Megakaryocytopoiesis

Platelets:
- anucleate blood cells
- circulates in amounts of 150-400 x109L; slightly higher in women than men
- triggers PRIMARY HEMOSTASIS on exposure to: endothelial, subendothelial
and plasma
procoagulants in blood vessel injury
- arise from MEGAKARYOCYTE

Megakaryocytes:
- polyploid: possess multiple chromosome copies w/in a single cell
- multilobulated nucleus w/ a granular cytoplasm
- cluster in the extravascular compartment adjacent to the abluminal
membrane (surface opposite the lumen)
- EMPEROPOIESIS faux phagocytosis; when myelocytic and erythrocytic
precursor cells crosses the megakaryocyte cytoplasm to reach the sinusoid
lumen
- also harvested in the LUNGS

Endomitosis
Megakaryocyte maturation mysterious form of mitosis lacks
telophase and cytokinesis
DNA replication proceeds to production of 8N, 16N, 32N ploidy
w/duplicated sets of chromosomes
Employ their copious DNA to synthesize abundant cytoplasm
Single megakaryocyte = 2000-4000 platelets
There are 108 megakaryocyte producing 1011 platelets a day
Key to endomitosis loss of spindle fiber orientation at the point of
telophase = chromosomes do not go to polar bodies but duplicate in
place in the equatorial plate

Megakaryocyte Progenitors
Megakaryocyteerythrocyte progenitor
Thrombopoietin (TPO);
IL-3
Burst-Forming Unit (BFU
Meg)

Colony-Forming Unit
(CFU-meg)

Light-Density
CFU (L-D-CFUMeg)

BFU-Meg and CF-Meg

Diploid and participates in normal mitosis
Maintains a pool of megakaryocyte progenitors

LD-CFU Meg
Little proliferative capacity and produces few cells
Begins to process through endomitosis to reach increased nuclear
ploidy
Transitional; promegakaryoblast; stage in w/c polyploidy is first
established
Enters terminal differentiation as they lose the ability to undergo
normal mitosis

Lab exams
Immunologic probes and flow cytometry
Usual flow cytometric progenitor markers:
o general stem cell marker CD34
o HLA-DR
o Platelet glycoprotein IIIa (GP IIb/II1, CD 41)
Platelet peroxidase
o Localized in the endoplasmic reticulum of the progenitors and
megakaryocytes
o Cytochemical stain in transmission electron microscopy

Terminal Megakaryocyte Differentiation

MK I Stage

MK II Stage

MK III Stage

megakaryobla
st

promegarkyocyt
e

megakaryocyt
e

MK-I stage/Megakaryoblast
May see plasma membrane blebs blunt projections from margin that
resembles platelets
Begin to develop most of its cytoplasmic ultrastructure
o Procoagulant-laden alpha granules
o Delta-granules (dense bodies)
o Demarcation system (DMS)
Additional immunologic probes:
o GP Ib part of GP Ib/IX/V von Willebrand factor adhesion receptor (CD
42)
o mpl TPO receptor
o cytoplasmic VWF detected by histochemical immunostaining
MK-II Stage

 Nuclear lobularity first become apparent as an indentation at the 4N

replication stage
CD 36 (GP IV) becomes visible

MK-III/Megakaryocyte
Nuclear is intensely indented and lobulated
Chromatin in variably condensed w/light and dark patches
Cytoplasm = azurophilic, granular and platelet-like

Thrombocytopoiesis (platelet shedding)

Megakaryocyte cultures usaing transmission electron microscopy
DMS dilates longitudinal bundles of tubules form cytoplasmic
extensions (proplatelet processes) develop transverse constrictions
appear throughout the processes
Proplatetelet processes pierce through or between sinusoid-lining
endothelial cells, extend into the venous bloos and release the
platelets

Hormones and cytokines of megakaryocytopoiesis

TPO

mRNA = kidney, liver, and smooth muscle cells

circulates in the plasma
binds a megakaryocyte and platelet membrane receptor, mpl
concentration in inversely proportional to platelet and megakaryocyte
mass
primary control mechanism for concentration = membrane binding and
disposal by platelets
induces stem cells to differentiate into megakaryocyte progenitors in
synergy w/cytokines
induces megakaryocyte progenitors to differentiate into
megakaryocytes
induces proliferation and maturation of megakaryocytes
induces platelet release
USES OF RECOMBINANT TPO:
o Elevates platelet count in healthy donors and in patients treated
for a variety of neoplasms including leukemia
o Commercial form NPlate (romilostim, Amgen) = effective in
raising the platelet count in immune thrombocytopenic purpura
Cell-derived stimulators of megakaryopoiesis
IL-3
o Acts w/TPO to nduce early differentiation of stem cells
IL 6 and IL 11
o Acts in the presence of TPO to enhance endomitosis,
megakaryocyte maturation and platelet release

IL 11 = Neumega (oprelvekin) = to stimulate platelet production

in patients w/chemotherapy induced thrombocytopenia
Other cytokines and hormones that participates synergistically w/TPO
and interleukins:
o Stem cell factor (kit ligand; mast cell growth factor)
o Granulocyte-macrophage colony stimulating factor (GM-CSF)
o Granulocyte colony stimulating factor (G-CSF)
o Erythropoietin (EPO)
Inhibits in vitro megakaryocyte growth:
o Platelet factor 4
o Beta thromboglobulin
o Neutrophil activating peptide 2
o IL 8
Reduction in the transcription factor FOG, GATA 1, and NF E2 diminish
megakaryocytopoiesis at the progenitor, endomitosis and terminal
maturation phase
o

PLATELETS
Diameter = 2.5 um
Circulating resting platelets = biconvex
EDTA = induces them to round up
Wright stain = circular to irregular, lavender and granular
Blood = even surface
Cluster w/erythrocyte near the center of the blood vessels
Platelets move laterally w/leukocytes into the white pulp of the spleen where
both are sequestered
Normal peripheral blood platelet count = 150-400 10 9/L
o Represents only 2/3 of the total circulating platelets since 1/3 is
sequestered by the spleen
Sequestered platelets = readily available in times of demand (acute
inflammation, after injury, major surgery or plateletpheresis)
Hypersplenism/splenomegaly = increased sequestration thrombocytopenia
During conditions of hemostatic needs:
o Become sticky and irregular
o Extend pseudopods
o Adhere to neighboring structures
o Aggregate w/each other
Reticulated platelets
stress platelets
Appear in compensation for thrombocytopenia
Larger; diameter exceed 6 um
Round up in EDTA
Citrated blood = cylindrical and beaded resembling megakaryocyte
proplatelet processes
Nucleic acid dyes (thiazole orange) binds w/RNA of the endoplasmic
reticulum = provides quantitative evaluation of reticulated platelet

production under stress; confounded by platelet dense granules w/c raises

the reticulated platelet count by taking up nucleic acid dyes