ABSTRACT: One of the major classes of adenosine diphosphate (ADP) receptor antagonists are thienopyridines. Thienopyridines compose a subcategory of antiplatelet medications, known as ADP receptor inhibitors, used commonly for the treatment of atherosclerotic cardiovascular disease. Thienopyridines, including ticlopidine, clopidogrel and prasugrel, are prodrugs administered orally that are further metabolized by hepatocytes to create active metabolites that irreversibly bind ADP receptors located on the platelet membrane. Thus, these selected drugs have an inhibitory effect for the duration of the platelet’s lifespan of 7–10 days. The goal of this manuscript is to review the currently available ADP receptor blockers with emphasis on chemical structure, mode of action and clinical use.
Keyword: clopidogrel; ticlopidine, prasugrel; acute coronary syndrome, antiplatelet , antagonist, antithrombotic, stenting
J INVASIVE CARDIOL 2009;21:406–412
Cardiovascular-related diseases remain one of the leading causes of mortality. The high prevalence of atherosclerosis has led to recent advances pertaining to the treatment of atherosclerotic heart disease. The use of platelet receptor antagonists has been one of the major pharmacological interventions in the treatment and prevention of myocardial infarction (MI), stroke and peripheral vascular disease. The role of platelets in hemostasis established by Bizzozero in 1882 initiated research investigating the relationship between platelet aggregation and atherogenesis.1,2 Platelet aggregation and interaction with a variety of factors ensures vascular integrity, however atherosclerosis and plaque rupture via vascular damage can cause platelet activation. Activated platelets undergo a series of steps including: shape change, adhesion to endothelial cells of vessels, aggregation and the secretion of granules that perpetuate the cycle.1–3 Within 1 minute of activation, the presence of fibrinogen and thrombospondin results in platelet aggregation through the linking of glycoprotein (GP) IIb/IIIa complexes.3 Fibrinogen and thrombospondin are secreted from a-granules. The release of dense granules results in the secretion of pro-aggregatory molecules such as adenosine diphosphate (ADP), serotonin and calcium. The shape of platelets changes from a discoid to spherical within seconds after activation once the concentration of ADP approaches 2–5 µM.5–7 ADP binds to specific ADP receptors located on the platelet membrane including P2Y1, P2Y12 and P2X1.1,4 Therefore, ADP is considered a natural agonist of platelet aggregation, as this molecule is involved in a positive feedback mechanism potentiating the process of platelet activation and thrombus formation.1 The role of ADP and ADP receptors has been the subject of much research over the past 30 years, leading to the development of drugs that block such interactions.
Aspirin is one of the oldest antiplatelet drugs, since the agent irreversibly inactivates platelet cyclooxygenase, causing platelet inhibition for the platelet’s life cycle.8–10 Shortly after the introduction of stents for coronary intervention, it was realized that aspirin was not sufficient to significantly reduce stent thrombosis to an acceptable clinical threshold.11 Stent thrombosis after intracoronary stenting occurred in 18% of patients treated with aspirin alone. The introduction of dual antiplatelet therapy involving the use of aspirin and ADP receptor inhibitors dramatically reduced the rate of thrombosis to 1%.12–15 This treatment modality is the most common indication for the use of ADP receptor antagonists. However, in patients with atherosclerosis, the addition of an ADP receptor antagonist to traditional aspirin therapy increases the bleeding risk.
One the major classes of ADP receptor antagonists is thienopyridines. Thienopyridines include ticlopidine, clopidogrel and prasugrel. These prodrugs, once administered orally, are further metabolized by hepatocytes to create active metabolites capable of irreversibly binding ADP receptors.17,18 Thus, these selected drugs have an inhibitory effect for the duration of the platelets’ lifespan of 7–10 days. Researchers continue to expand the current body of knowledge to elucidate the most effective doses, mode of action and the risks associated with various thienopyridine treatment regimens.
Ticlopidine
The FDA approved ticlopidine hydrochloride on October 31, 1991. Ticlopidine, commonly referred to as Ticlid® (Roche Laboratories, Nutley, New Jersey), is a prodrug administered orally to treat a variety of cardiac and vascular diseases. The drug must undergo hepatic biotransformation to form an active metabolite, thus the efficacy of platelet inhibition peaks after approximately 3–5 days of treatment.19,20 Some studies suggest a longer delay of inhibitory action, as 5 days of treatment were needed to attain 50% inhibition of platelet aggregation and 8–11 days to reach 60–70% inhibition.21 Normal aggregatory properties resume 4–8 days after the last treatment.22 Excretion of ticlopidine metabolites occurs primarily by the renal system (~60%), with only 23% of ticlopidine metabolites measured in feces. Patients with poor renal function should be placed on ticlopidine treatment with extreme caution, as data in this patient population are limited.9 Numerous trials on patients with transient ischemic attack (TIA),24 completed stroke,25,26 or intracoronary stents have revealed that ticlopidine alone at doses of 250 mg twice a day or in combination with aspirin is effective in reducing future events.27–29
The effectiveness of ticlopidine in comparison to aspirin as a treatment for patients with stroke precursors was analyzed in the Ticlopidine Aspirin Stroke Study (TASS). After 3 years of treatment with either 250 mg of ticlopidine twice daily or 650 mg of aspirin twice daily, a 12% reduction in non-fatal stroke and all-cause death, and a 21% reduction in fatal and non-fatal strokes were noted in those treated with ticlopidine in comparison to aspirin-treated patients. In the Canadian American Ticlopidine Study (CATS), patients with a completed stroke had a 30.2% reduction in death related to MI or stroke when treated with 250 mg of ticlopidine 2 times a day for up to 3 years compared to those taking a placebo.23 Based on the results of these randomized clinical trials, administration of 250 mg twice daily is recommended. The rationale behind administration of a loading dose is based on accelerated onset of platelet inhibition in studies where loading doses have been administered. However, the ability to administer loading doses of this drug has been tempered by the prevalence of gastrointestinal upset.
Although the efficacy of ticlopidine appears to be superior to the “gold-standard” treatment using aspirin, a major drawback to ticlopidine therapy is the increased risk of life-threatening side effects. Neutropenia, a disorder in which the neutrophil count drops to Clopidogrel
The documented health risks associated with ticlopidine treatment led to further research of possible medications that would prevent ADP-induced aggregation of platelets without such adverse side effects. Clopidogrel bisulfate (Plavix®), was developed by Sanofi Aventis U.S. (Bridgewater, New Jersey) and was approved by the FDA on November 17, 1997 based on clinical trials showing the efficacy of this drug.74,75 Clopidogrel is 6-times more potent than ticlopidine.76 The safety of the drug, as reported in the Clopidogrel Aspirin Stent International Cooperative Study (CLASSICS), revealed that the rate of health complications such as severe bleeding, thrombocytopenia and neutrocytopenia were 50% lower than rates associated with ticlopidine.4 Treatment with clopidogrel resulted in an 8.7% decrease in atherothrombotic episodes in comparison to aspirin.72 An aspirin-clopidogrel combination therapy was compared to standard aspirin treatment in the Clopidogrel in Unstable Angina to Prevent Recurrent Events (CURE) trial. After 3–12 months of treatment, the CURE trial revealed a 20% reduction in cardiovascular death, non-fatal MI and stroke among subjects receiving combination therapy compared to patients treated with aspirin only. Adverse side effects most commonly associated with clopidogrel are mild. Clopidogrel has not been studied in patients with significant hepatic disease.9 However, post-marketing studies have not shown any adverse effects of clopidogrel use in this population.
Clopidogrel, like ticlopidine, is a prodrug that must be metabolized by hepatic CYP450 in order to be active. Due to the need for biotransformation of clopidogrel, the clinical effects of treatment are not immediately observed. Clopidogrel is rapidly absorbed and the effects exerted by the active metabolite are both dose- and time-dependent. In addition, treatment can only reduce platelet aggregation by a maximum of 50–60%, as other mechanisms in addition to ADP-induced aggregation are responsible for platelet activation, adhesion and aggregation. A 52% reduction of platelet aggregation was recorded on the sixth day of treatment in healthy subjects prescribed a 75 mg daily dose of clopidogrel. The need for an immediate anti-aggregatory effect may require a high loading dose of clopidogrel, which can circumvent the latency period to a certain degree. Oral administration of 300–400 mg at least 6 hours prior to a coronary intervention, such as insertion of a stent or graft, was more effective than treatment after the procedure. A loading dose of 600 mg a few hours prior to a procedure was more effective in the prevention of MI than a 300 mg administration. Furthermore, a combination of a high loading dose and continuous maintenance doses of 75 mg once a day results in steady, maximal levels of the drug within 4–24 hours in comparison to 4–7 days without administration of a loading dose.75,82,83 Half of drug elimination is performed by the renal system, while 46% is excreted in feces.23
An emerging issue with clopidogrel treatment is the presence of interpatient variability and resistance.84 Clopidogrel resistance has been associated with increased risk for stent thrombosis.85,86 There are many definitions for clopidogrel resistance, making the definite diagnosis of clopidogrel resistance a challenge.87–89 One of the definitions of clopidogrel resistance includes less than a 10% reduction in platelet aggregation caused by 5 µmol/L of ADP in comparison to pretreatment measurements.90 The prevalence of clopidogrel resistance is in part determined by the definition applied, thereby limiting the comparison of data addressing the prevalence of clopidogrel resistance. Depending on the definition, the prevalence of non-responders to clopidogrel within the first 24 hours of treatment is between 4% and 30%.91
Inaccurate dosing and drug-drug interactions are extrinsic mechanisms that may result in some cases of resistance. In addition, intrinsic mechanisms may be involved, including variability in P2Y12 receptors, higher concentrations of ADP and increased activity of pro-aggregatory pathways.87
Another important mechanism for clopidogrel resistance is related to genetic polymorphisms of CYP450 isoforms, which may promote variability in platelet response to clopidogrel. In a recent study, Frere and coauthors studied the effect of CYP3A4, CYP3A5 and CYP2C19 gene polymorphisms on clopidogrel response and platelet reactivity assessed in patients presenting with acute coronary syndrome. They found that CYP2C19*2 polymorphism was significantly associated with ADP-induced platelet aggregation, but the CYP3A4*1B and CYP3A5*3 polymorphisms were not. After multivariate adjustment, they found that the CYP2C19*2 allele was more frequently present in clopidogrel non-responders. In another study, CYP3A4 metabolic activity was correlated to interpatient variability regarding platelet inhibition after clopidogrel administration, confirming the importance of the CYP450 enzyme genetic polymorphism as a contributor to clopidogrel resistance.94 The appearance of clopidogrel resistance is sparking more research into alternative thienopyridines and non-thienopyridine P2Y12 antagonists.
Clopidogrel structure and active metabolite. Clopidogrel is (+)-(S)-methyl 2-(2-chlorophenyl) - 2-(6, 7-dihydrothieno [3, 2- c] pyridin-5 (4 H)-yl) acetate sulfate (1:1). The empirical formula of clopidogrel bisulfate is C16H16ClNO2S•H2SO4, with a molecular weight of 419.9 g/mol. Clopidogrel has an absolute S configuration at carbon 7. The corresponding R enantiomer is completely inactive in terms of the drug’s anti-aggregatory property.95
Chemical structure of clopidogrel. The active metabolite could be one of 8 isomers due to the specific stereochemistry needed for function.
Clopidogrel is a thienopyridine prodrug that is metabolized by CYP450 in the liver into an active metabolite.96 The metabolic pathway of oxidation of clopidogrel has been investigated in detail and shows that only a small proportion of clopidogrel is metabolized by CYP450. About 85% of the drug is hydrolyzed by an esterase to create an inactive carboxylic acid derivative.92 CYP450 oxidizes the thiophene ring of clopidogrel to 2-oxoclopidogrel, which will undergo further hydrolysis by CYP450. Oxidation of the thiophene ring of clopidogrel by a CYP450- dependent mechanism, involving both CYP3A4 and CYP3A5, is necessary to generate 2-oxo-clopidogrel.97 The second reaction results in opening of the thiophene ring to form a thiol and carboxyl group.98,99 Subsequently, the thiol group forms a covalent disulfide bond with P2Y12 on platelets. Research has shown that clopidogrel selectively binds P2Y12, which is an adenylyl cyclic receptor, rather than all other ADP receptors (Scheme 1).100
Clopidogrel: Mode of action. Clopidogrel selectively and specifically inhibits the binding of the ADP platelet aggregator to its membrane receptors. By this mode of action it prevents the expression of GP IIb/llla receptors needed for the formation of fibrin bridges that link platelets. Platelets are key elements in hemostasis and thrombosis. ADP mediates platelet aggregation through its binding to P2Y12, which results in the expression of GP IIb/IIa which will trigger platelet thrombin interaction and aggregation.
Clopidogrel is similar in structure to ticlopidine and also undergoes hepatic metabolism, thus the overall mechanism is comparable.103 The active metabolites of both ticlopidine and clopidogrel are reported to form a disulfide bridge with specific cysteine residues on the extracellular portion of the P2Y12 receptor.68 However, researchers elucidated further details on the mode of action of the active metabolite of clopidogrel. P2Y12 is expressed in platelets, megakaryocytes and neuronal cells and its oligomers are located in lipid-rafts (microdomain-rich portions), whereas monomers and dimers were located in microdomain-free portions.58 The majority of P2Y12 receptors exist as oligomers in the resting state. In a dose-dependent manner, the addition of clopidogrel results in movement of P2Y12 to microdomain-free areas. Therefore, oligomer forms of the P2Y12 receptor associated with lipid-rafts are functional and able to interact with ADP molecules.70
P2Y12 has three active sites located in the intracellular domain, extracellular domain, and trans-membrane region.69,70 The Protein Databank identification of P2Y12 is 1VZ1, which indicates seven transmembrane regions. ADP binds with P2Y12 in three different areas: the head area, middle area and bottom area. In the head region, ADP forms a hydrogen bond between the hydroxyl hydrogen of the terminal phosphate group and the amide oxygen of leu87. In the middle region, there is a hydrogen bond between the N6 of the adenosine of ADP and the amide oxygen of asn43 in addition to other hydrogen bonds linking ADP with a serine residue of P2Y12. In the bottom region, there are two hydrogen bonds formed between ADP and P2Y12. The drug binds in three different areas of P2Y12. Figure 1 demonstrates the different interactions between clopidogrel and P2Y12.
P2Y12 also has two free cysteine residues located in the extracellular domain.68 There are also interactions between the drug and the receptor involving these two free thiol groups. Studies show that a disulfide bridge does not exist between these cysteine residues. Therefore, there is a possibility that the oxidized metabolite of clopidogrel will form a covalent disulfide bond with the free thiol groups of cys17 and cys270 of P2Y12 (Scheme 2). To achieve complete inhibition, the active metabolite should bind both cysteine residues. The need to bind both residues for optimal inactivation is the probable reason that low doses of clopidogrel are not as effective as higher doses.
Vasodilator stimulated phosphoprotein (VASP) phosphorylation, which effectively isolates the P2Y12 receptor, is an important mechanism for clopidogrel resistance. Furthermore, the availability of a VASP assay as a research tool has provided significant insights into the effect of thienopyridine therapy, specifically P2Y12 receptor inhibition.104,105 Low response to ADP-receptor antagonism by clopidogrel has been associated with a higher incidence of stent thrombosis.106 Blindt and coworkers assessed the phosphorylation status of VASP and ADP-induced platelet aggregation for the risk of stent thrombosis. They found that the mean VASP-platelet reactivity indices (VASP-PRI) were significantly higher in patients with stent thrombosis and VASP was the only independent predictor of stent thrombosis confirming the important role of VASP for clopidogrel response (Scheme 2).107
Prasugrel
With the development of clopidogrel and aspirin resistance in addition to the health risks associated with ticlopidine use, Daiichi Sankyo Company, Ltd, and Eli Lilly Company began research and production of another thienopyridine called prasugrel (Effient®). Recently approved by the FDA, prasugrel proves to be a promising treatment, as it is approximately 10 and 300 times more potent than clopidogrel and ticlopidine, respectively.108,109 The antiplatelet inhibition of prasugrel is dose-dependent and both 10 mg and 15 mg once daily treatments remain more effective than daily treatments with 75 mg of clopidogrel.110 In addition, a 60 mg loading dose of prasugrel compared to a dose of 300 mg of clopidogrel resulted in less patient pharmacodynamic variability.111 Four hours after administration of a 40 or 60 mg loading dose of prasugrel, inhibition of platelet aggregation was twice the value obtained from a 300 mg loading dose of clopidogrel.107 A combination treatment of prasugrel and aspirin had an additive effect on platelet aggregation and was well-tolerated in patients.112 Because aspirin and prasugrel function via different inhibitory mechanisms, combining these two treatments may create synergism.
The majority of observed side effects were mild, which coincides with the safety of clopidogrel. Reported events of bleeding and bruising associated with the lower doses of prasugrel (5 mg, 7.5 mg and 10 mg) were similar to those related to a 75 mg treatment plan with clopidogrel. Thus, prasugrel is not linked to the serious adverse health complications strongly associated with ticlopidine treatment.
An animal study revealed that rapid absorption and conversion to its active metabolite occurs only after oral administration.113 Prasugrel, like other thienopyridines, is a prodrug that must be metabolized by the liver in order for the treatment to be effective. The metabolites of prasugrel are excreted primarily in the urine (~70%).114 Clinically, prasugrel appears to be stronger and faster-acting than clopidogrel. Among patients with planned percutaneous coronary intervention, loading with 60 mg prasugrel resulted in greater platelet inhibition than a 600 mg clopidogrel loading dose, with an even greater effect when coupling the loading dose to a 10 mg per day maintenance treatment.115 In the recently published randomized Trial to Assess Improvement in Therapeutic Outcomes by Optimizing Platelet Inhibition with Prasugrel–Thrombolysis in Myocardial Infarction (TRITON-TIMI)-38 comparing prasugrel to clopidogrel, prasugrel was superior in reducing adverse cardiovascular events in patients undergoing coronary interventions, with only mild increases in bleeding risk.116 Based on this trial, prasugrel was submitted for FDA approval.
Chemical structure of prasugrel. Prasugrel is a racemic mixture with the formula of [(±)-2-[2-acetyloxy-6, 7-dihydrothieno [3, 2-c] pyridin-5(4H)-yl]-1-cyclopropyl-2-(2-fluorophenyl) ethanone].
Mode of action of prasugrel. Upon administration of prasugrel, the medication is hydrolyzed by carboxylesterases producing inactive thiolactone (R-95913). Thiolactone is metabolized via the CYP450-dependent pathway, primarily by CYP3A and CYP2B6, to develop an active, open-ring metabolite R-138727 with the formula of 2-[1-[2-cyclopropyl-1-(2-fluorophenyl)-2-oxoethyl]-4-mercapto-3-piperidinylidene] acetic acid.117 Both CYP2C9 and CYP2C19 may also be involved in the conversion of prasugrel into its metabolites.118 R-138727 contains two chiral atoms and its resulting four isomers have varying activity levels.
As a thienopyridine, the active metabolite of prasugrel binds the P2Y12 receptor, hence preventing ADP binding. The mechanism of prasugrel is the same as other thienopyridine drugs.
Conclusion
Thienopyridines compose a subcategory of antiplatelet medications that prevent aggregation through the binding of select, extracellular cysteine residues on the P2Y12 receptor located on the platelet membrane. Although these molecules possess similar structures, conversion processes and modes of action, they all have different outcomes within the human body. The identification of the harmful health complications caused by ticlopidine resulted in the shift toward greater use of clopidogrel. However, clopidogrel resistance (or non-response) emerged, creating a clinical need for further research into ADP-receptor antagonists that would be effective in this complex cohort of patients. With the development and testing of prasugrel, management of atherosclerosis and cardiovascular disease may become more effective and safe, as the active metabolite created from the biotransformation of prasugrel is more potent than ticlopidine and clopidogrel, while causing only mild side effects.
From the *University of Arizona Sarver Heart Center, the §Southern Arizona VA Healthcare System, Tucson, Arizona, the £Section for Thoracic and Cardiovascular Surgery, University of Nebraska Medical Center, Omaha, Nebraska, and ∞Whittier College, Whittier, California.
The authors report no conflicts of interest regarding the content herein.
Manuscript submitted September 4, 2008, provisional acceptance given September 29, 2008, final version accepted February 9, 2009.
Address for correspondence: Mehrnoosh Hashemzadeh, PhD, University of Arizona Sarver Heart Center, 1501 North Campbell Avenue, Tucson, AZ 85724. E-mail: mhashemzadeh@shc.arizona.edu
1. Peter K. Principles of Molecular Cardiology. Totowa, New Jersey: Humana Press, 2005.
2. Cazenave JP, Gachet, C. Anti-platelet drugs: Do they affect megakaryocytes? Bailliere’s Clinical Haemotology 1997;10:163–180.
3. Savi P, Herbert JM. ADP receptors on platelets and ADP-selective antiaggregating agents. Med Res Rev 1996;16:159–179.
4. Clappers N, Brouwer MA, Verheugt FW. Antiplatelet treatment for coronary heart disease. Heart 2007;93:258–265.
5. Colman RW. ADP receptors in platelets. Ann N Y Acad Sci 1990;603:198–209; discussion 110.
6. O’Brien JR, Heywood JB. Effect of aggregating agents and their inhibitors on the mean platelet shape. J Clin Pathol 1966;19:148–153.
7. Born GV. Observations on the change in shape of blood platelets brought about by adenosine diphosphate. J Physiol 1970;209:487–511.
8. Kidane AG, Salacinski H, Tiwari A, et al. Anticoagulant and antiplatelet agents: Their clinical and device application(s) together with usages to engineer surfaces. Biomacromolecules 2004;5:798–813.
9. Gonzalez ER. Antiplatelet therapy in atherosclerotic cardiovascular disease. Clin Ther 1998;20(Suppl B):B18–B41.
10. Lablanche JM, McFadden EP, Bonnet JL, et al. Combined antiplatelet therapy with ticlopidine and aspirin. A simplified approach to intracoronary stent management. Eur Heart J 1996;17:1373–1380.
11. Depta JP, Bhatt DL. Aspirin and platelet adenosine diphosphate receptor antagonists in acute coronary syndromes and percutaneous coronary intervention: Role in therapy and strategies to overcome resistance. Am J Cardiovasc Drugs 2008;8:91–112.
12. Goods CM, al-Shaibi KF, Liu MW, et al. Comparison of aspirin alone versus aspirin plus ticlopidine after coronary artery stenting. Am J Cardiol 1996;78:1042–1044.
13. Keighley CS, Urban P, Killer II, et al. Palmaz-Schatz coronary stent implantation without intravascular ultrasound and without subsequent anticoagulation: Clinical outcome. J Invasive Cardiol 1996;8:185–190.
14. Park SW, Park SJ, Hong MK, et al. Coronary stenting (Cordis) without anticoagulation. Am J Cardiol 1997;79:901–904.
15. Schatz RA, Baim DS, Leon M, et al. Clinical experience with the Palmaz-Schatz coronary stent. Initial results of a multicenter study. Circulation 1991;83:148–161.
16. Peters RJ, Mehta SR, Fox KA, et al. Effects of aspirin dose when used alone or in combination with clopidogrel in patients with acute coronary syndromes: Observations from the Clopidogrel in Unstable angina to prevent Recurrent Events (CURE) study. Circulation 2003;108:1682–1687.
17. DiPerri T, Pasini FL, Frigerio C, et al. Pharmacodynamics of ticlopidine in man in relation to plasma and blood cell concentration. Eur J Clin Pharmacol 1991;41:429–434.
18. Weber AA, Braun M, Hohlfeld T, et al. Recovery of platelet function after discontinuation of clopidogrel treatment in healthy volunteers. Br J Clin Pharmacol 2001;52:333–336.
19. Di Minno G, Cerbone AM, Mattioli PL, et al. Functionally thrombasthenic state in normal platelets following the administration of ticlopidine. J Clin Invest 1985;75:328–338.
20. Gent M, Blakely JA, Easton JD, et al. The Canadian American Ticlopidine Study (CATS) in thromboembolic stroke. Lancet 1989;1:1215–1220.
21. Puri RN, Colman RW. ADP-induced platelet activation. Crit Rev Biochem Mol Biol 1997;32:437–502.
22. Saltiel E, Ward A. Ticlopidine. A review of its pharmacodynamic and pharmacokinetic properties, and therapeutic efficacy in platelet-dependent disease states. Drugs 1987;34:222–262.
23. Jacobson AK. Platelet ADP receptor antagonists: ticlopidine and clopidogrel. Best Pract Res Clin Haematol 2004;17:55–64.
24. Albers GW, Amarenco P, Easton JD, et al Antithrombotic and thrombolytic therapy for ischemic stroke: American College of Chest Physicians Evidence-Based Clinical Practice Guidelines (8th Edition). Chest 2008;133(6 Suppl):630S–669S.
25. Crawford KM, Talbert RL. Antiplatelet therapy in secondary stroke prevention. Expert Opin Pharmacother 2001;2:1609–1613.
26. Fleck JD, Biller J. Choices in medical management for prevention of acute ischemic stroke. Curr Neurol Neurosci Rep 2001;1:33–38.
27. Hass WK, Easton JD, Adams HP Jr, et al. A randomized trial comparing ticlopidine hydrochloride with aspirin for the prevention of stroke in high-risk patients. Ticlopidine Aspirin Stroke Study Group. N Engl J Med 1989;321:501–507.
28. Ito E, Takahashi A, Yamamoto H, et al. Ticlopidine alone versus ticlopidine plus aspirin for preventing recurrent stroke. Intern Med 2003;42:793–799.
29. Ito MK, Smith AR, Lee ML. Ticlopidine: A new platelet aggregation inhibitor. Clin Pharm 1992;11:603–617.
30. Hass WK, Easton JD, Adams HP Jr, et al. A randomized trial comparing ticlopidine hydrochloride with aspirin for the prevention of stroke in high-risk patients. Ticlopidine Aspirin Stroke Study Group. N Engl J Med 1989;321:501–507.
31. Lubbe DF, Berger PB. The thienopyridines. J Interv Cardiol 2002;15:85–93.
32. Bhatt DL, Bertrand ME, Berger PB, et al. Meta-analysis of randomized and registry comparisons of ticlopidine with clopidogrel after stenting. J Am Coll Cardiol 2002;39:9–14.
33. Ono K, Kurohara K, Yoshihara M, et al. Agranulocytosis caused by ticlopidine and its mechanism. Am J Hematol 1991;37:239–242.
34. Muszkat M, Shapira MY, Sviri S, et al. Ticlopidine-induced thrombotic thrombocytopenic purpura. Pharmacotherapy 1998;18:1352–1355.
35. Steinhubl SR, Tan WA, Foody JM, Topol EJ. Incidence and clinical course of thrombotic thrombocytopenic purpura due to ticlopidine following coronary stenting. EPISTENT Investigators. Evaluation of platelet IIb/IIIa inhibitor for stenting. JAMA 1999;281:806–810.
36. Mataix R, Ojeda E, Perez MC, Jimenez S. Ticlopidine and severe aplastic anaemia. Br J Haematol 1992;80:125–126.
37. Ferrer F, De Arriba F, Moraleda JM, Vicente V. Aplastic anemia induced by ticlopidine after placement of coronary artery stents: Therapeutic role of granulocyte colony stimulating factor. Clin Drug Investig 1998;15:261–262.
38. Prescribing information: Ticlid (ticlopidine hydrochloride) tablets. In: Physicians Desk Reference. Montvale, New Jersey: Medical Economics Co., 2004.
39. McTavish D, Faulds D, Goa KL. Ticlopidine. An updated review of its pharmacology and therapeutic use in platelet-dependent disorders. Drugs 1990;40:238–259. Medical Economics Co., 2004.
40. Desager JP. Clinical pharmacokinetics of ticlopidine. Clin Pharmacokinet 1994;26:347–355.
41. Knudsen JB, Bastain W, Sefton CM, et al. Pharmacokinetics of ticlopidine during chronic oral administration to healthy volunteers and its effects on antipyrine pharmacokinetics. Xenobiotica 1992;22:579–589.
42. Picard-Fraire C. Ticlopidine hydrochloride: Relationship between dose, kinetics, plasma concentration and effect on platelet function. Thromb Res Suppl 1983;4:119–128.
43. Enjalbert R, Galy J. Structure of ticlopidine hydrochloride — A platelet antiaggregating agent. Acta Cryst 1992;48:1043–1045.
44. Dalvie DK, O’Connell TN. Characterization of novel dihydrothienopyridinium and thienopyridinium metabolites of ticlopidine in vitro: Role of peroxidases, cytochromes p450, and monoamine oxidases. Drug Metab Dispos 2004;32:49–57.
45. Yoneda K, Iwamura R, Kishi H, et al. Identification of the active metabolite of ticlopidine from rat in vitro metabolites. Br J Pharmacol 2004;142:551–557.
46. Ha-Duong NT, Dijols S, Macherey A, et al. Ticlopidine as a selective mechanism-based inhibitor of human cytochrome P450 2C19. Biochemistry 2001;40:12112–12122.
47. Richter T, Murdter TE, Heinkele G, et al. Potent mechanism-based inhibition of human CYP2B6 by clopidogrel and ticlopidine. J Pharmacol Exp Ther 2004;308:189–197.
48. Zhan C, Yang J, Dong XC, Wang YL. Molecular modeling of purinergic receptor P2Y12 and interaction with its antagonists. J Mol Graph Model 2007;26:20–31.
49. Mills DC. ADP receptors on platelets. Thromb Haemost 1996;76:835–856.
50. Jantzen HM, Gousset L, Bhaskar V, et al. Evidence for two distinct G-protein-coupled ADP receptors mediating platelet activation. Thromb Haemost 1999;81:111–117.
51. Kunapuli SP. Multiple P2 receptor subtypes on platelets: A new interpretation of their function. Trends Pharmacol Sci 1998;19:391–394.
52. Fabre JE, Nguyen M, Latour A, et al. Decreased platelet aggregation, increased bleeding time and resistance to thromboembolism in P2Y1-deficient mice. Nat Med 1999;5:1199–1202.
53. Foster CJ, Prosser DM, Agans JM, et al. Molecular identification and characterization of the platelet ADP receptor targeted by thienopyridine antithrombotic drugs. J Clin Invest 2001;107:1591–1598.
54. Zhang FL, Luo L, Gustafson E, et al. ADP is the cognate ligand for the orphan G protein-coupled receptor SP1999. J Biol Chem 2001;276:8608–8615.
55. Hollopeter G, Jantzen HM, Vincent D, et al. Identification of the platelet ADP receptor targeted by antithrombotic drugs. Nature 2001;409:202–207.
56. Ayyanathan K, Webbs TE, Sandhu AK, et al. Cloning and chromosomal localization of the human P2Y1 purinoceptor. Biochem Biophys Res Commun 1996;218:783–788.
57. Hechler B, Leon C, Vial C, et al. The P2Y1 receptor is necessary for adenosine 5'-diphosphate-induced platelet aggregation. Blood 1998;92:152–159.
58. Hechler B, Cattaneo M, Gachet C. The P2 receptors in platelet function. Semin Thromb Hemost 2005;31:150–161.
59. Bennett JS. Novel platelet inhibitors. Annu Rev Med 2001;52:161–184.
60. Savi P, Pereillo JM, Uzabiaga MF, et al. Identification and biological activity of the active metabolite of clopidogrel. Thromb Haemost 2000;84:891–896.
61. Leon C, Vial C, Gachet C, et al. The P2Y1 receptor is normal in a patient presenting a severe deficiency of ADP-induced platelet aggregation. Thromb Haemost 1999;81:775–781.
62. Humphries RG, Robertson MJ, Leff P. A novel series of P2T purinoceptor antagonists: Definition of the role of ADP in arterial thrombosis. Trends Pharmacol Sci 1995;16:179–181.
63. Cattaneo M, Gachet C. ADP receptors and clinical bleeding disorders. Arterioscler Thromb Vasc Biol 1999;19:2281–2285.
64. Baurand A, Eckly A, Bari N, et al. Desensitization of the platelet aggregation response to ADP: differential down-regulation of the P2Y1 and P2cyc receptors. Thromb Haemost 2000;84:484–491.
65. Ding Z, Kim S, Dorsam RT, et al. Inactivation of the human P2Y12 receptor by thiol reagents requires interaction with both extracellular cysteine residues, Cys17 and Cys270. Blood 2003;101:3908–3914.
66. Hoffmann C, Moro S, Nicholas RA, et al. The role of amino acids in extracellular loops of the human P2Y1 receptor in surface expression and activation processes. J Biol Chem 1999;274:14639–14647.
67. Savi P, Zachayus JL, Delesque-Touchard N, et al. The active metabolite of clopidogrel disrupts P2Y12 receptor oligomers and partitions them out of lipid rafts. Proc Natl Acad Sci USA 2006;103:11069–11074.
68. Bhatt DL, Scheiman J, Abraham NS, et al. Expert consensus document on reducing the gastrointestinal risks of antiplatelet therapy and NSAID use. A report of the American College of Cardiology Foundation Task Force on Clinical Expert Consensus Documents. Circulation 2008;18:1894–1909.
69. Hankey GJ, Sudlow CL, Dunbabin DW. Thienopyridine derivatives (ticlopidine, clopidogrel) versus aspirin for preventing stroke and other serious vascular events in high vascular risk patients. Cochrane Database Syst Rev 2000, CD001246.
70. Jarvis B, Simpson K. Clopidogrel: A review of its use in the prevention of atherothrombosis. Drugs 2000;60:347–377.
71. Feliste R, Delebassee D, Simon MF, et al. Broad spectrum anti-platelet activity of ticlopidine and PCR 4099 involves the suppression of the effects of released ADP. Thromb Res 1987;48:403–415.
72. CAPRIE Steering Committee. A randomized, blinded trial of SR25990C versus aspirin in patients at risk of ischemic events (CAPRIE). Lancet 1996;348:1329–1339.
73. Verstraete M, Zoldhelyi P. Novel antithrombotic drugs in development. Drugs 1995;49:856–884.
74. Yusuf S, Zhao F, Mehta SR, et al. Effects of clopidogrel in addition to aspirin in patients with acute coronary syndromes without ST-segment elevation. N Engl J Med 2001;345:494–502.
75. Patrono C, Bachmann F, Baigent C, et al. Expert consensus document on the use of antiplatelet agents. The task force on the use of antiplatelet agents in patients with atherosclerotic cardiovascular disease of the European society of cardiology. Eur Heart J 2004;25:166–181.
76. Thebault JJ, Kieffer G, Lowe GD, et al. Repeated-dose pharmacodynamics of clopidogrel in healthy subjects. Semin Thromb Hemost 1999;25(Suppl 2):9–14.
77. Pedersen AK, FitzGerald GA. Dose-related kinetics of aspirin. Presystemic acetylation of platelet cyclooxygenase. N Engl J Med 1984;311:1206–1211.
78. Gurbel PA, Bliden KP, Hiatt BL, O’Connor CM. Clopidogrel for coronary stenting: Response variability, drug resistance, and the effect of pretreatment platelet reactivity. Circulation 2003;107:2908–2913.
79. Muller I, Besta F, Schulz C, et al. Prevalence of clopidogrel non-responders among patients with stable angina pectoris scheduled for elective coronary stent placement. Thromb Haemost 2003;89:783–787.
80. Matetzky S, Shenkman B, Guetta V, et al. Clopidogrel resistance is associated with increased risk of recurrent atherothrombotic events in patients with acute myocardial infarction. Circulation 2004;109:3171–3175.
81. Vats HS, Hocking WG, Rezkalla SH. Suspected clopidogrel resistance in a patient with acute stent thrombosis. Nat Clin Pract Cardiovasc Med 2006;3:226–230; Quiz 232.
82. Gurbel PA, Lau WC, Bliden KP, Tantry US. Clopidogrel resistance: Implications for coronary stenting. Curr Pharm Des 2006;12:1261–1269.
83. De Miguel A, Ibanez B, Badimon JJ. Clinical implications of clopidogrel resistance. Thromb Haemost 2008;100:196–203.
84. Celik T, Iyisoy A, Gul H, Isik E. Clopidogrel resistance: A diagnostic challenge. Int J Cardiol 2009;131:267–268; Author Reply 268–269.
85. Harrison P, Frelinger AL 3rd, Furman MI, Michelson AD. Measuring antiplatelet drug effects in the laboratory. Thromb Res 2007;120:323–336.
86. Wiviott SD, Antman EM. Clopidogrel resistance: A new chapter in a fast-moving story. Circulation 2004;109:3064–3067.
87. Jaremo P, Lindahl TL, Fransson SG, Richter A. Individual variations of platelet inhibition after loading doses of clopidogrel. J Intern Med 2002;252:233–238.
88. Saw J, Steinhubl SR, Berger PB, et al. Lack of adverse clopidogrel-atorvastatin clinical interaction from secondary analysis of a randomized, placebo-controlled clopidogrel trial. Circulation 2003;108:921–924.
89. Frere C, Cuisset T, Morange PE, et al. Effect of cytochrome p450 polymorphisms on platelet reactivity after treatment with clopidogrel in acute coronary syndrome. Am J Cardiol 2008;101:1088–1093.
90. Lau WC, Gurbel PA, Watkins PB, et al. Contribution of hepatic cytochrome P450 3A4 metabolic activity to the phenomenon of clopidogrel resistance. Circulation 2004;109:166–171.
91. Savi P, Combalbert J, Gaich C, et al. The antiaggregating activity of clopidogrel is due to a metabolic activation by hepatic cytochrome P450-1A. Thrombos Haemost 1994;72:313–317.
92. Meunier B, de Visser SP, Shaik S. Mechanism of oxidation reactions catalyzed by cytochrome p450 enzymes. Chem Rev 2004;104:3947–3980.
93. Clarke TA, Waskell LA. The metabolism of clopidogrel is catalyzed by human cytochrome P450 3A and is inhibited by atorvastatin. Drug Metab Dispos 2003;31:53–59.
94. Savi P, Pereillo JM, Uzabiaga MF, et al. Identification and biological activity of the active metabolite of clopidogrel. Thromb Haemost 2000;84:891–896.
95. Nguyen TA, Diodati JG, Pharand C. Resistance to clopidogrel: A review of the evidence. J Am Coll Cardiol 2005;45:1157–1164.
96. Geiger J, Honig-Lied P, Schanzenbacher P, Walter U. Ligand specificity and ticlopidine effects distinguish three platelet ADP receptors. Eur J Pharmacol 1998;351:235–246.
97. Schroer K. Antiplatelet drugs: A comprehensive review. Drugs 1995;50:7–28.
98. Jin J, Kunapuli SP. Activation of two different G protein-coupled receptors is essential for ADP-induced platelet aggregation. Proc Natl Acad Sci USA 1998;95:8070–8074.
99. Hollopeter G, Jantzen HM, Vincent D, et al. Identification of the platelet ADP receptor targeted by antithrombotic drugs. Nature 2001;409:202–207.
100. Barone-Rochette G, Ormezzano O, Polack B, et al. Resistance to platelet antiaggregants: An important cause of very late thrombosis of drug eluting stents? Observations from five cases. Arch Cardiovasc Dis 2008;101:100–107.
101. Bonello L, Paganelli F, Arpin-Bornet M, et al. Vasodilator-stimulated phosphoprotein phosphorylation analysis prior to percutaneous coronary intervention for exclusion of postprocedural major adverse cardiovascular events. J Thromb Haemost 2007;5:1630–1636.
102. Hezard N, Metz D, Garnotel R, et al. Platelet VASP phosphorylation assessment in clopidogrel-treated patients: Lack of agreement between Western blot and flow cytometry. Platelets 2005;16:474–481.
103. Blindt R, Stellbrink K, de Taeye A, et al. The significance of vasodilator-stimulated phosphoprotein for risk stratification of stent thrombosis. Thromb Haemost 2007;98:1329–1334.
104. Niitsu Y, Sugidachi A, Ogawa T, et al. Repeat oral dosing of prasugrel, a novel P2Y(12) receptor inhibitor, results in cumulative and potent antiplatelet and antithrombotic activity in several animal species. Eur J Pharmacol 2008;579:276–282.
105. Sugidachi A, Asai F, Ogawa T, et al. The in vivo pharmacological profile of CS-747, a novel antiplatelet agent with platelet ADP receptor antagonist properties. Br J Pharmacol 2000;129:1439–1446.
106. Jernberg T, Payne CD, Winters KJ, et al. Prasugrel achieves greater inhibition of platelet aggregation and a lower rate of non-responders compared with clopidogrel in aspirin-treated patients with stable coronary artery disease. Eur Heart J 2006;27:1166–1173.
107. Brandt JT, Payne CD, Wiviott SD, et al. A comparison of prasugrel and clopidogrel loading doses on platelet function: Magnitude of platelet inhibition is related to active metabolite formation. Am Heart J 2007;153:66, e9–e16.
108. Jakubowski JA, Payne CD, Weerakkody GJ, et al. Dose-dependent inhibition of human platelet aggregation by prasugrel and its interaction with aspirin in healthy subjects. J Cardiovasc Pharmacol 2007;49:167–173.
109. Hagihara K, Kurihara A, Kawai K, et al. Absorption, distribution and excretion of the new thienopyridine agent prasugrel in rats. Xenobiotica 2007;37:788–801.
110. Farid NA, Smith RL, Gillespie TA, et al. The disposition of prasugrel, a novel thienopyridine, in humans. Drug Metab Dispos 2007;35:1096–1104.
111. Wiviott SD, Trenk D, Frelinger AL, et al. Prasugrel compared with high loading- and maintenance-dose clopidogrel in patients with planned percutaneous coronary intervention: The Prasugrel in Comparison to Clopidogrel for Inhibition of Platelet Activation and Aggregation-Thrombolysis in Myocardial Infarction 44 trial. Circulation 2007;116:2923–2932.
112. Wiviott SD, Braunwald E, McCabe CH, et al. Prasugrel versus clopidogrel in patients with acute coronary syndromes. N Engl J Med 2007;357:2001–2015.
113. Wiviott SD, Braunwald E, McCabe CH, et al; for the TRITON-TIMI 38 Investigators. Prasugrel versus clopidogrel in patients with acute coronary syndromes. N Engl J Med 2007;357:2001–2015.
114. Farid NA, Payne CD, Small DS, et al. Cytochrome P450 3A inhibition by ketoconazole affects prasugrel and clopidogrel pharmacokinetics and pharmacodynamics differently. Clin Pharmacol Ther 2007;81:735–741.
115. Rehmel JL, Eckstein JA, Farid NA, et al. Interactions of two major metabolites of prasugrel, a thienopyridine antiplatelet agent, with the cytochromes P450. Drug Metab Dispos 2006;34:600–607.
