Lesson 1: Platelets

PLATELETS LIPIDS
 Granular cytoplasm with a membrane but NO NUCLEAR MATERIAL
 Origin: Megakaryocytes Phospholipid
 Diameter: 2.5 um  Phosphatidylcholine and Sphingomyelin - neutral charge ; plasma layer (location)
 Mean Platelet Volume: 8-10 fL  If bilayer, outward facing: Plasma Layer ; inward facing: Cytosol Layer
 Circulating, resting platelets (not activated plt) are biconvex - if activated it is no  Phosphatidylinositol (Supplies: Arachidonic Acid), Phosphatidylethanolamine and
longer biconvex but rather pseudopods Phosphatidylserine (Supplies: Flips [Flippase], Coagulation Factors assemble) -
 Collection in EDTA causes them to round up anionic, polar, inner cytoplasmic layer (inner leaflet/cytosol layer)
 Blood Film: Circular to Irregular, lavender and granular  Since PS is negative in charge it attracts positive charge which is Calcium -
 Cluster with RBCs near the center of the blood vessel important in blood coagulation
 Move laterally with WBCs into the white pulp of the spleen to be sequestered (1/3 of the
plt are being sequestered) Cholesterol
 Storage Site/Organ of Plt: Spleen  Stabilizes membrane
 Normal Count: 150-400/450 x 109/L (two-thirds) - SI Unit  Maintains fluidity
 Under hemostatic need, they become irregular and sticky, extending pseudopods, and  Helps control passage of material
adheres and aggregates  Location: Fatty Acid Tail - hydrophobic region of phospholipid layer
 Splenomegaly - leads to decreased plt count (thrombocytopenia)
 Splenectomy - leads to increased plt count (thrombocytosis) GLYCOPROTEINS AND PROTEOGLYCANS
 Within the membrane
RETICULATED PLATELETS OR STRESS PLATELETS  Support surface glycosaminoglycans, oligosaccharides and glycolipids
 Thrombocytopenia  Glycolipids - if the carbohydrate is directly bound to the phospholipid bilayer
 Markedly larger (Diameter: > 6 um ; MPV: 12-14 fL)  2 Types of Proteins
 Citrated Whole Blood: Cylindrical and Beaded  Integral /Transmembranous Proteins - Spans the entire membrane
 Carry free ribosomes and fragments of RER - signals early and rapid release ; stained  Peripheral /Skeletal Proteins - Proteins found on the inner side of the
with thiazole orange membrane; provides shape and structure to the plasmam membrane

PLATELET ULTRASTRUCTURE Glycocalyx

 Membrane surface - carbohydrate coat
RESTING PLATELET PLASMA MEMBRANE  Absorbs albumin, fibrinogen and other plasma proteins
 Phospholipid bilayer  Thicker than RBC and WBC
 Selectively permeable  Adhesive
 Negative surface charge - contains Sialic Acid

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SURFACE-CONNECTED CANALICULAR SYSTEM (SCCS) Integrins

 Plasma membrane invades the platelet interior  Bind collagen to enable platelet to adhere to injured blood vessel
 Twists enabling platelets to store additional quantities of proteins  GP Ia/IIa - collagen
 Route for endocytosis and for secretion of granular contents - only alpha granules  GP Ic/IIa (a5B1) - laminin
 GP Ic/IIa (a6B1) - fibronectin
DENSE TUBULAR SYSTEM
 Parallel and closely aligned to the SCCS PLASMA MEMBRANE RECEPTORS FOR ACTIVATION: STRs
 Condensed remnant of RER Agonists
 Sequesters calcium and bears a series of enzymes that support platelet activation  Activate platelets
(phospholipase A2, cyclooxygenase, thromboxane synthase, phospholipase C)  Ligands for STRs
 Control center for platelet activation  Thrombin - Proetease-Activated Receptor I and 4
 Calcium - major ion needed for platelet activation  ADP -
 Epinephrine -a2-adrenergic
PLASMA MEMBRANE RECEPTORS FOR ADHESION  Thromboxane A2
 CAM Integrin Family
 CAM Leucine-Rich Repeat Family ADDITIONAL PLATELET MEMBRANE RECEPTORS
 CAM Immunoglobulin Gene Family  ICAMS “Intracellular Adhesion Molecules” (CD50, CD54, CD102) - inflammation and
 CAM Selectin Family immune reaction
 Seven-Transmembrane Receptor (STR) Family  PECAM “Platelet Endothelial Cell Adhesion Molecule” (CD31) - platelet to WBC and
platelet to endothelial cell adhesion
Electrophoresis Current Ligand Cluster Designation  FcyRIIA (CD32) - Heparin Induced Thrombocytopenia
Nomenclature Nomenclature  P-selectin (CD62) - platelet adhesion and aggregation, stored in a-granules
GP Ia/II/a Integrin: a2B1 Collagen CD29, CD49b
GP Ic/IIa Integrin: a5B1 Laminin CD29, CD49e PLATELET CYTOSKELETON
GP Ic/IIa Integrin: a6B1 Fibronectin CD29, CD49f
GP VI CAM of the Ig family Collagen Microtubules
GP Ib IX/V CAM of the leucine- VWF and Thrombin CD42a, CD42b,  Maintain platelet shape
Major receptor for rich repeat family bind GP Iba CD42c, CD42d  Thick circumferential bundle
cell adhesion Thrombin cleaves a Ratio: 2:2:2:1  Disassemble at refrigerator temperature or with colchicine
(arteries & site on HP V Bernard-Soulier  Cylindrical
arterioles) Syndrome  Contract on activation releasing contents of a-granules
GP IIb/IIIa Integrin: aIIbB3 Fibrinogen, VWF CD41, CD61  Calcium: Major substance for contraction
Major receptor for Glanzmann  Reassemble to provide rigidity to pseudopods
plt aggregation Thrombasthenia
GP IV Miscellaneous plt Collagen II and Microfilaments
receptor Thrombospondin  Composed of actin
 Contractile and anchors glycoproteins and proteoglycans
 Also present in cytoplasm
 As calcium increases, actin contracts

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 Intermediate Filaments Membrane-Bound Proteins

 Desmin and Vimentin Restricted to a-granule P-selectin Guanidine Monophosphate
 Connect with actin and tubules, maintaining platelet shape membrane 33
In a-granule and plasma GP IIb/IIIa Cap1
PLATELET GRANULES membrane GP IV CD9
GP Ib/IX/V PECAM-1
a-Granules
 50-80 in each platelet - most abundant Gray Platelet Syndrome
 Stain medium gray with osmium  Inherited absence of a-granule contents
 Filled with proteins (endocytosed, synthesized in megakaryocytes, membrane-bound)  Membranes and membrane-bound proteins are present
 When platelets activate, granule membrane fuse with SCCS and contents flow  Soluble proteins within are missing
 Root for secretion of a-Granules is via the SCCS  Platelets appear large and light gray (Wright Stain)
 Majority factor of a-Granules are Coagulation Proteins  Diminshed response to ADP, collagen, epinephrine and thrombin
 Mucocutaneous bleeding - bleeding for platelets
REPRESENTATIVE a-GRANULE PROTEINS  Aggregometry Test
Coagulation Proteins Noncoagulation Proteins
Proteins Present in Plasma and a-Granules δ-Granules
Endocytosed Fibronectina Albumin  2-7 per platelet
Fibrinogen Ig  Dense bodies
Megakaryocyte Factor V  Stain black with osmium
Synthesized  Stores endocytosed molecules
Thrombospondin  Migrate to plasma membrane and release their contents
VWF
Present in a-Granules but not plasma (Megakaryocyte Synthesized) δ-Granules (Dense Body) Contents
Megakaryocyte B-Thromboglobulin Endothelial Growth Factor  Nonmetabolic
Synthesized ADP  Supports neighboring platelet
High-Molecular-Weight- Multimerin aggregation by binding to ADP
Kininogen receptors P2Y1, P2Y12
 Unknown function
Plasminogen Activate Platelet-Derived Collagenase  ATP release is detected using firefly
Inhibitor I Inhibitor ATP luciferase luminescence as an in vitro
emasure of plt activation - a method
Plasminogen Platelet-Derived Growth called lumiaggregometry
Factor  Vasoconstrictor that binds endothelial
Platelet Factor 4 Transforming Growth Factor- Serotonin cells and plt membranes
B Calcium and Magnesium  Divalent cations support plt activation
Protein C Inhibitor Vascular Endothelial Growth and coagulation
Factor/Vascular Permeability
Factor

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Storage Pool Disorder PLATELET ACTIVATION

 Diminished δ-granule contents  Platelet activation or the three functions of the platelet:
 Inherited diseases characterized by albinism (Hermansky-Pudlak Syndrome)  Adhesion
 Does not affect morphology  Aggregation
 Failure to secrete when treated with thrombin  Secretion

Lysosomes
ADHESION
 Stain positive for arylsulfatase, B-glucuronidase, ACP and catalase
 Digest vessel wall matrix and autophagic debris  Platelets reversibly bind elements of the vascular matrix
 Cleans the mess in cells  For repair of minor injuries to blood vessels
 Require an actin contraction and formation of pseudopods
ADDITIONAL NOTES  Platelet to non-platelet surface
 Platelets - major cell involved in coagulation  Non-platelet surface is commonly collagen
 Resting Platelets - does not form extension/pseudopods  Collagen is the one exposed from endothelial cell
 Spleen - site of platelet sequestration
 Splenomegaly - decreased plt count Veins or Venules
 Splenectomy - increased plt count = all plt circulate  Platelets adhere directly to collagen through GP Ia/IIa, GP IV and GP VI
 Adhesion - able to attach to different surface ; plt to non-plt attachment
 Aggregation - plt to pkt Capillaries and Arterioles
 Cytosol - fluid portion, inner leaflet  First carpeted by VWF which binds with collagen, after which, platelets adhere
 Plasma - outer leaflet (GP Ib/IX/V)
 5 Types of Phospholipids  Turbulent bloodflow
 Phosphatidylcholine and Sphingomyelin - head phosphate = neutral  von Willebrand Factor - Serves as a mediator and a strong protein that acts as
 Phosphatidylinositol, Phosphatidylethanolamine and Phosphatidylserine - a bridge between collagen and the platelets
fatty acid tail = anionic  VWF will attach to the collagen and then the platelets will attach to the VWF
 3.2% Sodium Citrate - for coagulation  GP Ib/IX/V - major receptor for platelet adhesion (esp alpha component)/ VWF
 Reticulum - network of RNA
site
 VWF Disease - normal plt, missing VWF
 Enzymes involved in plt activation: Phospholipase A2, Cyclooxygenase,
Assessment
Thromboxane Synthase, Phospholipase C)
 Measurement of VWF Activity
 5H2A - Serotonin
 Quantitation of CAMs through flow cytometry
 Veins - fibroblasts release collagen
 Arteries - fibroblasts release collagen --> VWF
Normal ECM Suppresses Hemostasis:
 Prostacyclin
 Heparin sulfate
 Tissue factor pathway inhibitor

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 Nitric oxide  Prothrombinase - combination of factor 10 and 5

 Thrombomodulin
Assessment
AGGREGATION  Lumiaggregometry - most successful and uses the enzyme firefly luciferase to
identify ATP (delta-granules)
 Platelets irreversibly bind each other
 Assays for PF4 or B-thromboglobulin
 Extensive vessel damage
 Requires active GP IIb/IIIa (binds arginine-glycine-aspartic acid [RGD]
PLATELET ACTIVATION PATHWAYS
sequence of plasma proteins; fibrinogen)
 Fibrinogen - major protein that has RGD sequence G PROTEINS
 Includes pseudopod formation and redistribution of P-selectin to surface
 Control cellular activation for all cells at the inner membrane surface
membrane
 Significance: Activation of an enzyme
 Phosphatidylserine flips to the outer layer  Heterotrimer - made up of 3 units (alpha, beta and gamma)
 Membrane integrity is lost  Alpha monomer - acts as the enzyme that will phosphorylate
 Platelet to platelet  Zymogen - resting enzyme
 Inactive G-proteins - bound to guanosine diphosphate
Irreversible End Point  Active G-proteins - bound to guanosine triphosphate
 Platelets + VWF = White Clot/Hyaline Clot - inappropriate platelet
activation in arterioles and arteries (arterial thrombosis) EICOSANOID SYNTHESIS (PROSTAGLANDIN, CYCLOOXYGENASE,
 Platelet-VWF Plug THROMBOXANE PATHWAY)
 Fibrin + RBCs = Red Clot - inappropriate coagulation in venules and veins  Triggered by G protein
(venous thrombosis)  Major Enzyme Activated: Phospholipase A2
 Phospholipase A2 - first enzyme to act on phosphatidylinositol
Assessment  Enzymes from the Dense Tubular System:
 Platelet Aggregometry  Cyclooxygenase - convert arachidonic acid to Prostaglandin G2
 Lumiaggregometry  Peroxidase - convert Prostaglandin G2 to Prostaglandin H2
 Thromboxane Synthase - convert Prostaglandin H2 to form Thromboxane
SECRETION A2
 Activated platelets release granular contents  If in endothelial cell, Prostacyclin Synthase is used instead of
 Ligand binding integrins and STR triggers actin contraction Thromboxane Synthase
 Intermediate filaments and microtubules contract, compressing granules  Thromboxane A2 - activates platelet, supresses adenylate cyclase
 alpha-granules and lysosomes: secrete via SCCs; some are coagulation factors  Diminished action of adenylate cyclase - no converion of ATP to cAMP =
 delta-granules - secrete via plasma membrane; vasoconstrictors and platelet decreased cAMP
agonists  cAMP - sequesters calcium in the dense tubular system (active)
 PS Flips Outward - tenase and prothrombinase form supported by calcium  If there is greater amount of cAMP - calcium will be stored in DTS (inactive)
secreted by delta-granules  Prostacyclin Synthase - counterpart of TXA2 ; platelet inhibitor
 Tenase - combination of Factor 8 and 9

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 HEMATOLOGY 2 | LECTURE | PRELIMS

 Deficiency: Platelet release disorder or Aspirin-like disorder

INOSITOL TRIPHOSPHATE AND DIACYLGLYCEROL

 Second G protein-dependent pathway
 Enzyme: Phospholipase C
 Cleaves phosphodiester bond from carbon 3 of phosphatidylinositol
 Products: Diacylglycerol and Inositol Triphosphate
 Diacylglycerol - activates 2nd enzyme known as Phosphokinase C =
activating the protein pleckstrin = actin microfilament contraction
 Inositol Triphosphate - releases ionic calcium from DTS

ADDITIONAL NOTES
 Dense Tubular System - platelet organelle that sequesters ionic calcium and
binds a series of enzymes of the Eicosanoid Pathway
 Phosphatidylserine - platelet membrane phospholipid flips from the inner
surface to the plasma surface on activation and serves as the assembly point
for coagulation factors
 GP IIb/IIIa - platelet membrane receptor that binds fibrinogen and supports
platelet aggregation
 Prostacyclin - eicosanoid metabolite produced from endothelial cells that
suppresses platelet activity
 Serotonin - molecule stored in platelet granules (dense bodies)
 Von Willebrand Factor - plasma protein essential to platelet adhesion

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