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ALEXANDERIA UNIVERSITY

FACULTY OF MEDICINE
HEMATOLOGY DEP.
PRESENTATION:
PLATELETS & PLATELETS
DISORDERS
AYMEN OMER

NO
SHAPE
SIZE
STRUCTURE
MEMBRANE
RECEPTORS
ENZYMES
PROTIENS
HORMONES

PLATELETS

OVERVIEW
OVERVIEW

Platelets

Small
granulated
Non-nucleated
round or oval
2 4 in diameter
150000 450000/mm

life span average 7-11 days


Mean platelet volume of 7 to
9 fl.

Thrombopoietin
(THPO)
Megapoietin
MGDF
MPl ligand

fibroblasts
endothelial cells
certain leucocytes

60 75 % of platelets are present


in the circulating blood

Normally
25 40 % of platelets are
present in the spleen

MGKs polyploid cells, 30-50 m


diameter Repeated rounds of
endomitosis increasing rounds DNA
division without cellular proliferation
Largest cells in the BM Abundant
granular cytoplasm
Large multilobated nuclei
Polyploid DNA content may reach
32-64 times normal diploid cell
MGKs descend from pluripotent
hematopoietic progenitorsCFU-

tructure of platelets
Granules

Mitochondria

Golgi zone
Glycogen

Microtubules

Dense
body

Membrane
Dense
tubular
System

Microfilament
Dense body in
granule

Open canalicular
system

Structure of platelets

- Plasma membrane
*** plasma membrane contains
Phospholipids
glycoprotein receptors
Include
For
platelet factor 3

collagen

Accelerate clotting

reactions.
fibrinogen
vessel von-Willebrand Form precursors for
factor

thromboxane A

***
The
plasma
membrane
invaginates into the interior
of the platelets to form the
open canalicular system
serves as a pathway
uptake
of
extra cellular
calcium

release
intracellular
substances

of

*** There is also a coat of


glycoprotein on its surface which
helps its adhesion to injured
endothelium; but not to normal
endothelium

*** It is 6 nm thick and contain lipids


(phospholipids, cholesterol and
glycolipids),Carbohydrates(glycocalyx),
Proteins and glycoproteins .
Out of all glycoprotein and phospholipids
are functionally important.
*** Accelerate clotting reactions.
*** Form precursors for thromboxane A .

II Platelet cytoplasm
1- Contractile proteins

actin

thrombosthenine

myosin

Enable activated platelets to change


their shape
Responsible for the contraction of
platelets

2- A skeleton of microtubules that


keeps
the
disc
shape
of
platelets

3- Residuals of
endoplasmic reticulumGolgi apparatus
Synthesis
of enzymes

Calcium
store

4- Mitochondria and enzyme system for


synthesis of ATP and ADP

5- Lyzosomes containing hydrolytic


enzymes.

Glycogen granules for production of energy


anaerobically.

7- Enzyme system that synthesize


prostaglandins
from phospholipids of the Platelet
membrane.

8- 2 types of granules
A) Dense
granules

contain nonprotein substances

( ATP, ADP, Ca++


,Mg++ and
serotonin)

b) Alpha granules
contain the
secreted proteins

clotting factors
fibrin
stabilizing
factor
XIIIderived
platelet
growth factor

Plasma Membrane:

Number of specific receptors, often glycoproteins


(GPs) through which platelets interact.
Some important glycoproteins:
GP1a Collagen adhesion
GP1b Subendothelial microfibril adhesion
GP II b-IIIa Fibronogen binding, aggregation.
GP IV Thrombosponding binding
GP V Thromin binding, aggregation
GP IX Platelet adhesion: part of GP1b
complex

What is von Willebrand Factor (vWF)


Large multimeric protein synthesized in the
endothelial cells and megakaryocytes
It is constitutively synthesized by the
endothelial cells
It is present in the:
Subendothelium
Plasma
Alpha granules of platelets
Weibel-Palade bodies
The vWF present in plasma is of endothelial
origin
Function of vWF Mediate platelet
adhesion to the collagen in the
subendothelium

Platelet Adhesion
Adhesion Initial response to vessel wall injury
Independent of platelet activation
Passive (no energy required) Does not
require Ca2+
Mechanism of platelet adhesion Exposure of
subendothelium
vWF:GPIb/IX interaction mediates binding to
collagen at high shear rates
Gp Ia/IIa and GPVI mediate direct binding to
collagen at low shear rates
vWF induces signaling events that
include: a. cytosolic Ca2+
b.Activation of protein kinase C (PKC)
c.Phosphorylation events

Multistep Platelet Adhesion to Collagen


Injured vascular wall vWF
adheres to subendothelial
collagen
vWF binds to GPIb/V/IX
Rapidly formed bonds are
quickly broken and reestablished
Causes the platelet to roll along
the injured vessel
Rolling process slows down the
platelet
Allows platelet signaling
receptor GPVI to bind to collagen
Leads to a cascade of signals
activation of GPIIb/IIIa platelet
aggregation

Platelet Shape Change


Platelet shape change occurs
simultaneously with platelet
activation Flattened disc spiny
sphere with long pseudopod
projections
Reorganization of proteins in
structural zone Microtubules
contract Concentration of
organelles in center of cell
close to OCS
Increases platelet surface area
Biochemical reactions during
shape change Membrane GP
alteration GPIb/IX
GPIIb/IIIa
Granule secretion
Converts platelet from
adhesive state to an
aggregation state

Platelet Secretion
Platelet secretion ATP required for
centralization of organelles
Release of granule contents into OCS
Recruitment and activation of
additional platelets from the circulation
Released products promote
secondary hemostasis and repair
ADP, serotonin platelet activation
Ca2+ activation, assembly of
plasma coagulation factors
Growth factors & mitogens
stimulate vessel wall repair

Platelet Aggregation
Platelet aggregation is
absolutely dependent on
activation of GPIIb/IIIa
receptor complex
Primary functions of
GPIIb/IIIa Receptor for
fibrinogen (low shear)
vWF (high shear)
Requires Ca2+ + ATP
Activation upregulation of GPIIb/IIIa
receptors on to the
platelet membrane

Triggered by a
number of agonists
ADP, TXA2, Collagen,
Epinephrine, Thrombin,
Arachidonic Acid

GPIIb/IIIa

Platelet Aggregation
Agonists Collagen, thrombin,
arachidonic acid
Stimulate release of arachidonic acid
from membrane phospholipids by
PLA2 PC/PE + PLA2: AA
AA + COX (cyclo-oxygenase) : PGG2
(PGH2)
(Platelets)
PGG2 + thromboxane
synthetase TXA2 (thromboxane
A2) TXA2 platelet aggregation
TXA2 TXB2

(Endothelial cells)
PGG2 + PGI2 (prostacyclin synthetase)
PGI2 inhibits platelet aggregation

Platelets

Endothelial cells

Role of Platelets in Arterial Thrombosis

In healthy vasculature, circulating platelets


are maintained in an inactive state
HOW ?
By nitric oxide (NO) and prostacyclin
(PGI2) released by endothelial cells lining
the blood vessels.

In addition, endothelial cells also express


ADPase that degrades ADP released from
activated platelets.

Prostacyclin (PGI2) activates PGI2


receptors on Platelets.
This activation increases cAMP levels
and inhibits Platelet aggregation.
How???
cAMP inhibits release of ADP and 5HT
from dense granules of Platelets.
ADP and 5HT is involved in
Platelet aggregation !

The Cyclooxygenase
(Eicosanoid) Pathway in the
Endothelial Cell
TXA2 stimulates platelet
aggregation by decreasing
cAMP
calcium is released
from the DTS
stimulating
platelet aggregation and
secondary hemostasis. PGI2
inhibits platelet aggregation
by increasing cAMP
movement of calcium into DTS
This inhibits platelet activation

When the vessel wall is damaged,,,


Release of these substances is impaired
and subendothelial matrix is exposed.

Platelets adhere to exposed collagen via


2 1 and glycoprotein (GP)V1 (receptor on
their surface)
Platelets adhere to von Willebrand factor
(present in subendothelial matrix) via
GPIb and GPIIb/IIIa (IIb 3)

Adherent platelets undergo a change


in shape

Absence of Prostacyclin
cAMP

Secretion ADP from their dense


granules, and also there is synthesis
and release thromboxane A2.

Released ADP and thromboxane


A2 are platelet agonists, activate
ambient platelets and recruit
them to the site of vascular
injury

Disruption of the vessel wall also exposes tissue


factor( factor 3) present in subendothelieal region
Tissue factor initiates coagulation.
Also,,, the Activated platelets
potentiate coagulation by binding clotting factors.
supports the assembly of activation complexes.

This will enhance thrombin generation.

Thrombin
MOST potent platelet agonist , it activates
and recruits more platelets to the site.
HOW ??
Thrombin is a protease.
Platelet activation by thrombin is mediated
via Thrombin Receptors (on platelet surface)
called PARs (protease-activated receptors) :
PAR-1 and PAR-4

PAR-1 is the major human platelet


receptor
It exhibits 10100-times higher
affinity for thrombin when compared
with PAR-4.
-------------

Thrombin also converts fibrinogen to


fibrin.

After Recruitment,
Divalent fibrinogen or multivalent vWF
molecules bridge adjacent platelets
together to form platelet aggregates.

Fibrin strands then weave these


aggregates together to form a
platelet/fibrin mesh.

Platelet function assay


Expose platelets within citrated whole
blood to high shear (5,000 to 6,000/sec)
within a capillary tube and monitor the
drop in flow rate as the platelets form a
hemostatic plug within the center of a
membrane coated with collagen and
either ADP or epinephrine
Abnormal closure times are an indication
of platelet dysfunction, they are not
specific for any disorder
The test is coagulation factor independent
PFA-100 is more sensitive (>70 percent)
than the bleeding time (20 to 30 percent)
in detecting all subtypes of von
Willebrand's disease (vWD)

Platelet Function
Testing
PFA-100 System
Clinical Indications

PFA-100 Platelet Function Analyzer

Built-In Printer
LCD screen
Soft keys
Trigger solution container

Test Cartridge
Cassette

Carousel

PFA-100 Test Principle

before

after
p = -40 mBar

cup
aperture
150 M
800 l
blood

Filter
+
epinephrine
or ADP
collagen

capillary
200 M
flash
membrane

Platelet
plug

PFA-100 Test Principle


In Vivo Haemostasis

PFA-100

high
high shear
shear rate
rate
>5000
/s
>5000 /s
endothelial cell
collagen fibrils

Epinephrine
or
ADP

membrane with
collagen coating
von Willebrand Factor

von Willebrand
Factor
fibrinogen

erythrocyte
platelet

platelet

FLOW
erythrocyte
lumen

capillary 200m

To: Poujol, Nurden, Paponneau, et al.

Expected Normal Ranges

Expected Values
Col/Epi Col/ADP
3.8% (129mM) buffered Sodium citrate;
90% Central Interval (sec)**:

** Dade PFA-100 System Package Insert

85 - 165 71 - 118

Platelet function assay


Collagen/epinephrine closure
time (CEPI-CT).
Abnormal in Aspirin intake
Collagen/adenosine diphosphate
(CADT-CT).
Normal in aspirin intake

Petechiae 1-3mm
Purpura 3mm-10 mm
Ecchymosis >10mm