Finals of Platelets

8/6/2019 Finals of Platelets

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FUNCTIONS OF PLATELETS

BY DR komal makwana

2nd YEAR RESIDENT ,

DEPARTMENT OF PHYSIOLOGY,

MEDICAL COLLEGE BHAVNAGAR.


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OUTLINE

History

Formation Physical and Chemical Characteristics of Platelets

Functions- Platelet Plug Formation

Drug developed &Applied physiology(not asseparate part ,it will walk along with physiology.)


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HISTORY

ADDISON in 1841 described platelets asextremely 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 thehypothesis that platelets are derived from thecytoplasm of the megakaryocytes and basis ofthrombopoieses were established.


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Formationof

platelets


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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 ofcytoplasm and extruding them into thecirculation

Megakayocytes


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Physical andChemical

Characteristics

of Platelets


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Platelets (also called thrombocytes) are minutediscs 1 to 4 micrometers in diameter.(Greekword thrombus- plates. )

The normal concentration of platelets in theblood is between 150,000 and 4,00,000 permicro litre.


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Platelets are emerging as remarkable cellfragment with abundant metabolic capability

but minimal ability to synthesize proteinbecause it contains only law levels of RNAand lacks nucleas.

Stress platelets- are large and beaded.

Reticulated platelets- large, high RNA,recently released from the marrow.


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Distributions and kinetics of platelets

Between 60% and 75% of the platelets that havebeen extruded from the bone marrow are in thecirculating blood, and the remainder are mostly inthe spleen.

Splenic sequestrations Splenic cords are minutevessels, platelets get into it because of very smallsize, and have high transition time.(epinephrine -contraction of spleen, epinephrine- intracardiac

contraction of pulmonary vessels.)

Splenectomy- thrombocytosis.


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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 11.

Elimination by reticuloendothelial cells mainly

spleen.


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Platelet surface

Platelet plasma membrane

Glycocalyx

Membrane rafts or GEMS-Glycolipid enrichedmembrane domain

Surface connected canalicular system


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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 internally2. Hydrophobic: Fatty Acid chains, orients towardseach other, perpendicular to the plane of the membrane


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Separates intracellular environment fromextracellular, 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 Phosphatidylethanolamineand phosphatidylserine,



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Glycolipid enriched membranedomain(GEMS) or MEMBRANE RAFTS.

GEMS are associated outside to signalingproteins of immune receptor associatedpathways and attached to cytoskeletoninternally


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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


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Surface connected canalicular system

SCCS has less developed Glycocalyx it meansit has less glycoprotein like gpI-IX-V complex.

Act as reservoir of membrane to facilitateplatelet spreading and filopodias formationafter adhesion

Storage reservoir for membrane glycoproteinlike gp2b-3a that increases on platelet surfaceactivation.


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Route for granule release during plateletactivation and secretion.

Route for ingress and egress of molecules asthey translocate between plasma and platelet.


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Storage organs

Four types

granules

Dense bodies Lysosomes

microperoxisomes


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granules

Electron dense zones

Eccentrically

placed Rich in platelet

specific proteins

such as thromboglobulin.

less Electron density zone

Periphery

Vwf andmultimerine alongwith factor 5


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granules also have fibrinogen which isactively incorporated from plasma.

PDGF,VEGF(angiogenesisstimulator),endostatin(angiogenesis inhibitor)


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Dense bodies

3 to 5/cell

Bulls eye appearance electron dense

ATP , pyrophosphate, calcium, serotonin,GTP, GDP, magnesium.


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Circumferential microtubules maintains plateletshape

8-20 subunits of tubules made up of tubulin

Tubulin disassemble at refrigerator temperature orwith colchicine

Contract upon activation to allow the expression ofalpha granules

Assemble longitudinally to privide rigidity topseudopods

Platelet Cytoskeleton: Microfilamentsand Microtubules


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Microfilaments and Microtubules

In general functions for:

1. platelet shape changes2. extension of pseudopods

3. secretion of granule contents

Platelet Cytoskeleton


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Platelet Cytoskeleton: Mricrofilamentsand Microtubules

Microfilaments are thick meshwork of actinfilaments

Actin is contractile and anchors the membraneglycoproteins and proteoglycans

Actin also present throughout the cytoplasm,making 20-30% of platelet proteins

Platelet Ultrastructure


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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.


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Platelet biochemistry and metabolisam

Dry weight 60% proteins, 15% lipids, 8%carbohydrate .

Concentration of sodium and potasium 39and 138 mEq/l, respectively.

Maintained by active Na+/k+ ion pump, deriveenergy from membrane ATPase , which isoubain sensitive .


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Calcium pool in platelet

Unstimulated platelet maintain a low cytoplasmicca2+ concentration 100 to 500 nmol/l bylimiting ca2+ transport from plasma and

promoting active efflux. Two kinds of pool present , rapidly turning over

cytosolic pool regulated by a sodium-calciumantiporter in plasma membrane

more slowly exchanging pool regulated byca2+/mg2+ ATPase and sequestered in densetubular system.


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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 heartdisease

iv. COX Inhibitors (NSAIDs) (antiplateletdrugs)


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First we will see the events of hemostasis

superficially,

After getting the idea of major events we willstudy the role of platelet in detail.


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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 contentsand potentiate clotting


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Platelet adhesion , activation

aggregation


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GPIa/IIa and GPIb are platelet membrane proteins that bindto collagen and von Willebrand factor (vWF), causingplatelets to adhere to the subendothelium of a damagedblood vessel.

PAR1 and PAR4 are protease-activated receptors thatrespond to thrombin (IIa); P2Y1 and P2Y12 are receptors forADP (adenosine diphosphate); when stimulated by agonists,these receptors activate the fibrinogen-binding proteinGPIIb/IIIa and cyclooxygenase-1 (COX-1) to promoteplatelet aggregation and secretion.

Thromboxane A2 (TXA2) is the major product of COX-1involved in platelet activation. Prostaglandin I2 (PGI2),synthesized by endothelial cells, inhibits platelet activation.


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interactions among proteins of the "extrinsic"(tissue factor and factor VII), "intrinsic"(factors IX and VIII), and "common" (factorsX, V, and II) coagulation pathways

Factor xa bind to factor 5a on the surface ofactivated platelet , a specific surface protein to

which Factor X can bind, which is missing inscott syndrome.


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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


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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:


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1.Integrin GP Ia/Ila or 21: binds the adhesive

protein laminin and fibronectin

GP IIb/IIIa: ligand is Fibrinogen, VWF,vitronectin, fibronectin: target RGDamino acid sequence

forms a heterodime aIIbb3 afterencountering an inside out signalingmechanism

Glanzmann thrombasthenia

Platelet Plasma Membrane Receptors:ADHESION


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2. Immunglobulin Gene

GP VI: binds collagen and adhesive protein

Thrombospondin3. Leucine-rich Repeat Family

GP Ib/IX/V: 7 non covalently bound molecules withratios 2:2:2:1(will see them in figure)

(2) GP 1ba: accounts for VWF binding; binding ofthrombin

(2) GP IbB: crosses the membrane and interacts withactin binding protein: outside in signalling(we will

see what is the outside in signaling)


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3. Leucine-rich Repeat Family

(2) GP IX: associated withmucocutaneous bleeding disorderand Bernard Soulier Syndrome

(1) GP V


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Platelet Plasma Membrane Receptors:ACTIVATION: 7 transmembrane Rec

Thrombin, adenosine diphosphate, epinephrine andprostaglandin (eicosanoids) pathway productTXA2

PAR1, PAR4, P2Y1, P2Y12, adrenergic, PGI2


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Plt l t i 1b l


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Plt glycoprotein 1b complex- von

willebrand factor interaction and

signaling


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vWF

VWFgene : short arm of chromosome 12

VWFgene is expressed in endothelial cells andmegakaryocytes

vWF is produced as a propeptide which is extensivelymodified to produce mature vWF

Two vWF monomers bind through disulfide bonds

to form dimers Multiple dimers combine to form vWF multimers


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Vwf has A1 and A3 domains that binds tocollagen.

The A1 domain contains binding site for GP1bcomplex, primary role player in adhesion,changes its conformation in response to highshear forces, thus making high affinity ligand

for GPIb complex.


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GPIb complex signaling

GPIb signals lead to

Elevation ofintracellular calcium.

Activation of tyrosin kinase signaling pathway Activation ofinside out signaling through

IIb3integrin followed by platelet

aggregation.

Activation of protein kinase cPKC, protein

kinase GPKG, phosphoinositide 3 kinasePI3K.


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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 21 mainly adhesion

,gp vI- main player in collagen mediated

platelet activation


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GPVI and FcR chain signaling

FcR chain leads to

tyrosine

phosphorylation of the

immunoreceptortyrosin based activation

motifITAM


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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 C2(PLC2

C2).

PLC2 play critical role in aggregation and

secretion- generation of second messengers ITP& DAG- intracellular calcium rises & activation of

PKC.


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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 IIb3

supporting platelet plug formation, produce

red clot.


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Platelet ADP receptor and signaling

Two G protien coupled receptor are involved in

ADPinduced Platelet aggregation.

P2Y1 is associated with (phospholipase c

PLC)intra cellular calcium calcium mobilization,

shape change , and transient aggregation .

P2Y12- is associated with (adenylate cyclase-

convert ATPTO AMP)- do amplification ofaggregation and potentiation of secretion means

action of 2b3 (y means phosphotyrosinse.)


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Platelet ADP receptor and signaling


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TXA2 and TXreceptors

Arachidonic

acid

TxA2

GP IIb/IIIa

Epinephrine

CollagenThrombin

ADP


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. Mechanism of platelet aggregation to form a clot

ADP

releasedfrom

platelet

granule

receptor

Ca2+ influx

in other platelets

MembranePhospholipase

A2 activated

Phospholipid

Arachidonate

cyclooxygenaseacetylates

Aspirin

(drug)

PGH2

Thromboxane A2 (TxA2)

Diacylglycerol (DAG)+ inositol triphosphate

(IP3)

Phosphatidylinositol

MembranePhospholipase C

collagen

receptor

COLLAGEN

TxA2synthetase

Dazoxiben

(drug)

receptor

Ca2+ InfluxContraction

of microtubules

Granule

release

PLATELET

AGGREGATION

2O2


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Platelet aggregation, integrin receptor

and signaling


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SIGNAL srC- syk(tyrosin kinase) that bound to

tail of B3- adaptor protein SLP 76 Rac

GTPase vav.- all these lead to actin

reorganization.


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utside in platelet activation through ligandbinding to Integrins and Seven transmembranereceptors (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 Activation-Secretion


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Platelet in atherogenesis


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These drugs act by discrete mechanisms, andthus in combination their effects are additiveor even synergistic. Their availability has led

to a revolution in cardiovascular medicine,whereby angioplasty and vascular stenting oflesions now is feasible with low rates of

restenosis and thrombosis when effectiveplatelet inhibition is employed


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In platelets, the major cyclooxygenase product

is thromboxane A2, a labile inducer of platelet

aggregation and a potent vasoconstrictor.

Aspirin blocks production of thromboxane A2by acetylating a serine residue near the active

site of platelet cyclooxygenase (COX-1), the

enzyme that produces the cyclic endoperoxideprecursor of thromboxane A2.


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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 isachieved when 160 mg of aspirin is taken daily


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Dipyridamole interferes with platelet function byincreasing the cellular concentration of adenosine3,5-monophosphate (cyclic AMP). This effect ismediated by inhibition of cyclic nucleotide

phosphodiesterase and/or by blockade ofuptake ofadenosine, which acts at adenosine A2 receptors tostimulate platelet adenylyl cyclase. The only currentrecommended use of dipyridamole is in combinationwith warfarin for postoperative primary prophylaxis of

thromboemboliin patients with prosthetic heartvalves.


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P2Y1 and P2Y12 antagonist

Clopidogrel . Purinergic receptors respond to extracellularnucleotides as agonists. Platelets contain two purinergicreceptors, P2Y1 and P2Y12; both are GPCRs for ADP. The

ADP-activated platelet P2Y1 receptor couples to the Gq-PLC-IP3-Ca2+pathway and induces a shape change andaggregation. The P2Y12 receptor couples to Gi and, whenactivated by ADP, inhibits adenylyl cyclase, resulting inlower levels of cyclic AMP and thereby less cyclic AMP-dependent inhibition of platelet activation. Based on

pharmacological studies, it appears that both receptors mustbe stimulated to result in platelet activation , and inhibitionof either receptor is sufficient to block platelet activation


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Ticlopidine (TICLID) is a that inhibits theP2Y12 receptor. Ticlopidine is a prodrug thatrequires conversion to the active thiol

metabolite by a hepatic cytochrome P450enzyme. It permanently inhibits the P2Y12receptor by forming a disulfide bridge between

the thiol on the drug and a free cysteineresidue in the extracellular region of thereceptor and thus has a prolonged effect


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Abciximab. (REOPRO) is the Fab fragment of ahumanized monoclonal antibody directed againstthe aIIbb3 receptor. It also binds to the vitronectinreceptor on platelets, vascular endothelial cells,

and smooth muscle cells. The antibody is used in conjunction with

percutaneous angioplasty for coronarythromboses, and when used in conjunction with

aspirin and heparin, has been shown to be quiteeffective in preventing restenosis, recurrentmyocardial infarction, and death.


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Eptifibatide (INTEGRILIN) is a cyclic peptideinhibitor of the fibrinogen binding site onaIIbb3. It blocks platelet aggregation.

It is used to treat acute coronary syndromeand for angioplastic coronary interventions. its

benefit is somewhat less than that obtainedwith the antibody Abciximab , perhaps

because eptifibatide is specific for aIIbb3 anddoes not react with the vitronectin receptor.


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Platelet disorder


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THANK YOU

Documents

Finals of Platelets