HEMATOLOGY 2
HEMATOLOGY 2 LECTURE
Prof. Charito Bermido/ M1 Platelet Production, Structure, and Function
BY: MARIA VERONICA G. SANTIAGO, 3V (2023-2024)
I. PLATELETS
● at a concentration of 150 to 400 x 10^9/L
● average platelet counts slightly higher in women than in men
and slightly lower in members of both sexes who are older than
65 years.1
● trigger primary hemostasis on exposure to subendothelial
collagen or endothelial cell inflammatory proteins at the time of
blood vessel injury.
● arise from megakaryocytes
○ largest cells in the bone marrow and possess multiple
chromosome copies (polyploid).
○ also found in lungs
○ 30-50 um in diameter with a multilobulated nucleus
and abundant granular cytoplasm
○ account for less than 0.5% of all bone marrow cells
○ under the influence of stromal cell cytokines,
cluster with hematopoietic stem cells in vascular
niches adjacent to venous sinusoid endothelial cells.
(this just means environment sa loob ng vein next to
sinusoid endothelial cells)
○ arise from common myeloid progenitors
○ In response to thrombopoietin (TPO),
megakaryocyte progenitors are recruited from
common myeloid progenitors and subsequently
differentiate through several maturation stages.
○ The megakaryocyte progenitors extend proplatelet
processes, projections that resemble strings of
beads, through or between the endothelial cells and
into the venous sinuses, releasing platelets from the
tips of the processes into the circulation.
A. MEGAKARYOCYTE DIFFERENTIATION AND PROGENITORS
● Megakaryocyte progenitors
○ arise from the common myeloid progenitor under the
influence of the transcription gene product, GATA-1,
regulated by cofactor FOG1
○ GATA-1 suppresses MYB to balance
megakaryocytopoiesis with erythropoiesis
● Three megakaryocyte lineage-committed progenitor stages:
○ burst-forming unit (BFU-Meg) / least mature
○ intermediate colony-light0dednsity (CFU-Meg)
○ light-density CFU (LD-CFU-Meg)/ more mature
● all of these progenitor stages resemble lymphocytes and
cannot be distinguished by Wright-stained light microscopy.
● The BFU-Meg and CFU-Meg are diploid and undergo normal
mitosis to maintain a viable pool of megakaryocyte progenitors.
Their proliferative properties are reflected in their ability to form
hundreds (BFU-Megs) or dozens (CFU-Megs) of colonies in
culture
● LD-CFU-Meg, undergoes endomitosis
○ a partially characterized form of mitosis unique to
megakaryocytes in which DNA replication and
cytoplasmic maturation are normal but cells lose their
capacity to divide.
ENDOMITOSIS
● a form of mitosis that lacks telophase and cytokinesis
(separation into daughter cells).
● As GATA-1 and FOG1- driven transcription slows, another
transcription factor, RUNX1, mediates the switch from mitosis to
endomitosis by suppressing the Rho/ROCK signaling pathway.
In response to the reduced Rho/ROCK signal, inadequate levels
of actin and myosin (muscle fiber-like molecules) assemble in
the cytoplasmic constrictions where separation would otherwise
occur, preventing cytokinesis.7 Subsequently, under the
influence of transcription factor NF-E2, DNA replication proceeds
to the production of 8N, 16N, or even 32N ploidy with duplicated
chromosome sets.3 Although some megakaryocytes reach
128N, this level of ploidy is unusual and may signal hematologic
disease. Megakaryocytes employ their multiple DNA copies to
synthesize abundant cytoplasm, which ultimately differentiates
into platelets
TERMINAL MEGAKARYOCYTE DIFFERENTIATION
● As endomitosis proceeds, megakaryocyte progenitors leave the
proliferative phase and enter terminal differentiation
-a series of stages in which microscopists become able
to recognize their unique Wright-stained morphology in bone
marrow aspirate films or hematoxylin and eosin-stained bone
marrow biopsy sections. The morphologist, however, seldom
makes the effort to distinguish MK-I, MK-II, and MK-III stages
during a routine examination of a bone marrow aspirate smear.
● MK-I stage or megakaryoblast- what morphologists call the
least differentiated megakaryocyte precursor. Although they no
longer look like lymphocytes, megakaryoblasts cannot be
reliably distinguished from bone marrow myeloblasts or
pronormoblasts (also named rubriblast) using light microscopy
● The morphologist may occasionally see a vague clue: plasma
membrane blebs, blunt projections from the margin that
resemble platelets
● The megakaryoblast begins to develop most of its cytoplasmic
ultrastructure, including procoagulant-laden alpha-granules,
dense granules, and the demarcation system (DMS)
● The DMS
is a series of membrane-lined channels that invade from
the plasma membrane and grow inward to subdivide the entire
cytoplasm. The DMS is biologically identical to the
megakaryocyte plasma membrane and ultimately delineates the
individual platelets during thrombocytopoiesis.
● Nuclear lobularity first becomes apparent as an indentation at
the 4N replication stage, rendering the cell identifiable as an
MK-II stage, or promegakaryocyte, by light microscopy. The
promegakaryocyte reaches its full ploidy level by the end of the
MK-II stage. At the most abundant MK-III stage, the
megakaryocyte is easily recognized at 10x magnification on the
basis of its 30- to 50-um diameter.
● The nucleus is intensely indented or lobulated, and the degree of
lobulation is imprecisely proportional to ploidy. When necessary,
ploidy levels are measured using propidium iodide, a nucleic
acid dye in megakaryocyte flow cytometry. The chromatin is
variably condensed with light and dark patches. The cytoplasm
is azurophilic (lavender), granular, and platelet-like because of
the spread of the DMS and alpha-granules. At full maturation,
platelet shedding, or thrombocytopoiesis, proceeds.
THROMBOCYTOPOIESIS (PLATELET SHEDDING)
● During thrombocytopoiesis, a single megakaryocyte may
shed 2000 to 4000 platelets.
● In an average-size healthy human there are 10^8
megakaryocytes producing 10^11 platelets per day. The
total platelet population turns over in 8 to 9 days (the
so-called platelet lifespan).
● One cannot find reliable evidence for platelet budding or
shedding simply by examining megakaryocytes in situ, even
in well-structured bone marrow biopsy preparations.
However, in megakaryocyte cultures examined by
transmission electron microscopy, the DMS dilates,
longitudinal bundles of tubules form, proplatelet processes
 HEMATOLOGY 2
HEMATOLOGY 2 LECTURE
Prof. Charito Bermido/ M1 Platelet Production, Structure, and Function
develop, and transverse constrictions appear throughout the
proplatelet processes. In the bone marrow environment,
proplatelet processes are believed to pierce through or
between sinusoid-lining endothelial cells, extend into the
venous blood, and shed platelets Thrombocytopoiesis
leaves behind naked megakaryocyte nuclei to be consumed
by marrow macrophages.
Megakaryocyte Membrane Receptors and Markers
methods used to identify visually indistinguishable megakaryocyte
progenitors in hematologic disease.
● immunostaining of fixed tissue,
● flow cytometry with immunologic probes, and
● fluorescent in situ hybridization (FISH) with genetic probes
Flow cytometry can measure:
● MPL- the TPO receptor site present at all maturation stages, and
the stem cell and common myeloid progenitor marker CD34-
disappears as differentiation proceeds
● glycoprotein (GP) IIb/IIIa (CD41/CD61)- first appears on
megakaryocyte progenitors and remains present throughout
maturation, along with immunologic markers CD36 (platelet GP
4), CD42 (GP Ib) and CD62 (P-selectin).
● Cytoplasmic coagulation factor VIII, von Willebrand factor
(VWF), and fibrinogen may be detected in the fully developed
megakaryocyte by immunostaining
Hormones and Cytokines of Megakaryocytopoiesis
● The growth factor TPO is a 70,000 Dalton molecule that
possesses 23% homology with the red blood cell-producing
hormone erythropoietin.
● Messenger ribonucleic acid (mRNA) for TPO has been found in
the kidney, liver, stromal cells, and smooth muscle cells, though
the liver has the most copies and is considered the primary
source.
● TPO circulates as a hormone in plasma and is the ligand that
binds the megakaryocyte and platelet membrane receptor
protein identified earlier, MPL, named for v-mpl, a viral oncogene
associated with murine myeloproliferative leukemia
● The plasma concentration of TPO is inversely proportional to
platelet and megakaryocyte mass, implying that membrane
binding and consequent removal of TPO by platelets is the
primary platelet count control mechanism
● TPO, in synergy with other cytokines, induces stem cells to
differentiate into megakaryocyte progenitors and that it further
induces the differentiation of megakaryocyte progenitors into
megakaryoblasts and megakaryocyte.
● TPO also induces the proliferation and maturation of
megakaryocytes and induces thrombocytopoiesis
● Synthetic TPO mimetics (analogs) elevate the platelet count in
patients being treated for a variety of cancers, including acute
leukemia.
○ romiplostim (NPlate, Amgen), is a nonimmunogenic
oligopeptide that is effective in raising the platelet
count in patients with chronic immune
thrombocytopenic purpura (ITP)
○ eltrombopag (Promacta, Novartis), binds an MPL
site separate from romiplostim and is used in the
treatment of chronic ITP and in patients with
thrombocytopenia resulting from chronic hepatitis C or
severe aplastic anemia. They may have additive
effects.
○ interleukin-3 (IL-3), IL-6, and IL-11 - stimulate
megakaryocytopoiesis
■ IL-3- seems to act in synergy with TPO to
induce the early differentiation of stem cells
■ IL-6 and IL-11- act in the presence of TPO to
enhance endomitosis, megakaryocyte
maturation, and thrombocytopoiesis
■ IL-11 polypeptide mimetic, oprelvekin
(Neumega, Pfizer), stimulates platelet
production in patients with
chemotherapy-induced thrombocytopenia
● Other cytokines and hormones that participate synergistically
with TPO and the interleukins are stem cell factor, also called kit
ligand or mast cell growth factor; granulocyte-macrophage
colonystimulating factor (GM-CSF); granulocyte
colony-stimulating factor (G-CSF); and
acetylcholinesterase-derived megakaryocyte growth stimulating
peptide
● Platelet factor 4 (PF4), $-thromboglobulin, neutrophilactivating
peptide 2, IL-8, and other factors inhibit in vitro megakaryocyte
growth, which indicates that they may have a role in the control
of megakaryocytopoiesis in vivo.
● Internally, reduction in the transcription factors FOG1, GATA-1,
and NF-E2 diminish megakaryocytopoiesis at the progenitor,
endomitotic, and terminal maturation phases.2
PLATELETS
● The proplatelet process sheds platelets, cells consisting of
granular cytoplasm with a membrane but no nucleus, into the
venous sinus of the bone marrow.
● Although circulating, resting platelets are biconvex, the platelets
in blood collected using the anticoagulant
ethylenediaminetetraacetic acid (EDTA, lavender closure tubes)
tend to “round up.”
● On a Wright-stained wedge-preparation blood film, platelets
appear circular to irregular, lavender, and granular. Because of
their small size, it is difficult to examine the internal structure of
platelets using light microscopy. In the blood, the platelet surface
is even, and they flow smoothly through veins, arteries, and
capillaries.
● leukocytes - tend to roll along the vascular endothelium,
platelets cluster with the erythrocytes near the center of the
blood vessel. Unlike erythrocytes, however, platelets move back
and forth with the leukocytes from venules into the white pulp of
the spleen, where both become sequestered in dynamic
equilibrium.
● The normal peripheral blood platelet count is 150 to 400 X 109
/L.
● The platelet count decreases with increasing age, such that after
65 years of age, platelet counts of 122 to 350 ! 109 /L and 140 to
379 ! 109 /L are seen in men and women, respectively. This
count represents only two thirds of total body platelets;
● the remaining one third is sequestered within the spleen.
Sequestered platelets are immediately available in times of
demand— for example, in acute inflammation or after an injury,
after major surgery, or during plateletpheresis. In hypersplenism
or splenomegaly, increased sequestration may cause a relative
thrombocytopenia.
● Reticulated platelets, sometimes known as stress platelets,
appear in compensation for thrombocytopenia
● Reticulated platelets are markedly larger than ordinary mature
circulating platelets; their diameter in peripheral blood films
exceeds 6 "m, and their MPV reaches 12 to 14 fL.23 Like
ordinary platelets, they round up in EDTA, but in citrated
(blue-closure tubes) whole blood, reticulated platelets are
cylindrical and beaded, resembling fragments of megakaryocyte
proplatelet processes.
● Reticulated platelets carry free ribosomes and fragments of
rough endoplasmic reticulum, analogous to red blood cell
reticulocytes, which triggers speculation that they arise from
early and rapid proplatelet extension and release. Nucleic acid
dyes such as thiazole orange bind the RNA of the endoplasmic
reticulum. This property is exploited by hematology instruments
to provide a quantitative evaluation of reticulated platelet
 HEMATOLOGY 2
HEMATOLOGY 2 LECTURE
Prof. Charito Bermido/ M1 Platelet Production, Structure, and Function
production, a measurement that may be more useful than the
MPV.24 Platelet dense granule nucleotides, however, may
interfere with this measurement, falsely raising the reticulated
platelet count by binding nucleic acid dyes
● Reticulated platelets are potentially prothrombotic and may be
associated with increased risk of cardiovascular disease.
PLATELET ULTRASTRUCTURE
● Platelets, although anucleate, are strikingly complex and are
metabolically active. Their ultrastructure has been studied using
scanning and transmission electron microscopy, flow cytometry,
and molecular sequencing
RESTING PLASMA MEMBRANE
● platelet plasma membrane resembles any biologic membrane: a
bilayer composed of proteins and lipids, as diagrammed in
Figure 10.6. The predominant lipids are phospholipids, which
form the basic structure, and cholesterol, which distributes
asymmetrically throughout the phospholipids.
● The phospholipids form a bilayer with their polar heads oriented
toward aqueous environments—toward the blood plasma
externally and the cytoplasm internally. Their fatty acid chains,
esterified to carbons 1 and 2 of the phospholipid triglyceride
backbone, orient toward each other, perpendicular to the plane
of the membrane, to form a hydrophobic barrier sandwiched
within the hydrophilic layers
● The neutral phospholipids phosphatidylcholine and
sphingomyelin predominate in the outer blood plasma layer;
● the anionic or polar phospholipids phosphatidylinositol,
phosphatidylethanolamine, and phosphatidylserine predominate
in the inner, cytoplasmic layer.
● During platelet activation these phospholipids, especially
phosphatidylinositol, support platelet activation by supplying
arachidonic acid, an unsaturated fatty acid that becomes
converted to the eicosanoids, including the potent thromboxane
A2
● Cholesterol stabilizes the membrane, maintains fluidity, and
helps control the transmembranous passage of materials
through the selectively permeable plasma membrane
● Anchored within the membrane are glycoproteins and
proteoglycans that support surface glycosaminoglycans,
oligosaccharides, glycolipids, and essential plasma
surface-oriented glycosylated receptors that respond to cellular
and humoral stimuli, called ligands or agonists, transmitting their
stimulus through the membrane to activation organelles internal
to the platelet. The platelet membrane surface, called the
glycocalyx, also absorbs albumin, fibrinogen, and other plasma
proteins, in many instances transporting them to internal storage
organelles using a process called endocytosis
● maintaining a negative surface charge that repels other
platelets, other blood cells, and the endothelial cells that line the
blood vessels.
● ●
SURFACE- CONNECTED CANALICULAR SYSTEM
● plasma membrane invades the platelet interior, producing a
unique surface-connected canalicular system
○ SCCS twists sponge-like throughout the platelet,
enabling the platelet to store additional quantities of
the same hemostatic proteins found on the glycocalyx
○ also allows for enhanced interaction of the platelet with
its environment, increasing access to the platelet
interior as well as increasing egress of platelet release
products
○ e glycocalyx is less developed in the SCCS and lacks
some of the glycoprotein receptors present on the
platelet surface
○ the SCCS is the route for endocytosis and for secretion
of #-granule contents upon platelet activation.
DENSE TUBULAR SYSTEM
● Parallel and closely aligned to the SCCS
● condensed remnant of the rough endoplasmic reticulum
● sequesters Ca2% and bears a number of enzymes that support
platelet activation including phospholipase A2, cyclooxygenase,
and thromboxane synthetase, which support the production of
thromboxane A2, and phospholipase C, which supports
● production of the signaling molecules inositol triphosphate (IP3)
and diacylglycerol (DAG)
CYTOSKELETON: MICROFILAMENTS AND MICROTUBULES
● thick circumferential bundle of microtubules maintains the
platelet’s discoid shape
● circumferential microtubules parallel the plane of the outer
surface of the platelet and reside just within, although not
touching, the plasma membrane. There are 8 to 20 tubules
composed of multiple subunits of tubulin that disassemble at
refrigerator temperature or when platelets are treated with
colchicine. When microtubules disassemble in the cold, platelets
become round, but on warming to 37° C, they recover their
original disc shape. On cross section, microtubules are
cylindrical, with a diameter of 25 nm. The circumferential
microtubules could be a single spiral tubule
● microtubules move inward on platelet activation to enable the
expression of alpha-granule contents and subsequently
reassemble in long parallel bundles during platelet shape
change to provide rigidity to pseudopods
● Actin is contractile in platelets (as in muscle) and anchors the
plasma membrane glycoproteins and proteoglycans.
● Actin also is present throughout the platelet cytoplasm,
constituting 20% to 30% of platelet protein. In the resting
platelet, actin is globular and amorphous, but as the cytoplasmic
calcium concentration rises, actin becomes filamentous and
contractile
● The cytoplasm also contains intermediate filaments, ropelike
polymers 8 to 12 nm in diameter, of desmin and vimentin. The
intermediate filaments connect with actin and the tubules,
maintaining the platelet shape. Microtubules, actin
microfilaments, and intermediate microfilaments control platelet
shape change, extension of pseudopods, and secretion of
granule contents
PLATELET GRANULES: A-GRANULES, DENSE GRANULES,
AND LYSOSOMES
ALPHA GRANULES
● 50 to 80 !-granules in each platelet which stain medium gray in
osmium-dye transmission electron microscopy preparations
The #-granules are filled with proteins, some endocytosed, some
synthesized within the megakaryocyte and stored in platelets
● As the platelet becomes activated, #-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.
● employ the SCCS
DENSE GRANULES
● 2 TO 7 dense granules per platelet
○ These granules appear later than #-granules in
megakaryocyte differentiation and stain black (opaque)
when treated with osmium in transmission electron
microscopy
○ Small molecules are probably endocytosed and are
 HEMATOLOGY 2
HEMATOLOGY 2 LECTURE
Prof. Charito Bermido/ M1 Platelet Production, Structure, and Function
stored in the 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
#-granules—P-selectin, # IIb$3, and GP Ib/IX/V, for
instance—which implies a common source for the membranes of
both types of granules
● The contents of lysosomes probably digest vessel wall matrix
components during in vivo aggregation and may also digest
autophagic debris.
Plasma Membrane Receptors That Provide for Adhesion
● platelet membrane contains more than 50 distinct receptors,
including members of the cell adhesion molecule (CAM) integrin
family
○ (the CAM leucine-rich repeat family
○ CAM immunoglobulin gene family
○ the CAM selectin family
○ the seven-transmembrane receptor family
● Several integrins bind collagen, enabling the platelet to adhere
to the injured blood vessel lining. Integrins are heterodimeric
(composed of two dissimilar proteins). CAMs integrate their
ligands, which they bind on the outside of the cell, with the
internal cytoskeleton, triggering activation. GP Ia/IIa, or, using
integrin terminology, #2$1, is an integrin that binds the
subendothelial collagen that becomes exposed in the damaged
blood vessel wall, promoting adhesion of the platelet to the
vessel wall
● A5B1 and A6B1 bind the adhesive endothelial cell proteins
laminin and fibronectin- further promotes platelet adhesion
● GP VI
○ A collagen binding receptor
○ is a key collagen receptor that also binds the adhesive
protein thrombospondin
● GP Ib/IX/V
○ leucine-rich-repeat family CAM, multiple leucine-rich
domains
○ arises from the genes GP1BA, GP1BB, GP5, and
GP9
○ composed of two molecules each of GP Ib#, GP Ib$,
and GP IX
○ 1 molecule of GP V which are bound noncovalently
● The two copies of subunit GP Ib# bind VWF and support platelet
tethering (deceleration), necessary in capillaries and arterioles
where blood flow shear rates exceed 1000 s–1
● GP Ib$ molecules cross the platelet membrane and interact with
actin-binding protein to provide “outside-in” signaling
● Two molecules of GP IX and one of GP V help assemble the
four GP Ib molecules. The subunits of the integrin GP IIb/IIIa
(#IIb$3), are in a low affinity conformation (#IIb and $3) as they
are distributed across the plasma membrane, the SCCS, and the
internal layer of #-granule membranes of resting platelets.
● These form their active heterodimer, #IIb$3, after initiation of an
“inside-out” signaling mechanism triggered by agonist binding to
its receptor. Although various agonists may activate the platelet,
#IIb$3is a physiologic requisite because it binds fibrinogen,
generating interplatelet cohesion, called platelet aggregation.
The #IIb$3 integrin also binds other adhesive proteins that share
the target arginine-glycine-aspartate (RGD) amino acid
sequence with fibrinogen such as VWF, vitronectin, and
fibronectin.
The Seven-Transmembrane Repeat Receptors
● Thrombin, thrombin receptor activation peptide (TRAP),
adenosine diphosphate (ADP), epinephrine, serotonin, and
thromboxane A2 (TXA2) (and other prostaglandins) can function
individually or in combination to activate platelets
● These platelet “agonists” are ligands for seven-transmembrane
repeat receptors (STRs), so named for their unique
membraneanchoring structure. The STRs have seven
hydrophobic anchoring domains supporting an external binding
site and an internal terminus that interacts with G proteins to
mediate outside-in platelet signaling.
● Thrombin cleaves two STRs, protease-activated receptor 1
(PAR1) and PAR4, that together have a total of 1800 membrane
copies on an average platelet
● Thrombin also interacts with platelets by binding or digesting two
CAMs in the leucine-rich repeat family, GP Ib# and GP V, both of
which are parts of the GP Ib/IX/V VWF adhesion receptor.
● There are about 600 copies of the high-affinity ADP receptors
P2Y1 and P2Y12 per platelet
● These receptors are linked to different G-proteins and produce
distinct intracellular signals that have complementary effects on
platelet aggregation
● P2Y1 signaling leads to an increase in intracellular calcium
levels and contributes to initial platelet activation, shape change,
and the formation of small reversible aggregates, whereas
P2Y12 signaling leads to a decrease in cyclic adenosine
monophosphate (cAMP) levels and supports the formation of
irreversible platelet aggregates
● TP# and TP$ bind TXA2. This interaction produces more TXA2
from the platelet, a G-protein-based autocrine (selfperpetuating)
system that activates neighboring platelets.
● Epinephrine binds #2-adrenergic sites that couple to G-proteins
and open membrane calcium channels.
● The receptor site IP binds prostacyclin (prostaglandin I2, PGI2),
a prostaglandin produced by endothelial cells.
● The platelet membrane also contains STRs for serotonin,
platelet-activating factor, prostaglandin E2, PF4, and
B-thromboglobulin.
Additional Membrane Receptors
● The CAM immunoglobulin family includes the intercellular
adhesion molecules, or ICAMs (CD50, CD54, CD102), which
play a role in inflammation and the immune reaction
● platelet– endothelial cell adhesion molecule, or PECAM (CD31),
which mediates platelet-to-white blood cell and
platelet-to-endothelial cell adhesion
● Fc&IIA (CD32), a low-affinity receptor for the immunoglobulin Fc
portion that plays a role in a dangerous condition called
heparin-induced thrombocytopenia
● P-selectin (CD62) is an integrin that facilitates platelet binding to
endothelial cells, leukocytes, and one another.42 P-selectin is
found on the #-granule membranes of the resting platelet but
migrates via the SCCS to the surface of activated platelets.
P-selectin quantification by flow cytometry is a common means
for measuring in vivo platelet activation.
II. PLATELETS ACTIVATION
Adhesion: Platelets Bind Elements of the Vascular Matrix
● In vessels where the shear rate is more than 1000 s'1 , platelet
adhesion and aggregation require a defined sequence of events
that involves collagen, tissue factor, phospholipid, VWF, and a
number of platelet CAMs, ligands, and activators
● Damaged endothelial cells release VWF from cytoplasmic
storage organelles, which then adheres to sites of injury
● VWF becomes thread-like as it unrolls and exposes sites that
weakly bind the GPIb# portion of the platelet membrane GP Ib/
IX/V leucine-rich receptor
● reversible binding process that “tethers” thereby decelerating the
forward motion of the platelet.
● The interaction between platelet and VWF remains localized by
a liver-secreted plasma enzyme, ADAMTS13, also called
 HEMATOLOGY 2
HEMATOLOGY 2 LECTURE
Prof. Charito Bermido/ M1 Platelet Production, Structure, and Function
VWF-cleaving protease, which digests larger VWF multimers
into smaller, less biologically active forms
● At high shear rates, the VWF-GP Ib# tethering reaction is
temporary and the platelet rolls along the surface unless GP VI
comes in contact with the exposed ECM collagen
● Type I fibrillar collagen binding to platelet GP VI, which is
anchored in the platelet membrane by an Fc receptor-like
molecule, triggers internal platelet activation pathways, releasing
TXA2 and ADP, an “outside-in” reaction
● These agonists attach to their respective receptors: TP# and
TP$ for TXA2, and P2Y1 and P2Y12 for ADP, triggering an
“inside-out” reaction that raises the affinity of integrin #2$1 for
collagen. The combined effect of GP Ib/IX/V, GP VI, and #2$1
causes the platelet to become firmly affixed to the damaged
surface, where it subsequently loses its discoid shape and
spreads.
Aggregation: Platelets Irreversibly Cohere
● blood vessel injury exposes tissue factor expressed on
subendothelial smooth muscle cells and fibroblasts
● Tissue factor triggers the production of thrombin, which cleaves
platelet PAR1 and PAR4. This further activation generates the
“collagen and thrombin activated” or COAT platelet
● TXA2 and ADP are secreted from the platelet granules to the
microenvironment, where they activate neighboring platelets
through their respective receptors and trigger inside-out
activation of integrin #IIb$3 (GP IIb/IIIa receptor), enabling it to
bind RGD sequences of fibrinogen and VWF and support
platelet-to-platelet binding referred to as platelet aggregation
● P-selectin from the #-granule membranes moves to the surface
membrane to promote binding of platelets with leukocyte
● On further activation, in conjunction with aggregation, platelets
change in shape from discoid to round and extend pseudopods.
● As platelet aggregation continues, membrane integrity is lost,
and a syncytium or massive clump of platelets forms as the
platelets exhaust internal energy sources
● 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
● presence of 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. The risk of these cardiovascular events rises in
proportion to the number and avidity of platelet membrane #2$1
and GP VI receptors
● combination of polar phospholipid exposure on activated
platelets, platelet fragmentation with cellular microparticle
release, and secretion of the platelet’s #-granule and dense
granule contents triggers secondary hemostasis, called
coagulation
● 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.
Secretion: Activated Platelets Release Granular Contents
● Outside-in activation of the platelet through STRs (such as ADP
binding to P2Y12) and the immunoglobulin gene product GP VI
triggers actin microfilament contraction
● n. Intermediate filaments also contract, moving the
circumferential microtubules inward compressing the granules.
Contents of #-granules and lysosomes flow through the SCCS,
while dense granules migrate to the plasma membrane where
their contents are secreted. The dense granule contents are
small molecule vasoconstrictors and platelet agonists that
amplify primary hemostasis; most of the #-granule contents are
large molecule coagulation proteins that participate in secondary
hemostasis
● Phosphatidylserine is the polar phospholipid on which the factor
IX/VIII(tenase) and factor X/V (prothrombinase) complexes
assemble.
● The formation of both complexes is supported by ionic calcium
secreted by the dense granules
● The #-granule contents fibrinogen, factors V and VIII, and VWF
(which binds and stabilizes factor VIII) are secreted and increase
the localized concentrations of these essential coagulation
proteins, further supporting the action of tenase and
prothrombinase
Generation of Platelet Microparticles
● Microparticles are membrane-derived vesicles that form in
response to an activating stimulus that increases the platelet
intracellular concentration of calcium.
● . Elevated levels of intracellular calcium result in an inhibition of
the enzymes responsible for maintaining the asymmetric
distribution of phospholipids in the plasma membrane and an
activation of intracellular calpain, which cleaves the platelet
cytoskeleton
● Platelet microparticles, believed to be the most abundant
microparticles in the circulation, are formed after exposure of
platelets to strong agonists or shear stress, These vesicles are
made up of the plasma membrane and cytosolic material of the
parent cell from which they are derived and retain the cell
surface proteins found on the parent cell.
● microparticles have been found to modulate inflammation,
oxidative stress, angiogenesis, and thrombosis
● . The promotion of coagulation is the most studied platelet
function and results from the expression of phosphatidylserine
on the surface of the microparticles. Elevated levels of platelet
microparticles in patients with hypercoagulable conditions have
been shown to be predictive of adverse outcomes in some
settings
II. PLATELETS ACTIVATION PATHWAYS
G-PROTEINS
● G-proteins control cellular activation for all cells at the inner
membrane surface
● are #$& heterotrimers (proteins composed of three dissimilar
peptides) that bind guanosine diphosphate (GDP) when inactive
● Membrane receptor–ligand (agonist) binding promotes GDP
release and its replacement with guanosine triphosphate (GTP)
● The G# portion of the three-part G molecule briefly
disassociates, exerts enzymatic guanosine triphosphatase
activity, and hydrolyzes the bound GTP to GDP, releasing a
phosphate radical
● THe G-protein resumes its resting state, but the hydrolysis step
provides the necessary phosphorylation to trigger eicosanoid
synthesis or the IP3-DAG pathway.
Eicosanoid Synthesis
● eicosanoid synthesis pathway, alternatively called the
prostaglandin, cyclooxygenase, or thromboxane pathway, is one
of two essential platelet activation pathways triggered by
G-proteins found in platelets
● The platelet membrane’s inner leaflet is rich in
phosphatidylinositol, a phospholipid whose number 2 carbon
binds numerous types of unsaturated fatty acids, but especially
-eicosatetraenoic acid, commonly called arachidonic acid.
● G-protein activation triggers phospholipase A2, a membrane
enzyme that cleaves the ester bond connecting the number 2
carbon of the triglyceride backbone with arachidonic acid
● Cleavage releases arachidonic acid to the cytoplasm, where it
becomes the substrate for cyclooxygenase, anchored in the DTS
● Cyclooxygenase converts arachidonic acid to prostaglandin G2
 HEMATOLOGY 2

HEMATOLOGY 2 LECTURE
Prof. Charito Bermido/ M1 Platelet Production, Structure, and Function

and prostaglandin H2, and then thromboxane synthetase acts on

prostaglandin H2 to produce TXA2
● TXA2 binds membrane receptors TP# or TP$, inhibiting
adenylate cyclase activity and reducing cAMP concentrations,
which mobilizes ionic calcium from the DTS
● The rising cytoplasmic calcium level causes contraction of actin
microfilaments producing platelet shape change and further
platelet activation.
● The cyclooxygenase pathway in endothelial cells incorporates
the enzyme prostacyclin synthetase in place of the thromboxane
synthetase found in platelets
● The eicosanoid pathway end point for the endothelial cell is
prostaglandin I2, or prostacyclin, which binds the IP receptor
activating the IP3-DAG pathway, leading to an acceleration of
adenylate cyclase, an increase in cAMP, and a sequestration of
ionic calcium to the DTS. The unavailability of ionic calcium
shuts down platelet function.
● TXA2 has a half-life of 30 seconds, diffuses from the platelet,
and is spontaneously reduced to thromboxane B2, a stable,
measurable plasma metabolite.
● Thromboxane B2 is acted on by a variety of liver enzymes to
produce an array of soluble urine metabolites, including
11-dehydrothromboxane B2, which is stable and measurable

Inositol Triphosphate–Diacylglycerol Activation Pathway

● The IP3-DAG pathway is the second G-protein-dependent

platelet activation pathway
● Phospholipase C cleaves membrane phosphatidylinositol
4,5-bisphosphate (PIP2) to form IP3 and DAG, both second
messengers for intracellular activation. IP3 promotes release of
ionic calcium from the DTS, which triggers actin microfilament
contraction
● IP3 may also activate phospholipase A2. DAG triggers a
multistep process: activation of phosphokinase C, which triggers
phosphorylation of the protein pleckstrin, which regulates actin
microfilament contraction.

II. PLATELETS PROTEOME

●
Although platelets are anuclear, they contain a variety of
components required for protein synthesis, including ribosomes,
polyribosome complexes, and regulatory factors.57 It has also
been recently found that platelets contain microRNAs (miRNAs)
and template mRNAs.58 Proteomic analysis has indicated that
platelets contain thousands of unique transcripts that can be
translated in response to platelet activation and ligand binding to
the GPIIb/IIIa receptor. Such mechanisms allow platelets to alter
their phenotype in response to the level of activation.
Additionally, it is suggested that the protein synthesis pattern of
platelets can be altered by disease state