Antiplatelet drugs (c)

Question 1

Describe the structure of platelets.

Answer 1

Anuclear blood cells
Biconvex when inaactive
1-3um in diameter
When activated undergo conformational change to become more star shaped in appearance

Question 2

What are the normal blood counts for platelets, RBCs and WBCs?

Answer 2

Platelets - 150 to 450 x10^9/L
Rbcs - 3-6x10^12/L
WBC - 4.5 to 11 x10^9/L

Question 3

What is the lifecycle of a platelet?

Answer 3

Live for 5 to 10 days
Formed from magakaryocytes.

Question 4

What are the essential features in the ultrastructure of a platelet?

Answer 4

Alpha granules
Dense granules
Anuclear

Question 5

What are the two different types fo thrombosis?
How are they different?

Answer 5

Arterial thrombosis - white thrombus, high platelet count, normally triggered by sheer stress and endothelial injury, treated by aspiring and anti-platelet drugs. Cause MI and thrombotic stroke

Venous thrombosis - red thrombus, higher amounts of fibrin and rbcs, main contributor is coagulation from stasis, causes DVT and PE, treated by warfarin, heparin and DOACs.

Question 6

What is the clinical importance of platelets in thrombosis?

Answer 6

Arterial thrombosis
Coronary - MI
Cerebral - Ischaemic stroke
Approx: 30% mortality in west is due to arterial thrombosis.

Question 7

What is the function of platelets?

Answer 7

Adhere to damaged tissue particulary subendothelial collagen exposed in endothelial injury
Then undergo a conformational change due to actin reorganisation, will have pseudopodia, filopodia and lamellipodia this covers a greater area and patches up the damage
Will release functional mediators (see card)
Aggregate together due to cross links between fibrinogen and GPIIb/IIIa on platelet surface.
Them promote coagulation by expressing phosphatidylserine on surface aiding the coagulation cascade.

Question 8

What functional mediators do platelets release?

Answer 8

Stored mediators include:
Dense granules - ADP, ATP, seratonin
Alpha granules - fibrinogen, fibrinogen receptor

Synthesis de novo:
Thromboxane A2 from arachidonic acid

Question 9

How in a lab can we estimate platelet aggregation?

Answer 9

Platelet rich plasma is couldy (less light absorption)
When platelets aggregate they collect together and more light can pass through the mixture
In platelet poor plasma - the most light passes through.

We can measure absorption over time, is starts with low absorption = low platelet
Is starts with high absorption and rapidly decreases then high platelet aggregation.

Question 10

What are the features of integrin αIIb/β3?

Answer 10

Also known as fibrinogen receptor, GPIIb/IIIa or CD41/CD61
Are heterodimers with alpha and beta subunits
Only expressed in platelets, and in high quantities
Also found in alpha granules
Have high and low affinity forms with inside out signalling changing between the two.

Question 11

What are the features of fibrinogen?

Answer 11

Is a heterohexamer (mirror ends each containing 2 alpha, 2 beta and 2 gamma chains)
Synthesised in the liver
Found in the plasma and platelet alpha granules
Has two RGD binding motifes for GPIIb/IIIa on platelets.

Question 12

Describe how the GPIIb/IIIa becomes a high affinity form?

Answer 12

Platelet is activated
Talin binds to the cytoplasmic domain of receptor
Causes a conformational change - folds outside or inside out exposing binding site for fibrinogen at the junction between IIb and IIIa.

Question 13

What substances can activate platelets?

Answer 13

Collagen (found in ECM)
Thrombin (from coag cascade)
ADP (from dense granules)
Thromboxane A2 (de novo in platelets)
Platelet activating factor (de novo) minor role
Adrenaline (circulating hormone)
Seratonin (dense granules)

Question 14

What platelet activating receptors are important from a pharmacology perspective?

Answer 14

P2Y12 - bound to by ADP - is inhibited by clopridogel, ticagrelor, ticlopidine.
TP - bound to by thromboxane - COX inhibits reduce thromboxane
GpVI - bound to be collagen - developing glenzocimab to stop interaction
Alpha 2A - bound to be adrenaline - made worse by beta blockers.
PAR1 and PAR2 - bound to by thrombin.

Question 15

What platelet inhibitory receptors are important from a pharmacology perspective?

Answer 15

A 2A - bound to by adenosine - dipyradamole blocks ENT1 increasing effect of adenosine.
IP - bound to be PGI 2 - COX inhibitors reduce PGI 2

Question 16

What are some important platelet adhesion ligands?

Answer 16

Integrin alpha 2 beta 1 - binds to collagen for adhesion
Intergin Alpha IIb beta 3 - fibrinogen for aggregation
GpIb-V-IX - Von Willibrand Factor for adhesion

Question 17

What are the GPCR linked to inhibition of platelets?

Answer 17

IP from PGI2 and A 2A (adenosine)
Gs activates adenylyl cyclase
Increase cAMP

Question 18

What are the GPCRs linked to activation of platelets?

Answer 18

PAR1/4, TP, P2Y1are Gq subunits - activates PLCbeta2 to increase calcium ions
P2Y12 - Gi subunit - activates PLCbeta2 as above also activates PI3K decreasing cAMP

Question 19

Give the basic explanation as platelet activation as a result of endothelial cell injury.

Answer 19

Loss of endothelial layer exposed underlying collagen in the basement membrane and in deep tissues
Platelets adhere to exposed collagen by integrins of their surface
The platelet is activated by glycoprotein on surface binding to collagen and changes shape to spread over the damaged area.
Fibrinogen cross links many platelets together as fibrinogen receptor becomes high affinity form

Question 20

What is the basic process of platelet recruitement?

Answer 20

Activated platelets release ADP from dense granules and synthesise thromboxane A2.
These activate and recruit new arriving platelets.

Question 21

Describe how platelets form the primary haemostatic plug?

Answer 21

Platelet activation converts intregrin Alpha IIb Beta 3 from a low to high affinity state
In high affinity conformation is able to bind to fibrinogen
Fibrinofen has two binding sites per molecule so brings two platelets together
This aggregation forms the primary haemostatic plug.

Question 22

What are the unique properties of aspirin as an anti-thrombotic?

Answer 22

Clinical - used as secondary prevention for MI

Platelets contain COX-1 and thromboxane synthase
Irreversibly Inhibits COX-1 prevention conversation of arachidonic acid into Prostaglanding G2/ Prostaglanding H2.

Also inhibits PGI2 synthesis (less important see card)

Overall leads to decreased platelet activation and aggregation - prevents formation of an arterial thrombus prevents occlusion of coronary arteries.

Question 23

How is thromboxane produced?
What are its effects?

Answer 23

COX converts arachadonic acid to PGH2
Thromboxane synthase then converts to Thromboxane A2
Activates TP receptors (GPCR) on platelets -increases calcium
Synthesised by platelets
Pro-aggregatory and vasoconstrictor

Question 24

How is prostacyclin produced?
What are its effects?

Answer 24

COX enzymes converts arachidonic acid to PGH2
Prostacyclin synthase converts to PGI2
Mainly occurs in endothelium
Has anti-aggregatory and vasodilating effects
Acts via IP receptor (GPCR) on platelet surface Gs increase cAMP.

Question 25

How is aspirin anti-thrombotic if it inhibits the production of thromboxane (pro-aggregation) and PGI2 (anti-coagulation)?

Answer 25

Platelets cannot re-synthesises acetylated COX (hence less thromboxane), endothelial cells can resysnthesis COX (so PGI2 is still produced) do to presence of nucleus. Or highest concentration of aspirin is found in hepatic portal circulation (as given orally), platelets found here will circulate throughout body reducing systemic TXA2, endothelial cells remain put so PGI2 reduction low locally, aspirin inactivated so no longer has systemic effects.

Question 26

Why does COX-2 have a negative cardiovascular effect in atherosclerosis?

Answer 26

Atheroscloeross - inflammatory - more inducible COX-2 . Therefore selective COX-2 inhibitors can reduce PGI2 production in coronary arteries - prevent vasodilation in ischemic areas - reduce blood supply, can increase likelihood of an adverse cardiovascular event.

Question 27

What drugs inhibit platelet recruitement?

Answer 27

Aspirin inhibiting COX to reduced thromboxane A2 Ticagrelor, clopridogrel inhibiting P2Y12 to reduced affect of ADP.

Question 28

How does ADP cause platelet aggregation/adhesion? How is this utilised by pharmacology?

Answer 28

Bind to two GPCRs P2Y1 - causes transient aggregation P2Y12 - causes sustained aggregation P2Y12 is mainly only expressed in platelets and glial cells so is a good therapeutic target Therefore inhibitors of P2Y12 can be used as anti-thrombotics.

Question 29

What is the effect of increased calcium ions in platelet aggregation and platelet adhesion?

Answer 29

Increases expression of cell surface membrane receptors Including Fibrinogen receptor GPIIb/IIIa for aggregation And Alpha 2 beta 1 for adhesion to collagen.

Question 30

What is the basic mechanism of ADP antagonists?

Answer 30

Are thienopyridines (clopidogrel, ticlopidine, prasugrel) Are pro-drugs metabolised in vivo to active compound Covalently bind to P2Y12 receptors forming disulfide bone Causes irreversible inactivation of ADP receptor Some responses vary between patients due to variations in metabolism.

Question 31

What is the process of clopridogrel metabolism?

Answer 31

1. Clopridogrel is oxidised from ketone 2. Hydroxy group becomes a carbonyl group 3. Is hydrolysed to give a reactive -SH group to react with receptor. This is the same for prasugrel

Question 32

How does the efficacy of different ADP antagonists compare?

Answer 32

Ticlopidine - low efficacy and risk of haematological problems Clopridogrel - greater efficacy, improved safety profile, variable response in patient due to some having low CYP2C19 levels Prasugrel - greater efficacy, most consistent as CYP2C19 not required for oxididation.

Question 33

What is the use of ATP analogues in platelet aggregation and activation?

Answer 33

Is a weak and reversible inhibitor of ADP induced aggregation (Note clopidogrel and ticagrelor are derived from ATP) Reversible antagonist at P2Y12 Competitive is design but may bind allosterically More consistent than clopriodogrel Used for oral treatment for prevention of arterial thrombosis.

Question 34

What drugs primarily prevent platelet aggregation? When are they mainly used?

Answer 34

Fibrinogen antagonists mainly -fiban Abiciximab Eptifibatide Tirofiban Used IV in acute setting - for acute coronary intervention due to rapid onset, short half life and significant bleeding risk.

Question 35

What is the development around super aspirins?

Answer 35

Orally available GpIIb/IIIa antagonists -fiban All failed phase 3 clinical trials.