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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,
Platelet Plasma Membrane
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Platelet Plasma Membrane
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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61
. 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