FUNCTIONS OF PLATELETS

BY DR komal makwana 2nd YEAR RESIDENT , DEPARTMENT OF PHYSIOLOGY, MEDICAL COLLEGE BHAVNAGAR.

OUTLINE
History Formation Physical and Chemical Characteristics of Platelets Functions- Platelet Plug Formation Drug developed &Applied physiology(not as separate part ,it will walk along with physiology.)

HISTORY
ADDISON in 1841 described platelets as extremely minute granules in clotting blood. They were termed platelets by BIZZOZERO who observed their adhesive properties. They were microscopically examined by OSLER & SCHAEFER and by HAYEM in 19th century. In 1906 JAMES HOMER WRIGHT put the hypothesis that platelets are derived from the cytoplasm of the megakaryocytes and basis of thrombopoieses were established.

Formation of platelets

Megakayocytes
Platelets arise from megakaryocytes Largest cells in the body (30-50um) Polyploid Cluster on extravascular compartment The megakaryocytes, giant cells in the bone marrow, form platelets by pinching off bits of cytoplasm and extruding them into the circulation

Physical and Chemical Characteristics of Platelets

 Platelets (also called thrombocytes) are minute discs 1 to 4 micrometers in diameter.(Greek word thrombus- plates. ) The normal concentration of platelets in the blood is between 150,000 and 4,00,000 per micro litre.

 Platelets are emerging as remarkable cell fragment with abundant metabolic capability but minimal ability to synthesize protein because it contains only law levels of RNA and lacks nucleas. Stress platelets- are large and beaded. Reticulated platelets- large, high RNA, recently released from the marrow.

Distributions and kinetics of platelets

Between 60% and 75% of the platelets that have been extruded from the bone marrow are in the circulating blood, and the remainder are mostly in the spleen. Splenic sequestrations Splenic cords are minute vessels, platelets get into it because of very small size, and have high transition time.(epinephrine contraction of spleen, epinephrine- intracardiac contraction of pulmonary vessels.) Splenectomy- thrombocytosis.

Distributions and kinetics of platelets

normally have a half-life of about 5 days Normal platelet lifespan is 10 d. Every day, 1/10 of platelet pool is replenished. Daily turn over- 1.2 to 1.5 10 Elimination by reticuloendothelial cells mainly spleen.
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Platelet surface
Platelet plasma membrane Glycocalyx Membrane rafts or GEMS-Glycolipid enriched membrane domain Surface connected canalicular system

Platelet Plasma Membrane

Resting Platelet Plasma Membrane
Resembles a biologic membrane Bilayers composed of proteins and lipids Predominant lipid: Phospholipids, Cholesterol Phospholipids forms a Bilayer 2 parts: 1. Hydrophilic: Polar heads out to the plasma externally and cytoplasm internally 2. Hydrophobic: Fatty Acid chains, orients towards each other, perpendicular to the plane of the membrane

Platelet Plasma Membrane

Separates intracellular environment from extracellular, 20 nm thick. Conducts events like permeability, agonist stimulation, adhesion ,activation , secretion. ASSYMETRIC DISTRIBUTION Neutral Phospholipids present outside- : Phosphatidylcholine, Sphingomyelin - Inner cytoplasmic Layer(negative): Phosphatidylinositol Phosphatidylethanolamine and phosphatidylserine,

Platelet Plasma Membrane

Glycolipid enriched membrane domain(GEMS) or MEMBRANE RAFTS. GEMS are associated outside to signaling proteins of immune receptor associated pathways and attached to cytoskeleton internally

Glycocalyx 20-30 nm Thicker than WBCs and RBC. Platelets carries its functional environment with it to maintain a negative environment Anchored within the membrane are: 1. Glycoprotein 2. Proteoglycans Endocytosis: transport mechanism used by the glycocalyx

Surface connected canalicular system

SCCS has less developed Glycocalyx it means it has less glycoprotein like gpI-IX-V complex. Act as reservoir of membrane to facilitate platelet spreading and filopodias formation after adhesion Storage reservoir for membrane glycoprotein like gp2b-3a that increases on platelet surface activation.

 Route for granule release during platelet activation and secretion. Route for ingress and egress of molecules as they translocate between plasma and platelet.

Storage organs
Four types granules Dense bodies Lysosomes microperoxisomes

granules
Electron dense zones
less Electron density zone

Eccentrically placed Rich in platelet specific proteins such as thromboglobulin.

Periphery Vwf and multimerine along with factor 5

granules also have fibrinogen which is actively incorporated from plasma. PDGF,VEGF(angiogenesis stimulator),endostatin(angiogenesis inhibitor)

Dense bodies
3 to 5/cell Bulls eye appearance electron dense ATP , pyrophosphate, calcium, serotonin, GTP, GDP, magnesium.

Platelet Cytoskeleton: Microfilaments and Microtubules

Circumferential microtubules maintains platelet shape 8-20 subunits of tubules made up of tubulin Tubulin disassemble at refrigerator temperature or with colchicine Contract upon activation to allow the expression of alpha granules Assemble longitudinally to privide rigidity to pseudopods

Platelet Cytoskeleton
Microfilaments and Microtubules In general functions for: 1. platelet shape changes 2. extension of pseudopods 3. secretion of granule contents

Platelet Ultrastructure
Platelet Cytoskeleton: Mricrofilaments and Microtubules
Microfilaments are thick meshwork of actin filaments Actin is contractile and anchors the membrane glycoproteins and proteoglycans Actin also present throughout the cytoplasm, making 20-30% of platelet proteins

Platelet Ultrastructure
Platelet Cytoskeleton: Microfilaments and Microtubules
Intermediate Filaments: ropelike polymers; 8-12 nm in diameter; made up of desmin and vimectin Connects with actin and the microtublues and helps maintain the cell shape

Platelet dust or micro particles

New emerging concept Contribute to plasma coagulation specifically factor 10 and thrombin generation APPLIED deficiency of it seen in Scott syndrome associated with clinical bleeding.

Platelet biochemistry and metabolisam

Dry weight 60% proteins, 15% lipids, 8% carbohydrate . Concentration of sodium and potasium 39 and 138 mEq/l, respectively. Maintained by active Na+/k+ ion pump, derive energy from membrane ATPase , which is oubain sensitive .

Calcium pool in platelet

Unstimulated platelet maintain a low cytoplasmic ca2+ concentration 100 to 500 nmol/l by limiting ca2+ transport from plasma and promoting active efflux. Two kinds of pool present , rapidly turning over cytosolic pool regulated by a sodium-calcium antiporter in plasma membrane more slowly exchanging pool regulated by ca2+/mg2+ ATPase and sequestered in dense tubular system.

Platelet function:
A)Platelet function:
i. Blood clotting and stroke (anticoagulant drugs) ii. Prostaglandin and thromboxane formation from polyunsaturated fatty acids iii. Nutritional approach to prevent heart disease iv. COX Inhibitors (NSAIDs) (antiplatelet drugs)

 First we will see the events of hemostasis superficially, After getting the idea of major events we will study the role of platelet in detail.

First thing to learn

Platelet Plug Formation Adhesion - Platelets stick to injured vessel wall. Aggregation - Platelets stick to each other via fibrinogen bridges. Secretion - Platelets release granular contents and potentiate clotting

Platelet adhesion , activation aggregation

 GPIa/IIa and GPIb are platelet membrane proteins that bind to collagen and von Willebrand factor (vWF), causing platelets to adhere to the subendothelium of a damaged blood vessel. PAR1 and PAR4 are protease-activated receptors that respond to thrombin (IIa); P2Y1 and P2Y12 are receptors for ADP (adenosine diphosphate); when stimulated by agonists, these receptors activate the fibrinogen-binding protein GPIIb/IIIa and cyclooxygenase-1 (COX-1) to promote platelet aggregation and secretion. Thromboxane A2 (TXA2) is the major product of COX-1 involved in platelet activation. Prostaglandin I2 (PGI2), synthesized by endothelial cells, inhibits platelet activation.

Platelet interaction with coagulation system

 interactions among proteins of the "extrinsic" (tissue factor and factor VII), "intrinsic" (factors IX and VIII), and "common" (factors X, V, and II) coagulation pathways Factor xa bind to factor 5a on the surface of activated platelet , a specific surface protein to which Factor X can bind, which is missing in scott syndrome.

Receptors of platelet, heart of haemostasis

So first we will see which receptor binds to whom what is the mechanism of action associated disease due to deficiency of that receptor agonist and antagonist of that receptor

Platelet Plasma Membrane Receptors:

ADHESION and AGGREGATION
Cellular Adhesion Molecule 1. Integrin Family 2. Lucien-rich Repeat Family 3. Immunoglobulin gene Family 4. Selectin Family 5. Seven Transmembrane Family

Platelet Plasma Membrane Receptors: ADHESION

1.Integrin
GP Ia/Ila or 2 1: binds the adhesive protein laminin and fibronectin GP IIb/IIIa: ligand is Fibrinogen, VWF, vitronectin, fibronectin: target RGD amino acid sequence forms a heterodime aIIbb3 after encountering an inside out signaling mechanism Glanzmann thrombasthenia

2. Immunglobulin Gene GP VI: binds collagen and adhesive protein Thrombospondin 3. Leucine-rich Repeat Family GP Ib/IX/V: 7 non covalently bound molecules with ratios 2:2:2:1(will see them in figure) (2) GP 1ba: accounts for VWF binding; binding of thrombin (2) GP IbB: crosses the membrane and interacts with actin binding protein: outside in signalling(we will see what is the outside in signaling)

3. Leucine-rich Repeat Family (2) GP IX: associated with mucocutaneous bleeding disorder and Bernard Soulier Syndrome (1) GP V

 Platelet Plasma Membrane Receptors: ACTIVATION: 7 transmembrane Rec

Thrombin, adenosine diphosphate, epinephrine and prostaglandin (eicosanoids) pathway product TXA2 PAR1, PAR4, P2Y1, P2Y12, adrenergic, PGI2

Plt glycoprotein 1b complex- von willebrand factor interaction and signaling

VWF Function
Adhesion Mediates the adhesion of platelets to sites of vascular injury (subendothelium) Links exposed collagen to platelets Mediates platelet to platelet interaction Binds GPIb and GPIIb-IIIa on activated platelets Stabilizes the hemostatic plug against shear force

vWF VWF gene : short arm of chromosome 12 VWF gene is expressed in endothelial cells and megakaryocytes vWF is produced as a propeptide which is extensively modified to produce mature vWF Two vWF monomers bind through disulfide bonds to form dimers Multiple dimers combine to form vWF multimers

 Vwf has A1 and A3 domains that binds to collagen. The A1 domain contains binding site for GP1b complex, primary role player in adhesion, changes its conformation in response to high shear forces, thus making high affinity ligand for GPIb complex.

GPIb complex signaling

GPIb signals lead to Elevation of intracellular calcium. Activation of tyrosin kinase signaling pathway Activation of inside out signaling through IIb 3 integrin followed by platelet aggregation. Activation of protein kinase cPKC, protein kinase G PKG, phosphoinositide 3 kinasePI3K.

Platelet collagen interaction and signaling

Very imp activators of plt in vascular sub endothelium and vessel wall. Prime target for therapeutic intervention in patient of MI and stroke. 2 receptor , integrin 2 1 mainly adhesion ,gp vI- main player in collagen mediated platelet activation

GPVI and FcR chain signaling

FcR chain leads to tyrosine phosphorylation of the immunoreceptor tyrosin based activation motif ITAM

 ITAM lead to recruitment of the tyrosine kinase (Syk) through its tandem Src-homology(SH2) So activation of syk occurs, which lead to phosphorylation of phospholipase C 2(PLC 2 C 2). PLC 2 play critical role in aggregation and secretion- generation of second messengers ITP & DAG- intracellular calcium rises & activation of PKC.

 Why arteries have white clot? And veins have red? At high shear rate, arteries gpIb/v/IX receptors and vwf ligand play major role, fibrinogen is only stabilizing factor. Low shear rates , veins fibrinogen IIb 3 supporting platelet plug formation, produce red clot.

Protease activated receptor

PAR is G protein coupled receptor that convert extra cellular protein cleavage into intra cellular activation signal. Here the ligand is part of receptor and amino acid sequence here is SFLLRN

 THROMBIN comes and cleave the peptide bond between Arginine 41 and Serine42. These tethered ligand join irreversibly with the body of the receptor gives transmembrane signaling. subunits of G12,G13 platelet shape change subunits of Gq platelet secretion aggregation ,participates in activation of PIC PIC - calcium mobilization and PKC activation.

Platelet ADP receptor and signaling

Two G protien coupled receptor are involved in ADP induced Platelet aggregation. P2Y1 is associated with (phospholipase c PLC)intra cellular calcium calcium mobilization, shape change , and transient aggregation . P2Y12- is associated with (adenylate cyclaseconvert ATP TO AMP)- do amplification of aggregation and potentiation of secretion means action of 2b 3 (y means phosphotyrosinse.)

Platelet ADP receptor and signaling

TXA2 and TXreceptors

Collagen
Epinephrine
Arachidonic acid

Thrombin ADP

TxA2

GP IIb/IIIa

. Mechanism of platelet aggregation to form a clot

Phospholipid ADP released from platelet granule receptor Ca2+ influx in other platelets Membrane Phospholipase A2 activated Aspirin (drug) Arachidonate acetylates cyclooxygenase 2O2

Diacylglycerol (DAG) + inositol triphosphate (IP3)

PLATELET AGGREGATION Dazoxiben (drug) Granule release

PGH2 TxA2 synthetase

Phosphatidylinositol Membrane collagen receptor

Thromboxane A2 (TxA2) receptor

Phospholipase C

Contraction of microtubules COLLAGEN

Ca2+ Influx
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Platelet aggregation, integrin receptor and signaling

 SIGNAL srC- syk(tyrosin kinase) that bound to tail of B3- adaptor protein SLP 76 Rac GTPase vav.- all these lead to actin reorganization.

Platelet Activation-Secretion
utside in platelet activation through ligand binding to Integrins and Seven transmembrane receptors (STRs) Triggers actin microfilament contraction Intermediate filaments and microtubules contract = compression of granules Contents of the alpha granules and lysosomes flow through the SCCS Contents of delta granules are secreted through the plasma membrane

Platelet in atherogenesis

ANTIPLATELET DRUGS
Platelets provide the initial hemostatic plug at sites of vascular injury, participate in pathological thromboses that lead to myocardial infarction, stroke, and peripheral vascular thromboses. Potent inhibitors of platelet function have been developed in recent years.

 These drugs act by discrete mechanisms, and thus in combination their effects are additive or even synergistic. Their availability has led to a revolution in cardiovascular medicine, whereby angioplasty and vascular stenting of lesions now is feasible with low rates of restenosis and thrombosis when effective platelet inhibition is employed

 In platelets, the major cyclooxygenase product is thromboxane A2, a labile inducer of platelet aggregation and a potent vasoconstrictor. Aspirin blocks production of thromboxane A2 by acetylating a serine residue near the active site of platelet cyclooxygenase (COX-1), the enzyme that produces the cyclic endoperoxide precursor of thromboxane A2.

 Since platelets do not synthesize new proteins, the action of aspirin on platelet cyclooxygenase is permanent, lasting for the life of the platelet (7 to 10 days). repeated doses of aspirin produce a cumulative effect on platelet function. Complete inactivation of platelet COX-1 is achieved when 160 mg of aspirin is taken daily

 Dipyridamole interferes with platelet function by increasing the cellular concentration of adenosine 3,5-monophosphate (cyclic AMP). This effect is mediated by inhibition of cyclic nucleotide phosphodiesterase and/or by blockade of uptake of adenosine, which acts at adenosine A2 receptors to stimulate platelet adenylyl cyclase. The only current recommended use of dipyridamole is in combination with warfarin for postoperative primary prophylaxis of thromboemboli in patients with prosthetic heart valves.

P2Y1 and P2Y12 antagonist

Clopidogrel . Purinergic receptors respond to extracellular nucleotides as agonists. Platelets contain two purinergic receptors, P2Y1 and P2Y12; both are GPCRs for ADP. The ADP-activated platelet P2Y1 receptor couples to the GqPLC-IP3-Ca2+ pathway and induces a shape change and aggregation. The P2Y12 receptor couples to Gi and, when activated by ADP, inhibits adenylyl cyclase, resulting in lower levels of cyclic AMP and thereby less cyclic AMPdependent inhibition of platelet activation. Based on pharmacological studies, it appears that both receptors must be stimulated to result in platelet activation , and inhibition of either receptor is sufficient to block platelet activation

 Ticlopidine (TICLID) is a that inhibits the P2Y12 receptor. Ticlopidine is a prodrug that requires conversion to the active thiol metabolite by a hepatic cytochrome P450 enzyme. It permanently inhibits the P2Y12 receptor by forming a disulfide bridge between the thiol on the drug and a free cysteine residue in the extracellular region of the receptor and thus has a prolonged effect

 Abciximab. (REOPRO) is the Fab fragment of a humanized monoclonal antibody directed against the aIIbb3 receptor. It also binds to the vitronectin receptor on platelets, vascular endothelial cells, and smooth muscle cells. The antibody is used in conjunction with percutaneous angioplasty for coronary thromboses, and when used in conjunction with aspirin and heparin, has been shown to be quite effective in preventing restenosis, recurrent myocardial infarction, and death.

 Eptifibatide (INTEGRILIN) is a cyclic peptide inhibitor of the fibrinogen binding site on aIIbb3. It blocks platelet aggregation. It is used to treat acute coronary syndrome and for angioplastic coronary interventions. its benefit is somewhat less than that obtained with the antibody Abciximab , perhaps because eptifibatide is specific for aIIbb3 and does not react with the vitronectin receptor.

Platelet disorder

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