1.

01 PLATELETS
Professor Kerfelcel Retoriano HEMATOLOGY 2
A.Y 2022-2023

OUTLINE
• Platelets • Platelet System
A. Reticulated platelets A. Disorders of Platelet Adhesion
B. Platelet structure B. Disorders of Platelet
• Disorders Associated Aggregation
A. Quantitative Disorders C. Disorders of Platelet Release
B. Qualitative Disorders Reactions
• Megakaryocyte-Erythrocyte Progenitor • Hemostatic Plug Formation
• Platelet Maturation Sequence A. Platelet Adhesion
• Hormones and Cytokines of B. Platelet Secretion
Megakaryopoiesis C. Platelet Aggregation to
• Platelet Factors Platelet plug Formation

PLATELETS
• Also known as thrombocytes and is the largest of all hematopoietic cells.
• Originate from polypoid megakaryocytes
NV: 150 – 400 x 109/L
• Cytoplasm: light blue to purple Female > 65yrs: 120 – 350 x 109/L
• Size: 2-4 um Male > 65yrs: 140 – 379 x 109/L
• Granules: Red to violet, abundant
• 2 Parts:
o Chromomere – granular and located centrally
o Hyalomere – surrounds chromomere and non-granular; light blue appearance
• Thrombocytopoiesis – known as platelet shedding
• Functions:
o Maintain the integrity of blood vessels
o Form a hemostatic plug to stop blood loss from injured vessels, and in the
process, promoting coagulation of plasma factors.
 Platelets – involve in primary hemostasis – end product is the formation of a
platelet plug
▪ Interaction of platelets with collagen or subendothelial collagen triggers
the primary hemostasis.
Average platelets (OIO) 7 to 25 cells
Diameter 2.5 um
Mean platelet volume (MPV) 8 – 10fL
Single megakaryocytes produces 2000 – 4000 platelets
Average turnaround time 8 – 9 days
Lifespan 8 – 11 days
Maturation sequence 5 days
Megakaryocytes in bone marrow 2 – 4 megakaryocytes per 10x (LPO)
Appearance in Wright stain Irregular, lavender, granular

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1.01 PLATELETS

 Thrombopoietin – growth
factor for platelet
 SPLENOMEGALY – decrease
in platelet circulation
 SPLENECTOMY – increase
platelet in the circulation
 WRIGHT STAIN – best
anticoagulant to preserved
platelets

Reticulated Platelets
• Stress platelets
• A response to
thrombocytopenia
• 6um
• MPV: 12 – 14fL
 ↑sequestration – due to hypersplenism or splenomegaly that leads to
relative thrombocytopenia

Platelet Structure

Peripheral Zone
• Adhesion and aggregation
• Consists of the surface
coat or glycocalyx, plasma
membrane, and
submembrane area
• GLYCOCALYX – contains
glycoproteins or GPs

Glycoprotein Function
Ia Adhesion to collagen
Ib vWF binding, adhesion to subvascular subendothelium
IIb – IIIa Fibrinogen binding, vWF binding, platelet aggregation
IV Thrombospondin binding
V Thrombin binding, platelet aggregation
IX Adhesion together with Ib

• Plasma membrane – lies beneath the glycocalyx and the major component of
peripheral zone, and is rich in arachidonic acid
• Submembranous membrane – composed of platelet membrane receptor, ADP,
collagen, serotonin, epinephrine, thrombin, vWF, FV and FXa

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Sol-Gel Zone
• Provides a cytoskeletal system of platelets
• Microtubules – has tubulin → maintains shape of platelets
• Microfilaments – composed of actin (actomyosin/ thrombosthenin) and myosin
o Actin – responsible for clot retraction

Organelle Zone
• Contains three types of granules: alpha, dense bodies and lysosomes.
• Contains mitochondria – the metabolic center to influence platelet function.
Alpha granules Dense bodies Lysosomal granules
• PF4 - antiheparin • ADP • ACP
• B-thromboglobulin – for • ATP • Hydrolytic enzymes
tissue repair • Calcium
• PDGF • Serotonin
• Thrombospondin – for • Pyrophosphate
platelet adhesion • Magnesium
• vWF – involve in primary • Phosphate
hemostasis • Epinephrine
• Fibrinogen – secondary • 5-hydroxytryptamine (5-
hemostasis HT)
• Fibronectin – platelet
aggregation
• Factor V
• Plasminogen activator
inhibitor 1 (PAI-1)
• Fatcor XIII
• Protein S

Membrane System
• Contains a dense tubular system in which enzymatic system for the production of
prostaglandin is found
• It is the surface connecting system (open canalicular system) that acts as a canal
in releasing granules and involved in phagocytosis.

DISORDERS ASSOCIATED
Quantitative Disorders
A. Decreased platelet count
i. Distribution and dilution – includes Splenomegaly, Hyponatremia
and Transfusion
ii. Disorders of production – due to (1) Decrease in
megakaryocytes and (2) Ineffective production
iii. Disorders of destruction
a) Combined consumption – DIC and its causes
b) Isolated consumption – TTP

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1.01 PLATELETS

c) Immune destruction – ITP and drug induced (Quinidine and

Heparin)
B. Increased platelet count
i. IDA iv. Splenectomy
ii. Chronic inflammatory v. Rebound
disease thrombocytosis
iii. Malignancies

Qualitative Disorder
A. Surface membrane disorders – Glanzmann’s thrombasthenia and Bernard-
Soulier syndrome
B. Storage granule disorders
i. Hermansky-Pudlak syndrome
ii. Wiskott-Aldruch syndrome
iii. Chediak-Higashi syndrome
iv. Storage pool disease
C. Disorders of TXA2
D. Acquired – cardiopulmonary bypass (DIC and SLE)

 REMEMBER!!!
• Transcription gene product – this products promotes myeloid progenitor
differentiation
o GATA-1
o Cofactor FOG-1
 During endomitosis, GATA-1 & FOG-1 transcription is decreased.
 RUNX-1 – increased in endomitosis; signals shifting of mitosis to
endomitosis.
• MYB - inhibitor of common myeloid progenitor differentiation

MEGAKARYOCYTE-ERYTHROCYTE PROGENITOR
• TPO (thrombopoietin)
• Cytokines (IL-3, IL-6, IL-11)
• 3 megakaryocyte lineage committed progenitor stages:
o BFU-MEG (burst forming unit megakaryocyte)
o CFU-MEG (intermediate colony forming unit megakaryocyte)
 BFU-Meg and CFU-Meg → both are diploid and participate in
normal mitosis (I.P.M.A.T-C)
o LD-CFU-MEG (light density colony forming unit megakaryocyte)
 Loses its capacity to divide but retains DNA replication and
cytoplasmic maturation
• Cytochemical staining in TEM (platelet peroxidase)
• Flow cytometric progenitor markers [CD34, HLA-DR, Platelet glycoprotein iiia (GP
IIb-IIIa, CD41)]

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 Endomitosis – nuclear division without cell or cytoplasm division; lacks

telophase and cytokinesis.
 Promotes megakaryopoiesis: thrombopoietin, IL-3, IL-6, IL-11
 Thrombopoietin + IL-3 → induces early differentiation of stem cells
 Thrombopoietin + IL-6 + IL-7 → enhances endomitosis
• Inhibitor of megakaryocyte growth
o PF4 o IL-8
o B-thromboglobulin o Neutrophil activating peptide

MK-1 MK-2 MK-3 MK-4

Megakaryocyte /
Mature
Name Megakaryoblast Promegakaryocyte Granular
megakaryocyte
megakaryocyte
Size 10 – 24 mm 15 – 40 mm 20 – 90 mm 40 – 120 mm
2 – 32 lobes (8
Round/ oval or
Nucleus Indented lobes: most --
kidney-shaped
common)
Nucleoli 2–6 Variable -- --
Homogenous, Condensed;
Chromatin -- --
loosely organized coarse
Blue to pink;
Cytoplasm Basophilic Basophilic Coarse clumped
abundant
Absent by Wright Reddish blue;
Granules Present --
stain few to abundant
N/C ratio 3:1 1:2 Variable 1:1
5-10 per 10x
20% of 25% of
Bone objective,
megakaryocyte megakaryocyte --
marrow 1-2 per 50x
precursor precursor
objective
Peripheral
0% -- 0% --
blood
Usually reported
as adequate,
Largest cell in
increased, or
NOTES: -- -- the bone
decreased and
marrow
not as a
percentage

 Dwarf Megakaryocyte – is associated with myeloproliferative disorders and

myelodysplastic syndrome
 Megakaryocyte fragments – associated with CML, various form of cancer,
myelofibrosis, polycythemia vera, Hodgkin’s disease and leukocytosis due to
surgery or infection

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MK-1 MK-2 MK-3 MK-4

Size range 0 – 24 um 14 – 30 um 15 – 56 um 20 – 50 um
Cytoplasmic Deep blue Basophilic with Mostly pink Totally pink
staining (basophilic) pink center
Granules Rare Few Extensive Organized into
platelet fields
Nuclear Few compacted Lobes spread Many lobes Many
morphology lobes out in a spread out compacted lobes
horseshoe
shape
N:C ratio High Moderate Moderate Low

 Naked megakaryocyte – after it shed all the platelets it is consumed by bone

marrow macrophages.

PLATELET MATURATION SEQUENCE

• Maturation sequence takes about 5 days
• Platelets – produced directly from the megakaryocyte cytoplasm
• As megakaryocyte matures → clusters of granules aggregate → platelets

HORMONES AND CYTOKINES OF MEGAKARYOPOIESIS

• TPO (thrombopoietin)
o Primary source is the kidney; liver and smooth muscles
o IL-3 – megakaryocyte progenitors to megakaryocytes
o IL-6 and IL-11 – enhance endomitosis, megakaryocyte maturation, platelet
release (thrombocytopoiesis)
▪ IL-11 – stimulates platelet production in patients with chemotherapy-
induced thrombocytopenia.
• Platelet factor 4 (PF4), B-thromboglobulin, Neutrophil activating peptide 2 –
promoter of platelet growth in vitro
• IL-8 – inhibit megakaryocyte growth in vitro
• Other growth factors of platelet differentiation:
o Stem cell factor – includes kit ligand or mass cell growth factor
o Granulocyte – CSF
o Acetylcholinesterase derived-megakaryocyte growth stimulating peptide

PLATELET FACTORS
• Written in Arabic numerals to differentiate them from coagulation factors which are
designated as Roman numerals.

Platelet factor 1 Accelerates prothrombin conversion (recently discovered not to be a PF but

blood factor V absorbed on platelets).
Platelet factor 2 “fibronoplastic platelet factor” – facilitates the interaction of thrombin and
fibrinogen
Platelet factor 3 “thromboplastic platelet factor” – necessary for the generation of plasma
thromboplastin

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 Most important and probably the only one that is an intrinsic

component of platelets
Platelet factor 4 Anti-heparin factor

PLATELET SYSTEM
• Platelet adhesion • Provides the reaction surface for
• Platelet aggregation some coagulation system reactions.
• Release of platelet factors from the • Supports and maintains endothelial
granules lining.

Disorders of Platelet Adhesion

Bernard-Soulier • An inherited autosomal recessive disorder in which the GP 1b/IX
syndrome or Giant complex is missing from the platelet surface.
Platelet syndrome • Platelets on smear range from 5 to 8um up to 20um (giant
platelets)
• Platelet adhesiveness test is abnormal, the aggregation test with
Ristocetin is abnormal but normal with other agents like ADP,
collagen, epinephrine
von Willebrand • A quantitative or structural abnormality of vWF that diminishes
disease – vWD platelet adhesion. The hereditary type is an autosomal dominant
trait.
Type 1 vW The most common form of von
disease Willebrand disease characterized by
reduced levels of VIII:C, vWR:Ag,
vWR:RCo
Type 2 vW Characterized by reduced level of
disease vWR:Ag
Type 3 vW Characterized by reduced vWR:Ag,
disease vWR:RCo, and variable VIII:C

• Acquired von Willebrand disease – associated with autoimmune.

Disorders, lymphoproliferative disorders, myeloproliferative
disorders, pesticide exposure, hemolytic uremic syndrome (HUS),
etc.

Disorders of Platelet Release Reactions

Glanzmann thrombasthenia • Inherited autosomal recessive disorder caused by a
deficiency in platelet membrane GPIIb/IIIa complex.
• Laboratory findings: markedly prolonged bleeding time,
abnormal aggregation test with all agents like ristocetin,
ADP, etc, but with normal platelet count and morphology.
Acquired von Willebrand
disease
Thrombopathia Deficiency in ADP and abnormal platelet aggregate
Deficiency of Platelet Factor Abnormal platelet function particularly intravascularly hemolysis
3
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Disorders of Platelet Action

Alpha Granules
• Contents: fibrinogen, vWF, factor V, factor VIII, PF 4, BTG, PDGF, p-selectin
• 50 – 80 per platelet
• Stain medium gray in osmium dye
• SCCS (surface-connected canalicular system)
• Adhesion, aggregation, support plasma coagulation

Gray platelet • Inherited absence of alpha granule content.

syndrome • Presence of large light gray platelets in wright stained smear
• Decrease platelet aggregation response to ADP, collagen, epinephrine
and thrombin
• Moderate to severe lifelong mucocutaneous bleeding
Wiskott- • Lacks alpha and dense granules
Aldrich • Characterized by small platelets
syndrome

Dense Granules
• 2 to 7 per platelet • They migrate directly to the plasma
• Also known as dense bodies membrane
• Appear later than alpha granules • Contents: serotonin, ADP, ATP,
• Stain black in osmium (tem) calcium

Hermansky-pudlak (HPS)
Associated with albinism
Chediak-Higashi syndrome (CHS)
Non-albinism, x-linked
recessive granule deficiency
Storage pool Wiskott-Aldrich syndrome (WAS)
characterized by severe
disorders
eczema
Myeloproliferative neoplasms/ Both alpha and dense granules
myelodysplastic syndromes are reduced and abnormal in
size, shape and contents

HEMOSTATIC PLUG FORMATION

Platelet Adhesion
• Platelets adhere to subendothelial surface collagen
• Is possible because of vWF (present in the plasma and the subendothelium), GP
Ib and GP IIb-IIIa complex.
• Agonist – substance that stimulate platelet activation
• Platelets (disk-shaped in resting phase) → contraction of microtubules → platelets
activated (Spherical shape with extrusion of numerous pseudopods)
• Arachidonic acid + cyclohexogenase → prostaglandin + thromboxane synthase →
(TXA2) (for vasoconstriction and stimulate platelet secretion)

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Platelet Secretion
• Platelet granules → move towards platelet center → fuse with the open canalicular
system connected to the outside of the platelet → platelet secretion

Platelet Aggregation to Platelet plug Formation

• Other platelets are stimulated by ADP to undergo shape change
(disk→spherical→pseudopods) exposing the GP IIb-IIIa complex.
• GP IIb-IIIa – receptor for fibrinogen
• Fibrinogen binding links the platelets – first and reversible phase of aggregation
• After release reaction, the aggregation is irreversible.

Acquired disorder of platelet aggregation

Uremia Accumulation of toxic metabolites
Paraproteinemia Coating of platelet membrane with abnormal protein
Acute myelogenous Abnormal megakaryocyte
leukemia
Myeloproliferative Includes CML, myelofibrosis w/ myeloid metaplasia, PV,
disorders essential thrombocythemia
Drugs Common is ASPIRIN (inhibits cyclooxygenase)

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