Platelet Study Guide

Platelets (Chapter 13)

Description

• Smallest blood cells.
• Anucleated (no nucleus) with granulated cytoplasm.
• Consists of granular cytoplasm with a membrane but no nuclear material; granules are important for coagulation.
• Diameter: $2.5 \mu m$
• MPV (Mean Platelet Volume): 8 to 10 fL
• Shape: Biconvex when circulating and resting; becomes round in EDTA.
• Blood film: Circular to irregular, lavender, and granular (darker reddish-violet).
• Cluster with RBCs near the center of the blood vessel.
• Move laterally with WBCs into the white pulp of the spleen for sequestration (1/3 of platelets).
• Normal count: $150 \times 10^9/L$ to $400 \times 10^9/L$ (two-thirds circulate).
• Under hemostatic need, platelets become irregular and sticky, extending pseudopods and adhering (to blood vessels) and aggregating (attaching to each other).
• Splenomegaly leads to Thrombocytopenia.
• Splenectomy leads to Thrombocytosis.
• Reticulated platelets or Stress platelets:
  • Cause: Severe thrombocytopenia.
  • Size: Markedly larger.
  • Diameter: >6 \mu m
  • MPV: 12 to 14 fL
  • Citrated whole blood: Cylindrical and beaded.
  • Carry free ribosomes and fragments of RER.
  • Signals early and rapid release.
  • Stain: Thiazole orange

Platelet Size and Appearance

• Size: 2-4µm
• Nucleus: NA
• Cytoplasm: Light blue to colorless
• Granules: red to violet, abundant (darker = more granules)
• N/C Ratio: NA
• Reference Interval
  • Bone Marrow: NA (megakaryocytes only)
  • Peripheral Blood: 7-25 per 100x OIF (1000x magnification)
• Small compared to RBC & WBC
• Removal
• Early reticulocyte
• Stress reticulocytes or shift reticulocytes
• In shape

Ultrastructure of Resting Platelet

Plasma Membrane

• Phospholipid bilayer; selectively permeable

Lipids

1. Phospholipid

   • Phosphatidylcholine and sphingomyelin – neutral; outer plasma layer
   • Phosphatidylinositol, phosphatidylethanolamine, and phosphatidylserine – anionic; polar; inner cytoplasmic layer; PI (arachidonic acid); PS (flips; coagulation factors assemble)

2. Cholesterol

   • Stabilizes the membrane.
   • Maintains fluidity.
   • Helps control passage of material.
   • Location: fatty acid tails, in hydrophobic region

Glycoproteins and Proteoglycans

• Within the membrane
• Support surface glycosaminoglycans, oligosaccharides, and glycolipids

Membrane Surface (Glycocalyx)

• Glycocalyx – carbohydrate coat; has sialic acid (-)
• Absorbs albumin, fibrinogen, and other plasma proteins (+)
• Glycocalyx + plasma proteins = stick to each other
• Thicker than RBC and WBC
• Adhesive
• Negative surface charge

Surface-Connected Canalicular System (SCCS)

• Unique structure only in platelets
• Plasma membrane invades the platelet interior
• Twists enabling platelets to store additional quantities of proteins
• Route for endocytosis and for secretion of granular contents

Dense Tubular System (DTS)

• Major structure: Majority of enzymes needed for platelet activation and calcium are all stored in the DTS.
• Parallel and closely aligned to the SCCS
• Condensed remnant of RER
• Sequesters $Ca^{2+}$ and bears a series of enzymes that support platelet activation (phospholipase A2, cyclooxygenase, thromboxane synthase, phospholipase C)
• Control center for platelet activation (and calcium storage)

Plasma Membrane Receptors for Adhesion

• Found in outer surface
• Cell adhesion molecule (CAM) family
  • CAM integrin family
  • CAM leucine-rich repeat family
  • CAM immunoglobulin gene family
  • CAM selectin family
• Seven-transmembrane receptor (STR) family
• Integrins bind collagen to enable platelets to adhere to injured blood vessel (GP – glycoprotein)
  • GP Ia/IIa ($\alpha<em>2\beta</em>1$) – collagen (GP VI, IV)
  • GP Ic/IIa ($\alpha<em>5\beta</em>1$) – laminin
  • GP Ic/IIa ($\alpha<em>6\beta</em>1$) – fibronectin
  • GP Ib/IX/V (1b95): Major receptor for cell adhesion
  • GP IIb/IIIa ($\alpha<em>2\beta</em>3$): Major receptor for platelet aggregation
• Integral protein/transmembrane proteins
  • Those that span the entire membrane
• Peripheral proteins/skeletal proteins
  • Proteins found in the inner side of the membrane
  • They provide shape and structure in the plasma membrane
• Outer surface of the cell
  • Proteins + carbohydrates
  • GP - glycoprotein

Plasma Membrane Receptors for Activation: STRs

• Thrombin
• ADP (adenosine diphosphate)
• Epinephrine
• $TXA_2$ (thromboxane A2)
• PAR – protease activated receptor
• P2Y – purines

Additional Platelet Membrane Receptor

• ICAMS (CD50, CD54, CD102) – inflammation and immune reaction
  • ICAMS – intercellular adhesion molecules
• PECAM (CD31) – plt to WBC and plt to endothelial cell adhesion
  • PECAM – Platelet endothelial cell adhesion molecule
• $Fc\gamma RIIA$ (CD32) – HIT (Heparin-induced thrombocytopenia)
• P-selectin (CD62) – plt adhesion and aggregation; stored in α-granules of plt

Platelet Cytoskeleton – Maintain Integrity

Microtubules

• Skeletal framework
• Maintain plt shape
• Thick circumferential bundle
• Disassemble at refrigerator temp. or with colchicine
• Shape: Cylindrical
• Contract on activation (Ca) releasing contents of α-granules (Secretion)
• Reassemble to provide rigidity to pseudopods
• Flexible; disassembles when plt is activated

Microfilaments

• Composed of actin
  • Contractile; anchors glycoproteins and proteoglycans
  • Also present in cytoplasm
  • As calcium increases, actin contracts

Intermediate Filaments

• Desmin and vimentin
• Connect with actin and tubules, maintaining plt shape

NOTE: Contraction of microtubules and filaments when it encounters CALCIUM

Electrophoresis Nomenclature

• “Agonists”
• activate platelets
• ligands for STRs

Platelet Granules

α-Granules

• Most abundant
• 50 to 80 in each plt (small in size)
• Stain medium gray with osmium
• Filled with proteins (endocytosed thru SCCS; synthesized in megakaryocytes; membrane-bound)
• When plts activate, granule membranes fuse with SCCS (excreted) and contents flow
• Route of secretion: SCCS
• Majority of contents – coagulation proteins

Gray Platelet Syndrome

• Inherited absence of α-granule contents; giant
• Membranes and membrane-bound proteins are present
• Soluble proteins within are missing
• Plts appear large and light gray (Wright stain)
• Diminished response to ADP, collagen, epinephrine and thrombin
• Mucocutaneous bleeding (under skin, mucosal surfaces; ex: nosebleed, menorrhagia)

δ-Granules

• a.k.a dense bodies
• 2 to 7 per plt
• Stain black with osmium
• Stores endocytosed molecules
• Migrate to the plasma membrane and release their contents
• Route of secretion: Plasma membrane

Storage Pool Disorder

• Diminished δ-granule contents
• Inherited diseases characterized by albinism (e.g. Hermansky-Pudlak syndrome)
• Does not affect morphology
• Failure to secrete when treated with thrombin

Lysosomes

• Have hydrolytic enzymes to clean up debris
• Stain positive (+) for: arylsulfatase, β-glucuronidase, ACP, and catalase (enzymes)
• Digest vessel wall matrix and autophagic debris

Processes Involved in Platelet Activation

1. Adhesion
2. Aggregation
3. Secretion

Adhesion

• Platelet to non-platelet (collagen)
• Most common occurrence (minor injuries)
• Plts reversibly bind elements of the vascular matrix
• For repair of minor injuries to blood vessels
• Veins or venules:
  • Plts adhere directly to collagen through GP Ia/IIa, GP IV and GP VI
• Capillaries and arterioles:
  • Blood flow is more turbulent
  • First carpeted by VWF (mediator – strong protein as a bridge) which binds with collagen; after which, plts adhere (GP Ib/IX/V)
• Require actin contraction and formation of pseudopods
• Assessment:
  • Measurement of VWF activity
  • Quantitation of CAMs through flow cytometry

In normal blood vessels without injury, flow of blood is smooth and continuous.

3 functions of platelets; happen simultaneously

Normal ECM suppresses hemostasis:

• Prostacyclin
• Heparan sulfate
• Tissue factor pathway inhibitor
• Nitric oxide
• Thrombomodulin

Aggregation

• Platelet to platelet
• Plts irreversibly bind each other
• Extensive vessel damage
• Requires active GP IIb/IIIa (binds RGD sequence of plasma proteins; fibrinogen & VWF)
  • RGD: Arginine-glycine-aspartic acid
• Includes pseudopod formation and redistribution of P-selectin to surface membrane
• PS flips to the outer layer
• Membrane integrity is lost
• Irreversible end point:
  • “white clot” / hyaline clot – inappropriate plt activation in arterioles and arteries (arterial thrombosis)
    • platelet-VWF plug (platelet + VWF)
  • “red clot” = Fibrin + RBCs; inappropriate coagulation in venules and veins (venous thrombosis)
• Assessment:
  • Platelet aggregometry
  • Lumiaggregometry

Secretion

• Activated plts release granular contents once squeezed or contracted
• Trigger: Ligand binding integrins and STRs triggers actin contraction
• Intermediate filaments and microtubules contract, compressing granules
• α-granules and lysosomes: SCCS; some are coagulation factors
• δ-granules: Plasma membrane; vasoconstrictors and plt agonists
• PS flips outward: Tenase (Factor VIII & Factor IX, cleave Factor X) and prothrombinase (Factor X & Factor V, cleave Factor II – prothrombin) form supported by calcium secreted by δ-granules
• Assessment:
  • Lumiaggregometry – most successful; firefly luciferase (make fireflies glow) to identify ATP (from δ-granules)
  • Assays for PF4 or β-thromboglobulin

Platelet Activation Pathways

G Proteins

• All cells have this for platelet activation and intracellular communication & signaling.
• Control cellular activation for all cells at the inner membrane surface

Eicosanoid Synthesis Pathway

• Prostaglandin, cyclooxygenase, or thromboxane pathway (major enzymes involved)
• Triggered by G protein
• Major enzyme activated: Phospholipase A2
• Deficiency: Platelet release disorder or Aspirin- like disorder
• Corresponding Pathway in Endothelial cells:
  • thromboxane synthase → prostacyclin synthase
  • $TXA<em>2$ → $PGI</em>2$ (prostacyclin)
  • Prostacyclin: platelet inhibitor

cAMP sequesters calcium into DTS → decreased cAMP will cause $Ca^{2+}$ to go out of DTS → $Ca^{2+}$ released in cytoplasm → cause platelet activation

Inositol Triphosphate and Diacylglycerol

• Second G protein-dependent pathway
• From phosphatidylinositol
• Major enzyme involved: Phospholipase C (PLC)
  • PLC breaks down phosphatidylinositol into 2 fragments: diacylglycerol (DAG) & inositol triphosphate (IP3)
• DAG: activates phosphokinase → phosphorylate flextrin → cause direct actin contraction
• IP3: causes release of $Ca^{2+}$ from DTS → platelet activation

Summary: Platelet Characteristics