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© The European Society of Cardiology 2006. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

Pharmacology of thienopyridines: rationale for dual pathway inhibition

Borja Ibanez1,2, Gemma Vilahur1 and Juan J. Badimon1,*

1 Cardiovascular Biology Research Laboratory, the Zena and Michael A. Wiener Cardiovascular Institute, Box 1030, Mount Sinai School of Medicine, New York, NY 10029, USA
2 F. Conchita Rábago de Jiménez Díaz, Madrid, Spain

* Corresponding author. E-mail address: juan.badimon{at}mssm.edu


   Abstract
Top
 Abstract
Introduction
 Platelet biology
 Thienopyridines: P2Y12 receptor...
 Aspirin and clopidogrel: dual...
 New trends
 References
 
Atherothrombotic disease is the result of atherosclerosis progression, and its clinical manifestations, mostly secondary to atherosclerotic plaque disruption and subsequent thrombus formation. At the site of vascular lesions, platelets adhere to the exposed matrix proteins, prompting platelet activation, resulting in the secretion of multiple platelet agonists, mostly modulated by intracellular calcium release. Among them, ADP, thromboxane A2, thrombin, and others play a critical role in maintaining a ‘pro-platelet-activating’ environment. Aspirin has been considered the gold standard of antithrombotic therapy. Its antiplatelet activity is achieved mainly by inhibiting arachidonic acid pathway (blocking thromboxane A2 release from platelets). ADP binds to platelet P2Y12 receptor, although this receptor is important in platelet aggregation induced by other agonists. Thienopyridines (ticlopidine and clopidogrel) exert their antiplatelet activity by binding to the P2Y12 receptor, irreversibly modifying it. Oral clopidogrel loading dose of 300–600 mg clopidogrel produces significant inhibition of ADP-induced platelet aggregation 2 hours after administration, which becomes maximal after 6 hours. Both aspirin and clopidogrel are ‘selective platelet-receptor’ inhibitors, and therefore are weak and safe antiplatelet agents. The co-administration aspirin-clopidogrel results in enhancement of platelet inhibition, since the act via different platelet receptors. Current research is focused in reversible or more potent ADP antagonists.

Key Words: Acute coronary syndrome • ADP • Antiplatelet therapy • Aspirin • Clopidogrel • Thienopyridines • Thrombosis


   Introduction
Top
 Abstract
 Introduction
Platelet biology
 Thienopyridines: P2Y12 receptor...
 Aspirin and clopidogrel: dual...
 New trends
 References
 
Atherosclerosis is a diffuse process that starts early in life, asymptomatically progressing through adulthood, until clinically manifested.1 Atherothrombotic disease is the result of atherosclerosis progression, and its clinical manifestations [acute coronary syndromes (ACS), stroke, etc). These events are mostly secondary to atherosclerotic plaque disruption and subsequent thrombus formation.2,3 Atherosclerosis prevention is mainly focused on the management of the so-called ‘cardiovascular risk factors’; whereas thrombosis-related complications are mainly prevented and/or treated by antithrombotic therapies. In fact, the understanding of the processes of platelet activation/aggregation and the role of acute thrombus formation on the onset of ACS have led to a widespread use of antiplatelet therapy in cardiovascular disease. Because of its safety and effectiveness, aspirin is considered the gold standard of antithrombotic therapy in the prevention and treatment of cerebral, coronary, and peripheral arterial disease. The use of aspirin is associated with a 25% reduction in myocardial infarction, stroke, and vascular death. To improve the benefits associated with platelet inhibition, more potent antithrombotic agents are being developed. Thienopyridines are platelet adenosine diphosphate (ADP) receptor antagonists that were initially developed to provide new opportunities for those patients who are intolerant, resistant, or have failed to aspirin treatment. As discussed later, both aspirin and clopidogrel (the most widely used thienopyriridine) are considered weak and safe antiplatelet agents because they only block one of the multiple pathways involved in platelet activation. However, interestingly, their co-administration not only maintains the safety profile but displays synergistic and more potent antithrombotic effects than when administered as monotherapy.


   Platelet biology
Top
 Abstract
 Introduction
 Platelet biology
Thienopyridines: P2Y12 receptor...
 Aspirin and clopidogrel: dual...
 New trends
 References
 
Overview of platelet function
Platelets are major players in primary haemostasis and thrombus formation. Because they lack nucleus, for a long period of time they were considered passive organelles. Today we know that they play a critical role not only in the physiological haemostasis, but also in the vascular repairing process after endothelial dysfunction. Under normal conditions, platelets circulate in an inactivated state. Upon activation, they will adhere to the areas of endothelial dysfunction. The problems arise when their physiological function, formation of the haemostatic plug, is surpassed and leads to the formation of a luminal thrombus. Furthermore, besides playing a role in thrombus formation, platelets also play a role in the formation of the atherosclerotic plaque itself (e.g. local release of growth factors) and the inflammatory process within.4,5

General mechanism of platelet adhesion, activation, and aggregation: arterial thrombus formation
Platelet adhesion
At the site of vascular lesions, circulating von Willebrand factor (vWF) binds to the exposed collagen, which will subsequently bind to the glycoprotein (GP) Ib/IX/V receptor on the platelet membrane.6–8 Under pathological conditions and in response to changes in shear stress, vWF can be secreted from the storage organelles in platelets or endothelial cells, reinforcing the activation process. Although GPIb/IX/V-vWF interaction is enough to promote binding of platelets to subendothelium, it is highly transient, resulting in rapid dislocation of platelets to the site of injury. GPVI binding to matrix collagen has slower binding kinetics, but once initiated promotes a firm adhesion of platelets to the vessel surface.9 Finally, both GPIb/IX/V and GPVI also regulate platelet–leukocyte adhesion and are thereby implicated in other vascular process, such as inflammation and atherosclerosis.10–12

Platelet activation
Platelets can be activated by adhesion to arterial wall and/or by interacting with circulating agents such as epinephrine, thrombin, serotonin, thromboxane A2 (TXA2), and ADP via specific platelet surface receptors. Platelet activation leads to platelet shape change and calcium translocation within the platelet, crucial mediators for platelet activation. Subsequently, platelet free-ionic Ca2+ increase catalyses ADP liberation from dense platelet granules. This ADP release has an autocrine effect, promoting stable platelet aggregation by interacting with specific ADP receptors in the membrane, and also promotes a paracrine effect by binding to ADP receptors of neighbouring platelets, amplifying the activation process. Platelet activation also induces phospholipase A2 activation that triggers arachidonic acid metabolism. Platelet cyclooxygenase 1 (COX-1) catalyzes the conversion of arachidonic acid to prostaglandin G2/H2, and the latter is converted to TXA2. TXA2 is released to the circulation, where it binds TP-receptors, thus enhancing platelet activation and vasoconstriction. Figure 1 illustrates the major platelet receptors and the pathways of platelet activation.


Figure 0471
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  		Figure 1 Mechanisms and agonists involved in platelet adhesion, activation, and aggregation. PAR, protease-activated receptor; TP, thromboxane receptor.

 
Platelet aggregation
Secondary to Ca2+ increase is the conformational change of the integrin receptors {alpha}IIbß3 (GPIIb/IIIa) on the platelet surface. This ‘activation’ allows the binding of these receptors to adhesive proteins (primarily fibrinogen) favouring platelet–platelet interaction (aggregation) (Figure 1). In fact, platelet GPIIb/IIIa is not only activated downstream of adhesion receptors GPVI and GPIb/IX/V, but also by G protein-coupled receptors, such as thrombin (PAR-1 or PAR-4) or ADP receptors (P2Y1 or P2Y12) that reinforce GPIIb/IIIa-dependent platelet aggregation. A scheme of the platelet activation–aggregation process is depicted in Figure 2.


Figure 0472
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  		Figure 2 Healthy endothelium (left) presents antithrombotic properties because it is able to release vascular protective substances such as nitric oxide (NO), prostacyclin (PGI2), tissue plasminogen activator (tPA), and tissue factor pathway inhibitor (TFPi). On the contrary, dysfunctional endothelium (right) not only favours platelet adhesion, activation, and aggregation, but also promotes vascular lipid deposition, macrophage migration, and tissue factor (TF) expression (activation of the coagulation cascade). Following platelet adhesion, activation is characterized by platelet shape change. Activated platelets secrete different agonists, prompting activation of circulating platelets and a procoagulant environment. This pro-thrombotic milieu will favour thrombus formation and the subsequent clinical manifestations.

 
The ADP receptors (Purinergic receptors)
The P2Y1 receptor
The G{alpha}q-coupled P2Y1 receptor is responsible for inositol trisphosphate formation through activation of phospholipase C, leading to transient increase in the concentration of intracellular calcium, platelet shape change, and weak transient platelet aggregation.13–15 Pharmacological data have revealed an essential role for the P2Y1 receptor in the initiation of platelet ADP-induced activation, TXA2 generation, and platelet activation in response to other agonists.16

The P2Y12 receptor
This negatively coupled Gi receptor has extracellular cysteines and is responsible for completion of the platelet aggregation response to ADP.17 There are several signalling molecules downstream of P2Y12 activation such as cAMP, vasodilator- stimulated phosphoprotein (VASP) dephosphorylation, phosphoinositide 3-kinase, and Rap1B.18–22 Pharmacological approaches have shown a role for the P2Y12 receptor in dense granule secretion, fibrinogen-receptor activation, P-selectin expression, and thrombus formation, identifying it as a central mediator of the haemostatic response.23–27 However, this receptor is also important in platelet aggregation induced not only by ADP,28,29 but also by thrombin, immune complexes, adrenaline, serotonin,26 TXA2 and the PAR1-selective peptide agonist SFLLRN.28,29 Additionally, P2Y12 also directly contributes to exposure of phosphatidylserine at the surface of platelets where the coagulation factors bind to concentrate and to localize thrombin generation.30,31 Furthermore, both P2Y12 and P2Y1 are indirectly involved in platelet P-selectin exposure and formation of platelet–leukocyte conjugates, which leads to leukocyte-tissue factor exposure.30,32

It is of great importance to highlight that stimulation of each one of the P2Y receptor is not sufficient for full aggregation;33 in fact, there is a need for the concurrent activation of the P2Y1 (Gq) and P2Y12 (Gi) pathways for full platelet aggregation. Finally, although not activated by ADP, need is to say that platelets possess a third purinergic receptor (P2X1) which is a fast ATP-gated calcium channel receptor mainly involved in platelet shape change. Figure 3 shows in detail the platelet purinergic receptors.


Figure 0473
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  		Figure 3 P2Y receptors can be clearly divided into two subgroups: the Gq-coupled subtypes P2Y1 and the coupled to Gi P2Y12. P2Y1, which is responsible for platelet shape change and calcium mobilization, is coupled to Gq and activates phospholipase Cß (PLCß) that is responsible for the formation of inositol (1,4,5)-trisphosphate (IP3) and diacylglycerol (DAG), an activator of protein kinase C (PKC). IP3 causes calcium mobilization from internal stores. The P2Y12 receptor couples primarily to G{alpha}i2 inhibition of adenylyl cyclase (AC). The subsequent decrease in cAMP production leads, in turn, to a reduction in the activation of specific protein kinases (PKA), which can no longer phosphorylate the VASP; VASP phosphorylation is crucial for GP IIb/IIIa receptor inhibition. The subunit ß{gamma} activates the phosphatidylinositol 3-kinase, which is an important signalling molecule for P2Y12-mediated platelet-dense granule secretion and GP IIb/IIIa receptor activation. Finally, P2X1 is a gated cation channel protein activated by ATP. This activation leads to increased intraplatelet calcium, platelet shape change, and transient and weak platelet aggregation responses.

 

   Thienopyridines: P2Y12 receptor antagonists
Top
 Abstract
 Introduction
 Platelet biology
 Thienopyridines: P2Y12 receptor...
Aspirin and clopidogrel: dual...
 New trends
 References
 
Ticlopidine and clopidogrel
The thienopyridine derivatives, ticlopidine and clopidogrel, are antiplatelet agents that, by covalently binding to a cysteine residue of the P2Y12 receptor, irreversibly modify the platelet P2Y12 receptor.34 Consequently, platelets are affected for the remainder of their lifespan (7–10 days).35 However, only 60–70% of the ADP receptors are sensitive to the effects of thienopyridines. Ticlopidine was the first of these new class of antiplatelet agents.36 Two large-scale clinical trials, CATS37 and TASS,38 have demonstrated the effectiveness of ticlopidine reducing the risk of thrombotic events in patients with atherosclerotic diseases. Despite its efficacy as an ADP receptor antagonist, the wide use of ticlopidine was significantly affected by a rare but severe incidence of neutropenia (8{per thousand}). For these reasons, clopidogrel, a new agent structurally similar to ticlopidine, but with fewer side-effects (severe neutropenia in 0.5{per thousand}) emerged as new antiplatelet therapy. As a result, clopidogrel has almost replaced ticlopidine as a therapeutic antiplatelet agent, used alone or in combination with aspirin.39 Clopidogrel has proved useful for the prevention of ischaemic stroke, myocardial infarction, and vascular death in patients with symptomatic atherosclerosis.40 Beyond its anti-aggregatory effect, it reduces the formation of platelet–leukocyte conjugates in patients with ACS41 and decreases the expression of activated platelet-dependent inflammatory markers such as CD40 ligand (a potent stimulus of vascular inflammation) and CD62 P-selectin in patients undergoing percutaneous coronary intervention (PCI).42,43 In fact, clopidogrel, co-administered with aspirin, is being considered the treatment of choice for prevention of atherothrombotic complications.

Pharmacology
Pharmacokinetics and pharmacodynamics properties of thienopyridines
Metabolization
Thienopyridines are pro-drugs, and they require oxidation by the hepatic cytochrome P450 (CYP450) to become active antiplatelet compounds.44 The need for metabolization has a delayed effect in blocking P2Y12 platelet receptor and in their antiplatelet activity. Clopidogrel, via hepatic metabolization, leads to the formation of a thiol group34 that covalently binds to the extra-cellular cysteine residues of the P2Y12 receptor,45 thus precluding the binding of ADP.46 Although the activity of thienopyridines seems to depend on hepatic metabolism, clopidogrel has demonstrated a lack of interaction with various treatments commonly used in clinical practice.47–50 Conversely, in a recent study performed on a small number of subjects, it was claimed that atorvastatin reduced the activity of clopidogrel in vitro, reputedly through competitive inhibition of the CYP450 3A4 isoenzyme.51 However, this observation has not been clinically confirmed by detailed subanalysis of the CREDO trial52 or MITRA PLUS registry.53 In contrast, ticlopidine interactions with digoxin, theophylline, and cimetidine have been documented.54 Serum levels of thienopyridine metabolites are increased in patients with renal function impairment, but apparently unassociated with an excess prolongation of the bleeding time.55 Conversely, liver disease does not modify clopidogrel effect on ADP-induced platelet aggregation. However, clopidogrel should be used with caution in such patients because of a potential increased bleeding risk.

Dose and antiplatelet effects
Ticlopidine maintenance dose is 250 mg twice daily. Single loading doses of 500 mg have been tried but often cause abdominal cramping and discomfort.56 Following this maintenance dose, only 50% platelet aggregation inhibition is achieved at 5 days, and 60–70% at 8–11 days, limiting its usefulness in emergency settings. In contrast, an oral loading dose of 300–600 mg clopidogrel produces significant inhibition of ADP-induced platelet aggregation 2 h after administration, which becomes maximal (40–60%) after 6 h. This is of special interest in cases such as PCI in which rapid antiplatelet effect is needed. However, there are advocates for still higher clopidogrel loading doses; this hypothesis was tested by the ALBION trial (Montalescot G. EuroPCR, 2005). The results of this trial concluded that a loading dose of 900 mg of clopidogrel did not provide any incremental benefit over a 600 mg loading dose. If a loading dose of clopidogrel is not used, at least 3 repeated daily oral dose of 75 mg of clopidogrel is required to achieve a steady-state maximal platelet inhibition.57 The inhibition of platelet aggregation continues even after stopping the treatment with clopidogrel, and the rate at which normal aggregation is restored closely correlates with platelet production. As such, in healthy human volunteer studies, maximal inhibition of platelet aggregation causes a two-fold increase in the bleeding time, which returns to baseline values generally in about 5 days after treatment discontinuation.58 The long-term antiplatelet effects of clopidogrel could be considered as the ‘ying and yang’. On one hand, non-compliance of drug intake for 1 day has little or no effect on the antiplatelet effect; however, on the other hand, if an emergent surgery is required, clopidogrel should be withheld during 5 days in order to reduce the risk of bleeding complications. Interestingly, platelet reactivity in clopidogrel-treated subjects can be normalized by the infusion of regular plasma units (G. Vilahur and J.J. Badimon, submitted for publication). This suggests the possibility of counteracting the increased risk for bleeding during and after surgery in clopidogrel-treated patients by administering platelet units prior to the major surgeries.


   Aspirin and clopidogrel: dual antiplatelet therapy
Top
 Abstract
 Introduction
 Platelet biology
 Thienopyridines: P2Y12 receptor...
 Aspirin and clopidogrel: dual...
New trends
 References
 
It has been previously commented that aspirin and clopidogrel could be considered as weak antiplatelet agents with completely different mechanisms of action. This observation suggested the possibility of combining two weak and safe antiplatelet agents with the assumption of achieving stronger antiplatelet effect but still being safe. Aspirin exerts its antiplatelet effects by acetylating the serine moiety at position 529 of COX-1 and thereby irreversibly inhibiting the key enzyme required for the conversion of arachidonic acid to TXA2. As mentioned earlier, TXA2 is a potent platelet activating agent and results in platelet shape change, secretion of granular contents, and increased expression of GPIIb/IIIa receptors by binding to its cell surface receptor. Because platelets lack nuclei, they are unable to synthesize new COX-1 and are, therefore, permanently inhibited by aspirin. Despite being a relatively ‘weak’ antiplatelet agent, aspirin remains a frontline therapy with proven benefits in primary and secondary prevention of coronary artery disease (CAD). The important benefits of aspirin in ACS were first demonstrated in the ISIS-2 trial.59 Despite the proven benefits of aspirin, however, recurrent events remain high.60 As has been clearly depicted, there are many other platelet receptors different from the TXA2 receptor that can activate platelets and amplify platelet response upon agonist binding. Therefore, it is intuitive to think that other compounds that block these other receptors could exert additional benefits to aspirin monotherapy. This underscores the need to continue the search for new agents that can either replace or be used in addition to aspirin for short- and long-term management. In fact, inhibition of the platelet P2Y12 ADP-receptor by clopidogrel on a background of TXA2 inhibition by aspirin has proved an enhancement in platelet inhibition. Indeed, the CURE61 study demonstrated for the first time the benefit of adding clopidogrel to aspirin (additional 10% relative risk reduction) rather than using aspirin alone in patients having non ST-segment elevation ACS or unstable angina. Furthermore, patients who are resistant to aspirin (up to 10%) have higher rates of cardiovascular events and may derive special benefit from the combination therapy. On the other hand, the co-administration of different antiplatelet therapies that act at different targets also increases the risk of bleeding. This complex equilibrium has to be taken into account when dual antiplatelet therapy is planned. Despite all this, current clinical recommendations for patients undergoing PCI suggest loading dose regimens of 300–600 mg clopidogrel plus aspirin62 with a maintenance daily dose of 75 mg of clopidogrel. Although the benefits of this dual pathway inhibition therapy for high-risk patients are out of discussion, its use in low-risk patients has been recently challenged by the results of the CHARISMA trial.63 The data of CHARISMA should not be taken as disappointing, but as an indicator that for low-risk population, probably monotherapy (aspirin or clopidogrel) is enough to achieve equilibrium between antithrombotic effect and low bleeding risk.


   New trends
Top
 Abstract
 Introduction
 Platelet biology
 Thienopyridines: P2Y12 receptor...
 Aspirin and clopidogrel: dual...
 New trends
References
 
The most recent advances in antiplatelet therapy related to the inhibition of the P2Y12 receptors are focused on the availability of faster acting and reversible inhibitors. Several of these agents are being clinically investigated. AZD6140 is the first oral reversible ADP receptor antagonist64 that has been recently tested in the phase II ‘DISPERSE2’ Study (Cannon CP, AHA 2005). This anti-P2Y12 binds reversibly (24 h) to the P2Y12 receptor and, unlike the thienopyridines, AZD6140 is orally active without the requirement for metabolic activation. Cangrelor is an intravenous, selective, and specific antagonist of P2Y12 receptor whose initial experience in patients undergoing PCI has been recently reported.65 Finally, the JUMBO-TIMI 2666 trial has shown that the novel potent thienopyridine prasugrel has the potential to achieve higher levels of platelet inhibition than currently accepted conventional loading dose of clopidogrel. However, there is still a need for properly powered trials to answer whether this new antiplatelet approach will depict net clinical benefits (events reduction without excess bleedings).

Conflict of interest: J.J.B. has served as a consultant for Sanofi-Aventis. B.I. and G.V. have no potential conflict of interest.


   References
Top
 Abstract
 Introduction
 Platelet biology
 Thienopyridines: P2Y12 receptor...
 Aspirin and clopidogrel: dual...
 New trends
 References
 

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