PLATELETS

LEARNING OUTCOMES

¡ Describe the ultrastructure of resting platelets in

the circulation
¡ Define platelet function, including adhesion,
aggregation, and secretion.
¡ Explain the biochemical pathways of platelet
activation
RECALL:
MEGAKARYOCYTOPEISIS
¡ Megakaryocytopoiesis - refers to the process of the development and
maturation of megakaryocytes, the precursor cells responsible for
producing platelets.
Megakaryocytes
o the largest cells in the bone marrow
o possess multiple chromosome copies (polyploid).
o are 30 to 50 micrometers in diameter with a multilobulated nucleus and
abundant granular cytoplasm.
o account for less than 0.5% of all bone marrow cells
o two to four megakaryocytes per low-power field may be identified on a normal
Wright- stained bone marrow aspirate smear
RECALL:
MEGAKARYOCYTOPEISIS
Megakaryocytes
o cluster alongside other hematopoietic stem cells in vascular
niches adjacent to venous sinusoid endothelial cells

Rodak’s Hematology, 6th ed C10, p137, fig 10.1

RECALL:
MEGAKARYOCYTOPEISIS
Megakaryocytes
o extend proplatelet processes into the venous
sinuses, releasing platelets from the tips of the
processes into the circulation.

o are also found in the lungs.

Rodak’s Hematology, 6th ed C10, p137, fig 10.1

RECALL:
MEGAKARYOCYTOPEISIS

¡ Megakaryocyte progenitors arise from the common myeloid progenitor

v Stimulated GATA-1 (globin transcription factor-1) which is regulated by cofactor
FOG1 (“friend of GATA,”)
v suppressed by MYB (myeloblastosis)
¡ Three megakaryocyte lineage-committed progenitor stages,
1. burst-forming unit (BFU-Meg)
2. colony-forming unit (CFU-Meg)
3. light-density CFU (LD-CFU-Meg)
RECALL:
MEGAKARYOCYTOPEISIS

Endomitosis - a form of mitosis without telophase and cytokinesis

v Switch from mitosis to endomitosis is mediated by RUNX1 which suppresses the
Rho/ROCK signaling pathway, thus preventing cytokinesis.
vSubsequently, DNA replication proceeds to the production of 8N, 16N, or even
32N ploidy with duplicated chromosome sets. under the influence of transcription
factor NF-E2,
vMegakaryocytes employ their multiple DNA copies to synthesize abundant
cytoplasm, which ultimately differentiates into platelets.
 RECALL:
MEGAKARYOCYTOPEISIS

¡ TERMINAL MEGAKARYOCYTE DIFFERENTIATION

A. The megakaryoblast (MK-I) - has cytoplasmic “blebs” that resemble platelets
B. Promegakaryocyte (MK-II) – with abundant cytoplasm, is abundant, whil e the nucleus has minimal lobularity.
C Megakaryocyte (MK-III) - nucleus is lobulated, cytoplasm is granular, with evidence of the demarcation system
(DMS).
D. Terminal megakaryocyte shedding platelets from the proplatelet process.

The DMS
Ø membrane-lined channels from the plasma membrane growing inward to subdivide the cytoplasm
which will ultimately delineate the individual platelets during thrombocytopoiesis.

Rodak’s Hematology, 6th ed C10, p138, fig 10.3

RECALL:
MEGAKARYOCYTOPEISIS

Thrombocytopoiesis (Platelet Shedding)

• During thrombocytopoiesis, a single megakaryocyte may shed 2000 to 4000 platelets.
• In an average-size healthy human , there are 108 megakaryocytes, producing 1011
platelets per day.
• Total platelet population turnover (life span): 8 to 9 days
• Naked megakaryocyte nuclei are consumed by marrow macrophages
RESTING PLATELET
Resting plasma membrane
• resembles any biologic membrane
• With thicker glycocalyx
v During platelet activation phosphatidylinositol in the inner, cytoplasmic
layer of the phospholipid bilayer supplies arachidonic acid, an
unsaturated fatty acid that becomes converted to the eicosanoids,
including the potent thromboxane A2

Rodak’s Hematology, 6th ed C10, p141, fig 10.6

 RESTING PLATELET
Surface-Connected Canalicular System
¡ The plasma membrane extends in the interior, forming a unique surface-connected
canalicular system (SCCS)
• enables the platelet to store additional quantities of the same hemostatic proteins
found on the glycocalyx.
• allows enhanced interaction of the platelet with its environment
• Serves as the route for endocytosis and for secretion of alpha-granule contents
upon platelet activation.

Rodak’s Hematology, 6th ed C10, p142, fig 10.7

RESTING PLATELET

Dense Tubular System (DTS)

¡ Parallel and closely aligned to the SCCS
¡ condensed remnant of the rough endoplasmic reticulum
¡ sequesters Ca2+
¡ has several enzymes that support platelet activation including
phospholipase A2, cyclooxygenase, and thromboxane synthetase, which
support the production of thromboxane A2, and phospholipase C, which
supports the production of the signaling molecules inositol triphosphate
(IP3) and diacylglycerol (DAG).
Rodak’s Hematology, 6th ed C10, p142, fig 10.7
RESTING PLATELET

Cytoskeleton
v Microtubules, actin microfilaments, and intermediate microfilaments
control platelet shape change, extension of pseudopods, and secretion
of granule contents.

Rodak’s Hematology, 6th ed C10, p142, fig 10.7

RESTING PLATELET
Cytoskeleton
¡ A thick circumferential bundle of microtubules
• maintains the platelet’s discoid shape
• parallel to the plane of the outer surface of the platelet.
• move inward on platelet activation to enable the release of a-granule
contents and subsequently reassemble in long parallel bundles during
platelet shape change to provide rigidity to pseudopods.

Rodak’s Hematology, 6th ed C10, p142, fig 10.7

RESTING PLATELET

Cytoskeleton
¡ Microfilaments
• between the microtubules and the plasma membrane
• are composed of actin.
• Contractile
• anchors the plasma membrane glycoproteins and proteoglycans.
• In the resting platelet, actin is globular and amorphous, but as the cytoplasmic
calcium concentration rises, actin becomes filamentous and contractile.

Rodak’s Hematology, 6th ed C10, p142, fig 10.7

RESTING PLATELET

Cytoskeleton:
¡ Intermediate filaments
• connect with actin and the tubules, maintaining the platelet shape.

Rodak’s Hematology, 6th ed C10, p142, fig 10.7

RESTING PLATELET

Platelet Granules
a-granules:
• Each platelet has 50 to 80 a-granules
• Are filled with proteins, some endocytosed, some synthesized within the
megakaryocyte and stored in platelets

• During platelet activation, the membranes of alpha-granules merge with SCCS,

enabling the release of their contents into the surrounding microenvironment.
Which then contribute to platelet adhesion and aggregation, as well as to plasma
coagulation
Rodak’s Hematology, 6th ed C10, p142, fig 10.7
 RESTING PLATELET

Platelet Granules

Rodak’s Hematology, 6th ed C10, p148, tab 10.8

RESTING PLATELET

Platelet Granules
Dense granules
• Each platelet has two to seven dense granules
• Appear later than a-granules in megakaryocyte
• Small molecules are probably endocytosed and are stored in the dense
granules;
• In contrast to the a-granules, dense granules migrate to the plasma
membrane and release their contents directly into the plasma on platelet
activation.
• Membranes of dense granules support the same integral proteins as the a-
granules th
Rodak’s Hematology, 6 ed C10, p142, fig 10.7
RESTING PLATELET

Platelet Granules

Rodak’s Hematology, 6th ed C10, p143, tab 10.5

Rodak’s Hematology, 6th ed C10, p142, fig 10.7
PLASMA MEMBRANE
RECEPTORS THAT PROVIDE
FOR ADHESION

•The platelet membrane contains over 50 distinct

receptors, which play crucial roles in platelet function.
•Receptors belong to various families, including cell
adhesion molecule (CAM) family, integrin family, the
seven-transmembrane receptor family, and some
miscellaneous receptors
Rodak’s Hematology, 6th ed C10, p144, fig 10.8
PLASMA MEMBRANE
RECEPTORS THAT PROVIDE
FOR ADHESION

See table 10.6

Rodak’s Hematology, 6th ed C10, p145, table 10.6

PLASMA MEMBRANE
RECEPTORS THAT PROVIDE
FOR ADHESION

• Seven transmembrane receptors, also known as G protein-coupled receptors

(GPCRs), are a large family of cell surface receptors.
• They play diverse roles in cellular signaling, including in platelet activation and other
physiological processes.
• GPCRs have seven transmembrane alpha-helices that traverse the lipid bilayer of the
cell membrane.
• Extracellular loops connect the helices on the outer side of the membrane, while
intracellular loops connect them on the inner side.
PLASMA MEMBRANE
RECEPTORS THAT PROVIDE
FOR ADHESION

• GPCRs are activated by binding to specific ligands, which can be

hormones, neurotransmitters, or other signaling molecules.
• Upon activation, GPCRs interact with heterotrimeric G proteins located
on the cytoplasmic side of the membrane.
• This interaction triggers the dissociation of the G protein into its α, β,
and γ subunits, leading to the activation of various intracellular
signaling cascades.
• GPCRs play a crucial role in platelet activation and aggregation
PLASMA MEMBRANE
RECEPTORS THAT PROVIDE
FOR ADHESION

See table 10.7

Rodak’s Hematology, 6th ed C10, p146, table 10.7

 PLASMA MEMBRANE
RECEPTORS THAT PROVIDE
FOR ADHESION

• Other miscellaneous receptors play various roles in platelet function, inflammation,

immune reactions, and pathological conditions.
§ intercellular adhesion molecules (ICAMs) such as CD50, CD54, and CD102 which
mediate inflammation and immune reactions.
• Platelet-endothelial cell adhesion molecule (PECAM or CD31) facilitates platelet-
to-white blood cell and platelet-to-endothelial cell adhesion.
• FcgIIA (CD32) i- low-affinity receptor for immunoglobulin Fc portion involved in
heparin-induced thrombocytopenia.
• P-selectin - facilitates platelet binding to endothelial cells, leukocytes, and other
platelets.

Rodak’s Hematology, 6th ed C10, p146, table 10.7

THE PLATELETS

§ Normal peripheral blood platelet count:

• Range: 150 to 400 x 109/L
§ Age-related variations
• Decrease with increasing age
• Platelet counts after 65 years of age
• Men: 122 to 350 x 109/L
• Women: 140 to 379 x 109/L
THE PLATELETS

• Circulating platelets exhibit specific characteristics: biconvex

• Platelets collected with EDTA (ethylenediaminetetraacetic acid) tend to
change shape: more rounded or spherical
• Examination of platelets on a Wright-stained blood film reveals distinct
features.
• Appearance: circular to irregular
• Color: lavender
• Texture: granular
• Light microscopy offers limited insight into platelet internal structure.
THE PLATELETS

§ Platelets flow smoothly in the blood vessels.

§ Platelets vs. Leukocytes:
§ Platelets cluster with erythrocytes, while leukocytes roll along vascular
endothelium.
§ Platelets vs. Erythrocytes:
§ Platelets move back and forth with leukocytes into the white pulp of
the spleen, unlike erythrocytes.
THE PLATELETS
§ Platelet distribution
• 2/3 in peripheral blood
• 1/3 sequestered within the spleen
• Function of sequestered platelets
• Immediate availability in times of demand
• Acute inflammation
• After an injury
• After major surgery
• During plateletpheresis.
• Increased sequestration (hypersplenism/splenomegaly): may lead to relative
thrombocytopenia
THE PLATELETS
Reticulated platelets
§ also known as stress platelets, appear in response to thrombocytopenia.
§ are larger than ordinary mature circulating platelets.
• diameter in peripheral blood films > 6µm (normal platelet: 2.5µm)
• mean platelet volume (MPV) reaches 12 to 14 fL. (normal platelet: 8 to 10
femtoliter)
§ are potentially prothrombotic, may be associated with an increased risk of
cardiovascular disease.
§ cylindrical and beaded in citrated whole blood; resemble fragments of
megakaryocyte proplatelet processes
• May have arisen from early and rapid proplatelet extension and release.
PLATELET FUNCTION

¡ Platelet adhesion, aggregation, and activation are

crucial processes in hemostasis and thrombosis.
¡ They involve the recruitment, activation, and
aggregation of platelets at the site of vascular
injury.
PLATELET ADHESION

¡ Platelet adhesion is the initial step in hemostasis.

¡ It involves the binding of platelets to exposed subendothelial
matrix components such as collagen and von Willebrand factor
(VWF).
¡ Receptors involved in platelet adhesion include GP Ia/IIa (a2b1),
GP VI, and GP Ib/IX/V.
PLATELET ACTIVATION

¡ Platelet activation is triggered by various agonists released at the

site of injury, such as thrombin, ADP, and thromboxane A2.

¡ Activation leads to changes in platelet shape, secretion of granule

contents, and exposure of activation-dependent surface receptors.

¡ Key receptors involved in platelet activation include G protein-

coupled receptors (GPCRs) and integrins such as GP IIb/IIIa
(aIIbb3).
PLATELET AGGREGATION

• Platelet aggregation is the process by which activated platelets

adhere to each other via fibrinogen bridges.
• It results in the formation of platelet aggregates or clumps,
which strengthen the hemostatic plug.
• GP IIb/IIIa (aIIbb3) plays a central role in platelet aggregation by
binding fibrinogen and other adhesive proteins.
PLATELET ACTIVATION PATHWAYS

¡ The eicosanoid synthesis pathway, also known as the prostaglandin, cyclooxygenase,

or thromboxane pathway, is a key mechanism of platelet activation triggered by G-
proteins.
¡ The inner leaflet of the platelet membrane is rich in phosphatidylinositol, a
phospholipid that binds arachidonic acid (a type of unsaturated fatty acid) at the
number 2 carbon position.
¡ Membrane receptor-ligand binding activates phospholipase A2, an enzyme that
cleaves arachidonic acid from the phospholipid membrane.
PLATELET ACTIVATION PATHWAYS
¡ Arachidonic acid is converted to prostaglandin G2 and prostaglandin H2 by the
enzyme cyclooxygenase, which is anchored in the dense tubular system (DTS) of the
platelet.
¡ Thromboxane synthetase acts on prostaglandin H2 to produce thromboxane A2
(TXA2).
¡ TXA2 binds to membrane receptors TPa or TPb, inhibiting adenylate cyclase activity
and reducing cAMP concentrations.
¡ Reduced cAMP concentrations lead to the mobilization of ionic calcium from the
DTS.
¡ Elevated cytoplasmic calcium levels cause actin microfilament contraction, resulting
in platelet shape change and further activation.
PLATELET ACTIVATION PATHWAYS
§ Cyclooxygenase Pathway in Endothelial Cells
§ In endothelial cells, prostacyclin synthetase replaces thromboxane
synthetase.
§ The end product of the pathway is prostacyclin (prostaglandin I2),
which binds to the IP receptor, activating the IP3-DAG pathway.
§ Prostacyclin production by endothelial cells inhibits platelet activation
by increasing cAMP levels and sequestering ionic calcium to the DTS.
§ This creates a dynamic equilibrium with platelet activation in the intact
blood vessel.
PLATELET ACTIVATION PATHWAYS
§ Inositol Triphosphate–Diacylglycerol Activation
• The IP3-DAG pathway is the second G-protein-dependent platelet
activation pathway.
• G-protein activation triggers the enzyme phospholipase C, initiating a
cascade of intracellular events.
• Phospholipase C cleaves membrane phosphatidylinositol 4,5-
bisphosphate (PIP2) to form inositol triphosphate (IP3) and
diacylglycerol (DAG), serving as second messengers.
PLATELET ACTIVATION PATHWAYS
§ Inositol Triphosphate–Diacylglycerol Activation
• IP3 promotes the release of ionic calcium from the dense tubular system
(DTS) of platelets.
• Increased cytoplasmic calcium levels trigger actin microfilament contraction,
contributing to platelet activation.

• IP3 may also activate phospholipase A2, initiating further downstream

signaling events.
PLATELET ACTIVATION PATHWAYS
§ Inositol Triphosphate–Diacylglycerol Activation
• DAG triggers a multistep process:
• Activation of protein kinase C (PKC).
• PKC phosphorylates the protein pleckstrin, regulating actin
microfilament contraction.
• The IP3-DAG pathway orchestrates a series of intracellular events
leading to platelet activation and shape change.
REGULATION OF PLATELET FUNCTION

§ Activated platelets release factors like ADP and thromboxane A2,

which amplify platelet activation and aggregation.
o These factors recruit and activate additional platelets, promoting clot
formation.
§ Platelet function is tightly regulated to prevent excessive clotting.
§ Endothelial cells release factors like prostacyclin and nitric oxide to
inhibit platelet activation.
§ Antiplatelet drugs, e.g., aspirin and P2Y12 receptor antagonists, are used
to prevent thrombotic events.