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MTY1406 | HEMA2 (LAB)

WEEK 3 | PLATELET PRODUCTION, STRUCTURE, & FUNCTION (PART 2)

TABLE OF CONTENTS
I PLATELET ACTIVATION 1
A Adhesion: Platelets Bind Elements of 1
the Vascular Matrix
B Aggregation: Platelets Irreversibly 2
Cohere
C Secretion: Activated Platelets Release 3
Granular Contents
D Generation of Platelet Microparticle 4
II PLATELET ACTIVATION PATHWAYS 4
A G-Proteins 4
B Eicosanoid Synthesis 5
C Inositol Triphosphate-Diacylglycerol 5
Activation Pathway
III PLATELET PROTEOME 6

MAIN TOPIC
SUBTOPIC
SUB-SUBTOPIC
SUB-SUB-SUBTOPIC

PLATELET ACTIVATION
● NOTE:
o platelet adhesion, aggregation, and secretion
often occur simultaneously

ADHESION: PLATELETS BIND ELEMENTS OF THE

VASCULAR MATRIX
● As blood flows, vessel walls create stress, or shear
force
−1
o Measured in units: 𝑠
−1
o Venules and veins: 500 𝑠
o
−1
Arterioles and capillaries: 5000 𝑠
o Stenosed (hardened) arteries: 40,000 𝑠
−1
o In vessels where the shear rate is more than
−1
1000 𝑠
▪ platelet adhesion and aggregation require a
defined sequence of events that involves
collagen, tissue factor, phospholipid, Von
Willebrand Factor (VWF), and a number of
platelet CAMs, ligands, and activators
(Figure 10.9)
● Injury to the blood vessel wall disrupts the
collagen of the extracellular matrix (ECM) (Figures
10.9A and 10.9B)
● Damaged endothelial cells release VWF from
cytoplasmic storage organelles, which then adheres
to sites of injury (Figure 10.9C)
● This is a 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 VWF-cleaving protease,
which digests larger VWF multimers into smaller,
less biologically active forms.
● VON WILLEBRAND FACTOR (VWF)
o Molecular weight: 500,000 - 20,000,000 Daltons
o Circulates as a globular protein
o Under shear stress, VWF becomes thread-like as
it unrolls and exposes sites that weakly bind the
GPIbα portion of the platelet membrane GP Ib/
IX/V leucine-rich receptor.
▪ ‘Tethers’
▪ Reversible binding process
▪ Decelerates the forward motion of the
platelet
● ADAMTS13 (VWF-CLEAVING PROTEASE)
o liver-secreted plasma enzyme that plays a
crucial role in maintaining localized interactions
between platelets and von Willebrand factor
(VWF)
o Digests larger VWF multimers into smaller, less
biologically active forms.
● VWF-GPIbα TETHERING REACTION
o Temporary at high shear rates
o 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
o Anchored in the platelet membrane by an Fc
receptor-like molecule
o Triggers internal platelet activation pathways,
releasing TXA2 and ADP, an “outside-in”
reaction (Figure 10.8)

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MTY1406 | HEMA2 (LAB)
WEEK 3 | PLATELET PRODUCTION, STRUCTURE, & FUNCTION (PART 2)

● ADDITIONAL NOTES
○ GP Ib/IV/V: 2:2:2:1 complex of GP Iba
and IbB, GP IX, and GP V (recap)
○ Platelet plug is the end product of
hemostasis
○ Extrinsic tenase
■ TF bound to FVII will attach to
phosphatidyl serine -> Ca2+
■ FIX will be activated by extrinsic
factor

AGGREGATION: PLATELETS IRREVERSIBLY COHERE

● Blood vessel injury exposes tissue factor expressed
on subendothelial smooth muscle cells and
fibroblasts.
o Tissue factor triggers the production of
thrombin, which cleaves platelet PAR1 and
PAR4.
o This further activation generates the “collagen
and thrombin activated” or COAT platelet,
integral to the cell-based coagulation model
described in Chapter 35 (Figure 10.10)

● These agonists attach to their respective receptors,

triggering an “inside-out” reaction:
o TPα and TPβ for TXA2,
o P2Y1 and P2Y12 for ADP
● “INSIDE-OUT” REACTION
o Raises the affinity of integrin α2β1 for collagen
● COMBINED EFFECT OF GP Ib/IX/V, GP VI, AND α2β1
o Causes the platelet to become firmly affixed to
the damaged surface, where it subsequently
loses its discoid shape and spreads.
● PLATELET ADHESION

● PLATELET ACTIVATORS:
o TXA2
o ADP
▪ Secreted from the platelet granules to the
microenvironment.
▪ Activate neighboring platelets through their
respective receptors and trigger inside-out
activation of integrin αIIbβ3 (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.

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MTY1406 | HEMA2 (LAB)
WEEK 3 | PLATELET PRODUCTION, STRUCTURE, & FUNCTION (PART 2)

● P-SELECTIN ● Dense granules

o Released from the α-granule membranes o migrate to the plasma membrane where their
o Moves to the surface membrane to promote contents are secreted
binding of platelets with leukocytes. ● Dense granule contents
● On further activation, in conjunction with o small molecule vasoconstrictors and platelet
aggregation, platelets change in shape from discoid agonists
to round and extend pseudopods. o amplify primary hemostasis
o Allows platelets to cover more surface area ● Most of the α-granule contents
o Enhances platelet binding to other platelets o are large molecule coagulation proteins that
and foreign surfaces. participate in secondary hemostasis
o Membrane phospholipid asymmetry is lost
o Membrane integrity is lost
o Syncytium or massive clump of platelets
forms as the platelets exhaust internal energy
sources.
● PLATELET AGGREGATION
o Key part of primary hemostasis
o normal part of vessel repair
o arteries: formation of “white clot”
▪ “white clot” is composed primarily of
platelets and VWF (Figure 10.9D)
▪ Presence of white clots often implies
inappropriate platelet activation in
seemingly uninjured arterioles and
arteries
▪ Presence of white clot is the pathologic
basis for arterial thrombotic events (i.e.
acute myocardial infarction, peripheral artery
disease, and ischemic stroke.)
- risk of these cardiovascular events rises in
proportion to the number and avidity of
platelet membrane α2β1 and GP VI
receptors.
● SECONDARY HEMOSTASIS (COAGULATION)
o Triggered by the combination of:
▪ Polar phospholipid exposure on activated
platelets
▪ Platelet fragmentation with cellular
microparticle release
▪ Secretion of the platelet’s α-granule and
dense granule contents
● Fibrin and red blood cells deposit around and
within the platelet syncytium to form a bulky “red
clot.”
o 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.

SECRETIONS: 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 ● Presenting polar phospholipids on their
o triggers actin microfilament contraction membrane surfaces
● Intermediate filaments o platelets provide a localized cellular milieu that
o contracts, moving circumferential microtubules supports coagulation
inward compressing the granules ● Phosphatidylserine
● Contents of α-granules and lysosomes o the polar phospholipid
o flow through the SCCS

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MTY1406 | HEMA2 (LAB)
WEEK 3 | PLATELET PRODUCTION, STRUCTURE, & FUNCTION (PART 2)

o factor IX/VIII(tenase) and factor X/V - derived and retain the cell surface
(prothrombinase) complexes assemble proteins found on the parent cell.
▪ formation of both complexes is supported by o modulate inflammation, oxidative stress,
ionic calcium secreted by the dense granules angiogenesis, and thrombosis
● The α-granule contents ● Promotion of coagulation
o fibrinogen, factors V and VIII, and VWF (which o most studied platelet function
binds and stabilizes factor VIII) o results from the expression of
▪ secreted and increase the localized phosphatidylserine on the surface of the
concentrations of these essential coagulation microparticles
proteins ● Elevated levels of platelet microparticles in
▪ further supporting the action of tenase and patients with hypercoagulable conditions
prothrombinase o shown to be predictive of adverse outcomes in
● Platelet secretions some settings
o provide for cell-based, controlled, localized
coagulation PLATELET ACTIVATION PATHWAYS
● Additional α-granule secretion products that, G-PROTEINS
although not proteins of the coagulation pathway, ● Control cellular activation for all cells at the inner
indirectly support hemostasis (TABLE 10.8) membrane surface
● G proteins are ɑ γ heterotrimers that bind
guanosine diphosphate (GDP) when inactive
o ɑ γ heterotrimers are proteins composed of
three dissimilar peptides
● Membrane receptor-ligand (agonist) binding
o promotes GDP release and its replacement with
guanosine triphosphate (GTP)
● The Gɑ portion of the three part G molecule,
o briefly diassociates
o exerts enzymatic guanosine triphosphatase
activity
o hydrolyzes the bound GTP to GDP
▪ releasing a phosphate radical
● The G protein resumes its resting state
o but the hydrolysis step provides necessary
phosphorylation to trigger eicosanoid synthesis
or the IP3-DAG pathway

GENERATION OF PLATELET MICROPARTICLES

● Microparticles
o membrane-derived vesicles
o form in response to an activating stimulus that
increases the platelet intracellular
concentration of 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
- lead to the outward blebbing of the
plasma membrane and the formation of
platelet microparticles
▪ Platelet microparticles
- most abundant microparticles in the
circulation
- formed after exposure of platelets to
strong agonists or shear stress
▪ Vesicles
- made up of the plasma membrane and
cytosolic material of the parent cell

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MTY1406 | HEMA2 (LAB)
WEEK 3 | PLATELET PRODUCTION, STRUCTURE, & FUNCTION (PART 2)
EICOSANOID SYNTHESIS
● Alternatively called the prostaglandin,
cyclooxygenase, or thromboxane pathway
● One of two essential platelet activation pathways
triggered by G-proteins found in platelets
● Platelet membrane’s inner leaflet is rich in:
o Phosphatidylinositol
▪ a phospholipid whose number 2 carbon
binds numerous types of unsaturated fatty
acids
- especially 5, 8, 11, 14-eicosatetraenoic acid
or also called arachidonic acid
● Membrane receptor ligand binding and the
consequent G-protein activation triggers
phospholipase A2
o 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:
o Prostaglandin G2 and prostaglandin H2
▪ Then thromboxane synthetase acts on
prostaglandin H2 to produce TXA2
▪ TXA2 binds membrane receptors TPɑ or TP :
- inhibiting adenylate cyclase activity
- reducing cAMP concentrations
- which mobilizes ionic calcium from the
DTS
▪ The rising cytoplasmic calcium level causes
contraction of actin microfilaments
produces:
- platelet change and further platelet
activation
● Arachidonic acid used as an agonist
o It bypasses the membrane and directly enters
the eicosanoid synthesis pathway
● Cyclooxygenase pathway in endothelial cells
o Uses enzyme prostacyclin synthetase in place
of the thromboxane synthetase found in
platelets
o Endpoint: 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
- sequestration of ionic calcium to the DTS
NOTE: unavailability of ionic
calcium shuts down platelet
function
o Thus, endothelial cell eicosanoid pathway
suppresses platelet activation in the intact blood
vessel
5
▪ creating a dynamic equilibrium with the
eicosanoid pathway within the platelet
where platelet activation occurs.
● TXA2: half life of 30 seconds
o diffuses from the platelet and is spontaneously
reduced to thromboxane B2
▪ stable measurable plasma metabolite
● Production of clinical assay for plasma thromboxane
B2 as a measure of platelet activity have been
unsuccessful
o Because special specimen management is
required to prevent ex vivo platelet activation
with unregulated release of thromboxane B2
subsequent to collection
o Thromboxane B2 is acted on by a variety of liver
enzymes to produce an array of soluble urine
metabolites including:
▪ 11-dehydrothromboxane
- stable and measurable
INOSITOL TRIPHOSPHATE-DIACYLGLYCEROL
ACTIVATION PATHWAY (IP3-DAG)
● The second G-protein-dependent platelet
activation pathway.
● The G-protein triggers the enzyme phospholipase
C.
o Phospholipase C cleaves membrane
phosphatidylinositol 4,5 bisphosphate (PIP2) to
form IP3 and DAG, both second messengers for
intracellular activation.
o 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.
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MTY1406 | HEMA2 (LAB)
WEEK 3 | PLATELET PRODUCTION, STRUCTURE, & FUNCTION (PART 2)

PLATELET PROTEOME
● Despite a platelet being anuclear, they contain a
variety of components required for protein synthesis,
including ribosomes, polyribosome complexes,
and regulatory.
o Also, it contains microRNA (miRNAs) and
template mRNA.
● 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.
● These mechanisms allow platelets to alter their
phenotype in response to the level of activation.
● Furthermore, it is suggested that the protein
synthesis pattern of a platelet can be altered by
disease state.

REFERENCES
Rodak’s Hematology Clinical Principles and
Applications (6th Edition)

NOTE: MEMORIZE !!! ;-; eme

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MTY1406 | HEMA2 (LAB)
WEEK 3 | PLATELET PRODUCTION, STRUCTURE, & FUNCTION (PART 2)

PRACTICE QUESTIONS 9. What is the function of P-selection in platelet

1. What state in G-proteins provides necessary activation?
phosphorylation to trigger eicosanoid synthesis? A. Enhances platelet binding to fibrinogen
A. Resting B. Promotes platelet aggregation
B. Hydrolysis C. Facilitates binding of platelets with
C. Both A and B leukocytes
D. None of the above D. Triggers membrane phospholipid
2. The cyclooxygenase pathway in endothelial cells asymmetry
uses what enzyme in place of thromboxane 10. These are membrane derived particles that form
synthetase? in response to an activating stimulus which
A. Prostaglandin G2 increases the platelet intracellular concentration
B. Arachidonic acid of calcium.
C. Prostaglandin synthetase A. Phospholipase A2
D. Prostacyclin synthetase B. Microparticles
3. G proteins are ɑ γ heterotrimers. It binds C. G-proteins
guanosine diphosphate (GDP) when active. D. Intermediate filaments
A. First statement is true, second 11. An α-granule protein that has a property of
statement is false fibrinolysis control
B. First statement is true, second A. Plasminogen activator inhibitor-1
statement is false B. Platelet factor 4
C. Both statements are true C. Platelet derived growth factor
D. Both statements are false D. Plasminogen
4. Activation of phosphokinase C stimulates the 12. An α-granule protein that has a property of
phosphorylation of what protein? supporting mitosis of vascular fibroblasts and
A. Globular Protein smooth muscle cells
B. Fibronectin A. Endothelial growth factor
C. Pleckstrin B. Fibronectin
D. α-granule contents C. Platelet factor 4
5. An enzyme that cleaves membrane D. Protein C inhibitor
phosphatidylinositol 4,5 bisphosphate (PIP2) to
form IP3 and DAG?
A. Phospholipase C
B. Phospholipase A2
C. Thromboxane B2
D. Thromboxane B
6. Platelets contain ribosomes, polyribosome
complexes, nucleus. Protein synthesis pattern of
a platelet can be altered by disease state.
A. First statement is true, second
statement is false
B. First statement is true, second
statement is false
C. Both statements are true
D. Both statements are false
7. What is released from damaged endothelial
cells in response to injury to the blood vessel
wall?
A. Tissue factor
B. Collagen
C. VWF
D. ADAMTS13
8. What role does ADAMTS13 play in platelet-VWF
interactions?
A. Promotes platelet aggregation
B. Inhibits platelet adhesion
C. Digests VWF into smaller forms
D. Increases VWF molecular weight ANSWER KEYS:

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