College: Applied Medical Science

Department: Medical Laboratory Science

Platelets

Dr. Hytham Ahmed Abuagla

 PLATELETS

• Platelets are small, disk shaped, anucleated cells with a

diameter of about 1.5–3.5 μm.
• MPV 8-10 fl
• They are small fragments of cytoplasm derived from
megakaryocytes in B.M
• Life span: about 10 days.
• Normal blood contain: 150-450 x 103 platelets/µl
• They are circulate as free-floating cells and do not
adhere to normal vascular lining or to each other.
 Platelet structure
• Platelets are extremely small and discoid, 2.0 - 4.0 ×
0.5 μm in diameter, with a mean volume of 7 – 11 fl.
• Examination of platelet by electron microscope reveal
the following:
• 1. Glycocalyx: it is a layer composed of plasma proteins
and carbohydrate molecules that coat the platelet plasma
membrane.
• 2. Cytoplasmic membrane: It composed of a lipid bilayer. It
showed an extending into the interior of the cell what is
called the open canalicular system, that provides an
expanded reactive surface to which plasma clotting factors
are selectively adsorbed.
• 3. Microfilaments and microtubules: Directly beneath the
cell membrane is a series of submembrane filaments and
microtubules that form the cellular cytoskeleton.
• 4. Granules: There are three types of granules present in the
mature platelets, alpha granules, dense or delta granules,
and lysosomes.
• a. alpha granules: the most abundant and contain:
• heparin neutralizing platelet factor 4 (PF4)
• fibrinogen
• fibronectin
• thrombospondin
• von Willebrand factor
• thromboglobulin
• platelet derived growth factor.
 • b. dense granules: Named because of their appearance
when viewed by electron microscopy, contain:
• Serotonin.
• adenosine diphosphate (ADP).
• adenosine triphosphate (ATP).
• calcium.
• c. Lysosomes: Contain hydrolase enzymes.
• Extrusion of the contents of these storage granules
requires internal, cellular contraction.
• Secretions from the granules are released into the open
canalicular system.
• 5. Other cytoplasmic constituents:
• The platelet contains substantial quantities of the
contractile proteins, such as actomyosin
(thrombosthenin), myosin, and filamin.
• The cytoplasm of platelets also contains glycogen
and enzymes of the glycolytic and hexose pathways.
• Energy for metabolic activities and cellular contraction
is derived from aerobic metabolism in mitochondria
and anaerobic glycolysis-utilizing glycogen stores.
Microstructure of Platelets
 Dense(δ) granules PLT granule
α-granules
contents lysosomal granules

ATP PF4 Galactosidase

ADP β-Thromboglobulin Fucosidase
Calcium Fibrinogen Hexoaminidase
Serotonin Factor V Glucouronidase
Pyrophosphate Thrombospondin Cathepsin
P selectin Fibronectin Glycohydrolases

Transforming growth factor- PDGF others

beta (1)
Catecholamines PAI-1
(noradrenaline, adrenaline)

GDP/GTP Histidine rich

glycoprotein
Α2 macroglobulin
Thrombopoiesis
 Megakaryoblast

• Size: 15-50 µm in diameter .

• Round
• High nuclear cytoplasmic
(N/C) ratio.
Nucleus:
• compact with purple color
and several nucleoli (2-6)
Cytoplasm:
• a thin rim of basophilic
cytoplasm surround the
nucleus.
• No or few azurophilic
granules may be seen.
• Cytoplasmic pseudopodes
frequently seen.
 Promegakaryocyte

• Size: 20-80 µm in diameter

• N/C ratio: lower than megakaryoblast
(4/1-1/1)
Nucleus:
• Chromatin dark in color with increase
clumping.
• Nucleoli: 2 or more distinct nucleoli
may be seen.
Cytoplasm:
• Quantity: increased
• Basophilia: persist until full
maturation
• Pseudopodes are often visible
• Fine azurophilic granules are most
abundant adjacent to the nucleus.
 Megakaryocyte

• The largest cell in the B.M.

• It is the primary target of the thrombopoietin activity in
the day to day regulation of PLT production.
• Size: up to 160 µm in diameter
• N/C ratio: low (can be 1/12)
• Nucleoli: absent
• Nucleus: multi-nucleated (up to 32 nuclei), due to endo-
mitosis.
Megakaryocytes
 Platelet production

• Platelets are formed by fragmentation of megakaryocyte

cytoplasm.
• Platelet formation begins with the initial appearance of a
pink color in the cytoplasm of the megakaryocyte and
increase granularity near the border of the cell.
• The fully mature megakaryocyte shed PLTs explosively
from the edges of the cytoplasm.
• It has been estimated that each megakaryocyte can
shed 1000-5000 PLTs.
• After that, its nucleus and remaining cytoplasm are
phagocytosed by neighboring macrophages
 Thrombopoietin

§70 KD molecule with 23% homology to EPO

§Produced in the liver
§Concentration is inversely proportion to the platelet
and megakaryocyte mass
§Induces stem cell differentiation
 Interleukins

§Interleukin 3: acts in synergy with TPO; induces

early differentiation of stem cells
§Interleukin 6: acts only in the presence of TPO;
enhances endomitosis, Megakaryocyte maturation
and platelet release
 Platelet function in the hemostatic
process
• PLTs promote hemostasis in a number of ways:
1. After vascular damage, PLTs form a hemostatic plug to
seal the hole in the blood vessel.
2. PLTs provide a procoagulant surface on which blood
coagulation accelerated.
3. After platelet activation the membrane also expresses
binding sites for several coagulation proteins such as
factor XI and factor VIII.
4. PLTs inhibit the anticoagulant action of heparin by
secreting PF4.
 Primary hemostasis

• Primary haemostasis is a process whereby platelets

interact with elements of the damaged vessel wall,
leading to the initial formation of a “platelet plug”.
Formation of primary hemostatic plug

ØPrimary haemostatic plug formation can be

divided into three steps:
1. PLT adhesion
2. PLT activation and shape change (the release
reaction:
3. PLT aggregation
 Platelet adhesion
• Within seconds after blood vessel injury platelets
accumulate at the site of vascular damage and
begin to adhere to subendothelium or perivascular
connective tissue that has become exposed to
flowing blood.
• The major components to which PLTs adhere are
collagen fibrils.
• The PLT membrane is the site of interaction with
the plasma environment and with damaged vessel
wall.
• PLTs bind to collagen through von Willebrand factor
(vWF) and specific receptor on the platelet surface.
• vWf first adheres to collagen fibers and then changes
its conformation.
• This step is followed by the binding of PLTs to vWf via
PLT membrane glycoprotein Ib (GpIb) resulting in an
initial monolayer
Adhesion
Platelet activation (release reaction)
• After adhesion, PLTs undergo a shape change from that
of a disk to more spherical shape with extended
pseudopodes.
• At the same time, a secondary process occurs by which
certain substances stored in PLT granules are actively
discharged through the open canalicular system to the
outside of the adherent cells.
• These substances include: ADP, serotonin, βTG, PF4,
PDGF, and vWf.
• ADP induce adhesion of PLT to each others ”PLT
aggregation”
 Platelet aggregation
• This is characterized by cross - linking of platelets
through active GPIIb/IIIa receptors with fibrinogen
bridges
• Increases the size of a PLT hemostatic plug at the site
of injury.
• Also accompanied by the release of substances that
in turn recruit other PLTs to aggregate.
• In addition to ADP a wide variety of agents can cause
PLTs aggregation and secretion, include: epinephrin,
collagen, thrombin, PAF and Others
• Most of these agents act by binding to specific
receptors on the PLT surface membrane.
Platelet Aggregation
 Arachidonic acid metabolism
• Platelet aggregation may occur by at least two
independent but closely linked pathways.
1. Arachidonic acid metabolism pathway:
• Activation of phospholipase enzymes (PLA2) releases
free arachidonic acid from membrane phospholipids
(phosphatidyl choline).
• About 50% of free arachidonic acid is converted by a
lipo-oxygenase enzyme to a series of products including
leucotrienes, which are important chemoattractants of
white cells
• The remaining 50% of arachidonic acid is converted
by the enzyme cyclooxygenase into labile cyclic
endoperoxides, most of which are in turn converted
by thromboxane synthetase into TXA2.
• TXA2 has profound biological effects:
• Causes release of secondary platelet granule.
• Enhance local vasoconstriction.
• Increase local platelet aggregation.
• 2. The second pathway:
• The second pathway of activation and aggregation can
proceed completely independently from the first one
• Various platelet agonists, including thrombin, TXA2, and
collagen, bind to receptors and activate phospholipase C.
• This generates diacylglycerol which in turn elevate
intracellular calcium.
• Calcium is released from the dense tubular system
to form complexes with calmodulin
• This complex and the free calcium act as coenzymes
for the release reaction for the activation of:
1. Different regulatory proteins
2. Actin and myosin and the contractile system
3. The liberation of arachidonic acid from
membrane phospholipids and the generation of
TXA2.
Role of Platelets in fibrin formation

• Provide the procoagulant activity PF3 which is

demonstrable only on the activated PLTs and
represent a suitable surface on which the
activation of prothrombin to thrombin is catalyzed
by activated factor X (Xa).
• PTs provide a surface receptor(s) for factors Va and
Xa.
• Several coagulation factors including: fibrinogen,
and factor V are synthesized in megakaryocytes,
stored in PLTs and secreted during the release
process.