HEMATOLOGY 1: FINALS

Platelets/Thrombocytes
A. MEGAKARYOCYTIC SERIES
Responding to the growth factor thrombopoietin (TPO),
megakaryocyte progenitors are recruited from common
myeloid progenitors
1. BFU-Meg CFU-Meg

2. LD-CFU-Meg

3. Megakaryoblast / MK-I
− Size: 14-18um
− N/C ratio: 10:1
− Nucleus: Fine chromatin; multiple nucleoli
that generally stain blue
− Cytoplasm: Small to moderate amount; blue
(darker than myeloblast); nongranular,
plasma membrane blebs, blunt projections
− Begins development of alpha and dense granules

4. Promegakaryocyte/ / MK-II − Size: 15-40um

− N/C ratio: 4-7:1
− Nucleus: Irregularly shaped; may show slight
lobulation; coarse chromatin; multiple nucleoli
visible
− Cytoplasm: More abundant but less basophilic;
granules begin to from; Demarcation Membrane
System (DMS) develops.—gives rise to membrane
system/compartments of platelets

5. Granular Megakaryocyte / MK-III

− Size: 30-50um
− N/C ratio: 1-2:1
− Nucleus: Multiple nuclei or multilobulated
nucleus; coarser chromatin; no nucleoli nucleus
is intensely indented or lobulated, and the
degree of lobulation is imprecisely proportional
to ploidy.
− Cytoplasm: Abundant; pinkish-blue; diffusely
granular and has an irregular peripheral border
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Mature Megakaryocyte/Metamegakaryocyte
− Size: 40-120um
− N/C ratio: 1:1
− Nucleus: Multiple nuclei or multilobulated
nucleus; no nucleoli are visible
− Cytoplasm: Contains coarse clumps of
granules aggregating into bundles which bud
off from the periphery to become platelets
(2000-4000 platelets)
− Megakaryocyte

6.

7. Thrombocytes (cytoplasmic fragments)

− Size: 1-4um
− N/C ratio: --
− Nucleus: --
− Cytoplasm: Light blue to purple; very granular; consists of 2 parts: chromomere and hyalomere ▪
Outer hyalomere: nongranular pale blue that surrounds the centromere
▪ Inner chromomere

B. General Characteristics
− smallest of formed elements
− represents cytoplasmic fragments of megakaryocytes − Platelets trigger primary
hemostasis based on: o exposure to subendothelial collagen or o endothelial cell
inflammatory proteins at the time of blood vessel injury.
− resting platelets are biconvex
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− platelets in blood collected using EDTA tend to “round up.” On a Wright-stained wedge-preparation blood film,
platelets appear circular to irregular, lavender, and granular.
C. Kinetics
➢ Production: shed from the megakaryocyte cytoplasm into the venous sinuses ➢
Cytokines and Hormones:
o TPO
o IL-3 – induce early differentiation of stem cells o IL-6 and IL-11 – enhance
endomitosis, megakaryovyte maturation
➢ Distribution: 2/3 in the blood (Ave. value: 275x109/L; range of 150-400 x 109 /L), 1/3 in the
spleen ➢ Circulating Life Span: 8-12 days
➢ Destruction: removed by macrophages in the spleen and liver or through consumption in
daily coagulation mechanisms
− Platelet turnover rate = 35,000 ± 4,300 platelets/day ( 1% RBC everyday)
➢ Regulation of circulating numbers o Rebound thrombocytosis
− following platelet depletion due to increase megakaryoblast endomitosis o
Rebound thrombocytopenia
− Following platelet transfusion due to increase megakaryocyte endocytosis

D. Platelet Ultrastructure
1. Peripheral zone
A. Glycocalyx
− exterior fluffy coat
− rich in glycoproteins (Gp) which serves as membrane
receptors/adhesion for aggregagating agents and physiological
agonists
1. Gp lb
2. Gp IIb and IIIa
3. Gp Va

B. Cell Membrane
− contain phospholipids that play an important role in platelet function
− site of receptors for clotting factors and aggregating agents
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C. Submembranous area
− beneath Cell Membrane
− consists of an organized system of microfilaments
− site where messages from the external membrane are translated into chemical signals carrying platelet activation
and a change in the physical structure.

2. Sol-Gel Zone
Microtubules, actin microfilaments, and intermediate microfilaments control platelet shape change, extension of
pseudopods, and secretion of granule contents.
A. Microtubules
− structural support
− composed of CHON tubulin which maintains the discoid shape of platelets
B. Microfilaments
− composed of actin and myosin which upon stimulation are converted into actomyosin, a contractile CHON
important in clotting retraction
− mediates platelet contraction important in centralizations of granules and eventual release of granular
contents, and pseudopod formation

C. Intermediate filaments
− desmin and vimentin
− connect with actin at tubules – maintaining shape
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3. Organelle Zone

A. Granules
➢ α - granules
− most abundant
− As the platelet becomes activated, a-granule
membranes fuse with the SCCS. Their contents
flow to the nearby microenvironment, where
they participate in platelet adhesion and
aggregation and support plasma coagulation
− stain medium gray in osmium-dye transmission
electron microscopy preparations − contains
contact components:
▪ fibrinogen
▪ Platelet derived growth factor (PDGF)
▪ Platelet factor 4 (PF4) or heparin
neutralizing factor
▪ Protein S
▪ Protein C inhibitor
▪ β – thromboglobulin
▪ Factor V
▪ VWF
▪ Thrombospondin

➢ Dense Granules (delta granules)

− because of appearance and staining under electron microscope
- stain black (opaque) when treated with osmium in transmission electron microscopy
− migrate to the plasma membrane and release their contents directly into the plasma on platelet activation.
− contains non CHON components/chemicals which includes:
• ADP
• ATP
• 5-hydroxytryptamine
• Ca2+
• Mg2+
➢ Lysosomes
➢ Mitochondria

4. Membrane Systems A. Open Canalicular System

− provides direct communication between intracellular and extracellular environment
− intracellular compounds are released into the external environment
B. Dense Tubular System
− important for arachidonic acid metabolism
− acts as Ca sequestering pump which maintains cytoplasmic Ca level
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− The DTS sequesters Ca and bears a number of enzymes that support platelet activation

D. Platelet Functions
1. Platelet Adhesion
− Interaction between surface membrane receptor Gplb & VWF which links the platelet and the damaged vessel
following release of physiologic agonist, thrombin, epinephrines, platelet activating factor
− Response to certain agonists produced by damaged subendothelium or substances that platelets produce
− Degree or avidity of adhesion depends on extensiveness of damage

2. Platelet Aggregation
− Recruitment of more platelets and sticking to one another forming clumps
− Preceded by activation and shape change o platelets change in shape from discoid to round and extend
pseudopods. This allows platelets to cover more surface area and it enhances platelet binding to
other platelets and foreign surfaces.
− 3 main mediators of aggregation: o the platelet activators TXA2 and ADP are secreted from the platelet granules
to the microenvironment, where they activate neighboring platelets through their respective
receptors and trigger activation of (GP IIb/IIIa receptor), enabling it to bind fibrinogen and VWF and
support platelet-to-platelet binding
1. ADP
− Irreversible second wave of aggregation by changing conformation of IIb-IIa complex
− Main; particularly causes change in GpIIb-IIIa
2. Thrombin
− promotes ADP release
− stimulates ADP-induced secondary aggregation and promotes release of phospholipase
3. TxA2
− thromboxane A2
− promotes aggregation
− potent vasoconstrictor
− end product of arachidonic acid −
main mediator of aggregation
− platelet
(B), Trauma to the blood vessel wall exposes
adhesion. subendothelial collagen and tissue factor, triggerin
g
− (C), VWF serves as a bridge between subendothelial collagen and the platelet GP Ib/IX/V receptor.
Platelet interaction with collagen and GP VI receptors triggers release of TXA2 and ADP
− (D) platelet-platelet aggregation through binding of arginine-glycine-aspartate (RGD)–containing
ligands such as fibrinogen and VWF. ADP, Adenosine diphosphate, FG, fibrinogen; HS, heparan
sulfate; NO, nitric oxide; PGI2, prostacyclin; RBC, red blood cells; TF, tissue factor;

- Platelet aggregation is a key part of primary hemostasis, which in arteries may end with the formation
of a “white clot,” a clot composed primarily of platelets and VWF
- In abnormal conditions, white clots often implies inappropriate platelet activation in seemingly
uninjured arterioles and arteries and is the pathologic basis for arterial thrombotic events, such as
acute myocardial infarction, peripheral artery disease, and ischemic stroke.
- Fibrin and red blood cells deposit around and within the platelet syncytium to form a bulky “red clot.”
The red clot is essential to wound repair, but it may also be characteristic of inappropriate coagulation
in venules and veins, resulting in deep vein thrombosis and pulmonary embolism.
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3. Platelet release reaction/mechanism

− Involves platelet contraction mediated by the microfilament system
− Centralization of granules
− Subsequent release of granular contents which further promote platelet activation and aggregation o a-
granule contents are large molecule coagulation proteins that participate in secondary hemostasis o
The dense granule contents are small molecule vasoconstrictors and platelet agonists that amplify
primary hemostasis

4. Platelet coagulant activity

− Exposure of the negatively charged phospholipids
− Involves exposeure of Phosphatidyl serine (PS) expressed in inner leaflet of membrane. −
Scramblase

E. Quantitative disorders

1. Thrombocytosis
− Increase in number of platelets > 450, 000 /ul or 450x10 9/L
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a. Essential Thrombocytosis
− Aka Primary Thrombocytosis; primary BM defect
− PV and thrombocytopenia
− Always associated with bleeding tendencies accompanied by structural or functional abnormalities
− Caused by : clonal proliferation of hematopoietic cells
− Can be megakaryocytic series only or all series thrombocytopenia
b. Secondary thrombocytosis
− Do not manifest bleeding tendencies
− As a result of underlying condition
▪ IDA
▪ Chronic Inflammatory disease
▪ Splenectomy

2. Thrombocytopenia
− Decrease in platelet count <150x109/L (lower limit); < 100,000/ul
− Signs and symptoms: internal hemorrhage in form of petechiae, purpura and ecchymosis, prolonged bleeding
time and clot retraction
− Reticulated platelets, sometimes known as stress platelets, appear in compensation for thrombocytopenia o
markedly larger than ordinary mature circulating platelets; their diameter in peripheral blood
films exceeds 6 mm, and their MPV reaches 12 to 14 fL.

a. Decreased production
− Decrease in number of megakaryocytes
− Due to BM aplasia/hypoplasia
− Radiation
− Myelophthisis
− Drugs and toxin – alcohol, thiazide, aplastic anemia, chemotherapy except antifolate drugs

b. Ineffective production
− Vitamin B12 or Folate Deficiency
− Hereditary thrombocytopenia
− Di Guglielmo’s syndrome (erythroleukemia)
− PNH

c. Altered distribution
− Intravascular thymic splenic pool
− Hypothermia
− Transfusion of stored blood

d. Consumption
➢ Combined:
− DIC – Disseminated Intravascular hemolysis
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− toxicity
− Infections
− neoplasms
− Intravascular hemolysis (IV)
➢ Isolated (platelets only)
− Thrombotic thrombocytopenic purpura (TTP)
− Hemolytic Uremic Syndrome (HUS)
− Vasculitis – SLE

e. Immune destruction
− Production of antiplatelet Ab (alloantibodies and autoantibodies)
▪ Idiopathic thrombocytopenic purpura (ITP)
▪ post-transfusion purpura
▪ isoimmune neonatal purpura
▪ drug-induced
▪ HIV infection

F. Qualitative disorders

1. Surface membrane defects

− disorders affect Gps
− Genetics: both carriers

a. Bernard-Soulier syndrome
− Autosomal recessive disorder affecting the membrane receptor GpIb
− Characterized by:
▪ decreased platelet count
▪ Prolonged bleeding time
▪ Giant platelet
▪ Abnormal aggregation with ristocetin

b. Glanzmann’s thrombasthenia
− Aggregation disorder
− Autosomal recessive disorder affecting the receptor complex Gp IIb-IIIa preventing the binding of fibrinogen
▪ Normal platelet count and morphology
▪ prolonged bleeding time and clotting time
▪ Abnormal aggregation response to all agents except ristocetin

2. Granular defects/Storage Pool deficiencies

1) Dense Granule Deficiency/Delta - SPDs

a. Hermansky-Pudlak syndrome
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− Caused by mutation in the gene that controls the production of melanosomes, platelet dense bodies, and
lysosomes

b. Chediak-Higashi syndrome
− Characterized by giant cytoplasmic granules and decreased ADP release
− Manifesteations include ocular, neurologic, and skin abnormalities, possible neutropenia, normal platelet
count but diminished aggregation response

c. Wiskott-Aldrich syndrome
− X-linked disorder highlighted by thrombocytopenia, recurrent infections, eczema, and a predisposition to
secondary leukemia or lymphoma

2) Alpha Granule Deficiency

a. Gray Platelet Syndrome
− Characterized by the finding of large platelets that lack α – granules producing a gray appearance on a
Wright stained smear, prolonged BT, impaired aggregation response with ADP, collagen and
epinephrine, but normal response to thrombin and arachidonic acid

b. Quebec Platelet disorder

− Characterized by abnormal proteolysis of α – granules and selective defective aggregation with
epinephrine

3. Cyclooxygenase Deficiency

− Defect in thromboxane generation characterized by abnormal response to arachidonic acid; platelet count may be
normal

4. Membrane phospholipid deficiency

− Defect in platelet coagulant activity caused by impaired transmembrane migration of the procoagulant
− PS resulting in the absence of Xa-binding sites; aggregation and release mechanisms are normal

5. Miscellaneous disorders

a. Von Willebrand’s Disease

- Platelets will not adhere due to lacking of Von Willebrand’s factor

b. Afibrinogenemia
- No fibrinogen