PLATELETS IN HEALTH AND DISEASE

Dr.AnuPriya J

SCHEME

• Introduction • History • Formation • Structure • Properties • Functions• Role in hemostasis• Platelet function tests• Antiplatelet drugs • Platelet disorders • Transfusion

Introduction

• Blood is a complex fluid consisting of plasma and of formed elements – red blood cells, white blood cells & platelets.

• Platelets are small, anuclear, cytoplasmic fragments of megakaryocytes

• Normal platelet count – 1.5 to 4 L / mm3 of blood

• Platelets survive in circulation for about 8-10 days

• Removal -by the mononuclear phagocyte system -spleen plays a major role

• Half life – about 5 days

Introduction

History

• Hewson - in 1780 - very small undefined particles in blood.

• Max Schultze in 1865 - these particles must be degenerate and disintegrated leukocytes – as they showed a granular appearance.

• Riess in 1872 and Laptschinski in 1874 - leucocytes - mainly during infectious diseases – are the origin of the Schultze's corpuscles.

• George Hayem between the years 1878 and 1879 - related to erythrocytes - considered as their precursors - the term “haematoblasts”

• Neumann -1880 - stated they were artifacts derived from red cells following an incorrect technique of venipuncture .

History

• Giulio Bizzozzero - the first - in 1882 - clearly establish the significance of the particles- the third morphological element of blood, totally unrelated to white and red cells - gave a more precise description

• Bizzozzero named these elements “piastrine”, i.e. small plates

• Wright - Between 1906 and 1910 - identified bone marrow megakaryocytes as precursors of blood platelets – first to use the term “platelets”

History

• The invention of the aggregometer by Born in 1962 provided a valuable instrument to study platelet function and responsiveness to agonists

• In 1964, David-Ferreira published the first paper concerning platelet ultrastructure analyzed by means of electron microscopy.

History

• The late 20th century - the definition and characterization of many platelet receptors- the analysis of molecular mechanisms involved in signal

transduction - the introduction of anti-platelet treatments

• Recent – collection, storage & transfusion

History

Formation

Pluripotent stem cellsCommitted stem cells

CFU-MegaMegakaryoblast

Basophil megakaryocyteGranular megakaryocyteMature megakaryocyte

Platelets

Thrombopoiesis

CFU - GEMM

CFU-MegE

CFU-Mega

TPO

Formation • Thrombopoiesis – formation of platelets

• Regulated by - Thrombopoietin- Interleukin (IL-1,IL-3,IL-6,IL-11)- GM-CSF

• Each megakaryocyte produces between 1,000 to 3,000 platelets during its lifetime.

• An average of 1011 platelets are produced daily in a healthy adult.

•Small, granulated, anuclear, discoid

•Diameter - 2 to 3 μm

•Volume - 8 fl

•Size - One fourth of red cells

Structure

Granular contents

VWF

Properties

Adhesion

Activation& Release

Aggregation

Functions

• Temporary hemostasis• Blood coagulation• Clot retraction• Phagocytosis• Storage & transport• Wound healing & Vascular growth

Role in hemostasis

Role in hemostasis

Adhesion

• VWF – GpIb/IX/V • Collagen – GpIa/IIa• Fibronectin & Laminin – GpIc/IIa• Collagen – GpVI

Role in hemostasis

Role in hemostasis

Activation & Release Platelet agonists bind with specific membrane receptors ↓ G protein activation ↓ Activation of Phospholipase C ↓ Membrane inositol phospholipids ↓ IP3 , DAG

Role in hemostasis

IP3 • A calcium ionophore

• Causes calcium to enter the cytosol from the dense tubular system ( an internal platelet reservoir) and from the platelet exterior

• A rising cytosolic calcium concentration

Role in hemostasis

Calcium Rising cytosolic calcium concentration ↓ Activation of myosin light chain kinase ↓ Phosphorylation of myosin light chain ↓ Reorientation of cytoskeletal proteins ↓ Platelet shape change,secretion and contraction

Role in hemostasis

Calcium

• Contraction of the actin microfilaments – movement of granules to the open canalicular system – release

• Calcium and platelet agonists also activates phospholipase A2

Role in hemostasis

Activation & Release G protein activation and calcium ↓ Activation of Phospholipase A2

↓ Membrane phosphatidylcholine ↓ Arachidonic acid ↓COX PGH2 ↓ Thromboxane synthase TXA2

Role in hemostasis

• PGH2 – a cofactor enhancing the ability of collagen to function as a platelet agonist

• TXA2 – binds to a specific platelet membrane receptor – resultant activation of Phospholipase C – amplification of platelet activation through further generation of IP3,DAG

Role in hemostasis

• Activated platelets change shape from disc to tiny sphere with numerous projecting pseudopods

• Activated platelets exocytose the contents of their dense storage granules and alpha granules

• Platelet activating factor (PAF) – cytokine – neutrophils, monocytes & platelets ; produced from membrane lipids; acts via G proteins

Role in hemostasis

• Locally damaged red cells also release ADP which further activates the platelets

• Thrombospondin and Thrombonectin – facilitate contractile system activity – promote exocytosis of granular contents.

• Thrombospondin – binds to fibrinogen n GP IV receptor secondary phase of aggregation – larger, firmer aggregates

Role in hemostasis

Role in hemostasis

Role in hemostasis

Role in hemostasis

• Platelet activation – increased platelet coagulant activity

• Synthesize coagulation factor V

• Platelet phospholipids - Platelet factor 3 and 4 - accelerate the formation of Va, VIIIa

• Va – conversion of prothrombin to thrombin

• Platelets play a major role in formation of intrinsic prothrombin activator – clot formation

Role in hemostasis

XII XIIa

XI XIa

IXaIX

X Xa

VIIIaVIII

VII VIIa

X

Prothrombin

Intrinsic Pathway Extrinsic Pathway

VaV

Thrombin

Fibrinogen Fibrin monomer

Blood Coagulation Cascade

Collagen, HMW Kininogen, Kallikrein

↓

HMW Kininogen

Platelet phospholipidsCalcium

Platelet phospholipidsCalcium

Calcium

XIIIaXIIIStable fibrin polymer(clot)

Release of tissue factor(Tissue thromboplastin)

Platelet phospholipidsCalcium

Role in hemostasis

The activated platelets incorporated in the clot rearrange and contract their intracellular actin/myosin cytoskeleton.

The intracellular actin network - internal part of GpIIb/IIIa fibrinogen receptor.

The external part of GpIIb/IIIa - the fibrin network of the clot

As a result of platelet contractile force on the fibrin network, the formed clot will compact on itself and hence reduce its total volume.

Role in hemostasis Clot retraction

Platelet factor 4 Platelet derived growth factor(PDGF) Transforming growth factor β

• Chemoattractants for WBCs, Smooth muscle cells & fibroblasts.

• Activate these cells and accelerate wound healing.• PDGF – Potent mitogen for vascular smooth muscle.

Role in hemostasis Wound healing

Platelet function tests

• Platelet count• Bleeding time• Platelet aggregation test• Platelet adhesiveness test• Clot retraction time

Antiplatelet drugs

Bernard-Soulier Syndrome(Giant Platelet Syndrome)

• Discovered by Jean Bernard and Jean-Pierre Soulier, 1948

• Abnormality of the platelet glycoprotein Ib-IX-V complex, receptor for vWF – platelets cannot adhere

• Inherited in autosomal recessive manner

• Large platelets on peripheral blood smear• Normal count, Prolonged bleeding time• Decreased or absent glass bead retention• Platelets aggregate wt physiological agonists but fail to

aggregate wt ristocetin (similar to Von Willebrand disease)

Glanzmann's thrombasthenia

• Platelets lack GPIIb/IIIa; hence, no fibrinogen binding can occur

• Inherited in autosomal recessive manner

• Normal morphology and count • Platelets less able to aggregate ; defective clot retraction • Bleeding time is significantly prolonged• Platelets do not aggregate with all aggregating agents but

they aggregate with ristocetin.

Granule defects

• δ storage pool disease – dense body deficiency• α granule deficiency – grey platelet syndrome• αδ storage pool disease

• δ storage pool disease- Autosomal dominant- Absence of dense bodies- Seen in association with certain inherited disorders

Gray platelet syndrome

• A rare condition caused by a reduction or absence of the platelet alpha granules, or of the proteins contained in these granules

Pseudo gray platelet syndrome• Platelets in blood collected into EDTA appear gray and

agranular compared with platelets from citrated blood.• EDTA-exposed platelets show extensive activation, with loss of

storage granule contents and pseudopod formation

Von Willebrand disease (vWD)• Most common hereditary coagulation abnormality • Arises from a qualitative or quantitative deficiency of vWF• Hereditary – type 1, type 2, type 3.• Acquired

• Normal count, Prolonged bleeding time• Deficiency of factor VIII activity in the plasma• Platelets aggregate wt physiological agonists but fail to

aggregate wt ristocetin• Desmopressin – type 1 and 2 - stimulates release of VWF from

Weibel-Palade bodies of endothelial cells• vWF replacement therapy – type 3 disease

Thrombocytopenia• Pseudothrombocytopenia

• Decrease Production– Marrow Damage

• Aplasia• Drugs• Malignancy• Radiation

– Congenital Defects– Ineffective Production

• B12, Folic Acid Def

• Increase DestructionNon Immune• Disseminated Intravascular Coagulation• Thrombotic Thrombocytopenic Purpura• HELLP syndrome

Immune• Idiopathic Thrombocytopenic Purpura• Heparin Induced Thrombocytopenia• SLE, AIDS• Thrombotic Thrombocytopenic Purpura• Neonatal • Post transfusion

Thrombocytopenia

Idiopathic Thrombocytopenic Purpura

• Autoimmune• IgG autoantibody mediated platelet destruction• Thrombocytopenia with normal or increased number of

megakaryocytes • Diagnosis of exclusion• Childhood/Adult onset• Children – Acute; H/O viral infection , self-limiting• Adults – F > M; Chronic; longer course • 80% respond to Oral prednisolone – 3 months – if no

response – Splenectomy• Intravenous immunoglobulin or Immunosuppressive drugs

Dengue hemorrhagic fever

• Characterized by severe thrombocytopenia with bleeding manifestations

• Severity depends on the dengue virus subtype• Concomitant infection with more than one subtype• Dengue virus 2 – most severe – direct binding,

ultramicroscopic changes• Cytotoxic factor ; increased proinflammatory cytokines• Plasma leakage – decreased plasma proteins – decreased

fibrinogen

• PLATELET TRANSFUSION• FRESH FROZEN PLASMA

Transfusion

• Platelets collected by apheresis – intermittent/continuous flow cell separator.

• Single donor / Random donor platelet concentrate• Storage - 20-24ᵒ C under constant agitation – 5 days• Neither group specific or Rh specific – cross matching not

needed• Indications – thrombocytopenia • Contraindications – Immune mediated thrombocytopenia

Post transfusion

• 5-7 days after transfusion• Allo antibodies

• Anti-P1A1 [Antigen located on gp IIIa subunit]• Anti-Baka (Leka) [Antigen located on gp IIb subunit]

• Self – limiting ; 3-6 weeks

• High dose intravenous immunoglobulin – treatment of choice

References

• Boron & Boulpaep - Medical Physiology, 2nd Edition • Ganong's Review Of Medical Physiology, 24th Edition• Best & Taylor's Physiological Basis Of Medical Practice, 13/ E.• Dacie And Lewis Practical Haematology 11th Edition• de Gruchy's Clinical Haematology In Medical Practice, 5th Ed• Arthropod borne viral diseases – Current status and research

– D.Raghunath Rao, C.Durga Rao• Textbook of Medical Physiology G K Pal 2 E• Internet references

• Alfred Donne -1842 «globulin du chyle» (that is to say small globules derived from plasma) a sort of small globular, pale, opaline corpuscles visible in blood.

• Later described by Beale in 1850 as particles of «germinal matter» and by Zimmermann in 1860 as «small corpuscles»

Role in hemostasis

Clot retraction After the clot has been formed, the activated platelets

incorporated in the clot rearrange and contract their intracellular actin/myosin cytoskeleton.

The intracellular actin network is connected to the internal part of the integrin αIIbβ3 fibrinogen receptor. Following coagulation, the external part of αIIbβ3 will have bound to the fibrin network of the clot, and therefore, as a result of platelet contractile force on the fibrin network, the formed clot will compact on itself and hence reduce its total volume.

The mechanism is termed clot retraction.

Role in hemostasis