internal,medicine,RA,biotechnoogy,sepsis,airways and lungs, targeted therapy,platelet therapy

1. PLATELETS ACTIVATION IN NORMAL
CONDITIONS AND DURING ACUTE
THROMBOEMBOLIC DISEASES

2. STROKE
A thrombus originates
from carotid artery
and it extends directly
to the middle cerebral
artery. The thrombus
stops the blood flow
leading to ischemic
damage in the district
of brain tissue that
receives arterial blood
from the blocked
artery.

3. ACUTE MYOCARDIAL INFARCTION

4. PLATELETS
• Platelets originate for fragmentation of the cytoplasm of megakaryocytes
(large multinucleated bone marrow cells)
• The major regulator of platelet production is the hormone thrombopoietin
(TPO), which is synthesized in the liver. Their production is increased
during inflammation (IL-6).
• Normal platelets count in peripheral blood (PB) is between 150,000 and
450,000/ml, platelets are small (approximately 2-3 mm). Their average life
span in PB is 7 to 10 days. Approximately one-third of the platelets reside
in the spleen.
• Platelets are anucleate, but they contain mRNA and they have a limited
capacity to synthesize new proteins.
• Platelets contain three types of granules in their cytosol:
 Dense granules
 a-granules
 Lisosomial granules

5. PLATELETS GRANULES
• Dense granules: they contain substances that activate
platelets (i.e. ADP, ATP, serotonine)
• a-Granules: they contain proteins that increase their
adhesive capacity (fibrinogen, fibronectin, von Willebrand
factor)
• Lisosomial Granules: they contain glycosidases and
proteases but their functions are non completely elucidated

6. CYTOPLASMIC PROTEINS
ACTIN: is present in filamentous form (F-actin) and in globular
monomeric form (G-actin). During activation, F-actin molecules are
processed to obtain longer filaments that are anchored to the myosin and,
through the actin-binding proteins, to the GP Ib / IX present on the
cytoplasmic membrane.
CYTOSKELETON PROTEIN ASSOCIATED TO MEMBRANE:
several proteins including actin, filamin, talin, vinculin wich contribute
to maintain the discoid shape of quiescent platelets and contribute to the
structural change subsequent to activation. Some of these act as actin-
binding proteins.
MARGINAL BAND: constituted by a spiral of tubulin that surrounds
the perimeter of the platelet; during platelet activation the tubulin spiral
contracts and contributes to morphological changes.

7. PLATELETS RECEPTORS (I)
GLYCOPROTEIN Ia / IIa (a2 / b1 or VLA-2) is part of the integrin
family, it binds to collagen exposed on the vascular wall after endothelial
damage; it is responsible for platelet adhesion to the damaged vessel
wall.
GLYCOPROTEIN IIb / IIIa is part of the integrin family, it is
expressed on platelets surface, after activation of platelets it undergoes a
Ca2+ mediated conformational change that allows the binding of
fibrinogen and von Willebrand Factor. This change takes place thanks to
a process of "inside-out" signaling.
GLYCOPROTEIN VI this receptor binds collagen, its activation
induces platelet aggregation. It has also a minor role as adhesion
receptor.

8. P2Y1 and P2Y12: these G protein associated purinergic receptors are
activated by binding to ADP. The activation of P2Y1 determines the hydrolysis
of phosphatidylinositol, the formation of thromboxane A2, protein
phosphorylation and entry of Ca2+ into the cytosol. P2Y12 determines the
inhibition of cAMP synthesis.
TPa and TPb are receptors of thromboxane A2; their activation stimulates
phospholipase C (PLC) resulting in degradation of the PIP2 and formation of
IP3 and DAG.
GP Ib / V / IX receptor complex expressed on megakaryocytes and platelets;
has the role of allowing the initial adhesion of platelets to the damaged vessel
wall. The ligand of GP Ib is the von Willebrand factor
PAR 1 and PAR 4 (protease activated receptor) their ligand is thrombin, the
most potent activator of platelets. Their activation, started by fibrin, leads to
activation of the G protein.
PLATELETS RECEPTORS (II)

11. • Adhesion
• Activation and secretion
• Aggregation
• Interaction with coagulation factors
PLATELETS FUNCTIONS

12. PLATELETS ACTIVATION (I)
ADHESION
In normal conditions platelets in the blood stream are in contact only with
endothelial cells.
In an endothelial damage occurs, platelets come in contact with different
components of the vessel wall and, through interaction with specific
receptors, they adhere to the wall itself.
The initial adhesion is mediated by GP Ib / V / IX, whose main ligand is vWF.
The collagen in the connective tissue matrix also binds the platelets
receptor GPIa / IIa and, with a weaker link, GP VI.
Adhesion is followed by platelet activation.

13. ACTIVATION AND SECRETION
The first step of is the conformational change (platelets become flat, their
surface increases and they assume a dendritic shape) These changes
allows platelets to adhere more closely to collagen fibers. In addition
platelets degranulation leads to the release of ADP and thromboxane A2
into the circulation. These two substances amplify the reaction because
they are potent activators of circulating platelets enhancing the
recruitment of other platelets to be activated.
Platelets contain pre-mRNA of tissue factor (TF). Platelet activation leads to
the splicing of pre-mRNA and the synthesis of TF which activates the
coagulation cascade leading to the conversion of prothrombin to
thrombin and, finally, of fibrinogen to fibrin. These events determine the
stabilization of the thrombus.
PLATELETS ACTIVATION (II)

14. AGGREGATION
Activated platelets are able to bind fibrinogen. This protein, in fact, has two
binding sites for platelet integrin GPIIb / IIIa (also known as AIIb / b3),
abundantly expressed on the surface of platelets (40000-80000 receptors
per platelet). Fibrinogen represents a bridge between the platelets wich
form large aggregates.
INTERACTION WITH COAGULATION FACTORS
The platelet clot is stabilized by the formation of a fibrin network, the result of
the coagulation cascade. There is a synergistic action between platelets and
coagulation factors: the formation of thrombin, in fact, is due in large part
to the TF released from activated platelets.
PLATELETS ACTIVATION (III)

15. CONFORMATIONAL CHANGES

18. PLATELETS AGONISTS
STRONG (they activates inositol 3 phosphate hydrolysis)
THROMBIN
COLLAGENE
WEAK (they doesn’t activate inositol 3 phosphate hydrolysis)
ADP
EPINEPHRINE
THROMBOXANE A2

19. -THROMBIN-
Thrombin is PAR-1 and PAR-4 ligand
It is the most powerful activator of platelets: picomolar concentrations are
sufficient to stimulate platelets aggregation in vitro
The formation of thrombin occurs through the activation of coagulation which
starts from the interaction between TF and factor VIIa
Thrombin causes the receptor cleavage with the formation of a new n-terminal end
capable of binding the second extracellular loop of the receptor itself; this
determines the signal transmission through the membrane with activation of the G
protein.
This activation causes PIP2 degradation with formation of IP3 and DAG. The
resulting increase in thromboxane A2, phosphorylation of several proteins and
increased cytosolic Ca2+ concentration; through the activation of Rho also are
induced cytoskeletal changes that determines the conformational changes.

20. -COLLAGEN-
Collagen has an action similar to that of thrombin and determines IP2 hydrolysis,
thromboxane A2 formation, phosphorylation of several proteins and increase of
cytosolic Ca2+ concentration.
Its interaction with glycoprotein Ia / IIa allows the adhesion of platelets to the
damaged vessel wall.
Its interaction with the glycoprotein IV determines platelets aggregation and
activation; Glycoprotein IV has a minor role in determining adhesion to collagen.

21. -ADP-
ADP is stored in dense granules
It interacts with two platelet receptors linked to protein G:
• P2Y1 determines IP2 hydrolysis, formation of thromboxane A2,
phosphorylation of several proteins and increase of cytosolic Ca2+
concentration.
• P2Y12 inhibits adenylate cyclase with consequent reduction of the
cAMP concentration.

22. -THROMBOXANE A2-
It is synthesized by activated platelets starting from arachidonic acid (AA)
through cyclooxygenase (COX) activity.
It interacts with two platelet receptors coupled to protein G (TPA and TPB)
resulting in activation of PLC, degradation of PIP2 and formation of IP3
and DAG leading, respectively, to a rise of intracellular Ca2+ concentration
and to PKC activation.

23. SIGNAL TRANSDUCTION

27. INFLAMMATORY MEDIATORS
Platelets are able to produce pro-inflammatory substances that determine
alterations of the endothelial cells with consequent recall and chemotactic
activation of monocytes
CD40 ligand: a member of the TNF family, is stored in the cytoplasm of
quiescent platelets; following activation it is translocated on the platelet
membrane, and thereafter, the soluble active form is released; when it
reaches the endothelial cells it stimulates the production of peroxides, free
radicals, adhesion molecules, cytokines and TF.
Interleukin 1b: it is not stored in platelet granules, its synthesis is induced by
splicing of the mRNA content in platelets. It determines endothelial
activation with increased expression of adhesion molecules for monocytes
and polymorphonuclear cells and increased production of chemokines.
PF4, P-selectin, metalloproteinases, pro-angiogenic factors are other mediators
of inflammation whose production increases during platelet activation.

28. Figura 2 NEJM, Davì 2482
MEDIATORI DELL’INFIAMMAZIONE
PIASTRINICI

29. ANTIPLATELET DRUGS
Inhibition of the synthesis of thromboxane A2 (acetylsalicylic acid). Aspirin
inhibits COX-1 enzyme leading to inhibition of thromboxane A2
production. The effect of aspirin is limited to weak agonists; platelet
activation due to exposure to collagen and thrombin is preserved.
Inhibition of ADP receptor P2Y12 (clopidogrel, prasugrel, ticagrelor and
ticlopidine, these drugs, however, seem to also inhibit the binding of
fibrinogen to GPIIb / IIIa)
Inhibitors of GPIIb / IIIa: there are monoclonal antibodies (Abiciximab) and
angiotensin receptor blockers (tirofiban, eptifibatide). These drugs block
the final common pathway of platelet aggregation, related to the formation
of bridges of fibrinogen.

30. PLATELETS ISOLATION
PRP (platelet rich plasma)
Whole blood is centrifuged at low speed (120xg). This allows the separation
of plasma and platelets from WBC and RBC (the platelets remain in
suspension).
PPP (platelet poor plasma)
Starting from PRP, we proceed to a second centrifugation at high speed
(500xg) which separates the platelet pellet from PPP. The platelets are then
resuspended in a buffer containing glucose.
• PURIFICAZIONE PER GEL FILTRAZIONE
CENTRIFUGE PURIFICATION

31. MANUAL PLATELETS COUNT
Isolated platelets can be
counted in a Burker chamber
The platelets contained in 10 rectangles are
counted and then the following formula is
applied:
Sum of 10 rectangles x 0.02 = billion platelets /
mL
The platelets are then diluted to a concentration
optimal for the subsequent experimental phase

32. PRP

33. AGGREGATION TESTS
BORN AGGREGOMETER
LIGHT
The light passes with
difficulty through the
platelet suspension
(which is cloudy)
LIGHT
Agonist
As the platelets
aggregate, the light is
less hindered in its
passage

34. AGGREGAZIONE
Parameters:
Amplitude: max aggregation
Slope: is the slope of the curve
Lag time: latency time between
agonist addition and the beginning
of shape changes
Slope
Amplitude
Shape change

35. Platelet Function Testing
PFA-100®
System
Clinical Indications

36. PFA-100® Platelet Function Analyzer
Trigger solution container
Soft keys
LCD screen
Built-In Printer
Carousel
Cassette
Test Cartridge

37. PFA-100® Test Principle
before after
aperture
Ø150 µM
cup
Platelet
plug
Filter
+
epinephrine
or ADP
flash
membrane
800 µl
blood
capillary
Ø 200 µM
collagen
p = -40 mBar

38. high shear rate
>5000 /s
PFA-100® Test Principle
PFA-100®
capillary 200µm
Epinephrine
or
ADP membrane with
platelet
von Willebrand Factor
erythrocyte
FLOW
collagen coating
To: Poujol, Nurden, Paponneau, et al.
In Vivo Haemostasis
lumen
fibrinogen
platelet
collagen fibrils
erythrocyte
von Willebrand Factor
endothelial cell

39. í Simulates in-vivo conditions; high shear such as
present in small arteries (CVD)
í High shear increases the sensitivity to vWF
abnormalities
í Assesses the effect of anti-platelet agents under
physiological conditions*
*also recommendation of subcommittee on Biorheology - ISTH 1999
PFA-100® Test Principle -
summary

40. Col/Epi Col/ADP
3.8% (129mM) buffered Sodium citrate;
90% Central Interval (sec)**: 85 - 165 71 - 118
** : data based on testing of 127 samples with normal platelet function in Germany
** Dade
®
PFA-100
®
System Package Insert
Expected Normal Ranges
Expected Values

41. Comparison of the PFA-100® with skin Bleeding Time test
To : Mammen, Comp, Gosselin, et al..
0
1
0 1
Sensitivity
1 - Specificity
PFA-100®
Bleeding Time test
PFA-100®
Area Under Curve 0.98 0.70
Error 0.01 0.04
Bleeding
Time test
Populations:
206 normals
176 abnormals
PFA-100® Clinical Performance

42.  23% of 283 ACS patients Poulsen et al; ESC 2003
 38% of 129 post-AMI patients Andersen et al., 2003
 10% of 325 patients with CVD Gum et al., 2001
 27% of 89 patients with CVD, CAD Santos et al; ISTH 2001
 42% of 31 pts. with stable angina Crowe et al; ISTH 2001
 25% of 105 pts. with CAD von Pape et al; ASH 2000
 58% of 43 pts. undergoing PTCA von Pape et al; ASH 2000
 45% of 100 pts. with ACS Sambola et al; ISTH 2001
= 25% of low-responders or non-compliants!
Review of the literature on 1,105 patients on ASA
frequency of normal Col/Epi result:
PFA-100® and ASA resistance