Antithrombotic drugs

Question 1

Primary hemostasis

Answer 1

Hemostasis achieved via platelet adhesion and aggregation at the site of endothelial injury

1. Initiation: endothelial injury results in transient vasoconstriction. → exposure of subendothelial collagen → von Willebrand factor (vWF), which is a glycoprotein synthesized and stored in Weibel-Palade bodies of endothelial cells and α-granules of platelets, binds the exposed collagen
2. Adhesion (hemostasis): vWF and platelet GpIb receptors mediate the adhesion of platelets to the injured endothelium by forming pseudopodia; phospholipid is expressed on cellular membranes.
3. Activation: release of adenine diphosphate (ADP), thromboxane, calcium, and platelet activating factor (PAF), which assist in platelet aggregation, vasoconstriction and degranulation
4. Aggregation (hemostasis): mediated by GpIIb/IIIa-receptor and fibrinogen → formation of a white thrombus composed of platelets and fibrin

Question 2

Secondary hemostasis

Answer 2

Hemostasis achieved via the interaction of plasma coagulation factors (procoagulants): coagulation cascade
The coagulation cascade is a series of reactions, involving coagulation factor proteins, which constitutes the process by which blood changes from a liquid to a gel, forming a blood clot.

Injury to endothelium → activation of the extrinsic pathway (hemostasis)

• Tissue factor (factor III), which is present under the endothelium on fibroblasts, binds to and thus activates factor VII
• Factor VIIa and tissue factor form a complex (TF-FVIIa)* that activates factor X and factor IX.

Additionally, activation of the intrinsic pathway (hemostasis), especially through thrombin
- Thrombin activates factors XI and factor VIII.
Factor XIa activates factor IX
Factors VIIIa and IXa form a complex that activates factor X.
- This causes a positive feedback loop of factor X and thrombin activation via the intrinsic pathway

The common pathway (hemostasis) of the extrinsic and intrinsic pathways then follows:
Factor Xa and factor Va form a complex that cleaves prothrombin to thrombin (= factor II).
Thrombin cleaves fibrinogen (factor I) into insoluble fibrin (factor Ia) monomers.
Cross links of the fibrin network are stabilized by factor XIIIa → formation of a fibrin network → fibrin closely binds to the platelet plug, forming a stable fibrin clot (secondary or red thrombus, red because also RBCs bind to this clot)

Question 3

Factor IV

Answer 3

Calcium ions that are required in the coagulation cascade. the double positive charges link on one side to the coagulation factor (X, IX, VII, II (1972)), and to the negatively charged phospholipids on the other side.

Question 4

Conditions for platelets activation

Receptors responsible for the accomplishment of these conditions

Answer 4

Platelet once activate change in conformation. All this is possible if cAMP levels are low and calcium levels are high. If cAMP levels are high, platelets are no easily activated.

• P2Y12 receptors: purinergic receptors for ADP, coupled to Gi -> decrease in cAMP
• P2Y1 receptors: purinergic receptors for ADP, coupled to Gq -> PLC -> increase in calcium
• Thrombin receptors: coupled to Gq -> PLC -> increase in Calcium
• Thromboxane receptor: coupled to Gq -> PLC -> increase in calcium

Question 5

Factor VIII

Answer 5

It’s a cofactor, no proteolytic activity, it acts as a bridge between factor IX and factor X, allowing the first to activate the second.

Question 6

Hemophila A and its acquired form

Answer 6

• Hemophilia A: mutation in factor VIII, factor VIII can be administered
• Acquired form of hemophilia A: autoimmune disease. Tratment: Emicizumab, a bispecific monoclonal antibody with 2 different epitopes binding respectively factor IX and factor X, acting as a bridge and activating the mechanism of X activation.

Question 7

Role of thrombin

Answer 7

• cleave fibrinopeptides (FP) A and B from the α and β chains of the fibrinogen molecule, triggering spontaneous fibrin polymerization and the beginning of the fibrin clot
• amplifies the coagulation mechanism by activating cofactors V and VIII and factor XI by a positive feedback mechanism that serves to generate more thrombin
• activates factor XIII
• activates directly both endothelial cells and platelets
• bound to thrombomodulin activates the protein C pathway to suppress coagulation, and it activates TAFI to suppress fibrinolysis

Question 8

Antithrombin III action

Answer 8

Antithrombin III (ATIII) is a nonvitamin K-dependent protease that inhibits coagulation by neutralizing the enzymatic activity of thrombin and factors IXa, Xa, XIa, XIIa. 
Antithrombin III activity is markedly potentiated by heparin, the principal mechanism by which both heparin and low–molecular-weight heparin result in anticoagulation.

Question 9

Heparin and warfarin administration

Answer 9

Heparin: IV, oral is not possible
Walfarine: oral

Question 10

Role of vitamin K

Answer 10

Most coagulation facotrs are produced in the liver and need post- translational modifications to become functional.
Vitamin K serves as a cofactor of the enzyme gamma-carboxylase which is involved in the carboxylation of glutamic acid residues on precursor coagulation proteins. The carboxylation enables binding of these proteins to surface phospholipids to start the normal antithrombotic process.

Vitamin K gets oxidised at every cycle and it needs to be reduce by epoxide reductase in order to become active again.

Question 11

Warfarin (commercial name, pharmachology)

Answer 11

• Cumadin, Jatoven
• it competitively inhibits subunit 1 of the multi-unit vitamin K epoxide reductase complex 1 (VKOR1)
  → depletes functional vitamin K reserves, which in turn reduces synthesis of active clotting factors

Question 12

4 different novel anticoagulants in Europe

Answer 12

Dabigatran (Predaxa):

• Prevents thrombus development through direct, competitive inhibition of thrombin (thrombin enables fibrinogen conversion to fibrin during the coagulation cascade).
• Capsules, poorely absorbed (6-8%), it is a strong substrate of P-glycoproteins.
• It has a very efficacious antidote, a monoclonal antibody directed against Dabigatran, which immediately inhibits it.

Endoxaban, Rivaroxaban (Xarelto), Apixaban (Eliquis):

• Inhibitors of activated factor X.
• No antidote
• Substrate of P-glycoproteins but much less
• Therapeutic drug monitoring through drug concentration (no INR)

Question 13

Eliquis

Answer 13

Apixaban (Xa inhibitor)

Question 14

Xarelto

Answer 14

Rivaroxaban (Xa inhibitor)

Question 15

Cumadin

Answer 15

Warfarin (inhibitor of vitamin K activation)

Question 16

Aspirin (salicylic acid)

Answer 16

Irriversible inhibitor of COX1.
COX-1 is an enzyme embedded in the membrane and that always works as a dimer.
It releases an acetyl groupthe serine residue of the active site and covalently modifies the COX-1 enzyme. Thus, the conversion of arachidonic acid into tromboxane is no more possible.
Platelets are cells without a nucleus -> they can’t produce COX-1 again

The main target of NSAIDs is COX-1 in the stomach rather than in platelets -> often associated iwth gastrointestinal bleeding.
Inhibition of COX-1 in plt is thought to occur direclty in the portal vein

Question 17

Clopidogrel

Answer 17

Plavix

• Synthetic drug, a pure antagonist of P2Y12 purinergic receptor (ADP receptor) that is coupled to Gi. Antagonist -> high cAMP -> inactivation of platelets
• Prodrug, 15% will be activated, 85% is lost
• Cytochrome CYP2C19 (enterocytes and liver) activates it, carboxylesterases (foud all over the body) inactivate is

Question 18

Clopidogrel-PPIs interaction

Answer 18

Most of PPIs (omeprazole, lansoprazole, esomeprazole) are metabolized by CYP2C19, the enzyme that is suposed to activated clopidogrel, but it is unable to in this case because PPIs remain attached and occupy the enzyme.

Rabeprazole and pantoprazole are not metabolized by this enzyme so they can be used.

Question 19

Prasugrel

Answer 19

Son of clopidogrel

• Used in combination with Aspirin
• Less sensitive to carboxylesterase -> more stable
• Cost around 10 times more

Compared to clopidogrel, it is efficiently metabolized and achieves more complete plt inhibition, but also increases the risk of bleeding.

Question 20

Difference between aspirin and other COX inhibitors

Answer 20

• Apririn is not the most powerful inhibitor of COX-1
  BUT
• Aspirin causes an irriversible inhibition, the others a reversible one

Question 21

How much COX-1 needs to be inhibited to get a clinically significant antithrombotic effect? How should it be administered?

Answer 21

Approximately 95%.
Low dosage of aspirine (100-300 mg) every single day is enough to reach the desired level of inhibition. At this dosage the anti-inflammatory effet is negligible.

Question 22

COX-1 activity at basal levels

Which is the risk of giving COX-2 inhibitors to patients with high artherothrombotic risk?

Answer 22

Thromboxane is normally produce at basal sates in platelets, it is counteracted by NO and prostacylin. The latter is released by endothelial cells and it’s produced by COX-2.

In a non pathologic person levels of thromboxane and prostacyclin are at equilibrium.

Patients with high artherothrombotic risk have high levels of thromboxane (produced by plt) that are counterbalanced by high levels of prostacyclin (produced by endothelial cells). In these patients COX-2 inhibitors must be avoided. Thromboxane could lead to platelet activation.

Question 23

Aspirin and ibuprofen interaction

Answer 23

Ibuprofen blocks COX-1 and, by a smaller degree COX-2. But is blocks the action of aspirin on COX-1 and it doesn’t block COX-1 irriversibly.

Diclofenac and Ketorolac don’t block the action of Aspirin

Question 24

NSAIDs on gastric mucosa

Answer 24

COX-1 in the gastric mucosa is responsible for the production of prostacyclin and prostaglandins.
Prostaglandins inhibit acid secretion in the stomach and increase the production of bicarbonates and mucous.

Question 25

When aspirin should be suspended?

Answer 25

In a patient who takes aspirin due to an ischemic event in the past, suspending aspirin is extremely risky. It shouldn’t be done for small intervention.
If the patient is under double anticoagulat therapy (aspirin+clopidogrel), Clopidogrel may be suspended.
In patients with stent, this therapy goes on for at least 12 months, so if an intervention must be performed, it’s better to do it before starting the therapy or waiting till the end.

Question 26

Tirofiban

Answer 26

Direct antagonist of CPIIb/IIIa
Strong effect -> this drug is used for a short time, as a therapeutic bridge.
Risk of severe bleeding, including cerebral bleeding.

Question 27

Thrombolytic agents

Answer 27

Fibrinolytic therapy
- Fibrin-specific agents
→ Tissue plasminogen activator: Alteplase
→ Recombinant plasminogen activator (rtPA): produced by recombinant biotechnology techniques. Reteplase and Tenecteplase.
- Non-fibrin-specific agents
→ Streptokinase: a protein, produced by group A streptococci, that catalyzes the conversion of plasminogen to plasmin, which is responsible for clot breakdown.
→ Urokinase

Mechanism of action: directly or indirectly increase the conversion of plasminogen to plasmin

Indications: Early STEMI (< 12 hours), Early ischemic stroke (< 3 hours), Massive pulmonary embolism

Effects: ↑ PT, ↑ PTT, No change in platelet count

Contraindications to fibrinolytic therapy

• Prior intracranial hemorrhage
• Recent surgery
• Severe hypertension
• Active bleeding

Adverse effect: bleeding

Reversal of adverse effects
- Antifibrinolytics
→Tranexamic acid: a synthetic lysine analog and inhibitor of plasminogen with antifibrinolytic action
→ Aminocaproic acid: a lysine derivative and inhibitor of the plasminogen-plasmin cascade with antifibrinolytic action
- Fresh frozen plasma or cryoprecipitate (Cryoprecipitate is obtained from frozen blood plasma via centrifuge and contains more factor VIII and fibrinogen than fresh frozen plasma.)