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Introduction

The primary prevention strategy for acute coronary syndromes (ACS) after percutaneous coronary intervention (PCI) is mainly based on dual antiplatelet therapy (DAPT), whose standard regimen consists of aspirin in combination with a P2Y12 receptor inhibitor (eg, clopidogrel, prasugrel, or ticagrelor). As a widely used antiplatelet drug, clopidogrel can be used to treat coronary heart disease and stroke by inhibiting adenosine diphosphate (ADP) receptors to reduce platelet activation and aggregation. However, poor control of platelet activity, known as clopidogrel resistance (CR), may occur in some patients after clopidogrel therapy,¹ which may lead to in-stent thrombosis and myocardial infarction. In addition, the most common adverse effect caused by clopidogrel is the risk of bleeding, especially gastrointestinal bleeding. One study found that among patients who underwent PCI and drug-eluting stents (DES) and completed 12 months of DAPT, complications such as gastrointestinal bleeding and cerebral hemorrhage could be induced in both the aspirin and clopidogrel groups (incidence: 1.5% in the aspirin group vs 1.7% in the clopidogrel group, P=0.160), but the clopidogrel group was superior to the aspirin group in terms of secondary prevention.² Other studies have shown that P2Y12 receptor inhibitors, specifically clopidogrel, were superior in reducing the incidence of myocardial infarction (RR=0.77, 95% CI: 0.67–0.89) and hemorrhagic stroke (RR=0.53, 95% CI: 0.30–0.92) and did not significantly increase the risk of major bleeding (RR=0.96), as compared with aspirin alone (RR=0.96, 95% CI: 0.71–1.30).³ Notably, in ACS patients treated with PCI, ticagrelor and prasugrel were associated with a high risk of bleeding compared to clopidogrel, with prasugrel exhibiting a higher risk of short-term bleeding at 90 days (HR=1.66, 95% CI: 1.11–2.48).⁴ In recent years, several studies have found that with clopidogrel, patients may also suffer from rare adverse effects such as severe fatigue and bleeding from small bowel ulcers. In clinical individualised treatment, drug efficacy can be assessed by means of genetic polymorphism testing, platelet function testing and plasma concentration monitoring, so that dosage can be adjusted to achieve better therapeutic effects and reduce the occurrence of adverse reactions.^5–7

Although new antiplatelet agents are emerging, clopidogrel remains important in the treatment of cardiovascular disease (CVD). Current clinical practice guidelines (2023 ACC/AHA and 2023 ESC) point to clopidogrel as an alternative choice for patients at high bleeding risk and actively recommend genetic testing to guide clopidogrel use. As the precision medicine paradigm advances, dynamic dose adjustment based on pharmacogenomics may further reshape its clinical status, which provides an important direction for future research.

Pharmacokinetics and Pharmacological Effects of Clopidogrel

Clopidogrel, with the molecular formula C₁₆H₁₆CINO₂S, acts as an ADP receptor inhibitor and is an inhibitor of platelet aggregation, platelet inhibition results from irreversible binding of the P2Y12 receptor. Clopidogrel inhibits platelet aggregation by selectively inhibiting ADP binding to platelet receptors and inhibiting ADP-mediated activation of the glycoprotein IIb/IIIa (GPIIb/IIIa) complex.

Clopidogrel, a thienopyridine prodrug, is inactive in vitro and must be biotransformed in vivo to be effective, with a number of different genetically encoded metabolising enzymes involved in the multi-step biotransformation of clopidogrel.⁸ Clopidogrel is mainly metabolised by the liver after ingestion by two metabolic pathways, one is hydrolysed to inactive carboxylic acid derivatives via an esterase-dependent pathway (CES 1). The other is through the hepatic cytochrome P450-dependent pathway (CYP2C19: 44.9%, CYP2B6: 19.4%, CYP1A2: 35.8%), which first converts clopidogrel to the 2-oxo-clopidogrel intermediate, and then to clopidogrel-AM through CYP2C9, CYP2B6, CYP2C19 and CYP 3A 4/5 which ultimately acts systemically⁹ (Figure 1 and Table 1).

Table 1 Summary of Pharmacokinetics of Clopidogrel

Figure 1 Mechanism of action of clopidogrel in humans. Clopidogrel, as a precursor drug, needs to be metabolized by the liver before it can exert its antiplatelet effect. There are two metabolic pathways, one of which is hydrolyzed to inactive carboxylic acid derivatives by an esterase-dependent pathway (CES 1). The other is bioactivation via the hepatic cytochrome P450-dependent pathway (mainly catalyzed by CYP2C19, CYP2C9, and CYP3A4 enzymes) to produce the active metabolite clopidogrel-AM. This figure summarizes the key enzymatic steps and metabolic transformations that underlie the pharmacological activity of clopidogrel.

Clinical Use of Clopidogrel in Related Diseases

Coronary Artery Disease

Coronary artery disease (CAD) is defined as a condition with one or more of the following: a history of myocardial infarction, percutaneous coronary intervention (PCI) or coronary artery bypass grafting (CABG), coronary artery stenosis of ≥50%, and chest pain accompanied by myocardial ischaemia.¹⁰ CAD includes acute coronary syndrome (ACS) and stable coronary heart disease. According to the 2023 ESC Guidelines for the management of acute coronary syndromes, DAPT needs to be initiated as early as possible after ACS, and clopidogrel in combination with aspirin reduces the risk of early ischemic events. The guidelines emphasize that in the acute phase of ACS, ticagrelor or prasugrel are preferentially recommended for their more potent antiplatelet effects, but clopidogrel remains the logical choice for patients at high risk of bleeding or with contraindications, such as advanced age, prior history of bleeding, and inability to tolerate ticagrelor (eg, dyspnea) or prasugrel (eg, prior history of stroke). In patients requiring long-term anticoagulation after atrial fibrillation or mechanical valve replacement, clopidogrel in combination with an anticoagulant (eg, rivaroxaban) is associated with a lower risk of bleeding than ticagrelor/ prasugrel (Class I recommendation, Level of Evidence A).¹¹ In 2020, the safety and efficacy of clopidogrel, ticagrelor, and prasugrel were compared in patients with st-segment elevation myocardial infarction. All-cause mortality and ischaemic event rates were reduced with ticagrelor and prasugrel compared to clopidogrel. However, since clopidogrel is cheaper, it will still dominate in the clinic.^12,13 In 2023, Kim, MC et al¹⁴ found that in the treatment of patients with acute myocardial infarction with a high risk of bleeding, clopidogrel, as compared to ticagrelor, would reduce adverse clinical outcomes, such as bleeding, and was safer to use. In 2024, Kim, SH et al¹⁵ found that patients with acute myocardial infarction often experienced dyspnoea with the use of ticagrelor, and that switching to clopidogrel in patients with such symptoms resulted in an improvement in dyspnoea without increasing the risk of ischaemic events.In 2024, Li et al¹⁶ found that in dual-risk ACS patients who completed 9 to 12 months of DAPT after drug-eluting stent implantation (DAPT) and were free of adverse events for at least 6 months prior to randomization to a subgroup, a 9-month extension of the clopidogrel monotherapy regimen was superior to continuation of clopidogrel DAPT in reducing clinically relevant bleeding without increasing ischemic events.

In 2021, clopidogrel was found to reduce morbidity and mortality in stable coronary heart disease and has been widely used in such patients.¹⁷ According to the 2024 ESC Guidelines and the 2023 AHA/ACC/ACCP/ASPC/NLA/PCNA guidelines for the management of chronic coronary syndromes, clopidogrel monotherapy (75 mg/d) is the standard alternative (Class I recommendation) to aspirin in chronic coronary disease (CCS) for those who are not aspirin tolerant patients (Class I recommendation), and there is insufficient evidence to support the superiority of novel P2Y12 inhibitors over clopidogrel in long-term monotherapy, which is more suitable for long-term management because of its lower cost and lower bleeding risk. In patients at high bleeding risk for PCI, especially those with normal CYP2C19 genes, clopidogrel is the first choice for switching to monotherapy after a short period of dual resistance (1–3 months) (Class I recommendation).^18,19 Kang et al²⁰ found that clopidogrel monotherapy was superior to aspirin in reducing both thrombotic and bleeding risks in patients who were event-free 6–18 months after PCI and required long-term antithrombotic therapy, and that the advantages were independent of the patient’s risk of bleeding or the complexity of PCI, with a wide range of applicability and without the need to adjust treatment strategies.

Stroke

Stroke is defined as the sudden onset of a focal neurological deficit at a site consistent with the extent of the large cerebral arteries.¹² Currently, clopidogrel is increasingly used for secondary prevention of ischaemic stroke, and can be used as a reasonable monotherapy option in patients with non-embolic ischaemic stroke or as a 21-day dual antiplatelet therapy in combination with aspirin after minor ischaemic stroke or transient ischaemic attack (TIA) in patients at high risk of stroke recurrence.⁸ Carotid endarterectomy (CEA) improves carotid blood flow and reduces the risk of stroke. In 2023, a study found that patients with a history of symptomatic carotid artery disease were more likely to use clopidogrel at the time of surgery. From 2010 to 2017, a total of 1066 patients were treated with CEA. During the study period, clopidogrel use increased by 24.9% over these seven years, which equates to an annual increase of 11%, and clopidogrel was not associated with an increased risk of postoperative complications, including bleeding. These data suggest that clopidogrel should not be discontinued before CEA and should be considered part of the “optimal pharmacological treatment” for patients undergoing CEA.²¹

Central Retinal Vein Occlusion

Retinal vascular occlusions are the second most common vascular disorder in the retina after diabetic retinopathy.²² There are two major forms of retinal vein occlusion: branch retinal vein occlusion (BRVO) and central retinal vein occlusion (CRVO). Based on fundus fluorescein angiography (FFA), CRVO can also be classified into ischemic or nonischemic.²³ Al Ghaithi et al reported a case of a 54-year-old male with nonischemic CRVO and multiple systemic comorbidities, including diabetes, hypertension, and dyslipidemia. After initial treatment with aspirin failed, switching to clopidogrel demonstrated significant efficacy in improving visual acuity, resolving macular edema, and alleviating retinal vascular pathology, suggesting that clopidogrel may be superior to aspirin in select CRVO patients and providing strong evidence for its potential new indications in CRVO management.²⁴

Adverse Effects of Clopidogrel in Clinical Practice

Gastrointestinal Bleeding

Clopidogrel inhibits platelet aggregation and increases the risk of bleeding, the most common is gastrointestinal bleeding. One study found 2,252 reports of clopidogrel adverse events through the US Food and Drug Administration Adverse Event Reporting System (FAERS) database, and clopidogrel leads to the highest rate of hospitalised gastrointestinal bleeding events.²⁵ Clopidogrel does not directly damage the gastric mucosa, but on the one hand inhibits platelet production of vascular endothelial growth factor, which in turn inhibits the proliferative capacity of gastric mucosal epithelial cells. On the other hand, it induces apoptosis of gastric mucosal cells, which reduces the expression of intercellular tight junction proteins, thus affecting the repair ability of gastric mucosa. Therefore, clopidogrel also has a damaging effect on the gastric mucosa to some extent, which increases with increasing dose.²⁶

Liver Injury

Clopidogrel-induced liver injury has been seen in the form of hepatocellular or mixed cholestatic/hepatocellular patterns. The most common pattern of liver injury is mixed, followed by hepatocellular damage and cholestatic injury. It has been shown that hepatotoxicity occurs 35 days after initiation of clopidogrel (range 3–180 days) and the clinical presentation is more inclined to a dose-dependent response.²⁷

Neutropenia

A PubMed search for “clopidogrel” and “neutropenia” revealed 17 cases since 2000. Clopidogrel administration resulted in a significant decrease in haemoglobin and white blood cells, leading to an increased risk of bleeding. An 84-year-old female patient with a previous asymptomatic acute myocardial infarction, who had coronary stenting, experienced a significant decrease in haemoglobin and white blood cells approximately 40 days after clopidogrel administration. Subsequent replacement administration of ticagrelor resulted in a rise in markers after the switch.^28,29

Thrombotic Microangiopathy

Immune thrombocytopenic purpura, and thrombotic thrombocytopenic purpura are all thrombotic microangiopathies, which may be induced by the use of clopidogrel. Lizondo López, T et al³⁰ reported an example of thrombotic microangiopathy in a patient who developed microangiopathic haemolytic anaemia and thrombocytopenia after one month of treatment with clopidogrel and aspirin. After extensive clinical and laboratory investigations, it was shown that his thrombotic microangiopathy was induced by clopidogrel. Both Grossman, K and Távora, C et al^31,32 reported cases of clopidogrel-induced immune thrombocytopenic purpura, which can be considered to be caused by clopidogrel in patients presenting with isolated thrombocytopenia. Ndulue, CN et al³³ reported a case of thrombotic thrombocytopenic purpura triggered by clopidogrel in a Nigerian patient with chronic kidney disease (CKD), which resolved quickly after discontinuation of clopidogrel.

Rare Adverse Reactions

In addition to the common adverse reactions described above, many rare adverse reactions to clopidogrel have been reported. Severe fatigue syndrome is a rare but clinically significant side effect of overreaction to clopidogrel in patients undergoing neurovascular endovascular intervention. In 2021, Bass et al³⁴ assessed patient response to clopidogrel using the VerifyNow assay, which is expressed in terms of P2Y12 reaction units (PRUs), with lower PRU values equating to a greater degree of inhibition of the P2Y12 receptor and a lower presumed probability of platelet aggregation, with overresponse to clopidogrel defined as PRUs ≤60. The diagnosis of clopidogrel-induced severe fatigue was made when symptoms appeared after clopidogrel treatment and resolved with dose reduction.

To date, seven cases of insulin autoimmune syndrome (IAS) induced by clopidogrel have been reported, and the sulfhydryl group of clopidogrel metabolites can induce IAS with hypoglycemia as the main symptom. Shi Chen et al³⁵ conducted a meta-analysis of six trials involving a total of 61,399 participants and found that clopidogrel-associated hypoglycemia may occur in Asian participants. Du, W et al³⁶ reported recurrent hypoglycemic episodes in a patient after 23 days of antiplatelet therapy with clopidogrel, suggesting that physicians should be vigilant for hypoglycemia-related symptoms in clopidogrel users.

Clopidogrel-induced small bowel ulcers and bleeding are uncommon, and in 2021 Lee, SH. et al³⁷ reported a case of bleeding small bowel ulcers from clopidogrel use in an 86-year-old male clopidogrel-resistant patient, which was found to be expected to increase in older patients with risk factors. Rowell’s syndrome, a combination of polymorphic lupus erythematosus and lupus erythematosus, was first reported in the literature in 2024 in a 52-year-old woman with sjogren’s syndrome who was taking paquinimod for two months but developed this symptom when she mistakenly took clopidogrel for one week.³⁸ Clopidogrel induced arthritis is a rare instance, Faiza Javed et al³⁹ reported the development of inflammatory arthritis in a male patient after 5 days of clopidogrel, and after diagnosis by exclusion it was determined that clopidogrel was responsible for the development of the induced arthritis.

Impact of Genetic Polymorphisms on Clinical Use of Clopidogrel

Several genes are involved in the biotransformation of clopidogrel to the active drug, and their polymorphisms may interfere with the biotransformation, leading to a decrease or increase in the amount of active metabolite, thereby affecting the drug’s efficacy.

CYP2C19 Gene Polymorphisms

Some patients develop residual high platelet reactivity (HPR) during antiplatelet therapy. HPR has been shown to be significantly associated with thrombotic events. One of the major causes of residual HPR after antiplatelet therapy is the CYP2C19 polymorphism, the most common genetic variant associated with clopidogrel resistance. Recent studies have shown that patients with one or more CYP2C19 loss-of-function alleles (LoF, *2 or *3) with low levels of active metabolites have an increased incidence of HPR, clopidogrel resistance and major adverse cardiovascular events (MACE).⁴⁰ Clopidogrel efficacy decreases with increasing number of CYP2C19 LoF alleles. Individuals carrying one CYP2C19 LoF allele are referred to as intermediate metabolizers (IM) and those carrying two CYP2C19 LoF alleles are referred to as poor metabolizers (PM).^41,42 Several studies have shown that increasing the dose of clopidogrel improves platelet inhibition and overcomes resistance to clopidogrel in patients with IM (*2) but not in patients with PM (*2).⁴³ According to the Clinical Pharmacogenetics Implementation Consortium (CPIC) guidelines for clopidogrel treatment based on the CYP2C19 genotype, replacement therapy is recommended for patients who are PM.⁴⁴ There are significant racial and regional differences in the distribution of CYP2C19 polymorphisms, and it has been estimated that approximately 15% of white and black populations carry at least one LoF allele (*2 or *3) compared to 29–35% in Asian populations.⁸

Mohitosh Biswas et al^45,46 compared the clinical efficacy of clopidogrel versus other P2Y12 receptor antagonist treatments after PCI in CAD patients who inherited the CYP2C19 LoF allele. The results showed that CAD patients who inherited the CYP2C19 LoF allele and were treated with clopidogrel had a significant 62% increased risk of MACE compared with patients treated with other P2Y12 receptor antagonists such as prasugrel or ticagrelor. In another article, he assessed the overall risk of recurrent stroke in stroke or TIA patients who carried the CYP2C19 LoF allele and were taking clopidogrel, and the risk of recurrent ischemic stroke was significantly increased in Asian patients with stroke or TIA compared with non-carriers.⁴⁷ It has also been shown that in patients with coronary artery disease or stroke, clopidogrel users who carry the CYP2C19 LoF allele have a significantly higher risk of MACE than clopidogrel-treated individuals alone who do not carry a defect in this gene.⁴⁸

Fu et al⁴⁹ found that the proportion of clopidogrel-resistant individuals among carriers of one or more CYP2C19 loss-of-function alleles was estimated to be 71.7%, which was significantly different from 32.1% among non-carriers. Similarly, Jia et al⁵⁰ demonstrated a higher incidence of platelet hyperreactivity during clopidogrel treatment of ischemic stroke in CYP2C19 *2 or *3 carriers than in non-carriers.

Liu et al⁵¹ demonstrated that the risk of clopidogrel resistance was higher in carriers of the CYP2C19*2 allele than in non-carriers, but did not increase the risk of vascular events or recurrence rates.

A gain-of-function (GoF) allele, CYP2C19*17, was first described in 2006.⁵² This variant allele increases gene transcription, thereby increasing enzyme activity and clopidogrel responsiveness. Individuals carrying one or both *17 alleles have been described as ultra-rapid metabolizers (UM) based on the genotype and phenotype of CYP2C19.^41,42 A meta-analysis by Li, Y et al⁵³ showed that carriers of the CYP2C19*17 allele had better clinical outcomes with clopidogrel and a lower risk of cardiovascular events, but carriers of CYP2C19*17 had a higher risk of bleeding. In 2010, the US Food and Drug Administration (FDA) recommended that individuals should be considered for alternative antiplatelet agents or higher doses of clopidogrel depending on their CYP2C19 genotype or poor metabolism genotype.⁵⁴

CYP3A4 Gene Polymorphisms

The CYP3A4 enzymes plays a role in the metabolism of the clopidogrel prodrug molecule and is primarily responsible for the bioconversion of approximately 40% of 2-oxoclopidogrel (the inactive metabolite) to the active metabolite cis-thiol-clopidogrel, which is responsible for P2Y12 receptor inhibition.^55,56 The expression of the CYP3A4 enzyme is derived from the CYP3A4 gene, and the CYP3A4*1G mutation is thought to be a protective factor against clopidogrel resistance in ischemic stroke patients, as this mutation increases the concentration of the active clopidogrel-related substance, thereby increasing platelet inhibition. Liu⁵¹ showed that the estimated risk of clopidogrel resistance was significantly lower in CYP3A4*1G carriers than in non-carriers.

ABCB1 Gene Polymorphisms

The p-glycoprotein (P-gp) encoded by the ABCB1 gene regulates the absorption of clopidogrel in the small intestine.⁵⁷ P-gp is a transmembrane protein whose main function is to pump drugs out of cells and into the circulation, and this pumping mechanism may affect the bioavailability of the drug. Mega et al⁵⁸ found that ABCB1 gene polymorphisms affect the degree of platelet inhibition, which is strongly correlated with the risk of MACE. Simon et al⁵⁹ reported an association between adverse events in patients taking clopidogrel and the ABCB1 (C3435T, rs1045642) polymorphism, with carriers of the GG and AA genotypes showing a significantly increased incidence of adverse events. ABCB1 polymorphisms have been found to influence ADP-induced platelet aggregation, and carriers of the G allele are more prone to exhibit hyporesponsiveness to antiplatelet therapy. The ABCB1 C3435T polymorphism could be considered as a potential genetic biomarker for the risk of MACE in CAD patients on clopidogrel after receiving PCI. One study confirmed that patients carrying the ABCB1 C3435T double mutation (TT) had a significantly increased risk of MACE compared with patients carrying the CC genotype or the CC+CT genotype.⁶⁰

However, there are studies that suggest otherwise, Mugosa, S et al⁶¹ conducted a study related to ABCB1 gene polymorphisms in a population in Montenegro, Europe, but found no significant correlation between ABCB1 gene polymorphisms and clopidogrel efficacy and safety.

CYP1A2 Gene Polymorphisms

A Korean study found an enhanced response to clopidogrel in smokers, which has been referred to as the “smoker’s paradox”. However, this phenomenon is not universal and was only observed in carriers of the cytochrome P450 CYP1A2 allele, suggesting a genotype-dependent effect of smoking on clopidogrel response.⁶² Cresci et al⁶³ recruited 2732 patients diagnosed with myocardial infarction and taking clopidogrel and evaluated the correlation between long-term clinical efficacy and safety of clopidogrel in patients carrying the CYP1A2*1c allele. The results showed that patients carrying the CYP1A2*1c allele were significantly more responsive to clopidogrel, but they had a worse prognosis and a significantly higher mortality rate due to major bleeding events.

PON1 Gene Polymorphisms

The paraoxonase 1 (PON1) gene may play an important role in clopidogrel resistance. The PON1 gene is involved in high-density lipoprotein (HDL) antioxidant processes such as platelet-activating factor acetylhydrolase and lecithin-cholesterol acyltransferase, which has the ability to hydrolyse oxidised low-density lipoprotein (LDL) cholesterol and cleave phospholipid peroxidation adducts, leading to potential prevention of atherosclerosis. Several studies have shown that reduced PON1 activity affects serum glucose, increases the risk of diabetes and reduces platelet inhibition.^64,65 PON1 is also involved in the esterification and subsequent inactivation of clopidogrel, which is more likely to lead to clopidogrel resistance.⁶⁶

It has been shown that single nucleotide polymorphisms in the PON1 gene are associated with lower clopidogrel responsiveness in patients with atherosclerosis, and that the PON1 Q192R polymorphism is associated with clopidogrel biotransformation. However, the above conclusions have been challenged by a large number of studies that have failed to replicate these results, possibly due to epigenetic changes.^67,68 Mohitosh Biswas et al⁶⁹ in assessing the overall risk of MACE associated with harboring the PON1 Q192R gene variant in patients taking clopidogrel, found that the PON1 Q192R gene polymorphism did not have a significant effect on the risk of MACE or bleeding events in patients treated with clopidogrel.

KDR Gene Polymorphism

The KDR gene is responsible for the transcription of vascular endothelial growth factor receptor 2 (VEGFR2), which plays an important role in cardiovascular disease and platelet aggregation. Al Awaida, W. et al⁷⁰ found that the KDR (rs1870377) gene correlates with CR in cardiovascular disease (CVD) patients admitted for percutaneous coronary intervention as a potential genetic biomarker.

Table 2 Clopidogrel-Related Gene Polymorphisms and Their Clinical Significance

Clopidogrel-related gene polymorphisms and their clinical significance are shown in Table 2. A panel of experts assembled by the American College of Cardiology Foundation (ACCF) and the American Heart Association (AHA) has suggested that the current methodologies for genotyping are still a matter of considerable debate.⁷¹ The main advantage of genotypic testing is that it does not require measurement after drug administration.⁷² Proponents argue that there are common genetic polymorphisms that have been shown to affect platelet response to clopidogrel and its clinical efficacy in randomized clinical trials and registration experience, and that genotypic assessment is justified. In contrast, opponents argue that the available evidence does not demonstrate that CYP2C19 carriers can improve efficacy by adjusting clopidogrel dosage or using alternative antiplatelet agents. Furthermore, in clinical practice, genotype-guided antiplatelet therapy selection strategies do not yield immediate results, which severely limits the usefulness of these data in the acute care setting.⁵⁰ In addition, further studies have recommended treating patients based on their clinical indications rather than genetic testing.⁷³ In conclusion, there are limited examples of pharmacogenomic testing requirements or recommendations in detailed clinical practice guidelines (CPGs), and the presence of a standardized method for assessing the evidence for clinical application of pharmacogenomic testing could increase the recommendation of pharmacogenomic testing in CPGs to some extent. Consistent recommendations for pharmacogenomic testing in CPGs may enhance the clinical utilization of testing, provide more effective treatments, and benefit society.⁷⁴

Monitoring Methods

Platelet Function Test

The Platelet Function Test (PFT) provides a rapid assessment of platelet function and an estimate of the degree of platelet inhibition. Thus, the test is applicable for assessing clopidogrel-treated patients at elevated thrombotic risk to gauge antiplatelet therapy efficacy and residual platelet activity, guiding optimal medication and dosing selection.¹³ Wadhwa et al⁷⁵ evaluated the economic viability of platelet function testing (PFT) in dual antiplatelet therapy (DAPT). They recommended PFT, followed by a switch from clopidogrel to prasugrel in combination with aspirin if resistance is identified, citing the reasonable cost of PFT for tailoring DAPT regimens to individual patients. The primary PFTs for clopidogrel include the VerifyNow assay, light transmission aggregometry (LTA), thromboelastography (TEG) platelet labelling system, rotational thromboelastography (ROTEM) and vasodilator-associated stimulated phosphoprotein (VASP) assay.

VerifyNow Assay

The VerifyNow assay is a whole blood point-of-care test that measures P2Y12 receptor activity by turbidimetric optical detection of platelet aggregation. The test is rapid bedside assay, completed in less than 5 minutes, which is an advantage over LTA and VASP phosphorylation assays. The VerifyNow assay also allows direct monitoring the effect from clopidogrel on P2Y12 receptors, helping determine the dosage of clopidogrel in patients due to undergo coronary artery stenting. In addition, the analysis is technically simple and the results are easy to interpret.⁷⁶ The results are expressed in PRUs and the effective therapeutic window for clopidogrel is 85 < PRU < 208.⁷⁷

Light Transmission Aggregometry (LTA)

LTA is considered gold standard in all PFTs. In LTA, the optical density of platelet aggregates in a sample is detected in the optical channel, whereas in the impedance aggregometry method, the change in resistance between two electrodes is measured. Although LTA has been included in many studies, it has been criticized for being time-consuming and lacking standardization and reproducibility.^78,79 The results of LTA are expressed as the maximum platelet aggregation rate. A study has shown that a maximum platelet aggregation rate greater than 40% is the optimal LTA platelet function threshold for clopidogrel when it is used to treat intracranial aneurysm shunts and prevent thromboembolic events.⁸⁰

Thromboelastography (TEG) Platelet Labelling System and Rotational Thromboelastometry (ROTEM)

Thromboelastography combined with platelet mapping (TEG-PM) produces more complex data than PRU. The output of this test is four real-time curves of clot development as a function of time, showing clot initiation, expansion, and lysis. The values extracted from these curves represent multiple aspects of the coagulation cascade, with enzyme-promoted clot formation, intensity of clot formation, and rate of clot breakdown all being part of the underlying TEG curve. Under these conditions, platelet aggregation and the resulting clot intensity are determined exclusively by ADP-dependent platelet aggregation, mediated by the P2Y12 ADP receptor.⁸¹ The rate of ADP-induced platelet inhibition was calculated from TEG to determine the incidence of CR and the factors influencing it.⁸² Using the TEG-PM assay, the following reference values were formulated under the effective therapeutic window for clopidogrel, with amplitude (MA) values of 31 mm-47 mm; arachidonic acid (AA) greater than 50% and ADP greater than 30%. Individualized antiplatelet therapy based on TEG-PM results reduces the risk of ischaemic events in patients with non-cardioembolic ischaemic stroke without increasing the risk of bleeding events and mortality.⁸³

Conventional TEG techniques can overproduce thrombin and therefore their lack of ability to determine ADP receptor inhibition is considered a major problem associated with conventional TEG. However, ROTEM is able to monitor clopidogrel without thrombin production and Schultz-Lebahn, A et al⁸⁴ used ROTEM to analyse platelet function.

Vasodilator-Associated Stimulated Phosphoprotein (VASP) Assay

The most pharmacologically specific assay for assessing clopidogrel therapy is VASP, which targets only the P2Y12 receptor.^79,85 The VASP assay is based on a physical principle: quantitative flow cytometry measures levels of phosphorylated and dephosphorylated VASP, which correlate with inhibition or activation of P2Y12. However, we must consider that VASP phosphorylation is regulated by a number of factors, which are impaired in certain comorbidities. In addition, VASP assays are time-consuming.^86,87 Aleil et al⁸⁸ implemented the first VASP assay in 2005 for the detection of clopidogrel-resistant patients with ischaemic cardiovascular events. The effective therapeutic window for clopidogrel is formulated with a reference platelet reactivity index (PRI) of 16–50 and requires the study of scatter plots to interpret the conditions.⁷⁶

Therapeutic Drug Monitoring

High-performance liquid chromatography (HPLC) is usually used for the identification and quantification of compounds, and the determination of drug levels in patients’ plasma using HPLC becomes difficult due to the low levels of prodrugs in plasma after clopidogrel ingestion and the instability of sulfhydryl derivatives. Therefore, liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been used to assay the blood of three patients with coronary artery disease who underwent stent implantation, which allowed the simultaneous determination of clopidogrel, 2-oxo-clopidogrel and clopidogrel thiol metabolites in human plasma.⁸⁹ In 2024, Li et al⁹⁰ studied 100 patients with ischemic cerebrovascular disease diagnosed by a neurologist as needing clopidogrel therapy, and all the patients were genotyped for CYP2C19, the plasma concentration and plasma clopidogrel clearance of different groups of patients before and after clopidogrel treatment were detected by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS), the platelet aggregation rate of patients with different genotypes was determined by turbidimetric assay, and CR and stent thrombosis were analyzed in all the groups after 3 months of treatment.

Clinically, the genotypes of the patients were tested before administration, and after the end of the administration, the results of the PFT test were used to reflect the clinical results together with the plasma concentration determined by HPLC-MS/MS to provide a basis for judgement of the clinical administration of clopidogrel.⁹¹

Discussion

The clinical application of clopidogrel, as a major drug in antiplatelet therapy, requires a balance between optimization of efficacy and risk control. In this paper, we synthesized the available evidence that genetic polymorphisms and individual differences in drug metabolism are key factors contributing to CR and adverse events.The CYP2C19 LOF allele has been identified as a major genetic marker affecting the production of active metabolites of clopidogrel, and gene-directed therapy significantly reduces the risk of in-stent thrombosis and myocardial infarction, especially in patients with CAD.⁸⁸ However, the association of PON1 Q192R polymorphism with CR remains controversial, and a recent Meta-analysis showed that it had no significant effect on the risk of MACE and bleeding,⁶⁵ suggesting that future studies need to focus on epigenetic regulation and combined multigene effects.

In terms of clinical monitoring strategies, the combination of PFT and blood drug concentration provides a double guarantee for individualized treatment.VerifyNow and VASP assays can dynamically assess the level of P2Y12 receptor inhibition, whereas HPLC-MS/MS technology provides a direct basis for dosage adjustment by quantifying the concentration of active metabolites of clopidogrel.^71,72,86,87 Notably, the lack of harmonized standards for PFT thresholds and the limitations of VASP assays to be interfered by comorbidities need to be carefully considered in clinical interpretation. In addition, the applicability of genotype-guided immediate testing (eg, bedside CYP2C19 rapid typing) for patients with acute PCI still needs to break through technical bottlenecks.⁵⁰

The gene-efficacy relationship shows heterogeneity across the disease spectrum: switching to ticagrelor in CYP2C19 LOF carriers in stroke patients reduces the risk of recurrence, whereas the genetic evidence for peripheral arterial disease (PAD) is not yet sufficient, suggesting that clinical decision-making needs to be combined with disease-specific evidence.⁸⁸ Notably, while novel P2Y12 inhibitors circumvent some of the metabolic defects, their bleeding risk (eg, prasugrel short-term bleeding HR=1.66)⁴ limits the advantages of their use in older or high-risk bleeding populations, highlighting the unique position of clopidogrel in balancing thrombotic/bleeding risk.

Conclusion

Recent studies have shown that platelet aggregation plays a key role in both the initiation and progression of thrombosis. Despite the emergence of new antiplatelet agents, clopidogrel remains the basic drug in current clinical practice due to its favorable safety profile, including a low risk of bleeding and high tolerability. However, the individual variability of this drug is of concern-gene polymorphisms leading to clopidogrel resistance can increase the risk of thrombotic events, suggesting the importance of precise dosing. In CAD, secondary prevention of ischemic stroke, gene-directed therapeutic strategies have shown significant benefits, with carriers of the CYP2C19 LOF allele switching to ticagrelor or prasugrel experiencing a lower incidence of major ischemic events (myocardial infarction, in-stent thrombosis, etc.) compared with conventional regimens, while non-carriers continue to use clopidogrel to not only achieve comparable thrombotic efficacy to that achieved with the newer P2Y12 inhibitors but also significantly reduce the risk of major hemorrhage and significantly reduced the risk of major bleeding. Of particular note, in areas with limited medical resources, targeted genetic testing may be more cost-effective than “empirically reinforced antithrombotic” regimens. Clinical assessment of clopidogrel efficacy is currently based on the determination of optimal thresholds for platelet inhibition by light turbidimetric assay (LTA), platelet function assays such as VerifyNow, and in vivo measurement of active metabolite concentrations by HPLC-MS/MS. However, the standardization of these methods still faces challenges, with a lack of uniform consensus on critical values for functional assays and effective concentration thresholds for pharmacokinetic monitoring still requiring large-scale cohort validation.

In summary, this review aims to help achieve individualized and precise drug administration, reduce or avoid the occurrence of clinical adverse events, reduce the severity of the corresponding symptoms, and ensure that patients’ treatment progresses smoothly, as well as to improve the quality of life and confidence of patients during the treatment period. Therefore, it has significant social and economic benefits.

Abbreviations

ADP, adenosine diphosphate; CR, clopidogrel resistance; GPIIb/IIIa, glycoprotein IIb/IIIa; CAD, Coronary artery disease; PCI, percutaneous coronary intervention; CABG, coronary artery bypass grafting; ACS, acute coronary syndrome; TIA, transient ischaemic attack; CEA, carotid endarterectomy; BRVO, branch retinal vein occlusion; CRVO, central retinal vein occlusion; FFA, fundus fluorescein angiography; FAERS, the US Food and Drug Administration Adverse Event Reporting System; CKD, chronic kidney disease; PRUs, P2Y12 reaction units; IAS, insulin autoimmune syndrome; HPR, high platelet reactivity; LOF, loss-of-function; GOF, gain-of-function; UM, ultra-rapid metabolizers; FDA, the US Food and Drug Administration; P-gp, p-glycoprotein; MACE, major adverse cardiovascular event; HDL, high-density lipoprotein; LDL, low-density lipoprotein; CVD, cardiovascular disease; CPGs, clinical practice guidelines; PFT, platelet function test; DAPT, dual antiplatelet therapy; LTA, light transmission aggregometry; TEG, thromboelastography; ROTEM, rotational thromboelastography; VASP, vasodilator-associated stimulated phosphoprotein; AA, arachidonic acid; HPLC, High-performance liquid chromatography; LC-MS/MS, liquid chromatography-tandem mass spectrometry; HPLC-MS/MS, high-performance liquid chromatography-tandem mass spectrometry; CYP2C19, cytochrome P450 Family 2 Subfamily C Member 19 gene; CYP3A4, cytochrome P450 Family 3 Subfamily A Member 4 gene; ABCB1, ATP Binding CassetteSubfamily B Member 1 gene; CYP1A2, cytochrome P450 Family 1 Subfamily A Member 2 gene; PON1, paraoxonase 1; KDR, Kinase Insert Domain Receptor.

Acknowledgments

This work was supported by Wu Jieping Medical Foundation (320.6750.2023-26-3).

Disclosure

The authors report no conflicts of interest in this work.

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