Platelets
PRODUCTION, STRUCTURE
AND FUNCTION
JOANN M. MACATUGGAL, RMT, MPH
I. MEGAKARYOCYTOPOIESIS
A special type of production wherein it is designed for the production of megakaryocytes
that will later on shed out platelets
Two Phases:
a. PROLIFERATIVE PHASE
PROGENITORS
resemble lymphocytes and cannot be distinguished by Wright-stained light
microscopy
Arise from the common myeloid progenitor under the influence of the
transcription gene product, GATA-1 ( globin transcription factor-1), regulated by
cofactor FOG1 (friend of GATA)
Megakaryocyte differentiation is suppressed by another ranscription gene
product, MYB, so GATA-1 and MYB act in opposition to balace
megakayocytopoiesis in one arm with differentiation to the RBC line in another
arm(erythropoiesis).
Burst forming unit-Meg (BFU-Meg) - least mature specific progenitor; diploid and
able to perform mitosis
Colony forming unit-MEG (CFU-Meg) - intermediate; diploid and able to perform
mitosis
Light density CFU-Meg (LD-CFU-Meg) - more mature progenitor; cannot divide but
it retains DNA replication and cytoplasmic maturation for endomitosis
ENDOMITOSIS - a form of mitosis that lacks telophase and cytokinesis( separation into
daughter cells)
B. TERMINAL DIFFERENTIATION PHASE
PRECURSORS
As endomitosis proceeds, megakaryocyte progenitors leave the proliferative
phase and enter terminal differentiation
MK-I stage (megakaryoblast)
resembles the myeloblast and pronomoblast (rubriblasts)
identification by morphology alone is inadvisable.
has cytoplasmic blebs that resemble platelets
MK-II stage (promegakaryocyte)
cytoplasm is abundant, nucleus shows minimal lobularity
MK-III stage (megakaryocyte)
the nucleus is lobulated with basophilic chromatin
the cytoplasm is azurophilic and granular, with evidence of the
demarcation system
Thrombocytopoiesis (platelet shedding)
At full maturation, platelet shedding, or thrombocytopoiesis, proceeds.
The total platelet population turns over in 8 to 9 days.
A single megakaryocyte may shed 2,000 to 4,000 platelets.
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. Megakaryocyte is adjacent to
endothelial cell of the bone marrow and extends a proplatelet process
through or between the endothelial cells into the vascular sinus.
MEGAKARYOCYTE MEMBRANE RECEPTORS AND MARKERS
Immunostaining of fixed tissues, flow cytometry with immunologic probes, and FISH with
genetic probes are used to identify visually indistinguishable megakaryocyte progenitors.
There are several megakaryocyte membrane markers that can be measured including
MPL and CD34 (disappear as differentiation proceeds)
The platelet membrane glycoprotein IIb/IIIa first appears on megakaryocyte progenitors
and remains present throughout maturation along with CD36, CD42, and CD62.
Cytoplasmic coagulation factor VIII, von Willebrand factor and fibrinogen may be
detected in the fully developed megakaryocyte by immunostaining.
HORMONES and CYTOKINES of MEGAKARYOCYTOPOIESIS
THROMBOPOIETIN (TPO)
70,000 dalton molecules that possesses 23% homology with the RBC producing
hormone, erythropoietin
mRNA for TPO has been found in the kidneys, liver, stromal cells, and smooth
muscle cells.
The liver is considered as the primary source of TPO.
The plasma concentration of TPO is inversely proportional to platelet and
megakaryocyte mass; 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 megakaryocytes.
TPO also induces the proliferation and maturation of megakaryocytes and induces
thrombocytopoiesis.
Other cytokines that function with TPO to stimulate megakaryocytopoiesis
includes IL-3, IL-6 and IL-11
IL-3 seem to act in synergy with TPO to induce the early differentiation of stem
cells whereas IL-6 and IL-11 in the presence of TPO to enhance endomitosis,
megakaryocyte maturation, and thrombocytopoiesis.
Other cytokines and hormones that participate synergistically with TPO and ILs are
kit ligand or mast cell growth factor, GM-CSF, G-CSF and acetylcholinesterase-
derived megakaryocyte grown stimulating peptide.
Platelet factor 4, Beta-thromboglobulin, neutrophil activating peptide 2, IL-8, and other
factors inhibit in vitro megakaryocyte growth which indicates that they may have a role in
the control of megakaryocytopoiesisin vivo.
PLATELETS (Thrombocytes)
Shape of platelets:
Resting and circulating platelets: biconvex or disk-shaped
Activated platelets: spherical with pseudopods
Round: In EDTA, when at cold/ref temperature and treated with colchicine
Cylindrical and beaded that resembles fragment of megakaryocyte proplatelet: in
citrated blood
Produced directly from the megakaryocyte cytoplasm
Each megakaryocyte produces between 2000 to 4000 platelets
Important in both primary and secondary hemostasis
Described as cells with granular cytoplasm but no nuclear material
Platelets cluster with the RBCs near the center of the blood vessel
Platelets move back and forth with the WBCs from venules into the white pulp of the
spleen
LIFE SPAN: 9-10 days; Maturation time is 5 days
On a Wright-stained PBS:
platelets are spread throughout the RBC monolayer (7-21 cells per 100x field)
platelets have an average diameter of 2.5 um (or 2 to 4 um)
Reticulated platelets
Stress platelets
appear in compensation for thrombocytopenia
newly released from megakaryocytes and still contain RNA
markedly larger than the usual platelets (diameter in PBS exceeds 6um where MPV
reaches 12 to 14 fL)
clinical use:
can help differentiate bone marrow failure from peripheral destruction in
thrombocytopenia
early predictor of bone marrow recovery after chemotherapy and
transplantation
Potentially prothrombic (maybe associated with increased risk of
cardiovascular diseases)
Size of platelets:
Normal: average of 2.5 um
Mean Platelet Volume (MPV)
reference range: 6.8 to 10.2 fL
LOW MPV: Small platelets; HIGH MPV: Bigger platelets
EDTA causes swelling of platelets (causes approximately 20% increase in
MPV during the first hour)
Cytoplasm
On a Wright-stained PBS, platelets appear lavander and granular
two general parts on light microscope
chromomere (granulomere)
centrally located, granular
hyalomere
peripherally located, non- granular
⅓ ofthe platelets are found in your spleen; ⅔ are on the circulation
Composed about 60% protein, 30% lipid, 8% carbohydrate, various mineral, water and
nucleotides
Glucose/dextrose is the major source of energy of platelets.
PLATELET STRUCTURE
I. PERIPHERAL ZONE
The outer layer, composed of membranes and responsible for platelet adhesion and
aggregation
known as the transmitter region/stimulus or transmitter zone
this is where substances bind or attach at the platelet, where substances enter or leave
the platelet
GLYCOCALYX
outermost part, fuzzy coating, primarily composed of glycoproteins including
coagulation factors V, VIII and fibrinogen
Unique among the cellular components of the blood
Composed of plasma proteins and carbohydrate molecules that are related to
the coagulation, complement, and fibrinolytic systems.
The glycoprotein receptors of the glycocalyx mediate the membrane contact
reactions of platelet adherence, change of cellular shape, internal contraction,
and aggregation.
PLASMA MEMBRANE
composed of phospholipids and cholesterol
Phospholipids forms the basic structure of platelets which makes the
membrane selectively permeable (phospholipid bilayer); supports activation of
platelets and allows to form clots
Phosphatidyl inositol - support platelet activation with the help of
thromboxane A2
Phosphatidyl serine - helps clotting factors attach to platelet to form
clot; binding site for clotting factors
Phosphatidyl choline
Sphingomyelin
Cholesterol helps to stabilize cell membrane; maintains fluidity for the
permeability of platelet membrane
SUB-membranous area
where messages from external membrane are translated into chemical signals
causing the activation and physical change in platelet
II. SOL-GEL ZONE
Forms the cytoskeleton of the platelet
Composed of microfilaments (actin and myosin), intermediate filaments (desmin and
vimentin), and microtubules (tubulin)
Provides the structure for maintaining the circulating discoid shape, and also maintains
the position of the organelles
Microtubules, actin microfilaments, and intermediate microfilaments control platelet
shape change, extension of pseudopods, and secretion of granules
MICROTUBULES
8-20 tubules per platelets
major responsible for platelet disc shape and maintains platelet disc shape
on cross section, microtubules are cylindrical, with a diameter of 25nm. The
circumferential microtubules could be a single spiral tubule
dissassemble when at refrigerator temperature or when platelets are treated
with colchicine
when microtubules dissassemble in the cold, platelets become round, but on
warming to 37deg C, they recover their original disc shape
MICROFILAMENTS
composed of actin and myosin or actomyosin (thrombosthenin), a contractile
protein for platelet contraction
Actin is present throughout the platelet cytoplasm, constituting 20% to 30% of
platelet protein
resting platelet actin is globular and amorphous
activated platelet actin becomes filamentous and contractile
INTERMEDIATE FILAMENTS
composed of desmin and vimentin
they are ropelike polymers 8 to 12 nm in diameter
they connect with actin and the tubules, maintaining the platelet shape
III. ORGANELLE ZONE
Composed of mitochondria, alpha granules, dense/delta granules, and lysosomes
Mitochondria- for energy production / ATP synthesis
ALPHA GRANULES
300 to 500 nm in diameter eg. Platelet factor 4, platelet derived growth factor,
thrombospondin, vwf, fibrinogen, fibronectin, factor V
there are 50- 80 alpha granules in each platelet
stain medium gray in osmium-dye transmission electron microscopy
as the platelets becomes activated, alpha granule membranes fuse with surface
connect canalicular system or open canalicular system
DENSE GRANULES/ DELTA Granules
250 to 350 nm in diameter eg. Calcium, ADP, Pyrophosphate, ATP, Serotonin,
magnesium
there are 2 to 7 dense granules per platelet
Stain black (opaque) when treated with osmium in transmission electron
microscopy
in contrast to your alpha granules which employ the SCCS, dense granules
migrate to the plasma membrane and release their contents directly into the
plasma on platelet activation
LYSOSOMAL GRANULES (LYSOSOMES)
contains acid hydrolases, ACP and hydrolytic enzymes
300 nm in diameter granules that stain positive for arylsulfatase, b-
glucuronidase, acid phosphatase, and catalase
digest vessel wall matrix components during in vivo platelet aggregation
digest autophagic debris
they do have clearing responsibilities to remove infectious agents and cellular
debris
SUBSTANCES SECRETED BY PLATELETS AND THEIR ROLE IN HEMOSTASIS
IV. MEMBRANOUS SYSTEM
composed of surface connected canalicular system and dense tubular system
DENSE TUBULAR SYSTEM (DTS)
derived from rough ER and sequesters or hold calcium for platelet activation
process and prostaglandin synthesis
The DTS sequesters calcium and bears a number of enzymes that support
platelet activation including phospholipase A@, cyclooxygenase, and
thromboxane synthetase, which supports production of the signaling molecules
inositol triphosphate (IP3) and diacylgycerol (DAG)
Control center for platelet activation
SURFACE CONNECTING CANALICULAR SYSTEM or OPEN CANALICULAR SYSTEM
(OCS)
An invaginationof plasma membrane, acts as a canal for the release of granule
and also involve in platelet phagocytosis
it is the system that forms the invaginated, sponge-like portion of the cell that
provides an expanded reactive surface to which plasma clotting factors are
selectively adsorbed
Twists sponge-like throughout the platelet
store additional quantities of the same hemostatic proteins found on the
glycocalyx
allows for entrance interaction of the platelets with its environment, increasing
access to the platelet interior as well as increasing egress of platelet release
products
route for endocytosis and canal for secretion of a alpha granule contents
PROPERTIES of PLATELETS
1. ADHESIVENESS
The property of sticking to a rough surface
During injury of bloof vessel, endothelium is damaged and the subendothelial collagen is
exposed.
While coming in contact with collagen, platelets are activated and adhere to collagen
Other factors which accelerate adhesiveness are collagen, thrombin, ADP, thromboxane
A2, calcium ions, P selectin, and vitronectin
2. AGGREGATION
The grouping of platelets
During activation, the platelets change their shape wth elongation of long filamentous
pseudopodia which are called processes or filopodia
Filopodia help the platelets aggregate together. Activation and aggregation of platelets is
accelerated by ADP, TXA2 and platelet-activating factor
3. AGGLUTINATION
The clumping together of platelets
Aggregated platelets are agglutinated by the action of some platelet-agglutinins and
platelet activating factors
FUNCTIONS of PLATELETS
Blood clotting
Clot retraction
Hemostasis
Repair ruptured blood vessel
Defense system
ACTIVATORS of PLATELETS
Collagen
Von Willebrand factor
Thromboxane A2
Platelet-activating factor
thrombin
ADP
Calcium ions
P selectin
Convulxin
INHIBITORS of PLATELETS
Nitric Oxide
Clotting factors II,IX,XI, XII
Prostacyclin
Nucleotidases