Clinical consequences of aspirin and clopidogrel resistance: an overview

Authors


M. D. Mijajlovic, Neurology Clinic, Clinical Center of Serbia and School of Medicine, University of Belgrade, Belgrade, Serbia

Dr Subotica 6, 11000 Belgrade, Serbia

Tel.: +381641929401

Fax: +381112684577

e-mail: milijamijajlovic@yahoo.com

Abstract

The aim of this review is to introduce the concept of personalized medicine in secondary stroke prevention with antiplatelet medication. In the last years, many studies have been conducted regarding aspirin resistance and genotyping of clopidogrel metabolism. A review of the currently published data on this issue emphasizes the importance of focusing on the individualizing approach in antiplatelet therapy to achieve maximal therapeutic beneficial effect. However, many authors suggest that, before new information from ongoing trials become available, good clinical practice should dictate the use of low dose of aspirin that was shown to be effective in the prevention of stroke and death in patients with ischemic cerebrovascular disease, because higher doses do not have significantly better efficacy than lower doses in secondary stroke prevention, but lower-dose aspirin is associated with less side effects. On the other hand, many factors are associated with clopidogrel resistance, and recent genetic studies showed that the CYP2C19*2 genotype (loss-of-function allele) is related to poor metabolism of clopidogrel, but larger studies are needed to definitively confirm or rule out the clinical significance of this genetic effect. The aim of personalized approach in secondary stroke prevention is to take the most appropriate medicine in the right dose in accordance with the clinical condition of the patient and associated risk factors.

Introduction

Antiplatelets agents are the major players for secondary stroke prevention. It is necessary to emphasize that the goal of stroke preventive therapy is to lower the risk – not to make the risk of stroke zero. In patients with transient ischemic attack (TIA) or ischemic stroke, antiplatelet drugs are able to decrease the risk of stroke by 11–15%. Platelets play a major pathogenic role in thrombus formation. Excessive platelet activation in case of plaque erosion or rupture leads to generation of unwarranted levels of thrombin, which initiates thrombosis and formation of thrombi at sites of plaque disruption. Platelet activation can be induced by the cooperative action of multiple factors, including serotonin, epinephrine, thrombin, ADP, and TXA2 [1]. However, platelet reactivity is variable and patients do not respond uniformly to antiplatelet therapy, which influences the outcomes of secondary prevention. The most widely used antiplatelet drugs for stroke prevention are aspirin and clopidogrel. Despite the regular antiplatelet treatment, some patients experience thromboembolic events. Those patients are clinically designated as aspirin or clopidogrel resistant or non-responders.

Aspirin

Aspirin (acetylsalicylic acid, ASA) is a salicylate drug and one of the most important and widely used drugs for the primary and secondary prevention of atherothrombotic diseases. Aspirin is a relatively safe, easy to administer, and readily available drug. Also, in evidence-based guidelines, aspirin is the drug of first choice for secondary stroke prevention.

Aspirin irreversibly inhibits cyclooxygenase-1 enzyme (COX-1) in platelets by acetylating its serine-529 residue in the active enzyme site, thereby blocking thromboxane A2 (TXA2), the most significant trigger for platelet activation, and other eicosanoide production from arachidonic acid. COX-1-dependent TXA2 inhibition lasts for the period of a platelet's lifespan (7–10 days); therefore, the effects of aspirin are maintained with daily dosing intervals. Aspirin induced COX-1 inhibition is rapid and most importantly irreversible [1]. After a single 325 mg dose of ASA, platelet COX-1 activity is completely inhibited and recovers for approximately 10% per day, due to nascent platelet release in the circulation. After a single dose, a serum peak level is reached in about 1 h and then declines gradually [2]. Salicylates are eliminated mostly by the kidney, as salicyluric acid (75%), free salicylic acid (10%), salicylic phenol (10%), acyl glucuronides (5%), gentisic acid (<1%), and 2,3-dihydroxybenzoic acid [3]. When small doses (less than 250 mg in an adult) are taken, all pathways proceed by first-order kinetics, with an elimination half-life of about 2.0–4.0 h [4]. When higher doses of salicylate are ingested (more than 4 g), the half-life becomes much longer (15–30 h), because the biotransformation pathways become saturated [4]. The overview analysis of an indirect comparisons between the various doses of aspirin suggests that aspirin doses as low as 30 mg per day to as high as about 1300–1500 mg per day have the same efficacy in recurrent stroke prevention, but lower-dose aspirin usage is associated with less side effects. Some experts suggest an aspirin dose of 75–81 mg per day, because it provides the best safety and efficacy balance for cardio-cerebrovascular disease prevention [5].

The Aspirin Trialists' Collaboration suggests that the use of aspirin provides an overall 25% risk reduction in secondary thrombotic events [6]. Despite strong evidence in preventing thrombotic events, a lot of patients still fail to respond to aspirin therapy. Also, aspirin resistance in stroke patients is not uncommon [7]. The aspirin resistant patients are at about a fourfold increased risk of non-fatal and fatal cardiovascular, cerebrovascular, or vascular events while taking aspirin than their aspirin sensitive counterparts [8].

In a recent meta-analysis of 20 studies, which included 2930 patients with cardiovascular diseases, 810 patients (28%) were classified as aspirin resistant, using a variety of platelet function assays. Patients were inclouded in study if they were receiving aspirin therapy as an antithrombotic medication and also they were classified prospectively as aspirin sensitive or aspirin resistant before the ascertainment of any clinical outcome, or were grouped on the basis of clinical outcome and then classified for aspirin status. Patients were considered to be aspirin sensitive if their platelets responded as expected to aspirin treatment, and platelet function, however measured, was inhibited, and were considered to be aspirin resistant if their agonist induced platelet response was not inhibited by aspirin as expected. In addition, the investigators were blinded to the patient's aspirin sensitive and aspirin resistant status. A cardiovascular-related event occurred in 41% of aspirin resistant patients (OR 3.85, 95% CI 3.08–4.80), death in 5.7% (OR 5.99, CI 2.28–15.72), and an acute coronary syndrome in 39.4% (OR 4.06, CI 2.96–5.56). Also, aspirin resistant patients did not benefit from any other antiplatelet treatment. The results of the meta-analysis suggest that patients who were resistant to aspirin were at a greater risk of clinically important cardiovascular event than patients who were sensitive to aspirin. The prevalence of non-responsiveness to aspirin was statistically higher (< 0.5) in those patients who suffered recurrent cerebral ischemia while taking aspirin compared with patients who remained without new brain ischemic symptoms [8].

So far, there is no generally accepted universal definition of aspirin resistance. The failure of aspirin to prevent clinical events or to inhibit platelet aggregation, ex vivo and in vitro is known as aspirin resistance. There are several factors associated with aspirin resistance (Table 1).

Table 1. Factors associated with aspirin resistance
Factors associated with aspirin resistance
Female sex
Increased age
Diabetes
High plasma triglycerides
Low hemoglobin level
Simultaneous administration of other non-steroidal anti-inflammatory drugs
Elevated norepinephrine levels
Cigarette smoking
Hypercholesterolemia
Polymorphisms affecting COX-1 (e.g. 50T), COX-2 (-765C), TXA2 synthase
Transient increase of platelet COX-1/COX-2 expression in new platelets: platelet turnover is accelerated in response to stress, for example, following coronary artery bypass grafting

The term clinical aspirin failure (clinical ‘resistance’, treatment failure) refers to those patients who had recurrent ischemic events while on aspirin therapy. The term platelet non-responsiveness to aspirin (laboratory ‘resistance’, biochemical aspirin resistance) describes the inability of aspirin to inhibit arachidonic acid and/or collagen-induced platelet aggregation [9, 10].

The prevalence of clinical aspirin failure has been reported with frequencies ranging from 5.5% to 60% of treated patients [11-16]. This wide range is due to clinical differences in the case mix of the patients, different doses of aspirin used, and differences of the methodology used to assess responsiveness to aspirin therapy.

The aspirin resistance can be measured by several laboratory methods. The historic gold standard for assessment of platelet function is optical (or light transmittance) platelet aggregometry (OPA), also used to predict the risk of recurrent ischemic events [17]. The platelet aggregometry has major disadvantages, including poor reproducibility, large sample volume, slow assay time, the need for sample preparation, and a skilled technician [18]. It is shown as well that results of OPA are not indicative of the risk of recurrent vascular events [19]. There are also two point-of-care devices that evaluate platelet function, and they have become available: the PFA-100 analyzer (Dade-Behring, Marburg, Germany) and the VerifyNow System (Accumetrics, San Diego, CA, USA). PFA-100 device measures the closure time of a microscopic aperture in a membrane/cartridge coated with collagen/epinephrine or collagen/ADP using whole blood anticoagulated with sodium citrate. Using this test, aspirin resistance is defined as a closure time for a collagen/epinephrine cartridge of <164 s despite regular aspirin intake. The PFA-100 test has a good sensitivity and reproducibility. The test needs to be carried out within 3–4 h after blood collection and depends upon plasma's von Willebrand factor and hematocrit.

The VerifyNow System is a turbidimetric-based optical detection system for measuring platelet-induced aggregation, that is, the VerifyNow System measures platelet function by the rate and extent of light changes in whole blood as platelets aggregate over time in response to agonists that are specific to various antiplatelet medications. Within the test device compartments, the instrument measures the increase in light transmittance over time. A patient's blood sample that exhibits inhibited platelet function produces low light transmittance, whereas a sample with normal platelet function produces high light transmittance. This measurement is based on the principles of optical aggregometry and has been shown to correlate well with that method.

Other laboratory methods are based on the measurement of thromboxane A2 pathway end products – serum thromboxane B2 and urinary 11-dehydrothromboxane B2 [13, 20]. The last one is frequently used in large trials on aspirin resistance [13, 21].

In a systematic review, the prevalence of biochemical aspirin resistance varied widely: from 6% (95% CI 0–12%) with optical aggregometry to 26% (95% CI 21–31%) with PFA-100 analyzer [22].

Till now, there is not enough evidence to designate only one laboratory method to determine aspirin resistance. That is why some advocates multiple parameters to detect aspirin resistance [23].

The possible causes of aspirin resistance may be divided in two groups: first one related and second one unrelated to the COX-1 pathway thromboxan A2 production. Poor patient compliance, failure to prescribe aspirin properly, concomitant use of anti-inflammatory drugs or co administration of proton pump inhibitors, advanced age and overweight, as well as polymorphisms of COX-1 gene are COX-1-related causes of aspirin resistance [24]. It is important to educate patients about the aspirin mechanisms of action [25]. The COX-1-unrelated causes include up-regulation of COX-2 pathway of thromboxan A2 production by platelets, monocytes, macrophages, and increased production of prostaglandin F2-like compounds by lipid peroxidation of arachidonic acid. The genetic factors include polymorphism of glycoprotein IIIa and glycoprotein Ia/IIa collagen receptor genes [26-30]. More evidences about prognostic significance of gene polymorphism are required.

Kojuri et al. prospectively studied the effect of aspirin on platelet function, and they founded that frequency of aspirin resistance is not dependent on the dose of aspirin [31].

Aspirin resistant patients may benefit in dual antiplatelet therapy with clopidogrel or in addition of dipyridamol. Until new information from ongoing trials become available, good clinical practice should dictate the use of the lowest dose of aspirin that has already been effective in the prevention of stroke and death in patients with ischemic cerebrovascular disease, that is, 75–100 mg daily [32].

Clopidogrel

Clopidogrel is taken by about 40 million patients worldwide to prevent secondary vascular event [33].

Clopidogrel is an inactive prodrug that requires two-step oxidation by the hepatic cytochrome P450 (CYP) system to generate its active compound, which irreversibly inhibits the adenosine diphosphate (ADP) P2Y purinoceptor 12 on circulating platelets. The platelet inhibition induced by clopidogrel is dose and time dependent. Platelet inhibition is dose-related up to a single dose of 400 mg of clopidogrel, with no further increase with 600 mg [34]. The maximum inhibition with a single 400-mg dose is achieved after 2–5 h, whereas a dose of 75 mg per day produces the same level of inhibition after 3–7 days [34, 35]. This fact, especially time dependence, should be taken into account when starting clopidogrel for stroke prevention therapy.

Pharmacokinetic and pharmacodynamic response to clopidogrel depends on genetic polymorphisms [36, 37]. The genome-wide association study of clopidogrel response has reported that the CYP2C19*2 genotype (loss-of-function allele), the most common genetic variant, is associated with poor metabolism of clopidogrel with diminished platelet response and poorer cardiovascular outcome [36]. The CYP2C19*2 genotype is common in diverse populations. In white populations, approximately 24% of people have at least 1 CYP2C19*2 allele. The frequency of this allele is 18% in Mexican Americans, 33% in African Americans, and much higher in Asian populations – 51% (with at least 1 copy) [38].

In a recent study (= 2208) of patients with recent myocardial infarction, it was examined whether any of the known allelic variations of genes that modulate clopidogrel's absorption (ABCB1), metabolic activation (CYP3A4/5 and CYP2C19), or biologic activity (P2RY12 and ITGB3) is associated with a risk of death from any cause, non-fatal stroke, or myocardial infarction. It was found that none of the selected single-nucleotide polymorphisms, CYP3A5, P2RY12, or ITGB3, were associated with a risk of an unwanted outcome, while patients carrying the ABCB1 variant allele (genotype CT or TT) had a higher hazard ratio for an outcome event than those with the ABCB1 wild-type allele (genotype CC). Patients with any two CYP2C19 alleles (*2, *3, *4, or *5) that result in loss-of-function had a higher risk of major event (death, myocardial infarction, or stroke) than patients with one or none loss-of-function allele [39]. On the basis of these findings and related pharmacokinetic and pharmacodynamic data (ClinicalTrials.gov number, NCT01123824), the Food and Drug Administration (FDA) has issued a black box warning about the reduced effectiveness of clopidogrel in patients who are carriers of two loss-of-function alleles (so-called poor metabolizers) and they suggested that carriers of these alleles should receive a higher dose of clopidogrel or an alternative antiplatelet agent [40].

However, recent study conducted among 5059 genotyped patients (genotyped for three single-nucleotide polymorphisms: *2, *3, *17, that define the major CYP2C19 alleles) with acute coronary syndromes, compared the effect of clopidogrel with placebo in poor metabolizers. The clopidogrel therapy significantly reduced the rate of cardiovascular events, irrespective of the genetically determined metabolizer phenotype. The effect of clopidogrel in reducing the rate of cardiovascular events was nearly the same in patients who were homozygous or heterozygous for loss-of-function alleles and in those who were not carriers of the alleles. In conclusion, the authors stated that among patients with acute coronary syndromes, the effect of clopidogrel as compared with placebo is consistent, regardless of CYP2C19 loss-of-function carrier status [41].

Feher et al. compared the characteristics (risk profile, previous diseases, medications, hemorheologic variables, plasma von Willebrand factor, and soluble P-selectin levels) of patients with normal clopidogrel platelet inhibition with those patients in whom clopidogrel was not effective in providing platelet inhibition (defined by Carat TX4 aggregometer; Carat Diagnostics Ltd, Budapest, Hungary). Study included 157 patients with chronic cardio- and cerebrovascular diseases (83 males, mean age 61 ± 11 years, 74 females, 63 ± 13 years) taking 75 mg clopidogrel per day (not combined with aspirin). They showed that patients with effective clopidogrel inhibition of platelet function have a significantly lower body mass index (26.1 vs 28.8 kg/m2; < 0.05) and that they are probably more likely to be using benzodiazepines (25% vs 10%) and selective serotonin reuptake inhibitors (28% vs 12%) (< 0.05). On the other hand, there was no significant difference in the rheologic parameters and in the plasma levels of von Willebrand factor and soluble P-selectin between the examined groups. Based on these results, authors suggested that clopidogrel therapy should be weight-adjusted [42].

There are several factors associated with clopidogrel resistance: blood glucose level, diabetes, and high systolic and diastolic blood pressure. The incidence of low response or non-response to clopidogrel ranges between 5% and 30% [43, 44]. It should be taken into consideration that aspirin-resistant patients may have a high rate of clopidogrel resistance as well.

Recent studies showed that clopidogrel non-responsiveness, that is, resistance is probably dependent on the administered dose. The common daily dose of clopidogrel is 75 mg, if a bolus is provided, therapeutic levels of platelet inhibition can be achieved within hours [34]. The clopidogrel resistance can be managed in some patients by increasing the given dose. In the pharmacodynamic study of Gurbel et al. comparing 300-mg and 600-mg clopidogrel loading doses, treatment with a 600-mg loading dose during elective percutaneous coronary interventions reduced clopidogrel non-responsiveness to 8% compared with 28–32% after a 300-mg loading dose [45]. Nearly, the same increased responsiveness was observed in the ISAR-CHOICE study (Intracoronary Stenting and Antithrombotic Regimen: Choose Between 3 High Oral Doses for Immediate Clopidogrel Effect), where the highest effect of platelet inhibition was observed with a 600-mg clopidogrel loading dose, while a non-significant increase in platelet inhibition was noticed with a 900-mg loading dose [46].

The CREDO study demonstrated that patients who received a 300-mg clopidogrel loading dose at least 6 h before percutaneous coronary intervention, had a 38.6% reduction in death, myocardial infarction, or urgent target vessel revascularization at 1 month [47]. Another studies also showed that 600-mg loading dose had lower rate of resistance, lower rate of recurrent, and periprocedural ischemic events [47-50]. A recent meta-analysis demonstrated that the 600-mg clopidogrel loading dose was associated with a lower rate of cardiovascular death or non-fatal myocardial infarction during 1-month follow-up (OR 0.54; 95% CI 0.32–0.90; = 0.02) and with no increase in major (OR. 1.88; 95% CI. 0.24–14.8; = 0.55) or minor (OR 0.99; 95% CI 0.49–2.0; = 0.98) bleeding, compared with a 300-mg loading dose [51, 52].

Patients with clopidogrel resistance may benefit more from the third-generation thienopyridine as prasugrel, or ticagrelor and cangrelor. In a recent study, Wiviott et al. compared prasugrel, a new thienopyridine, with clopidogrel. They randomly assigned 13.608 patients with moderate-to-high-risk acute coronary syndromes who were scheduled for percutaneous coronary intervention to receive prasugrel (a 60-mg loading dose, followed by 10 mg/day maintenance dose) or clopidogrel (a 300-mg loading dose, followed by 75 mg/day maintenance dose), for 6–15 months. Death from cardiovascular causes, non-fatal myocardial infarction, or non-fatal stroke occurred in 12.1% of patients receiving clopidogrel, but only in 9.9% of patients receiving prasugrel (< 0.001). Moreover, prasugrel therapy was associated with significantly lower rates of cerebral ischemic events, but with an increased risk of major bleeding, including bleeding with fatal outcome. However, total mortality did not differ significantly between treated groups [53]. In a subset of diabetic patients, the overall benefit of this drug was more significant, with a better efficacy and yet with a lower rate of major bleeding [54]. Several other new P2Y12 inhibitors are currently at different stages of clinical development, including those which reversibly inhibit the P2Y12 receptor, either by oral (Ticagrelor) or intravenous (Cangrelor) administration [55, 56].

The larger studies will be needed to definitively rule out a genetic effect of the loss-of-function alleles in population to better define responsiveness to these antiplatelet drugs.

What to do next-practical recommendations

If a patient presents with a new vascular event like TIA, stroke, or myocardial infarction in spite of antiplatelet therapy, we suggest to do the following:

  1. Assess whether patient was compliant, and if not, consider to continue original antiplatelet therapy.
  2. Assess possible drug interactions, for example, aspirin and ibuprofen, and if yes, try to avoid drug interactions.

But if patient is compliant and there is no evidence of possible drug interactions, the question what to do next remains. If the patient is only on the aspirin antiplatelet therapy, increasing the dose of aspirin is probably not a best solution, due to higher risk of bleeds and no guarantee for more protection against ischemic events, because large aspirin doses have not been associated with proportionally greater benefit [57]. Other solution could be switching to another antiplatelet drug, for example from aspirin to clopidogrel or aspirin plus extended-release dipyridamole, but unfortunately, there have been no clinical trials to indicate that switching antiplatelet agents reduces the risk of subsequent events [58]. There are ongoing trials trying to demonstrate that switching of the original antiplatelet therapy to an alternative strategy in patients who had a recurrent ischemic event despite the original antiplatelet therapy will reduce the risk of new vascular events (e.g., SWITCH study: Secondary prevention WITh a CHange in antiplatelet regime). Until results from these trials become available, the physician must take a notice on the individual patient's risk factors and tolerance.

Conclusion

The concept of personalized medicine for secondary stroke prevention depends primarily on the genes and drug effects. There is no doubt that in near future, a huge progress in pharmacogenetics and pharmacogenomics will be made in this field.

The goal of personalized approach in secondary stroke prevention is to take the most appropriate medication in a right dose for the right patient.

Acknowledgements

This work was partly granted by the Ministry of Education, Science and Technological Development of the Republic of Serbia (Project No. 175022).

Conflict of interest

Authors have no conflict of interest to declare.

Ancillary