Platelets

VEENA SHRIRAM
Platelets
Formed in bone marrow, 150-350,000 /µl
Sequestered in spleen (30%)
2-4 m in diameter, life span 8-12 days, no nucleus

Active cytoplasm
actin + myosin
enzyme synthesis + storage of calcium
synthesis of prostaglandins
dense granules containing ADP and ATP
a-granules (fibrinogen, PDGF, vWF, fibronectin)
fibrin stabilizing factor
 THROMBOPOEISIS
Pluripotent hemopoitic stem cell
CSF
Megakaryoblast

Premegakaryocyte
Thrombopoietin
Promegakaryocyte FIVE
DAYS
Megakaryocytes
Endomytosis
Protoplatelets
Platelets
Each megakaryocyte produces between 5,000 and 10,000 platelets.
Structure and composition
Non nucleated
1-4 µm
Colourless,Refractile
Spherical or Oval
Discoid shape
Normal count – 1.5 to 4.5 lac / cumm
Three pools – BM , Blood stream , Spleen
Life span – 7 to 9 days
Leishman’s stain : appear as cell with
faint bluish cytoplasm
with reddish-purple granules
 Electron microscopic structure :

 Cell membrane
 Microtubules
 Cytoplasm
and Organelles
 Cell membrane
P  6 nm ,trilaminar
 Similar to plasma membrane
 Lipid ,carbohydrates and Proteins

• Phospholipid
Plat. Factor 3 – Thx A2 – Aggregation
• GLP
Glycocalyx ( Repellant,Adhesion )
• Receptors :
- IbIx – VWF – Adhesion
- II b IIIa – Fibrinogen – Aggregation
- P2Y1 and P2Y2 – for action of ADP
 Microtubule :
p
• Encircle the whole cytoplasm
• Made by polymerised protein
(Tubulin)
• maintained discoid shape
 CYTOPLASM
Endoplasmic reticulum Contractile protein
• Metabolism Actin
• Protein synthesis. Myosin
Thrombosthenin
Golgi apparatus
Ca++ storage α – Granules
PDGF, factor V, VIII
Fibrinogen
Mitochondria Thrombospondin
- ATP Dense granules
Serotonin ,Ca++, ADP
 Properties of Platelets,

 Adhesion
 Activation
 Aggregation
 Retraction
…………role in Haemostasis
 Haemostasis
Definition :
Steps :
1) Vasoconstriction
2) Platelet plug formation
3) Clot formation
4) Clot retraction
Vasoconstriction
 myogenic spasm, neural involvement,serotonin

 Importance in daily small vessel injuries

 Role of platelets
Platelet plug formation
:
 Adhesion
 Activation
 Aggregation
 Adhesion

vWF
IbIx

Endothelium
 Activation

Biochemical changes Shape changes

Platelet Exposed to collagen

Activation of ‘G’ surface protein

Formation of Inositol triphosphate
( acts as Ca++ inopore )
Increase intracellular Ca++
Activation of contractile protein
( shape changes )
And activation of phospholipase A2
 Activation of phospholipase A2

Liberation of Arachidonic acid

from Platelet membrane
Cyclo-oxygenase Lipo-oxygenase
pathway (80%) pathway( 20%)

Thromboxane A2 PGH2

Amplification of Platelet activation and Aggregation

Shape changes :

Phosphorylation of myosin light chain

and actin filaments polymerization

Dissolved microtubules

Loss of discoid shape

with pseudopod formation
 Aggregation
Bridge between two activated platelets leads to aggregation

Liberation of factors from activated FIBRIN

platelets

Platelet exposure to these agonists

like- - ADP ,TX-A2 ,Thrombin
Fibrinogen receptors ( GP Iib, IIIa )
are exposed on activated platelets Tx-A2 ADP

Bridge formation by fibrin

Platelet Aggregation
And plug formation
Functions of platelets :

1) Role in haemostasis - in first two steps

2) Role in repairing of injured blood vessel

3) Role in clot retraction

4) Transport function – 5 HT
Applied :

Platelets are involved in various stages of haemostasis

1) Vasoconstriction

2) Platelet plug formation

 Integrity of blood vessel

-Maintained by various chemical components invoved with collagen

Vessel wall diseases

Hereditary :
Marfan syndrome
Hemorrhagic telangiectasia

Metabolic and Inflammatory

Henoch-Schonlein
Senile purpura
Rickettsial Diseases
Scurvy
Lab investigations

1) For Vascular integrity

Capillary fragility test :

 Platelet plug formation
Platelet -disorders

Abnormal Count Abnormal function

( Normal count 1.5 to Von willebrand disease

4.5 lac / cumm) Bernard soulier
Thrombocytopenia Thromboasthenia
Thrombocytosis ( > 4.5 ) (Glanzman’s disease)
Thrombocytopenia :
- Platelet. Count <1.5 lac / cu mm

Causes :
- Decrease production
- Bone marrow depression
- Increase destruction
- post splenomegaly
- auto A/B
- Drugs ( quinine,linid and sulfa )
- Diseases ( Dengue,HIV ,HIT)
Platelet function tests :

1) Bleeding time :
- Duke’s method or IVY method
 Platelet count

Manual : Automated :
- PBS ( Qualitative ) - with machines
- with Neubaur’s chamber ( more accurate )
( Quantitative )
Platelet count with Neubaur’s chamber

Fluid : REES – ECKER fluid

-sodium citrate 3.8 gm
+ Brilliant cresyl blue 0.1 gm
+ Formaldehyde 2 ml
+ Distilled water 100 ml
Using RBC square for calculation

Normal range – 1.5 to 4.5 lac /cumm

3) Clot retraction time :

Incubating a test tube of clotted blood

clot retraction normally occurs

within 20 to 60 min .

abnormally increased when

integrity of contractile protein is lost

Also, seen with thrombocytopenia ( < 50,000 count )

DISORDERS :

1) Dengue fever :

Three possible trigger to induce in thrombocytopenia

a) Direct lysis - D V Ag attached to Plt.
b) Immune mediated – Demonstrated D V Ag –Ab

complex on Plt surface

c) Infection of the endothelial cell causes
modulation
endothelial cells
and used up platelets extra clot formation
2) Immune thrombocytopenia ( ITP ) :

- isolated thrombocytopenia with normal bone marrow

- Childhood ( common ) and Adult (rare) forms
- Auto antibodies against surface Ag – IbIx / IIbIIIa
- A/B covered platelets – cleaved by microphage
- Treatment : Steroid + Thrombopoetin receptor agonist
3)Thrombotic thrombocytopenia :

- Rare disorder with extensive microvascular

thrombi
- A/B against the metaloproteinase that cleaves the
VWF
- Increase free VWF – Increase Plt.Adhesion
–increase clots
- Clinical presentation
– purpura or end organ failure

- Treatment
: Plasma pheresis,
4) Heparin induced thrombocytopenia ( HIT )

- Rare disorder
- When IG g A/B develops against (heparin + Plt. Factor
4)
- This complex – Activates platelets,monocytes and
endothelial cells – induced clot formation
and used up platelets
- Treatment : Direct Thrombin inhibitor
– Argatroban,Lepirudin
Qualitative / functional abnormality

 Inherited thrombocytopenia
 Aquired (drugs)

Inherited

1) Von Willebrand disease

2) Bernard soulier
3) Thromboasthenia
 Functional platelet abnormalities :
Von will brand disease :
- Autosomal dominant
-Three types 1) partial quantitative deficiency
2) partial qualitative deficiency
3) total VWF deficiency

VWB
IbIx

Endothelium

ADHESION DEFECT
Ristocetin agglutination test :

- Ristocetin is an antibiotic previously used to treat

streptococcal infections,
 -It is no longer used clinically because
it causes thrombocytopenia and platelet clump formation.
 
In an unknown fashion, the antibiotic ristocetin
causes Von Willebrand factor to bind the platelet
receptor glycoprotein Ib (GpIb), so when
ristocetin is added to normal blood, it causes
platelet clumps

- if ristocetin is added to blood lacking the factor

 Bernard soulier disease
ADHESION DEFECT

VWB
IbIx

Endothelium
Thromboesthenia or Glanzman’s disease
IIbIIIa

Aggregation defect
 Drugs :
Activation of phospholipase A2
Aspirin
Liberation of Arachidonic acid
from Platelet membrane
Cyclo-oxegenase Lipo-oxygenase
pathway (80%) pathway( 20%)

Thromboxane A2 PGH2

Amplification of Platelet activation and Aggregation

Clopidogrel :

- irreversibly blocks the ADP receptors on platelets

- ADP mediated further Platelet activation stopped

c c
Abciximab
And
Tyrofiban

competitive inhibitor of GP IIb/IIIa receptors,

exerting its effects via the prevention
of the binding of fibrinogen 
 PLATELET TRANSFUSION

Indication :
1) platelet count < 20,000
2) platelet count < 40,000
with acute bleeding

( indicated to treat acute hemorrhage secondary to thrombocytopenia

or to provide prophylaxis from hemorrhage in patients
with bone marrow aplasia.)
Platelet transfusion :
 collection and storage:

Whole blood collection

Centrifuge ( 22
degree )
Platelet rich plasma

Collection in separate bag

by compressor

Storage at 22 degree
in AGGITATOR
LIFE SPAN OF STORED PLATELETS IS ONLY 5 DAYS )
RDP :
- Random donated platelet
- Blood collection from various donors
and separate the platelets
- also includes collection of other
component ( PCV ,plasma) from same
donated blood
Actiaaavators of platelets

Collagen and microfibrillar proteins

ADP released from damaged RBCs and activated
platelets
Thromboxane from activated platelets
Platelet activating factor from basophils
Epinephrine (stress)
Thrombin
Platelets
Membrane
Receptors: thrombin, ADP, epi, serotonin
Adhesion proteins: vWF, fibronectin, collagen, fibrinogen

coat of glycoproteins adhesion to injured areas

phospholipids activation of intrinsic pathway
adenylate cyclase cAMP activate other platelets
Platelets
Vessel injury or atherosclerotic plaque rupture
subendothelial protein layer exposed
platelets bind to subendothelial vWF, and collagen via
surface glycoproteins.
platelets swell
release platelet agonists from granules
generate thrombin
activation of new platelets
crosslinking of platelet aggregate by surface glycoprotein
contractile elements pull fibrin threads
 Platelets

Leukotrienes (chemoattractants)
Thromboxane (release more granules)
Release of thrombospondin (stabilizes platelet-fibrin)
Release of PDGF (stimulates smooth muscle proliferation)
Release of ADP (attracts more platelets)
Platelet inhibition
Anti-platelet agent Mechanism of action Agonist pathway
affected

Aspirin Irreversibly inhibits ADP, collagen,

COX TXA2

NSAID reversibly inhibits ADP, collagen,

COX TXA2

GP IIb-IIIa receptor Inhibits fibrinogen ADP, collagen,

and vWF binding thrombin, epi,
serotonin, TXA2
Coagulation Defects

Thrombocytopenia
bleeding small capillaries and blood vessels
mucosal, skin
low number of platelets
ITP- autoimmune (common)
The first step in the extrinsic coagulation pathway is
(A) Activation of factor X
(B) Activation of factor XII
(C) Conversion of prothrombin to thrombin
(D) Release of tissue thromboplastin
(E) Conversion of fibrinogen to fibrin
Which of the following is the strongest activator of platelet?
a. Thrombin
b. Serotonin
c. Thromboxane A2
d. Epinephrine
A healthy 6-years-old girl is brought to the emergency department because of a
severe, uncontrolled nose bleed. She has numerous small pin point
hemorrhages (blotches) on her arms & legs. Her blood values are as follows:
◦ Hb = 13 g/dl, Leukocytes count = 7000/cubic mm, Platelet count = <
10,000/cubic mm

a) Give her provisional diagnosis.

b) Give the pathophysiology of her disease.

c) Mention her treatment. (1+3+1 marks)

key: (Reference: p 465, Guyton, 11th Ed.)

a) DIAGNOSIS: Thrombocytopenic Purpura (1mark)
b) PATHOPHYSIOLOGY: She has a very low number of platelets in the circulating blood,
so has a tendency to bleed more from many small venules or capillaries leading to
small punctate hemorrhages throughout all the body tissues. (1 mark)
Her skin displays many small, purplish blotches, giving the disease the name
thrombocytopenic Purpura (0.5mark)
Platelets are important for repair of minute breaks in capillaries and other small
vessels, but she is deficient in them leading to nose & cutaneous bleed. (0.5mark)
Bleeding will not occur until the number of platelets in the blood falls below
50,000/ml, rather than the normal 150,000 to 300,000. Levels as low as 10,000/ml are
frequently lethal. (1 mark)
c) TREATMENT: Giving fresh whole blood transfusions (can control bleeding for 1-4
days) that contain large numbers of platelets. (0.5mark)
Splenectomy is often helpful, because the spleen normally removes large numbers of
platelets from the blood. (0.5mark)
Rashid was started on blood transfusion pre operatively to improve the hemoglobin prior to surgery. He
began to feel irritability, chills and palpitation just within 5 minutes after the start of transfusion. But the
transfusion was continued and the patient developed fever, shock-like state with fall of blood pressure and
scanty urine output. a. i) What was the possible reason for Rashid’s symptoms?
ii) Give the mechanisms in detail for development of these symptoms?
iii) What steps you will take to prevent such situation? (0.5+1+1 Marks)
iv)Enumerate other hazards of blood transfusion reaction.
Key: a. i) Mismatched blood transfusion. 0.5 mark
ii) Hemolysis of donor’s red cells after agglutination by corresponding agglutinin in receipient’s plasma
results release of toxins from hemolysed cells causing irritability, chills and palpitation due to circulatory
shock. 0.5 mark
Immediate hemolysis and phagocytosis of RBCs results in release of excessive Hb in circulation, much of the
excess leaks through the glomerular membranes into the kidney tubules, recipitates. Renal vasoconstriction,
circulatory shock, and renal tubular blockage together cause acute renal shutdown. Less or no urine.0.5 mark
iii) Blood group typing: ABO and Rh 0.5 mark
Cross matching 0.5 mark
b. What happens when a specific allergen is injected directly into circulation? Give the
sequence of events that may follow? (2.5 marks)
KEY:b • When a specific allergen is injected directly into the circulation, it reacts with
basophils of the blood & mast cells in the tissues located immediately outside the small
blood vessels. (0.5 mark)
• Widespread allergic reaction occurs throughout the vascular system & closely associated
tissues. This is called anaphylaxis. (0.5 mark)
• Histamine is released into the circulation causes: • body-wide vasodilation (0.25 mark)
• Increased permeability of the capillaries with resultant marked loss of plasma from the
circulation. (0.25 mark)
• An occasion person dies of circulatory shock within a few min unless treated with
epinephrine to oppose the effects of the histamine. (0.5 mark)
• Mixture of leukotrienes called slow-reacting substance of anaphylaxis released from the
activated basophils & mast cells cause spasm of the smooth muscle of the bronchioles,
eliciting an asthma-like attack. (0.5 mark)
A young man received a cut during shaving which started bleeding but after sometime the bleeding stopped
spontaneously.
a. Give mechanism involved in the spontaneous arrest of bleeding in this man.
key: a} Hemostasis in this man would involve the following mechanisms:
Vascular constriction: Trauma to vessel wall instantaneously causes it to contract.
Platelet plug: When platelets come in contact with collagen in the vessel wall they undergo certain changes
which ultimately lead to formation of platelet plug by the following mechanism
1. Platelets become sticky and adhere to collagen in the tissues
2. They secrete large quantities of ADP and their enzymes form thromboxane A2 which act on nearby
platelets to activate them and the stickiness of these additional platelets causes them to adhere to the
original activated platelets
3. In this way at the site of damaged vessel wall more and more platelets are activated causing them to
adhere to each other and forming platelet plug
Extrinsic pathway: Damage to the blood vessel will initiate extrinsic pathway of coagulation. (Fig. 36.3
Guyton 11th Ed)
b. What is the process of spontaneous arrest of bleeding called? (1)
key. b} The process is called hemostasis
Q. A 43 years old male pale looking man presents with complaint of difficulty in breathing on exertion. He has
history of Inflammatory Bowel Disease followed by surgical removal of terminal portion of Ileum 3 years back. His
blood picture reveals:
Hemoglobin 8.7 gm/dl MCV (Mean Corpuscular Volume) 106 fl (normal range 80-100 fl)
a) Why he suffered from breathing difficulty? (1) b) What is type of anemia in this case? (1)
c) Which nutrient is deficient in this patient? (1) d) Describe the pathogenesis of his disease. (2)

Key: a) Decreased hemoglobin concentration in this patient results in tissue hypoxia. Hypoxia stimulates respiratory
centre leading to increased rate of respiration. As a result, the respiratory muscles become over-worked leading to
exhaustion resulting into difficulty in breathing. (1)
b) Megaloblastic anemia. (1) c) Vitamin B12 is deficient. (1)
d) Maturation Failure of RBCs, caused by poor absorption of Vitamin B12, (which is required for RBC maturation)
from the Gastrointestinal Tract due to surgical removal of ileum (from where B12 is normally absorbed) as a
treatment of inflammatory bowel disease. (1)
3 to 4 years of defective B12 absorption are usually required to deplete its stores & cause maturation failure
anemia. (1)