Study Notes on Platelet Production, Structure, and Function

• Platelets are nonnucleated blood cells that originate from megakaryocytes, the
largest cells in the bone marrow.

• Circulates at a concentration of 150 – 450 x 109 /L

• Platelets: ↑ women ↓ men ---------------- men & women > older than 65 years

• Platelets trigger primary hemostasis (stop bleeding) at time of blood vessel injury

I. Megakaryocytopoiesis

• Platelets arise from unique bone marrow cells called megakaryocytes, which are the
largest cells in the bone marrow and possess multiple chromosome copies
(polyploid).

• Megakaryocytes account for less than 0.5% of all bone marrow cells and localize
adjacent to venous sinusoid endothelial cells within vascular niches.

• On a Wright-stained bone marrow aspirate smear, each megakaryocyte is 30 to 50

μm in diameter.

• Bone marrow stromal cells release an array of cytokines that influence

megakaryocyte maturation, including thrombopoietin (TPO), the key regulator of
megakaryocytopoiesis.

• Megakaryocytes can also be found in the lungs, where they may contribute to
overall platelet production.

• Megakaryocyte Differentiation and Progenitors

o Megakaryocyte progenitors rise from common myeloid progenitors under

transcription factors like GATA-1 and FOG1.

▪ GATA-1: Regulates megakaryocyte differentiation. (from box 11.1 GATA

1 is Globin transcription factor-1, a protein product of the X chromosome
gene GATA1)

▪ FOG1: Friend of GATA (From box 11.1 It is a product of the ZFPM1 (zinc
finger protein multitype 1) gene.)

o Megakaryocyte differentiation is suppressed by MYB, which regulates

differentiation between platelet and RBC lineages.

▪ MYB: A transcription factor that suppresses megakaryocyte

differentiation, balancing erythropoiesis and megakaryocytopoiesis.
 o Three megakaryocyte lineage-committed progenitor stages (from common
myeloid progenitor:

▪ BFU-Meg (Burst-forming Unit Megakaryocyte): The least mature

megakaryocyte progenitor stage that forms hundreds of colonies in
culture. (normal mitosis)

▪ CFU-Meg (Colony-forming Unit Megakaryocyte): An intermediate

progenitor stage that forms dozens of colonies in culture. (normal mitosis)

▪ LD-CFU-Meg Light-Density Colony-forming Unit Megakaryocyte): The

most mature megakaryocyte progenitor stage, which undergoes
endomitosis.

• Endomitosis:

o A unique mitosis lacking telophase and cytokinesis.

o Switch mediated by transcription factors RUNX1 and NF-E2.

▪ RUNX1: A transcription factor that mediates the switch from mitosis to

endomitosis by suppressing the Rho/ROCK signaling pathway.

▪ NF-E2: A transcription factor that promotes DNA replication during

endomitosis to increase ploidy levels.

o Results in polyploid cells (8N to 32N).

• Terminal Megakaryocyte Differentiation

o A series of stages exhibiting unique Wright-stained morphology in bone marrow

aspirate smears or hematoxylin & eosin-stained bone marrow biopsy sections.

o Includes stages MK-I, MK-II, and MK-III, each showing distinct morphological
characteristics:

▪ MK-I (Megakaryoblast): Least differentiated megakaryocyte precursor.

Cytoplasm is basophilic, and the formation of the demarcation
membrane system (DMS) begins. Although no longer lymphocyte-like,
MK-I cannot reliably be distinguished from myeloblasts or pronormoblasts
by light microscopy. Plasma membrane blebs resembling platelets may
project from the margin.

▪ MK-II (Promegakaryocyte): Nuclear lobularity becomes apparent with

moderate condensation of chromatin. Increases ploidy levels.

▪ MK-III (Mature Megakaryocyte): The cell’s diameter increases to 30-50

μm. The nucleus becomes intensely indented or lobulated, and the
cytoplasm appears filled with platelets. Cytoplasm becomes more
abundant, granular, and azurophilic, reflecting the synthesis of α-
granules and dense granules. Platelet shedding begins at this stage.

o Demarcation Membrane System (DMS): This unique structure subdivides the

cytoplasm into areas that eventually become individual platelets during
thrombocytopoiesis.
• Thrombocytopoiesis (Platelet Shedding)

o Each megakaryocyte releases 2,000-4,000 platelets, producing up to 1011

platelets per day in an average-sized adult.

o Platelet turnover rate: 8–9 days in circulation, with mass regulated by pro- and
anti-apoptotic proteins (e.g., Bak, Bcl-xL).

o Proplatelet Processes: These long cytoplasmic extensions penetrate through or

between sinusoid-lining endothelial cells into the venous blood, where platelets
are shed.

o The process leaves behind residual megakaryocyte nuclei, which are

phagocytized by marrow macrophages.

o DMS Role in Platelet Shedding: The DMS dilates, and proplatelet processes form
longitudinal tubules and transverse constrictions before platelet release.

• Megakaryocyte Membrane Receptors and Markers

o Markers:

▪ MPL: The thrombopoietin (TPO) receptor present at all stages of

maturation.

▪ CD34: A stem cell marker expressed in early stages of differentiation,

disappearing as maturation progresses.

▪ CD41/CD61: Glycoproteins IIb/IIIa involved in adhesion, first appears on

megakaryocyte progenitors and is expressed throughout development.

o Cytoplasmic Contents:

▪ Coagulation factors VIII and VWF are synthesized during maturation and
stored for subsequent platelet function.

▪ Coagulation factors VIII and VWF present in mature cells.

• Hormones and Cytokines

o Thrombopoietin (TPO):

▪ A 70,000-Dalton growth factor primarily synthesized in the liver. It

stimulates the proliferation, differentiation, and maturation of
megakaryocytes, stimulate RBC production, and regulates platelet
production by binding megakaryocyte to the MPL receptor.

▪ Circulating TPO levels are inversely proportional to platelet and

megakaryocyte mass due to binding and removal by platelets as a
primary mechanism for controlling platelet count.
 ▪ TPO also induces the proliferation and maturation of megakaryocytes
and induces thrombocytopoiesis

o Interleukins:

▪ IL-3: Acts synergistically with TPO during early differentiation.

▪ IL-6 and IL-11: Enhance endomitosis, megakaryocyte maturation, and

thrombocytopoiesis.

o Synthetic TPO Receptor Agonists:

▪ Medications like romiplostim and eltrombopag are used to stimulate

platelet production in thrombocytopenic patients.

II. Platelets

• Platelets are anucleate, granular cytoplasmic fragments derived from

megakaryocytes.

• Respond to vascular injury, aggregating at the site to prevent bleeding.

• Average diameter: 2.5 µm.

• Appear circular to irregular on Wright-stained blood films and are distributed among
the RBC monolayer at 7 – 21 cells per 100x field.

• Mean Platelet Volume (MPV): Ranges from 7 to 12 fL.

o Indicates subpopulations, including large platelets.

o Random size variation due to release volume, not age or vitality.

• Resting platelets:

o Biconvex shape.

o Smooth flow in vessels; cluster near erythrocytes in the bloodstream.

• EDTA-anticoagulated blood:

o Platelets "round up."

• Spleen:

o Platelets dynamically move between blood and spleen, maintaining

equilibrium.

• Normal platelet count:

o Peripheral blood: 150–450 × 10⁹/L.

o Declines with age:

 ▪ Men (≥65 years): 122–350 × 10⁹/L.

▪ Women (≥65 years): 140–379 × 10⁹/L.

o Two-thirds circulate in the blood; one-third is sequestered in the spleen.

• Reticulated (Stress) Platelets:

o Compensatory production for thrombocytopenia.

o Larger size (≥6 µm diameter; MPV of 12–14 fL).

o Contain RNA and fragments of rough endoplasmic reticulum.

o Potentially prothrombotic; linked to conditions like sepsis, coronary artery

disease, and preeclampsia.

o Measured using nucleic acid dyes (e.g., thiazole orange), but dense granule
nucleotides may interfere with accuracy.

o Indicative of rapid proplatelet release during thrombocytopenia.

III. Platelet Ultrastructure

• Resting Platelet Plasma Membrane:

o A bilayer composed of phospholipids, cholesterol, and transmembrane

glycoproteins.

o Glycocalyx: A 20–30 nm thick external layer containing glycosylated receptors

and adhesive proteins (e.g., fibrinogen).

▪ Facilitates protein endocytosis and platelet adhesion by supporting

plasma protein transport to internal organelles.

o Outer layer: Neutral phospholipids (e.g., phosphatidylcholine, sphingomyelin).

o Inner layer: Charged phospholipids (e.g., phosphatidylinositol,

phosphatidylserine, phosphatidylethanolamine).

• Surface-Connected Canalicular System (SCCS):

o Invaginations of the plasma membrane extending deeply into the cytoplasm,

resembling a spongy network.

o Store hemostatic proteins, allow for endocytosis and secretion.

o Secrete contents from α-granules to the platelet's exterior during activation.

o Enhance the release of hemostatic proteins during activation.

• Dense Tubular System (DTS):

o A remnant of the rough endoplasmic reticulum.

 o Sequesters Ca²⁺ and contains enzymes like phospholipase A2 and
thromboxane synthase, critical for activation.

• Cytoskeleton:

o Microtubules:

▪ Maintain discoid shape.

▪ Composed of 8–20 tubulin subunits arranged in cylindrical structures (25

nm diameter).

▪ Reorganize during activation, facilitating granule release and

pseudopodia formation.

o Actin Microfilaments:

▪ Aid in contraction and activation.

▪ Constitute 20–30% of total platelet protein.

▪ Resting state: Globular actin polymerizes into contractile filaments upon

calcium activation.

o Intermediate Filaments:

▪ Composed of desmin and vimentin.

▪ Interact with actin and tubulin to maintain platelet shape and support
structural changes during activation.

• Platelet Granules:

o α-Granules:

▪ Contain ∼50–80 granules per platelet.

▪ Store coagulation proteins (e.g., fibrinogen, VWF), adhesion molecules

(e.g., thrombospondin), and growth factors (e.g., platelet-derived
growth factor).

▪ Release contents via fusion with the SCCS during activation.

o Dense Granules:

▪ Contain ∼2–7 granules per platelet.

▪ Store small molecules like ADP, ATP, calcium ions, and serotonin.

▪ Amplify platelet activation by promoting further aggregation and

vasoconstriction.

o Lysosomes:

▪ Contain hydrolytic enzymes (e.g., acid phosphatase) to digest

extracellular matrix components during aggregation.
• Plasma Membrane Receptors That Provide for Adhesion

o Platelet adhesion depends on glycoprotein receptors, integrins, and cell

adhesion molecules (CAMs) that interact with ligands like VWF, fibrinogen, and
collagen.

o GPIb/IX/V Complex:

▪ Member of the leucine-rich repeat (LRR) family of adhesion receptors.

▪ Binds VWF and enables platelet tethering under high shear stress in
arterioles and capillaries.

▪ Stabilizes platelet adhesion to subendothelial surfaces and mediates

rolling along the vessel wall.

o GPVI:

▪ A major receptor for subendothelial collagen exposed during vascular

injury.

▪ Activates intracellular signaling cascades that enhance integrin

activation and platelet spreading.

▪ Works in conjunction with integrins like α2β1 to maintain stable adhesion.

o GPIIb/IIIa (Integrin αIIbβ3):

▪ Central to platelet aggregation by binding soluble fibrinogen and VWF.

▪ Undergoes conformational change during "inside-out" signaling,

increasing ligand-binding affinity.

▪ Facilitates interplatelet cross-linking to form stable platelet plugs.

o Integrins:

▪ Mediate "outside-in signaling" by transmitting extracellular signals to the

platelet cytoskeleton.

▪ Integrin α2β1 binds collagen and enhances adhesion under low shear
stress.

▪ Provide structural support for cytoskeletal rearrangements, enabling

platelet spreading and clot stability.

• Seven-Transmembrane Repeat Receptors

o Seven-Transmembrane Repeat Receptors (STRs) are integral plasma

membrane proteins involved in signal transduction.

o These receptors feature seven hydrophobic transmembrane domains

anchored to the lipid bilayer, enabling them to interact with intracellular G-
proteins.
 • Key Receptors and Ligands:

o PAR1 and PAR4: Cleaved by thrombin, initiating activation of at least two

physiologic pathways.

o P2Y1 and P2Y12: ADP receptors that regulate intracellular calcium levels and
promote irreversible platelet aggregation.

o TPα and TPβ: Receptors for thromboxane A2 that enhance autocrine platelet
activation.

• Additional Membrane Receptors

o PECAM-1 (CD31): Mediates adhesion between platelets, endothelial cells,

and leukocytes.

o P-selectin (CD62P): Facilitates interactions with leukocytes by binding P-

selectin glycoprotein ligand-1 (PSGL-1).

o FcγRIIa (CD32): Engages immune complexes, contributing to inflammatory

and immune responses.

IV. Platelet Function

Platelet activation is key to preventing blood loss and is integral to both primary and
secondary hemostasis. Platelet function includes platelet adhesion, aggregation, and
secretion.

• Aggregation: Platelets Irreversibly Cohere

o Platelets adhere and form clusters to prevent blood loss at injury sites.

o Mechanisms:

▪ Platelets tether to exposed subendothelial collagen via von Willebrand

Factor (VWF).

▪ GPIb/IX/V interaction with VWF enables deceleration of platelets in

high shear environments.

▪ GPVI binds directly to collagen, triggering activation pathways.

▪ Activation of integrins, including GPIIb/IIIa, enhances fibrinogen-

mediated aggregation.

▪ ADP and thromboxane A2 (TXA2) amplify aggregation by activating

neighboring platelets.

o Key Receptors:

▪ GPIbα: Binds VWF and mediates tethering.

▪ GPVI: Key receptor for fibrillar collagen.

 ▪ Integrins (e.g., α2β1 and GPIIb/IIIa): Essential for firm adhesion and
aggregation.

o Dysregulated aggregation contributes to thrombosis in myocardial infarction

and stroke.

• Secretion: Activated Platelets Release Granular Contents

o Granule Types and Their Contents:

▪ α-Granules: Contain adhesive proteins (e.g., VWF, fibrinogen) and

growth factors like PDGF.

▪ Dense Granules: Contain ADP, ATP, calcium, and serotonin.

▪ Lysosomes: Contain enzymes like acid phosphatase.

o Mechanism:

▪ Outside-in signaling via receptors (e.g., GPVI) triggers cytoskeletal

contraction.

▪ Dense granules release their contents through direct plasma

membrane fusion.

▪ α-Granules release contents via the surface-connected canalicular

system (SCCS).

o Role in Coagulation:

▪ Polar phospholipids on the platelet surface localize coagulation

factors.

▪ Dense granule calcium supports formation of tenase and

prothrombinase complexes.

▪ α-Granule proteins like fibrinogen stabilize clot formation.

• Generation of Platelet Microparticles

o Formation:

▪ Platelet activation causes increased intracellular calcium and

cytoskeletal cleavage by calpain.

▪ Membrane blebbing leads to microparticle release.

o Characteristics:

▪ Composed of plasma membrane and cytosolic contents.

▪ Express phosphatidylserine and retain parent cell proteins.

o Functions:

▪ Promote coagulation through phosphatidylserine.

 ▪ Modulate inflammation and oxidative stress.

o Elevated microparticles in conditions like disseminated intravascular

coagulation (DIC).

V. Platelet Signaling Pathways

• G-Proteins

o Structure: Heterotrimeric proteins with α, β, and γ subunits.

o Activation:

▪ Ligand binding induces GDP-to-GTP exchange on the Gα subunit.

▪ GTP-bound Gα dissociates, activating downstream effectors.

o Signaling Outcomes:

▪ Activation of eicosanoid synthesis and the IP3-DAG pathway.

• Eicosanoid Synthesis

o Steps:

▪ Phospholipase A2 liberates arachidonic acid from membrane

phospholipids.

▪ Cyclooxygenase (COX) converts arachidonic acid to prostaglandin H2.

▪ Thromboxane synthase produces TXA2.

o Role of TXA2:

▪ Enhances calcium mobilization and granule secretion.

▪ Promotes aggregation via autocrine and paracrine effects.

o Clinical Relevance: Aspirin inhibits COX to reduce thrombotic risk.

• Inositol Triphosphate-Diacylglycerol (IP3-DAG) Pathway

o Process:

▪ Phospholipase C cleaves PIP2 to form IP3 and DAG.

▪ IP3 mobilizes calcium from the dense tubular system.

▪ DAG activates protein kinase C, leading to pleckstrin phosphorylation.

o Effects:

▪ Cytoskeletal contraction and granule release.

▪ Amplification of platelet activation.

VI. Platelets and Inflammation

Platelets interact with immune complexes via FcγRIIa and express Toll-like receptors to
recognize pathogens.

• Proinflammatory Mediators:

o Platelet factor 4 (PF4), RANTES, and interleukin-1β.

• Interactions:

o P-selectin mediates platelet-leukocyte aggregation.

o CD40 ligand enhances monocyte and endothelial activation.

• Functions:

o Link between hemostasis and immune response.

o Contribution to chronic inflammation in atherosclerosis.

Platelet Proteome

• Despite lacking a nucleus, platelets synthesize proteins using RNA transcripts stored in
their cytoplasm.

• Proteins produced dynamically in response to environmental signals enhance

platelet functionality.

• Contains mRNA, ribosomes, and translational machinery.

• Platelets synthesize proteins like GPIIb/IIIa upon activation.

• Key Proteins:

o Fibrinogen, thrombospondin, and plasminogen activator inhibitor-1.

• Alterations in the proteome are markers for diseases like sepsis and cancer.

SUMMARY (see from the book)