Title: “Clopidogrel resistance in patient with myocardial infarction and ischemic stroke.”
Principal Investigator:
INDIAN side:
Dr. Renu Saxena
Professor & Head of Department
Deptt. Of Hematology
All India Institute of Medical Sciences
U.S side
Dr. Gundu Rao
Professor, Lillehei Heart Institute
Department of Laboratory Medicine & Pathology
University of Minnesota
Co- Investigator:
INDIAN side:
Dr. Rakesh Yadav
Deptt. Of Cardiology
All India Institute of Medical Sciences
Dr. Madhuri Behari
Professor & Head of Department
Deptt. of Neuroscience
All India Institute of Medical Sciences
U.S side
Specific Aims & Objectives
1) To determine the prevalence of Clopidogrel resistance and expression of activation markers such as P-selectin and CD40 in the normal healthy population, and patients with vascular diseases such as coronary artery disease and stroke.
2) To correlate platelet receptor polymorphisms with the occurrence of drug resistance in the patient population.
Funding Agency
Indian Council of Medical Research/National Institute of Health
Background and Significance
Platelets play an integral role in the process of thrombosis, both physiological and pathophysiological. Activation and aggregation of platelets has a central role in the propagation of intracoronary thrombi after spontaneous atherosclerotic plaque disruption resulting in myocardial ischemia or infarction in the acute coronary syndromes (ACS), or after the mechanical disruption that results from percutaneous coronary intervention (PCI). Hence antiplatelet agents like Aspirin and Thienopyridines (Clopidogrel and Ticlopidine) are used in
n Prophylaxis of patients undergoing vascular grafting or PCA
n Management of ACS
n Long term prevention of cardiovascular and cerebrovascular events
However in recent years, concern over resistance to the effect of anrtiplatelet agents has emerged. Several mechanisms have been proposed and studied including platelet receptor polymorphisms. Platelets initially adhere to collagen and von Willebrand factor at the site of the disrupted plaque, resulting in an initial platelet monolayer. After activation, platelets release secondary agonists such as thromboxane A2 and adenosine diphosphate (ADP), which in combination with thrombin generated by the coagulation cascade result in stimulation and recruitment of additional platelets. Hence, antiplatelet therapy is a cornerstone of the management of patients with ACS, especially those undergoing PCI and also for long term prevention of cardiovascular and cerebrovascular events.
Antiplatelet drugs:
Aspirin inhibits cyclooxygenase (COX) by irreversible acetylation, which prevents the production of thromboxane A2. The antithrombotic effect of aspirin results from the decreased production of this prothrombotic, vasoconstrictive substance. The thienopyridines irreversibly inhibit ADP binding to the P2Y12 receptor on the platelet surface. By blocking this receptor, these agents interfere with platelet activation, degranulation, and—by inhibiting the modification of the glycoprotein IIb/IIIa receptor—aggregation.
Thienopyridines include ticlopidine and clopidogrel. Both agents are rapidly absorbed prodrugs that are modified hepatically to active metabolites Both the agents have similar platelet effects and have been shown to be clinically efficacious. However, clopidogrel has largely replaced ticlopidine because of an improved safety profile, with a lower incidence of hematologic complications (neutropenia pancytopenia and thrombotic thrombocytopenic purpura) than ticlopidine. The effects of clopidogrel are time and dose dependent, with a ceiling effect at approximately 50% to 60% inhibition of platelet aggregation. Loading doses of clopidogrel of 300 to 600 mg reach near steady-state levels of platelet aggregation by 4 to 24 hours, whereas daily maintenance dosing with 75 mg daily without a preload results in steady-state levels within 4 to 7 days.
Resistance to anti-platelet drugs:
Although antiplatelet agents reduce arterial thrombosis, "resistance" to their effects can occur. Resistance implies the failure of an agent to achieve its pharmacological effect. Several mechanisms of clopidogrel resistance are possible. Extrinsic mechanisms include inappropriate dosing or underdosing of clopidogrel, drug–drug interactions, including a possible interaction between clopidogrel and atorvastatin, CYP3A4 activity (measured by erythromycin breath test), variable absorption of the prodrug and clearance of the active metabolite. Intrinsic mechanisms include P2Y12 receptor variability, increase in number of receptors, increased release of ADP, or upregulation of other platelet activation pathways.
To identify the failure of an agent to achieve a pharmacological effect, one must be able to measure it reliably. Several assays are available to measure platelet function and effects of antiplatelet agents. A commonly used test of platelet function measures platelet aggregation by light transmittance (optical aggregometry) in platelet-rich plasma in response to an agonist (arachidonic acid, ADP, collagen, epinephrine, or a thrombin receptor–activating peptide). This mechanism allows monitoring of different drug effects by allowing choice of agonist (eg, ADP for thienopyridines). Because of inter- and intra-patient variability results are often reported as a percentage of a baseline value. Other methods include the cone and plate (let) analyzer, a rapid test that measures whole blood platelet aggregation under conditions of high shear stress and the ultegra rapid function assay. Similarly assessment of platelet activation can be done by flow cytometric detection of GpIIb/IIIa and P-Selectin.
Clopidogrel resistance:
Muller et al have reported prevalence of Clopidogrel “non-responders” among patients with stable angina scheduled for coronary stent replacement (93). They evaluated the effect of 100mg/day aspirin with a loading dose of 600mg of Clopidogrel. They defined Clopidogrel non-responders by an inhibition of ADP (5and 30umol/L) induced platelet aggregation to less than 10% of base line values after the ingestion of the drug. They found 5-11% of the patients as non-responders and 9-26% semi-responders. Lau et al studied both normal subjects and patients undergoing stent replacement to determine the prevalence of drug resistance. Since Clopidogrel is activated by hepatic cytochrome P450 (CYP) 3A4, they monitored the activity of this enzyme in both the test subjects. They concluded that some patients are resistant to these drugs because of biological variability and drug-drug interactions. They also attributed the difference due to variations in (CYP) 3A4 activity. Angiolillo et al compared the effect of standard dose of 300mg with a high dose of 600mg Clopidogrel after coronary stenting. They concluded that use of higher dose optimizes platelet inhibitory effects. Serebruany et al evaluated the effect of Clopidogrel in a large heterogenous population using ex vivo measurements of platelet aggregation, Prevalence of hypo-responsiveness and hyper-responsiveness to Clopidogrel in these patient population was 4.2% and 4.8% respectively. A Polish study with a small sample size (n=31) studied the effect of Clopidogrel in patients with stable angina using a rapid whole blood platelet function assessment. They conclude that the clinical importance of Clopidogrel resistance is still debatable. In view of the fact that data are not available on large number of clinical trials, their conclusion is reasonable. Over recent years, variable platelet response and potential resistance to therapy has emerged with thienopyridines. Studies have shown a dose- and time-dependent variability in response to clopidogrel as measured by optical platelet aggregometry in response to ADP.
n Clopidogrel non-responder
· Inhibition of ADP (5 & 20 µmol/L) induced platelet aggregation <10% when compared to baseline values 4, 24, 48 hrs after clopidogrel intake
n Semi-responder
· Inhibition of 10-29 %
n Responders
· Inhibition > 30%
Nonresponders are defined as those with <10% reduction in platelet aggregation to ADP and semiresponders as those with 10% to 29% reduction 4 hours after 600-mg clopidogrel load,11 as no additional effect was seen with this treatment regimen at 24 hours. In this study, prevalence of clopidogrel non –responder was 5-11% and semi responders was 9-26%. However there is lack of any Indian data regarding the prevalence of clopidogrel resistance.
Alternatively another method of evaluating platelet activation could be to look at Platelet activation markers like P-Selectin and CD40 by Flow cytometry. In patients on clopidogrel in whom these markers show evidence of platelet activation, clopidogrel resistance will be considered.
Platelet ADP Receptors:
One of the most important mediators of hemostasis and thrombosis is adenosine diphosphate (ADP).Two platelet ADP receptors, P2Y1 and P2Y12, have been shown to initiate platelet activation when stimulated in concert. Both are heterotrimeric G-protein-coupled receptors Stimulation at P2Y1 via activation of the Gq pathway leads to intracellular calcium mobilization and platelet shape change and a rapidly reversible wave of platelet aggregation. Stimulation at P2Y12 via activation of the Gi pathway leads to inhibition of adenylyl cyclase and activation of phosphoinositide-3 kinase.The end effect is affinity modulation of the glycoprotein IIb-IIIa (GPIIb-IIIa) receptor for fibrinogen, resulting in fibrinogen binding and slowly progressive and sustained platelet aggregation. P2Y12 thus appears to have a pivotal role in the irreversible wave of platelet aggregation that occurs on exposure to ADP. The importance of P2Y12 is emphasized by the fact that it is the target of the thienopyridine drugs ticlopidine and clopidogrel. However there is substantial interindividual variation in platelet response to ADP which may be genetically controlled because of variation in the P2Y1 and/or P2Y12 genes.
P2Y12 gene polymorphism:
P2Y1 and P2Y12 genes are located on chromosome 3. The P2Y1 gene spans 4 kb and is made up of a single exon of 3122 base pairs encoding a 372-aa protein. The P2Y12 gene spans 47 kb and is made up of 3 exons and 2 introns.
In their study, Fontana et al analyzed the P2Y12 gene in the study population and found 4 single-nt polymorphisms (SNPs) and a single-nt insertion polymorphism.Two variations were located 139 nt and 744 nt after the 5_ intron start site, consisting of a C-to-T (i-C139T) and a T-to-C (i-T744C) transition, respectively. Another polymorphism consisted of a single-nt insertion (A) at position 801 of the intron (iins801A). The remaining 2 polymorphisms were found in exon 2 and consisted of a C-to-T transition (C34T) and a G-to-T transversion (G52T), respectively, 34 nt and 52 nt after the 5_ start site of exon 2; neither modified the encoded amino acid (Asn6 and Gly,12 respectively). As the i-C139T, i-T744C, i-ins801A, and G52T polymorphisms were in complete linkage disequilibrium in their population, they designated H1 as the major haplotype (a C in position 139, a T in position 744, and absence of the i-ins801A in the intron, as well as a G in position 52 of exon 2) and H2 as the minor haplotype (a T in position 139, a C in position 744, presence of the i-ins801A in the intron, and a T in position 52 of exon 2). The respective frequencies of haplotypes H1 and H2 were 86% and 14%. The H2 haplotype was associated with higher maximal aggregation in response to ADP, with median values of 34.7% in subjects carrying none of the H2 alleles, 67.9% in subjects carrying 1 H2 allele and 82.4% in the 3 subjects carrying 2 H2 alleles The increased maximal platelet aggregation in response to ADP in the H2 haplotype was related to differences in the mechanism regulating cAMP inhibition by ADP. An increase in the number of receptors on the platelet surface was proposed to explain the association between the H2 haplotype and platelet responsiveness to ADP. Indeed, the H2 haplotype could be linked to a sequence variation in the promoter region that could increase transcription efficiency. Because thienopyridines only provide partial P2Y12 blockade, and aspirin does not inhibit P2Y12-mediated amplification of platelet responses, carriers of the H2 allele may have less protection of these platelet inhibitors.
In another study association of gene sequence variations in P2Y12 and occurrence of neurological adverse events in patients with symptomatic peripheral artery disease (PAD) during clopidogrel treatment was tested. The 34C>T polymorphism in exon 2 which is not linked to the rest 4 polymorphisms and 52G>T, both located in the coding region of the P2Y12 gene were selected to study the effect on the response to clopidogrel treatment. They found that subjects undergoing current clopidogrel treatment, who were carriers of the P2Y12 polymorphism 34C>T, had a 4-fold higher risk to have an adverse neurological event, defined as ischemic stroke and/or carotid revascularization within a 2-year observation period than subjects carrying the wild-type genotype in another study, P2Y12 H2 haplotype was found to be associated with PAD and the possibility of Theinopyridine resistance in carriers of H2 haplotype was suggested.
Background and Preliminary Work:
Role of platelets in the pathogenesis of atherosclerosis, thrombosis and stroke is well documented . Therefore, there is a great need for developing specific and effective drugs for modulating platelet function. A thorough understanding of the signaling mechanisms involved in the regulation of platelet function will facilitate the development of better anti-platelet drugs. Agonists interact with the platelet at specific receptor sites on the plasma membrane and initiate a series of signaling events capable of modulating shape change, adhesion, aggregation, secretion of granule contents and expression of activation markers on the membrane.Platelet aggregates are formed when the GP11b/111a receptors get activated and bind fibrinogen and recruit other platelets to form clumps. This phenomenon plays a significant role in the formation of effective haemostatic plug as well as growth of thrombus. Weak agonists such as epinephrine, ADP require the production of pro-aggregatory prostaglandin endoperoxides and thromboxane to cause platelet aggregation and secretion. Aspirin is a specific inhibitor of cyclooxygenase and prevents the formation of pro-aggregatory PG endoperoxides.
Data from large number of clinical studies have demonstrated that at any given risk, irrespective of the disease state, aspirin at low to medium concentration is as effective as any other drug. Although ability of a plant bark (bark of willow, Salix alba) product to reduce fever was discovered two hundred years ago, the mechanism of action of aspirin remained elusive till late 1900. Nobel laureate Sir John R. Vane and his associates at the Royal College of Surgeons, in London, in 1971 proposed the first satisfactory mechanism as to how aspirin works. Within a short period of time extensive work was done by various groups to elucidate the mechanism of action of aspirin like compounds. At the same period, another Nobel Laureate Dr. Bengt Samuelsson of the Karolinska Institute, Stockholm, Sweden, discovered that the prostaglandin synthase produces transient bioactive prostanoids like PGG 2 /PGH2 and thromboxane A2 from the substrate arachidonic acid. These findings revolutionized the research in platelet physiology and pharmacology.
Platelet Physiology:
Blood platelets interact with a variety of soluble agonists such as epinephrine (EPI), adenosine diphosphate (ADP), thrombin and thromboxane (TXA2); many cell matrix components, including collagen, laminin, fibronectin, and vonWillebrand factor and biomaterials used for construction of invasive medical devices. These interactions stimulate specific receptors and glycoprotein-rich domains (integrin and non-integrin receptors) on the plasma membrane and lead to the activation of intracellular effector enzymes. Agonist–mediated activation of platelets stimulates Phospholipase C (PLC) and it then triggers the hydrolysis of Phosphatidyl inositol 4, 5-bisphosphate, the formation of second messengers such as 1, 2-diacyl glycerol and inositol 1, 4, 5 trisphosphate. Diacylglycerol activates protein kinase and inositol trisphosphate facilitates the mobilization of free calcium from the storage sites. The majority of regulatory events appear to require free calcium. Ionized calcium is the primary bioregulator, and a variety of biochemical mechanisms modulate the availability of free calcium (27). Elevation of cytosolic calcium stimulates Phospholipase A2 and liberates arachidonic acid (AA). Free AA is transformed to a novel metabolite thromboxane, a potent platelet agonist. This is the major metabolite of AA metabolism and plays an important role in platelet recruitment, granule mobilization, secretion of granule contents, and expression of activated GP11b/111a (ά11b β3) receptors). Up regulation of activation signaling pathways, will increase the risk for clinical complications associated with thromboembolic episodes.