PLATELETS

STRUCTURE & FUNCTIONS

INTRODUCTION

• Platelets are smallest blood cells, colourless

• Size : 2-4 μm

• Volume : 5.8 μm3

• Shape : Spherical, ovoid discoid structures

• Leishmans staining : faint bluish cytoplasm, reddish purple granules

• Nucleus : absent

STRUCTURE OF PLATELET

CELL MEMBRANE

• Thick trilaminar membrane

• Consists of lipids, carbohydrates, proteins & glycoproteins

SALIENT FEATURES OF CELL MEMBRANE

• Glycoproteins : surface coat – prevents adherence to normal endothelium

• Phospholipids : contains PF-3 – activates clotting process

• Canalicular system

• Receptors : combines with collagen, fibrinogen & von Willebrand factor (vWF)

• Precursors : TxA2, PGs, LT & PF-3,4

CYTOPLASM
 • Golgi apparatus

• Endoplasmic reticulum

• Mitochondria

• Microtubules

• Microvesicles

• Granules

• Glycogen

• Lysosomes

• Proteins

• Enzymes

• Hormones

MICROTUBULES

• Made up of tubulins (α and β)

• Form a compact bundle

• They provide stability to the membrane and maintain discoid shape of circulating platelets

PROTEINS

• Contractile proteins :

 Actin

 Myosin

 Thrombosthenin

• Other proteins :

 Fibrin stabilizing factor

 Platelet Derived Growth Factor (PDGF)

 vWF

GRANULES

• Two types of granules are present in the cytoplasm of platelets :

i. Dense granules

ii. α granules
 • Dense granules (3-8): contain substances like

 Phospholipids, TG, cholesterol

 ATP, ADP, Serotonin

 α granules (50-80): contain secreted proteins

 Clotting factors, vWF

 PDGF, PF – 3,4

NORMAL COUNT & VARIATIONS

• Normal count : 1.5 lakhs – 4.5 lakhs /cumm of blood

• Average count : 2.5 lakhs/cumm of blood

• Life span 8-12 days

• Destruction – mainly in spleen

FUNCTIONS OF PLATELETS

• Temporary hemostatic plug

• Blood coagulation

• Clot retraction

• Thrombolysis

• Phagocytosis

• Storage & Transport

• Vascular growth

HEMOSTASIS

Definition: The term hemostasis means prevention of blood loss or spontaneous arrest of bleeding.

It is achieved by mechanisms such as:

1. Vasoconstriction

2. Formation of platelet (temporary hemostatic) plug

3. Formation of blood clot (definitive hemostatic) plug

4. Clot retraction

5. Fibrinolysis

1. VASOCONSTRICTION

• Immediately after a blood vessel is cut, vessels constrict reducing the blood flow.

• It is produced by nervous reflexes, local myogenic spasm & local humoral factors.

• Platelets release thromboxane A2 - vasoconstrictor

2. PLATELET PLUG FORMATION

• When platelets come in contact with a damaged vascular surface/exposed collagen:

1. Adhesion

2. Aggregation

3. Activation & Release

1. PLATELET ADHESION

• The damage to blood vessel exposes underlying portion of the vessel wall that are normally
concealed from the circulatory platelets by intact lining of the endothelium

• Platelets have high affinity to adhere to the exposed vascular wall, which is called platelet
adhesion
 • Especially, exposed collagen and von Willebrand factor in the injured vessel wall attract
platelets as platelets have receptors for them on their cell membrane

2. PLATELET AGGREGATION

• Platelet adhesion is immediately followed by platelet aggregation at the site of injury.

• Fibrinogen, GpIIb-IIIa and thrombin promote platelet aggregation

• Simultaneously, platelets are activated, and activated platelets release chemicals like ADP and
platelet activating factor (PAF) further facilitate platelet aggregation.

• Thus, accumulation of more platelets at the site of vascular injury facilitates the process of
temporary hemostatic plug (platelet thrombus) formation.

• Then, formation of clot is initiated.

3. ACTIVATION & RELEASE
Through platelet activating factor (PAF)

• Platelet adhesion activates platelets. Activation of platelet is facilitated by PAF, acts via G-
proteins and activates phospholipase-C and diacylglycerol (DAG).

• DAG increases cytoplasmic calcium by promoting calcium release from dense tubular systems
that are present close to open canaliculi.

Through platelet activating factor (PAF) (Contd..)

• Calcium causes contraction of microfilaments that helps in movement of granules to the open
canalicular system.

• Membranes of granules fuse with the membranes of canaliculi and the contents of granules
are released into the canaliculi by exocytosis.

• From canaliculi, chemicals are transferred to the exterior of the cell.

• During this process, platelets change their shape and exhibit movement.

MECHANISMS OF PLATELET RELEASE….

Through thromboxane and serotonin

• Formation of DAG (as described above) activates the enzyme phospholipase A2, which
converts membrane phospholipid into arachidonic acid in turn form thromboxane A2 (TX-A2).

• TX-A2 is a potent vasoconstrictor and also promotes platelet aggregation

Serotonin released from platelet produces vasoconstriction and promotes hemostasis.

MECHANISMS OF PLATELET RELEASE….

Through thrombospondin and thrombonectin

• Thrombospondin and thrombonectin released from platelet granules facilitate the activity of
contractile system and further promote exocytosis of granules.

• If the rent in a vessel is small – platelet plug itself can stop the blood loss.

• If it is a large hole – platelet plug + blood clot

IMPORTANCE OF PLUG FORMATION

• Closes many small holes due to rupture of small blood vessels occurring everyday in the body
(capillary integrity).

• In thrombocytopenia, hemorrahgic spots appear under skin – purpura (Petechial spots)

3. BLOOD COAGULATION

• Many substances found in the blood affect blood coagulation.

 • Some promote coagulation – procoagulants

• Some inhibit coagulation – anticoagulants

STEPS OF CLOTTING MECHANISM

• Prothrombin activator is formed in response to rupture of the vessel or damage to the blood.

• Prothrombin activator converts prothrombin to thrombin

• Thrombin converts fibrinogen to fibrin

COMMON PATHWAY

CONVERSION OF PROTHROMBIN TO THROMBIN

• Prothrombin activator in the presence of ionic Ca++, converts prothrombin to thrombin

• Platelets accelerate this conversion.

• Rate limiting step – formation of prothrombin activator

PROTHROMBIN

• It is alpha2 – globulin with molecular weight of 68,700.

• Formed in the liver

• Vitamin K in essential for formation of prothrombin (+ other clotting factors)

CONVERSION OF FIBRINOGEN TO FIBRIN

FIBRINOGEN

• HMW protein (MW = 3,40,000)

• Formed in the liver

• Thrombin converts fibrinogen to fibrin monomers which polymerize to form fibrin.

 • Fibrin – stabilising factor – strengthens the reticulum.

BLOOD CLOT

• It is composed of meshwork of fibrin fibers entrapping blood cells, plasma & platelets.

• It adheres to the vascular opening and prevents further blood loss.

VICIOUS CIRCLE OF CLOT FORMATION

Blood clot formation

↓
Thrombin
↓
Activates more amount of prothrombin and clotting factors (VIII, IX, X, XI and XII)
↓
Still more blood clotting

FORMATION OF PROTHROMBIN ACTIVATOR

• It is formed by

• Extrinsic pathway that begins with injury to the vessel wall & surrounding tissues.

Intrinsic pathway that begins when blood is exposed to collagen fibres

EXTRINSIC PATHWAY

EXTRINSIC PATHWAY

• It begins with the traumatized vascular wall or extra vascular tissues.

1. Release of tissue factor or thromboplastin

2. Activation of factor X – It is activated by factor VII, tissue factor & Ca ++ ions

3. Activated factor X forms prothrombin activator – factor V, Ca++ & tissue factor are needed.

4. Prothrombin activator splits prothrombin to thrombin in the presence of Ca ++ ions

INTRINSIC PATHWAY
INTRINSIC PATHWAY

• It begins with blood trauma or exposure of blood to collagen.

1. Blood trauma causes activation of factor XII & release of platelet phospholipids.

2. Activation of factor XI – it requires HMW kininogen and prekallikrein.

3. Activation of factor IX

4. Activation of factor X – It requires activated factor VIII & platelet phospholipids.

5. Activated factor X forms prothrombin activator – It requires activated factor V & platelet
phospholipids.

ROLE OF CALCIUM IONS

• Ca++ ions are required for acceleration of both

• extrinsic

• intrinsic (except for the first 2 steps) blood clotting mechanisms.

EXTRINSIC Vs INTRINSIC PATHWAY

• Clotting occurs by both pathway simultaneously

• Extrinsic pathway is explosive (15 sec) & limited by factors III,VII, X and V

• Intrinsic pathway is slow to proceed (1-6 min)

4. CLOT RETRACTION

• After a clot is formed, it begins to contract expressing a fluid within 20-60 min.

• The fluid expressed is called serum.

• Serum = plasma – fibrinogen and clotting factors.

• Platelets are essential for this process.

 5.FIBRINOLYTIC SYSTEM

FIBRINOLYTIC SYSTEM

• It is the process of dissolution of blood clot.

• The inactive plasminogen is converted to plasmin by the action of thrombin, t-PA & u-PA.

• Plasmin lyses fibrin threads.

• Human t-PA, urokinase & streptokinase are the enzymes used for the treatment of early MI.

PREVENTION OF BLOOD CLOTTING- Intravascular Anticoagulants

1. Smoothness of the endothelium – prevent contact activation of intrinsic system.

2. A layer of glycocalyx on the endothelium - repels clotting factors & platelets

3. Thrombomodulin binds with thrombin

• It removes thrombin & slows the clotting process.

• It also activates protein C which inactivates activated factors V and VIII

4. Fibrin fibers
5. Antithrombin III
6. Heparin - highly –vely charged.
1. It combines with anti-thrombin III & removes thrombin .
2. Heparin + anti-thrombin III complex removes activated factors IX,X, XI and XII.

ANTICOAGULANTS

• Anticoagulants prevent blood from clotting. They are used for

1. Collection of blood samples for lab investigations.

2. Preserving blood for transfusion

 3. Anticoagulation therapy

INVITRO ANTICOAGULANTS

1. EDTA (ethylene diamine tetra-acetic acid)

• Sodium & potassium salts of EDTA.

• It acts by chelating effect on the blood Ca ++

2. Sodium, ammonium & potassium citrate

• It prevents clotting by inactivating Ca ++ ions.

3. Double oxalate

• Ammonium and potassium oxalates.

• It forms insoluble complex with Ca++ thereby preventing coagulation.

4. Sodium fluoride

• It is the inhibitor of glycolytic enzymes & prevents loss of glucose

5. Oxalates

• Oxalates of sodium, potassium & ammonium form insoluble complexes with Ca 2+

6. Heparin

• It is a powerful anticoagulant first isolated from liver.

• It is secreted by granules of basophils & mast cells

• Functions:

• It neutralizes thrombin.

• It facilitates action of antithrombin III & inhibits the active forms of IX, X, XI & XII.

• Destroyed by heparinase

ANTICOAGULANTS FOR ANTICOAGULATION THERAPY

They are used for treatment or prevention of thrombosis.

• Oral anticoagulants

• Intravenous anticoagulants.

ORAL ANTICOAGULANTS

• Vitamin K antagonists – Coumarin derivatives eg., Warfarin.

• Mode of action: Plasma levels of II, VII, IX, X and protein C begin to fall. It competes with
vitamin K for the active sites in enzymatic processes.
 • Protein C and protein S

• Mode of action: Inactivates activated factors V & VIII

INTRAVENOUS ANTICOAGULANTS

• Heparin – facilitates thrombin inhibition with AT-III

• Hirudin – Most specific inhibitor of thrombin.

BLEEDING DISORDERS

• Hemophilia A

• Christmas disease

• Vitamin K deficiency

• Thrombocytopenia purpura

• Disseminated intravascular coagulation

1. HEMOPHILIA A

• Cause – deficiency of factor VIII. It occurs only in males.

It is a sex-linked recessive hereditary disease (women are carriers)
• Diagnosis: CT & normal BT
Clinical features
Bleeding tendency occurs
Treatment
• Fresh blood transfusion
• Injection of factor VIII or thromboplastin

2. CHRISTMAS DISEASE
(Hemophilia B)

• Cause - deficiency of factor IX.

• It is a sex linked recessive disease.

• Hematoma & bleeding episodes occur.

• Treatment – replacement of factor IX.

3. VITAMIN K DEFICIENCY

• It is important for liver formation of factors II , VII, IX, X and protein C (Vit - K dependent
factors)

• Sources: Green leafy vegetables, cereals, animal tissue & bacterial flora.

• It is absorbed from small intestine in the presence of bile salts.

 TREATMENT

• Injection of vitamin K

4. THROMBOCYTOPENIA PURPURA

• It is characterised by spontaneous haemorrhages beneath the skin, mucous membrane and

internal organs.

• TYPES:

• Primary (idiopathic) – congenital or hereditary & occurs in children.

Secondary – due to allergies & infections.

DIAGNOSIS

• CT is normal

• BT is prolonged

• capillary endothelium resistance leads to increased capillary fragility

• platelet count

Poor clot retraction & poor constriction of injured blood vessels

TREATMENT

• Injection of ACTH or corticosteroids

• Splenectomy helps by decreasing platelet destruction in spleen.

5. DISSEMINATED INTRAVASCULAR COAGULATION

• There is widespread intravascular coagulation.

• Cause - traumatized tissue releases tissue factor into the blood.

• The clots plug the small blood vessels.

DIAGNOSIS

• Thrombocytopenia

• ↑ Fibrin degradation products

• TREAMENT

• Elimination of precipitating factors

• Replacement of coagulation factors & platelets.

TESTS OF HEMOSTASIS

• Indications:

a) Spontaneous bleeding

b) Excessive bleeding after injury

• Tests for defects in

a) Temporary hemostatic plug

b) Definitive hemostatic plug

TESTS FOR TEMPORARY HEMOSTATIC PLUG

1. Bleeding time

Normal - 2-3 min by Dukes method

2. Capillary fragility test

BP cuff pressure of 100mm Hg for 5min.

More than 10 petechiae in forearm indicates capillary weakness or thrombocytopenia

3. Platelet adhesion test

4. Platelet aggregation test

TESTS FOR DEFINITIVE HEMOSTATIC PLUG

1. Clotting time (CT)

2. Prothrombin time (PT)

3. Activated partial thromboplastin time (APTT)

1. CLOTTING TIME

Common methods

a) Capillary tube method (Normal 2-8min)

b) Lee and white method (Normal 5-12 min)

• Prolonged clotting time more than 10 min indicates absence or deficiency of clotting factors.

2. PROTHROMBIN TIME

• Rabbit brain emulsion (Tissue thromboplastin) is added to plasma in the presence of calcium

• Time required to form clot – prothrombin time

• Normal value 12-16 sec

 • Prolonged prothrombin time- Deficiency of factors II,V,VII & X

3. ACTIVATED PARTIAL THROMBOPLASTIN TIME (APTT)

• Partial thromboplastin, kaolin, and Ca 2+ are added to plasma

• Normal APTT – 35 to 45 sec

• APTT is prolonged in deficiency of XII, XI, IX & VIII