction.
PDGF (help in repair of damaged blood vessels)

Dense granules : (5-HT, Serotonin, ADP, ATP)

 α granules : (clotting factors V, VIII,vWF, PDGF)
 Platelets
Physiological variation-
• Age – less in infants
• After meals, after severe muscle exercise, at high
altitude increased.

• Pathological :
• Thrombocytosis - after spleenectomy,
after haemmorhage, severe injury,
stress.
• Thrombocytopenia - idiopathic, Bone Marrow
depression, acute leukemia, small pox, dengue fever,
toxaemia, septicaemia, uraemia.
 Properties of Platelets

1. Adhesiveness – Platelets on contact with surface,

become activated & stick to surface.

2. Activation - Platelets stick to each other. ADP &

TxA2 (Thromboxane A2)

3. Aggregation - Platelets, clumping together. (PAF)

Properties of Platelets
 Functions of Platelets
1. Role in Haemostasis -
Spontaneous arrest of bleeding by physiological
process.
a) Vasoconstriction,
b) Temporary haemostatic plug &
c) Definitive haemostatic plug formation.

2. Role in Clot Formation

3. Role in Clot Retraction

 Functions of Platelets
4. Role in repair of injured blood vessels –
PDGF (platelet derived growth factor from α granules ).

5. Role in defense mechanism-

Modify inflammatory response (by interacting with
WBC & secreting cytokines, chemokines, PDGF. )
Help in
phagocytosis of viruses & immune complexes.

6. Transport & storage function-

Can take up -HT (serotonin) from GIT , store it &
transport it to site of injury.
 Haemostasis
 Spontaneous arrest (stoppage) of
bleeding from
injured blood vessel by physiological
process.

 It is initiated immediately following

injury to blood vessel by several steps.
 Steps for Haemostasis

1. Vascular spasm/Vasoconstriction

2. Formation of temporary haemostatic plug

3. Formation of definitive haemostatic plug

4. Growth of fibrous tissue into blood clot to close

hole in vessel permanently.
 1. Vasoconstriction
 Vasoconstriction results from
• Nervous reflexes,
• local myogenic spasm,
• local humoral factors from traumatized tissue &
platelets.

 Degree of spasm proportionate to degree of trauma to

blood vessel.

 Initial vasoconstriction transient later maintained for

several minutes or even hrs by humoral facilitation.
 1. Vasoconstriction
 Following injury to blood vessel platelets get adhered to
damaged endothelium & exposed collagen.

 Platelets release 5HT & other vasoconstrictors like

endothelin which increase initial vasoconstriction.

 Usually arterioles & small arteries respond by

Vasoconstriction & decrease blood loss from damaged
blood vessel.
Vasoconstriction
 2. Formation of Temporary Haemostatic plug
a) Platelet Adhesion-
 Following injury to vessel wall platelets come in
contact with exposed collagen, laminin,
von- Willebrand factor on endothelial cells of vessel
wall.
 Promoted by Ca2+ & ADP

 Von- Willebrand factor – Glycoprotein, produced by

endothelial cells. Important role in haemostasis by
promoting platelet adhesion & regulates circulating
level of factor VIII. Deficiency leads to Von- Willebrand
disease
Temporary Haemostatic plug
2. Formation of Temporary Haemostatic plug
b) Platelet Activation (ADP, thrombin & TxA2 )
 Platelet binding to exposed collagen initiates its
activation.
 Activated platelets
1. Change their shape - swell with large no. of
pseudopodia.
2. Release granules
3. Platelets become sticky & adhere to each other

 Vicious cycle initiated. Further activation & adherence

of large no. of platelets forming a platelet plug.
Platelet Activation Pathways (1)
COLLAGEN

THROMBIN
ADP

GpIIb/IIIa
GpIIb/IIIa
GpIIb/IIIa
GpIIb/IIIa
GpIIb/IIIa Aggregation

Adrenaline Platelet
Adhesion
vWF

Endothelium

Exposed Collagen
• Gp IIb/IIIa (also K/a integrin complex) found on
platelets is a receptor for Fibrinogen & vWF & aids
platelet activation.
• Platelet activation by ADP, leads to
conformational change in platelet
receptors that induces binding to fibrinogen.
• Thereby helps in platelet activation, aggregation
Platelet Activation Pathways (2)

Thrombin

Platelet
Fibrinogen Binding Site
Fibrinogen

Platelet Aggregation
Herbert. Exp Opin Invest Drugs 1994;3:449-455.
 [Link] of Temporary Haemostatic plug
c) Platelet aggregation
 Platelet adherence & aggregation ultimately lead to
formation of platelet plug
 Inhibition of furthur plug formation by Prostacycline
which inhibits TxA2 formation & this localizes platelet
plug .
 Platelet aggregation
Platelet aggregation
(activates )

Phospholipase C
(activates )

Phospholipase A2 releases Arachidonic

acid from membrane Phospholipid

Converted to

Thromboxane A2
Prostacyclin
Inhibits TxA2, w/c inhibits
esPlatelet aggregation
Platelet aggregation

TxA2 causes Platelet aggregation, w/c along with Platelet adhesion helps in
formation of temporary haemostatic plug which stops bleeding.
Prostacyclin inhibits TxA2 formation and keeps platelet plug localised,
prevents intravascular spread of clot
 Role of Aspirin

• Aspirin by inhibiting Thromboxane A2

formation prevents Platelet aggregation.

• Therefore aspirin in low doses prevents

myocardial infarction,
stroke, transient ischaemic attack
[Link] of Temporary Haemostatic plug
 3. Formation of definitive haemostatic plug

 By process of clot formation which involves complex

series of events.

 Clotting or coagulation of blood occurs by

 Extrinsic pathway Intrinsic pathway

3. Formation of definitive haemostatic plug
 Haemostasis
4. Fibrous organization /dissolution of blood clot

 Once a clot has been formed it can either be

invaded by fibroblasts or can dissolve .
Fibrous organization in 1-2 wks.

 When clots occur where not needed, substance

within the clot activated & dissolve it.
 Haemostasis
Injury to vessel wall

Release of 5HT & other Endothelium damaged & Release of Tissue

vasoconstrictors Collagen exposed Thromboplastin
Attracts platelets
Platelets Adhesion (adhere to Via Intrinsic Via Extrinsic
exposed collagen) pathway pathway

Release ADP & TxA2

Activation of coagulation
Platelet Activation (platelets
change shape,release granules)
Formation of Fibrin
(Loose) Platelet Aggregation

3. Definitive
2. Formation of temporary
haemostatic plug
haemostatic plug
1. Vasoconstriction Can be removed by
Cannot be removed by
anticoagulants anticoagulants
 Limiting reactions (prevention of IV coagulation)

1. Removal of clotting factors IX a, Xa ,XIa, XIIa from

circulation by Antithrombin III.

2. Decrease in supply of clotting factor.

3. Presence of natural anticoagulants i.e. Heparin &

protein C.

4. Interaction between TxA2 & Prostacyclin.

 Blood coagulation
• When blood looses its fluidity within few minutes
& gets converted into a jelly like mass it is known
as clot & process known as Coagulation/clotting.

• It prevents excessive bleeding from wounds.

 Blood coagulation

• Blood clotting factors play major role.

• Most are inactive forms of proteolytic enzymes. When

active cause cascade of reaction of clotting process.

• Positive feedback mechanism.

 Coagulation Factors
• Factor I Fibrinogen
• Factor II Prothrombin
• FactorIII TissueThromboplastin
• Factor IV Calcium ions
• Factor V LabileFactor, Proaccelerin
• Factor VII StableFactor, Proconvertin
• Factor VIII Antihaemophilic Factor- A
• Factor IX ChristmasFactor/AHF- B
• Factor X Stuart Prower Factor
• Factor XI Plasma Thromboplastin Antecedent (C)
• Factor XII Hageman Factor
• Factor XIII Fibrin stabilizing Factor
• HMWK (Fitzgerald) Prekallikrein (Fletcher)
 Coagulation Cascade
• FEATURES
• Enzymatic cascade (amplification)

• Coagulation Factors are proteins, present in plasma

( represented by Roman numerals )
• Requires Ca 2+

• Localized to site of injury

• Is Reversible (via production of plasmin)

 Mechanism of clot formation

• Rupture of vessel or damage to blood itself leads to

cascade of reaction involving dozen of coagulation
factors.

• Takes place in 3 steps-

 Mechanism of clot formation
• Takes place in 3 steps
1. Formation of Prothrombin Activator (PA)

2. Formation of Thrombin (PA catalyzes conversion of)

Prothrombin PA Thrombin

3. Formation of Fibrin (Thrombin catalyzes conversion of)

Fibrinogen Thrombin Fibrin fibers which
enmesh blood cells & plasma, to form clot.
 Clot formation (cont)

Prothrombin Activator , rate limiting factor in

causing blood coagulation .

Formed in 2 ways by

Extrinsic pathway &

Intrinsic pathway.
 Prothrombin (Factor II)
 It is a plasma protein, α2 globulin (15mg/dl).

 Formed by liver

 Vitamin K required for its formation.

 lack of Vitamin K or liver disease prevents its

formation & increases PT leading to bleeding tendency.
 Role of thrombin
1. Fibrinogen to Fibrin

2. + ve feedback role – accelerates rate of

formation of PA by activating factor V (5) &
furthur conversion of Prothrombin to
Thrombin .

3. Also activates protein C.

 Fibrin Stabilizing Factor (XIII)
• Released from entrapped platelets.

• Activated by Thrombin & causes covalent

bonding between fibrin monomers &
multiple cross linking between fibers

• Adding strength to fibrin meshwork.

 Blood clot & Clot Retraction
• Blood clot - clot adheres to vascular opening &
prevent blood loss

• Clot Retraction - within few minutes after clot is

formed, it begins to contract & expresses most of fluid
from clot within 20-60 mins. Fluid expressed is serum.

• Platelets are necessary for Clot Retraction .

Failure of CR means no. of platelets is low.
 Intrinsic & Extrinsic Pathway
• Blood Trauma or contact with collagen
Injury to vessel wall &
XII XIIa other body tissues
HMWK, Prekallikrein v
III
XI XIa
Ca2+
IX IXa VIIa VII

VIII VIIIa
III
X Xa X
PL,Factor3 Ca2+
Factor V
rate limiting factor PA
PT Thrombin
Ca2+

Fibrinogen Fibrin
III XIII, Ca2+
Fibrin Threads
 Role of calcium ions in coagulation

• Except 1st two steps of Intrinsic Pathway Ca2+

required for acceleration of all blood clotting
reactions.

• In its absence clotting by either pathway does not

occur.
 Imp. difference between Extrinsic & Intrinsic pathway is
1. Factor III initiates Extrinsic pathway

2. Extrinsic pathway is limited by Tissue Factor,

Factor V,VII,X in blood. It is explosive (15-20 sec)

Contact of Factor XII & Platelets with collagen

initiates Intrinsic pathway.

• Intrinsic pathway much slower to proceed

(1-6mins)
 Prevention of blood clotting in normal vascular sysytem
1. Endothelial factors
(a)smoothness of endothelial lining.
(b)-vely charged particles over endothelial lining
repels clotting factors.

2. Presence of natural anticoagulants like Heparin &

Protein C
• &
• Activated Protein C inactivates Va, VIIIa (5,8 )
 Prevention of blood clotting in normal vascular system
3. Velocity of circulation .

4. Simultaneous activation of fibrinolytic system. (Tissue

Plasminogen Activator (t-PA) by endothelial cells.)
along with clotting.

5. Antithrombin III that removes IXa, Xa, XIa, XIIa

clotting factors from circulation.
• Functions of platelets

• Haemostasis

• Coagulation cascade (intrinsic & extrinsic)

• Prevention of clotting in normal vascular

system

• Aspirin in low doses prevents TIA, stroke, MI

 Fibrinolytic System
• This reopens many small vessels blocked by clots and
thus prevents occlusion.

• Plasmin lyses fibrin & fibrinogen to Fibrin Degradation

Products. (FDP)

• FDP inhibits thrombin.

• It is much slower process than clotting.

 Fibrinolytic System
releases
Plasminogen ([Link] blood)
Tissue damage t-PA Thrombin
(tissue plasminogen activator )
inhibits
Plasmin
• Fibrin , Fibrinogen FDP

• Human t-PA, Urokinase, Streptokinase are fibrinolytic

&
• Used in Treatment of early Myocardial Infarction &
Transient Ischaemic attack (TIA).
 Factors affecting - fibrinolysis
• Promoted by –
1. Stress, strain e.g. violent exercise, surgery

2. Epinephrine, Corticosteroids, Phenformin

3. Tissue activators - Urokinase in urine

 Factors affecting-fibrinolysis
• Inhibited by-
E-amino caproic acid, Aprotinin

• Significance –
1. Removes clots & prevents minute vessels from being
blocked.

2. Plays important role in cell movement & ovulation,

Producing defects in growth & infertility .
 Haemorrhagic/Bleeding Disorders
• Classified as
a) Defective blood clotting due to
1. Deficiency of Clotting Factor (1,2,5,8,9,10)
2. Deficiency of Vitamin K (2,7,9,10)
3. Anticoagulant overdose

b) Platelet Disorders
1. Deficiency of platelets
2. Functional disorders of platelets

c) Vascular Disorders
 Haemophilia A (Classical Haemophilia)

• Bleeding disorder, due to deficiency of factor VIII

• Inherited Recessive, X-linked disease

• Occurs exclusively in males.

• Females are generally carriers i.e. transmit

disease.
 Haemophilia A
• If Haemophilic male, marries carrier Haemophilic
female possible to have female Haemophilic child.

 Clinical features not apparent since birth

 But generally start early in life within first 3 yrs.

 Tendency of bleeding into

• soft tissue, muscles, joints, GIT,
Urinary tracts, nose.
 Haemophilia A
• It is characterized by

• Bleeding Time is Normal

• Platelet count & Prothrombin Time (PT) is

normal

• Marked increase in Clotting Time

• PTT prolonged. (partial thromboplastin time

40secs)
 Haemophilia A
• Treatment:
1. Injection of purified factor VIII (FFP- fresh frozen
plasma)

2. Injecting Thrombin or Thromboplastin

3. Fresh blood transfusion because factor VIII lost

rapidly on storage.
 Haemophilia B
 Also k/a Christmas disease.

 Deficiency of factor IX.

 Recessive , X-linked disease

 Occurs in males &

 Females are carriers.

 Haemophilia C

• Deficiency of factor XI (PTA) (plasma

thromboplastin antecedent.)

• Inheritance Mendelian Dominant.

• Male, female both affected.

 Defective blood clotting due to- def. of Vitamin K
• Absorption of vitamin K in Small intestine requires bile
salts.
• It is required for the synthesis of Factor 2,7,9,10 in
Liver.

• CT & PT prolonged & serious haemorrhages may

occur.

• CT- Clotting Time PT- Prothrombin Time

 Causes of vitamin K deficiency

1. Obstructive jaundice
Absence of bile decreases fat absorption & therefore
decreases Vit. K absorption.

2. Chronic Diarrhoeas-
decreased fat absorption & therefore Vit. K absorption
also decreased.
 Causes of vitamin K deficiency

3. Liver Disease

4. Premature babies (immaturity of liver)

 Treatment – injection of vitamin K because gut

flora not develop satisfactorily (recovery in 48 hrs)
 b) Platelet Disorders
1) Deficiency of platelets

2) Functional disorders of platelets

1. Thrombocytopenic Purpura

a) Idiopathic TP b) Secondary TP
 1. Thrombocytopenic Purpura-
 Purpura means purple colored petechial
haemorrhages & bruises in skin.

 a) Idiopathic TP - cause not known

 b) Secondary TP-
• bone marrow depression due to cytotoxic drugs,
irradiation,
• hypoplastic & aplastic anemia;
• septicaemia, uraemia.
 Thrombocytopenic Purpura
 Platelet count low,

 Clot Retraction is deficient

 Poor constriction of ruptured blood vessels

 Clot is soft, friable, does not retract well.

 Easy bruisability

 Multiple Sub Cutaneous Haemorrhages .

Thrombocytopenic purpura

Petechiae
 Platelet Disorders
• Diagnosis –
1. CT normal (3-10 mins)

2. BT increased (1-6mins)

3. Capillary fragility increases

4. Capillary resistance decreases

 2) Functional disorders of platelets
Thrombosthenic Purpura -
functional disorder of platelet characterised by
increased BT with normal platelet count.

• Causes – drug induced defect by Aspirin , Penicillin,

other disease like Uraemia, Cirrhosis, Leukemia.

• Treatment -
• a)ACTH in primary TCP
• b) Spleenectomy
 Anticoagulants
• The substances that inhibit coagulation are known
as Anticoagulants.

Natural Anticoagulants-
a) Heparin (inhibits active forms of CF IX, X, XI, XII ).
b) Antithrombin III
c) Protein C ( inhibits active forms of CF V & VIII )
Function-
• Maintains fluidity
• Prevents Intra Vascular Thrombosis.
 Synthetic Anticoagulants

1. Heparin

2. Calcium Sequesters/Decalcifying Agents-

3. Vitamin k Antagonists

4. Defibrination Substances
 HEPARIN
• Acts both in vivo & in vitro.

• 1st isolated from liver, Polysaccharide in nature

• Secreted by Basophils & Mast cells, destroyed by

Heparinase.

• Mechanism
• Facilitates action of Antithrombin III thereby
inhibits active forms of CF IX, X, XI, XII.
 Antithrombin III
• Present in plasma as well as in vascular
endothelium.

• It inactivates Clotting Factors IX, X, XI, XII.

& removes them from circulation

• Its action is facilitated by Heparin.

 Protein C
Synthesized in liver & is a plasma protein.
Thrombomodulin (natural anticoagulant)activates it.

• Mechanism-
• Inactivates factor Va & VIIIa & inhibits clotting
mechanism.
• Increases fibrinolysis by promoting Plasmin
formation.
 Protein C
• Thrombin
Thrombomodulin (binds thrombin)
Protein C Activator
Protein-S
Protein C Activated protein C

Inactivates Factor Inactivates inhibitor of t-PA

Va & VIIIa
Tissue PA
Plasminogen Plasmin
Fibrinolysis
 Synthetic Anticoagulants

1. Heparin

2. Calcium Sequesters/Decalcifying Agents-

3. Vitamin k Antagonists

4. Defibrination Substances
 2)Calcium Sequesters/Decalcifying Agents
 Calcium is removed from blood

a) By additon of substances forming insoluble salts

e.g. Na Citrate, Na Oxalate (sequester – to hold)

b) Chelators - binds Ca 2+
e.g. EDTA
 3) Vitamin k Antagonists
 Effective orally
Includes Coumarin derivatives-
 Dicoumarol, Warfarin, Phenindione,
Nicoumalone

 Mechanism-
Substrate competitive inhibition.

• (Vit. K def. results in deficiency of Clotting Factor

2,7,9,10)
 4) Defibrination Substances

• Cause destruction of fibrinogen.

• Snake Venom causes defibrination & stimulates

fibrinolysis.
 Lab Test In Bleeding Disorders
1. Bleeding time

2. Clotting Time

3. Capillary Fragility Test of Hess/ Tourniquet Test

4. Platelet Count

5. Prothrombin Time ( 11-16 sec )

6. Partial thromboplastin time (PTT) (40sec)

7. Clot Retraction Test

 1) Bleeding time
Time interval between skin prick & arrest of
bleeding is known as Bleeding Time.

It depends on depth of wound, degree of

hyperemia.

Normal BT indicates
• Platelet count,
• its function &
• capillaries are normal.
 Bleeding time
Dukes method

• Normal BT by Dukes method varies from

1-6mins.

Ivy’s method
• BT - 6mins.

• BT is prolonged in Purpura,
• Normal in Haemophilia
 2) Clotting time
• Time taken by fresh blood to get coagulated
by formation of Fibrin threads .

Capillary tube method

- 6 mins.

Modified Lee & White test tube method

• Normal C T 6-10 mins.
 Clotting time
Reduced
Menses,
Before & after child birth.

Increased
Hemophilia,
Liver disease, Afibrinogenemia,
Vitamin K deficiency DIC.
 Lab Test In Bleeding Disorders

• Capillary Fragility Test - Hess/ Tourniquet Test

• Appearance of 10 or > petechiae is +ve test.

• E.g. purpura, vessel wall defect.

 Lab Test In Bleeding Disorders
Prothrombin Time
 To oxalated or citrated plasma are added Tissue
Thromboplastin & CaCl2 solution

 Incubated at 370C. End point conversion of fluid

plasma into a gel (due to fibrin formation)

 Normal Prothrombin Time 11-16 sec.

 Average 12 sec.
 Lab Test In Bleeding Disorders
• Partial thromboplastin time (PTT)
• 40sec

• To monitor heparin therapy.

• Prolonged in
• Haemophilia,
• Von Willebrand disease
 Clot Retraction Test
Indicates Platelet count & its function.

 (In both EP & IP , Prothrombin Activator catalyze

conversion of PT to Thrombin.
 Thrombin acts on Fibrinogen to form Fibrin clot. )

After 20 mins to 60mins, Clot retracts or

contracts down to 40% of its original volume
with liberation of yellowish fluid k/a serum.
 Clot Retraction Test
• Contraction of clot is activated by Thrombin &
calcium stores in ER,GA, Mitochondria.

• It is completed in 24hrs.

• When clot contracts it closes the vessel still

furthur & prevents blood loss.

• CR retarded in thrombocytopenia (platelet

count low)
 Thrombosis
• Formation of clots inside blood vessels is called
Thrombosis.

• The bits of thrombus when break off & travel in

blood stream is k/a Emboli.

 Types of Thrombus-
• a) Red Thrombi, b) White Thrombi,
c) Mixed Thrombi
 Effects of Thrombi

1. Ischemia & Infarction

2. Thromboembolism
Cardiac & venous Thrombi produce Emboli more frequently
than arterial Thrombi.

E.g. Pulmonary embolism (calf veins),

Cerebral embolism (heart or carotid artery)
 Prevention of thrombus
• Prevention of Thrombus by administration of

1. Drugs that decrease platelet adhesion

e.g. Aspirin, Dipyridamole

2. Anticoagulants
low doses of Heparin (inhibits active CF 9,10,11, 12)

Dicoumarol (Vitamin K antagonist) CF 2, 7, 9, 10

 Prevention of thrombus
3. Intermittent Compression/Electric Stimulation
of calf muscles to prevent post operative venous
thrombosis in addition to drugs.

4. Use of t-PA or Streptokinase

In Treatment of Intra Venous clots.
Given within first hr or so after occlusion.
It can be life saver in pulmonary embolism.
 Disseminated IV Coagulation(DIC)
• Occasionally clotting mechanism becomes activated in
widespread areas of circulation giving rise to DIC.
• Cause - large trauma or dying tissue in body

Increased Tissue Factor released into blood

Small but numerous blood clots

plug peripheral blood vessels

decreased delivery of O2 & other nutrients to tissues

excerbates the situation.
• Effect – bleeding because of excessive use of clotting factors.
 Anti haemostatic Mechanism

1. Factors preventing platelet aggregation

prostacyclin, Aspirin

2. Circulating Anticoagulants- Heparin,

Antithrombin III, Protein C

3. Fibrinolytic mechanism- Plasminogen(by

liver), Plasmin, t-PA, Urokinase.
Thanks