Clopidogrel Resistance in Ischemic Stroke Patients

Abstract

Stroke remained the second leading cause of death globally in 2019. The antiplatelet drug clopidogrel is used to keep blood clots from forming in people who have experienced a stroke. Although most people find clopidogrel to be safe and beneficial, there is inevitably a range in how each patient responds. The review covers about Clopidogrel resistance in stroke patients, their risk factors and the methods to identify it. Clopidogrel resistance is characterized as the drug’s inability to prevent the target enzyme from acting. The prognosis of patients with ischemic stroke and the responsiveness to clopidogrel are significantly impacted by the various genetic polymorphism CYP2C19 genotypes. The two primary mutant alleles, CYP2C19 *2 and CYP2C19 *3, have been found to be the most prevalent genotypes. Better mRS scores six months after treatment showed a higher response rate in patients without these CYP2C19 variant alleles. Other factors are drug-drug interaction (proton pump inhibitors), demographics (age, sex, social history), comorbid conditions, etc. Blood samples for testing platelet reactivity were drawn one month after discharge from a peripheral blood sample. Several methods are used to identify the clopidogrel resistance. Some of them are ADP-Induced platelet aggregation, Platelet Reactivity Index VASP, Verify Now Assay, TEG Analyzer, Plasma microRNA-223. Drugs that are not prodrugs and whose metabolism is not dependent upon CYP2C19 can be selected as a superior alternative in case of CR. Ticagrelor is one such effective substitute. Proton pump inhibitors and clopidogrel should only be used concurrently in patients with reliable clinical indications.

Introduction

Stroke is described as “rapidly developing clinical signs of focal (or global) disturbance of brain function, that lasts more than 24 hours or results in death, with no clear cause other than that of vascular origin” by the World Health Organization. Stroke is primarily caused by either a blood vessel rupture resulting in hemorrhage or a blockage of blood flow resulting in ischemia in the surrounding tissue. Clinically, it is classified into two subtypes based on the mechanism: hemorrhagic and ischemic. Approximately 15%–20% of stroke cases are hemorrhagic, whereas 80%–85% are ischemic.[1] Stroke remained the second leading cause of death globally in 2019. In addition, the majority of all new strokes (62.4% of all stroke events in 2019) continued to be caused by Ischemic Stroke (IS).[2] The antiplatelet drug clopidogrel is used to keep blood clots from forming in people who have experienced a heart attack, stroke, or other symptoms. Before bioconversion, clopidogrel is a prodrug without antiplatelet action.[3] Clopidogrel is an irreversible P2Y12 receptor antagonist prescribed for patients with recent stroke, myocardial infarction, or established peripheral artery disease, who are treated for arteriosclerotic episodes. Although most people find clopidogrel to be safe and beneficial, there is inevitably a range in how each patient responds. The US Food and Drug Administration’s black box warning for clopidogrel states that CYP2C19 genotyping now enables clinicians to customize a patient’s treatment plan. The prognosis of patients with ischemic stroke and the responsiveness to clopidogrel are significantly impacted by the various CYP2C19 genotypes.[4] The present review was conducted to identify the reasons for clopidogrel resistance (CR) among stroke patients and to assess various methods to evaluate CR.

Mechanism of Clopidogrel

Clopidogrel is a second-generation thienopyridine.[5] Clopidogrel, a prodrug which is an adenosine diphosphate (ADP) receptor antagonist, must be oxidized to become an active metabolite by the liver enzyme cytochrome P450 (CYP450). Only a limited amount of clopidogrel is metabolically active.[6,7] An inactive carboxylic acid derivative is produced when esterases hydrolyze 85% of the clopidogrel that is absorbed from the stomach. The remaining 15% is broken down by the liver’s CYP450 enzyme into the intermediate metabolite 2-oxo-clopidogrel. A range of isoenzymes further oxidize this intermediate metabolite, resulting in the production of an extremely unstable active thiol derivative and an inactive carboxyl group. Clopidogrel’s active metabolite is known to exist in four isomers, denoted as H1–H4. It has been shown, nevertheless, that the therapeutically meaningful properties of the active clopidogrel metabolite are exclusive to the H4 isomer.[8]

Clopidogrel Resistance

CR is characterized by the drug’s inability to prevent the target enzyme from acting. By comparing ADP-induced platelet aggregation before and after treatment, residual posttreatment P2Y12 activity serves as a proof of CR.[9] CR is defined as either less than a 10% change in maximum platelet aggregation rate (MPAR) on day 5 or an MPAR (day 5) greater than 50% of the baseline magnitude (day 0).[4] Antiplatelet resistance is categorized as “clinical” or “laboratory.” The typical indicator of “laboratory” antiplatelet resistance is persistent platelet activity despite platelet inhibitory treatment. Treatment failure is referred to as “clinical” antiplatelet resistance. Poor patient compliance, insufficient dosage, medication interactions, and higher platelet turnover can all contribute to clinical drug resistance.[10] High on-treatment platelet reactivity (HTPR) is characterized as high CR or responsiveness. Because the etiology of HTPR is the same as the cause of inadequate response and resistance, HTPR may represent the pharmacodynamics of clopidogrel on platelets.[11] CR represents a significant clinical challenge in the management of stroke, posing risks of treatment failure and recurrent ischemic events.

Clinical Insights and Applications

Patients who encounter recurrent stroke episodes or stent thrombosis while undergoing clopidogrel therapy, as well as those who do not attain the desired level of platelet inhibition from the medication may be at risk for developing CR. People who have specific traits or health issues may be more susceptible to developing CR. This includes those who are elderly, have diabetes, have genetic variations that impact clopidogrel metabolism, and those taking drugs that interact with clopidogrel concurrently (such as proton pump inhibitors [PPIs]). Determining CR in these situations can help to guide treatment choices, improve patient outcomes, personalize medicine, reduce adverse events, and support current research initiatives in this area.

Factors Causing CR

Several risk factors were considerably greater in the CR group compared to the clopidogrel-sensitive (CS) group, including hypertension, diabetes, utilization of Calcium Channel blocking (CCB) agents, and PPIs, as well as creatinine, homocysteine (HCY), fasting blood glucose, hs-CRP, and triglyceride levels.[12]

A) Genetic polymorphism

The most significant factor affecting the pharmacodynamic and pharmacokinetic responses to clopidogrel is the CYP2C19 genotype. The two primary mutant alleles, CYP2C19*3 and CYP2C19 *2, were found to be the most prevalent genotypes in Asian cultures. Allelic variations of CYP2C19*2 or CYP2C19*3 increase the risk of CR. The risk of bleeding is elevated by the CYP2C19*17 allele.[13] Gene frequencies of alleles *2 and *3 were considerably greater in the CR group compared to the CS group, and high HCY levels were strongly linked to CR. High HCY levels, mutant heterozygous genotypes, and mutant homozygous genotypes were all substantially linked to CR.[2]

Four distinct CYP2C19 activity phenotypes can be produced by combining two wild-type, Loss of Function (LOF), and/or Gain of Function (GOF) alleles. They are as follows:

1. Carrier of alleles with two LOF CYP2C19 or a wild-type allele combined with the LOF CYP2C19 gene. These people respond less well to clopidogrel’s antiplatelet effects.

2. Reduced to normal metabolizers are people who have one copy of CYP2C19, that is, LOF, and one copy, that is, GOF. These people respond to clopidogrel with a reduced to normal antiplatelet response.

3. People who carry the wild-type gene in two copies are referred to as normal metabolizers. These patients respond to clopidogrel’s antiplatelet effects normally.

4. The enhanced metabolizer phenotype is linked to carriers of two GOF copies or one wild-type allele and one GOF CYP2C19 allele. These people respond to clopidogrel with a normal to elevated antiplatelet response.[14]

The clinical outcomes were worse when the P2Y12 and CYP2C19 gene polymorphisms were combined than when either the P2Y12 or the CYP2C19 gene polymorphism was present alone. Genetic variants are crucial for clopidogrel efficiency, as evidenced by the rise in polymorphism numbers and the corresponding rise in medication resistance. Recurring stroke episodes may be brought on by CYP2C19 and P2Y12 gene polymorphisms and connected to an inadequate response to Ischemic Cardiovascular Disease (ICVD) treatment.[15] The NR1I2 and CYP2C19 single-nucleotide polymorphisms have been linked to CR and long-term clinical outcomes in patients with acute IS. Poor outcomes were more frequent in persons with the CC genotype of NR1I2 rs 13059232 than in subjects carrying the T allele. Clinical results were significantly better for patients with the CC genotype who frequently took aspirin compared to those who remained on clopidogrel.[16] Better Modified Rankin Score (mRS) scores 6 months after treatment showed a higher response rate in patients without these CYP2C19 variant alleles.[17]

B) Proton pump inhibitors

PPIs are commonly used together with clopidogrel, an antiplatelet medication intended to prevent cerebrovascular illnesses. In general, taking PPIs and clopidogrel together results in negative medication interactions. The liver’s CYP450 is used in the metabolic process of both clopidogrel and PPIs. Due to their interactions with one another’s metabolism, taking these medications at the same time may reduce their antithrombotic effects.[18] Esomeprazole and omeprazole, two PPIs that primarily utilize CYP2C19 and CYP3A4 as their major metabolites and may lessen clopidogrel’s effectiveness, should not be used together.[19] Ilaprazole is a newer PPI, and it is well known that it has little interaction with CYP450. Ilaprazole is partially metabolized by the CYP450 family members CYP3A4 and CYP2C19. Ilaprazole is verified to be metabolized via a nonenzymatic process, while the precise mechanism is still unknown. So, it is assumed that the action and metabolism of clopidogrel are unaffected by the combination therapy of ilaprazole and clopidogrel.[20]

C) Demographics

Bivariate studies revealed that several risk factors, including female gender and height, are linked to CR. They also revealed that women are more likely than males to develop this resistance.[11] The percentage of clopidogrel that is converted into its active form is reduced in elderly patients with diminished CYP450 function, which affects the efficacy of clopidogrel. As a result, the average age is higher in CR patients. Alcohol inhibits Carboxylesterase 1 (CES1), which can have an impact on how other medications work by interfering with CES1’s function in the liver. Rather than being hydrolyzed, clopidogrel is transesterified into ethyl clopidogrel in the presence of alcohol. However, CES1 is not metabolized when alcohol consumption rises, and both hydrolysis and transesterification metabolism slow down. Because alcohol consumption increased the amount of clopidogrel in its parent form and increased the quantity of clopidogrel that converted into the active form in comparison to the alcohol-free condition, the CR-negative group consumed more alcohol than the control group.[20] Smoking, which increases P450 system activity and is one of the primary risk factors for ischemic stroke, also improves clopidogrel’s effectiveness. Patients taking Dual antiplatelet therapy (DAPT) with aspirin and clopidogrel had a decreased stroke occurrence than nonsmoking patients. Current smokers had reduced incidence of High on-treatment Platelet Reactivity (HPR) and CR.[21] It has also been proposed that the link between antiplatelet resistance and hypertension is caused by hypertensive individuals’ increased arterial tone, endothelial dysfunction, and shear stress. It has been noted that nifedipine, verapamil, and diltiazem all have a platelet-inhibiting action. Researchers have discovered that CR and hypertension are related. Recurrent strokes are at risk due to hypertension. The non-responsiveness to clopidogrel in patients with hypertension could be explained by the increased aggregability and adhesivity of platelets.[2] According to a different study, high blood pressure raises the shear stress in blood vessels, which could reactivate platelets and lead to CR.[22]

Resistance to clopidogrel is the result of a confluence of pharmacokinetic, clinical, and hereditary variables. It is well known that genetic variations in the CYP2C19 gene, specifically loss-of-function variants, contribute to decreased clopidogrel efficacy. Impaired clopidogrel response has also been linked to clinical variables such as diabetes mellitus and advanced age. Clopidogrel metabolism and effectiveness are further impacted by pharmacokinetic interactions, particularly those with PPIs and certain statins. Comprehending these variables is crucial in discerning individuals who exhibit an increased susceptibility to CR and in formulating tailored treatment approaches to maximize antiplatelet therapy effectiveness and enhance patient results [Table 1].

Table 1.

Indicators to suspect clopidogrel resistance

Indicators	Reason
Genetic factors	Presence of certain genetic polymorphisms, particularly variations in the CYP2C19 gene associated with reduced clopidogrel metabolism
Recurrent cardiovascular events	Patients experiencing recurrent cardiovascular events, such as stroke, despite being on clopidogrel therapy may raise suspicion of clopidogrel resistance
Clinical characteristics	Patients with clinical factors known to influence clopidogrel metabolism or response, such as diabetes mellitus, advanced age, or concomitant use of medications that interfere with clopidogrel metabolism (e.g., proton pump inhibitors)
History of non-response	Individuals with a history of poor response to clopidogrel, evidenced by previous cardiovascular events despite therapy or suboptimal platelet inhibition on laboratory testing
High-risk procedures	Patients undergoing high-risk procedures such as PCI or CABG may be at increased risk of thrombotic events if they exhibit clopidogrel resistance

CABG: Coronary artery bypass grafting, PCI: Percutaneous coronary intervention

Methods to Identify CR

ADP-induced platelet aggregation

Clopidogrel’s inhibitory effects were limited to ADP, while proaggregatory stimuli such as collagen, thrombin, or the thromboxane A2 agonist U46619 did not reduce platelet aggregation. Furthermore, clopidogrel’s selectivity for ADP-induced platelet activation was confirmed by means of the ADP-degrading enzyme apyrase. If clopidogrel has no apparent effect on ADP’s ability to reverse prostaglandin E1 (PGE1)-stimulated cyclic AMP, it inhibits ADP-induced platelet aggregation, which is time dependent.[23]

To determine platelet reactivity, collect the blood samples 1 month following discharge and centrifuge the mixture of blood–citrate for 5 min at 120 g while platelet-rich plasma (PRP) is produced. Store it at room temperature for use within 1 h. After determining the platelet count in PRP, it is adjusted to 2.5 × 108 mL^-1 using homologous platelet-poor plasma (PPP). ADP (10 mol/L) is used to excite platelets, and aggregation is measured by a PAP4 aggregometer. By using PPP as a standard, the aggregate is described as a variation of light transmittance percentage from the baseline. The maximum intensity of platelet aggregation in response to ADP has a coefficient of variation of 6.5%. ADP-induced platelet aggregation (ADP-Ag) >70% is used to identify nonresponse to clopidogrel.

Platelet reactivity index vasodilator-stimulated phosphoprotein (PRI VASP), a standardized flow cytometric method, is utilized to identify the VASP phosphorylation state of whole blood. Based on the equation PRI VASP = [MFI (PGE1) MFI (PGE1 + ADP)/MFI PGE1] 100, a platelet reactivity index (PRI VASP) is computed using the median fluorescence intensity (MFI) of samples incubated with PGE1 or PGE1 plus ADP. The nonresponse to clopidogrel is defined by PRI VASP >50%.[24]

Thromboelastography analyzer

To examine a patient’s platelet aggregation, a thromboelastography (TEG) analyzer is employed. This method is dynamic, quick, and accurate, and it may be used in real time. As a result, it can assess several altering processes, such as platelet aggregation, fibrinolytic activity, and blood coagulation.[13]

Point-of-care VerifyNow assay

VerifyNow is an easy and rapid way to determine CR, which interferes with effective antithrombotic actions.[20] VerifyNow P2Y12 is made to test a drug’s direct impact on the P2Y12 receptor. PGE1 and ADP are both used in the VerifyNow-P2Y12 assay to raise intraplatelet cyclic adenosine monophosphate. The P2Y12 Reaction Units (PRU) and percent inhibition of P2Y12 by thrombin receptor activating peptide (% inhibition P2Y12) from baseline activation are the units used to express the results of the VerifyNow-P2Y12 assay. This agglutination is measured by the VerifyNow analyzer as an increase in light transmittance. The nonresponse to clopidogrel is defined with a PRU >240 AU.[24] In comparison to TEG, this test is quicker and does not require a lab setting. The amount of blood needed is less than the amount needed for TEG.[25]

Plasma microRNA-223

Inflammatory markers such as C- Reactive Protein (CRP) monomers and platelet-derived microvesicles, as well as the expression level of microRNA-223, may serve as indicators of the efficiency and safety of antiplatelet medications. An increase in the risk of clopidogrel HTPR is shown by a drop in plasma miR-223 expression levels both before and after treatment in acute patients. Patients with Acute Ischemic Stroke (AIS) may benefit from the use of combined plasma miR-223 and CYP2C19 gene polymorphism detection to predict the development of clopidogrel HTPR.[26]

Measuring CR involves employing a range of laboratory assays and clinical assessments to evaluate platelet function, pharmacodynamic response, and genetic factors. Platelet function assays, such as VerifyNow-P2Y12 assay and light transmission aggregometry, directly measure platelet reactivity and response to clopidogrel. Clinical assessments, including TEG and aggregometry-based tests, offer additional perspectives on platelet function and antiplatelet drug efficacy. Combining these methods allows for comprehensive evaluation of CR, facilitating risk stratification and guiding personalized treatment approaches in clinical practice.

Conclusion

Clopidogrel is a commonly used antiplatelet drug that is given as monotherapy or in combination with aspirin for stroke prevention. Some patients presumably develop secondary stroke due to CR. CR is caused due to various factors such as genetic factors, drug–drug interaction, demographics (age, gender, social history), and comorbid conditions. The significant interindividual variation in clopidogrel’s antiplatelet action is related to both variations in the drug’s absorption and variations in the genes that encode for the hepatic cytochrome CYP2C19 enzyme, specifically CYP2C19*2 and CYP2C19*3 polymorphisms. Females have a higher chance to be resistant to clopidogrel than males. Several methods are used to identify CR. Some of them are ADP-Ag, VerifyNow assay, TEG analyzer, PRI VASP, and plasma microRNA-223. Ilaprazole can be given in combination with clopidogrel, as other PPIs cause CR if given in combination. Drugs that are not prodrugs and whose metabolism is not dependent upon CYP2C19 can be selected as a superior alternative in case of CR. One such effective substitute is ticagrelor. PPIs and clopidogrel should be used together only in patients who have clear clinical indications.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.