Pharmacokinetic, pharmacodynamic, and pharmacogenetic assays to monitor clopidogrel therapy

Abstract Clopidogrel is the most common and widely used antiplatelet agent for patients with coronary artery disease following confirmation by electrocardiographic studies. The nonresponsiveness of patients to clopidogrel and the possibility of testing for clopidogrel resistance by platelet function assays (PFA) are contentious issues. Light transmission aggregometry (LTA) is considered as the gold standard test among all PFA. In this review, the most commonly used PFA used for monitoring the effect of clopidogrel, LTA, vasodilator‐stimulated phosphoprotein assay phosphorylation, rotational thromboelastometry (ROTEM) delta and ROTEM platelet, thromboelastography, PFA‐100, VerifyNow P2Y12 assay, Multiplate analyzer, Plateletworks assay and pharmacogenetic studies, are comparatively discussed including their principles of action, advantages, and disadvantages. VerifyNow P2Y12 assay can be accepted as the ideal point of care test out of the discussed assays. However, modified assays are required which could overcome the limitations associated with currently available assays.


| INTRODUC TI ON
Clopidogrel is a second-generation thienopyridine drug which acts as an inhibitor of platelet aggregation and hence used as an effective medication for coronary artery disease (CAD) and percutaneous coronary intervention (PCI). 1,2 However, clopidogrel resistance among patients has become a significant concern. Hence, it has become important to decide whether the patient is clopidogrel resistant or not. 3 If resistant, then the optimum dose or changing/adding a different antiplatelet drug needs to be considered. In order to resolve this problem, platelet function tests (PFTs) play an important role in providing information for physicians regarding management of patients on clopidogrel therapy. This article reviews the role of platelet function assays (PFA) in monitoring clopidogrel therapy, together with the principles, applications, and limitations of the different tests discussed comparatively. Further, the future prospects of PFTs are also discussed.

| AC TI ON OF CLOPIDOG REL
Antiplatelet therapy is an essential pharmacological therapy given to patients with atherothrombotic disease to inhibit the platelet aggregation by blocking the platelet receptors involved in adhesion.

Genetic variations between patients and delayed onset of action of
clopidogrel result in the increase in resistance to clopidogrel therapy. Major reason for clopidogrel resistance was identified as the cytochrome P450 CYP2C19 polymorphism. 4,5 When clopidogrel is ingested, 85% of prodrug is absorbed by liver and converted to its carboxylic acid derivative by carboxylic esterase. This is known as CLPM (clopidogrel metabolite), which is a major inactive metabolic product circulating in the blood and helps to determine the pharmacokinetics of the prodrug. Fifteen percent will be converted to thiol metabolite of clopidogrel (CTM) by the hepatic cytochrome isoenzymes (CYP P450 1A2, CYP2B6, CYP2C9, CYP2C19, and CYP3A4). CTM involves in specific and irreversible blocking of P2Y12 receptors, inhibiting the adenosine diphosphate (ADP)-induced platelet aggregation. CTM consists of four isomers; H1-H4, where H3 (inactive form) and H4 (active circulating form) are mainly considered in monitoring the action of clopidogrel ( Figure 1). [6][7][8][9][10][11] Thiol will irreversibly bind to P2Y12-ADP receptor on platelets via a permanent disulfide bond with two cysteine residues (cys 17 and cys 270) on the receptor, thus inhibiting the ADP binding permanently and thereby activating adenylyl cyclase enzyme to increase cyclic adenosine monophosphate (cAMP). Thus, protein kinase level increases, stimulating phosphorylation of vasodilator-stimulated phosphoprotein (VASP). Hence, inhibiting the activation of receptor complex gp IIb/III. Consequently, no thrombosis will occur. Furthermore, clopidogrel reduces secretions from dense granules in platelets and reduces arachidonic acid, collagen, and thrombin-induced platelet activation. Clopidogrel also decreases the enzymatic activation of coagulation pathway, decreasing thrombin formation.
Clopidogrel action solely depends on dose and time. Usually, 400-600 mg of dose is given and will take 2-5 hours for the platelet inhibition with 400 mg dose. Seventy-five milligram of daily dose will take about 7 days for maximum platelet inhibition and management of atrial fibrillation. Half-life of clopidogrel active metabolite is approximately 6 hours. [12][13][14][15]

| CLOPIDOG REL RE S IS TAN CE
Although clopidogrel is widely used, it shows increased resistance/response variability among thrombosis patients as it is highly susceptible to drug interactions and CYP gene single nucleotide 15% Hepatic cytochrome isoenzymes polymorphisms, thus reducing the in vitro enzyme activity inhibiting/reducing the conversion of clopidogrel into its active thiol metabolites. Therefore, it is difficult to decide the relevant dose without performing a PFT. 16,17 Degree of clopidogrel resistance may vary based on the laboratory method used. 14 Prevalence of resistance with clopidogrel and aspirin has been studied by using different laboratory techniques. Expected prevalence of aspirin resistance by those studies was about 5.5% to 60%, while clopidogrel resistance was 16.8% to 21%. Hence, dual antiplatelet therapy or triple therapy along with another potent agent was required. 18 Clopidogrel resistance is mainly associated with CYP2C19*2 genotype loss-of-function allele. Genetic variations regarding conversion to active metabolite by CYP P450 enzymes also play a major role in resistance. 17,19,20 Conferring to the studies, clopidogrel resistance in population was about 4%-30%, and the variation was due to the use of different platelet function studies. 21 Further, when citrated blood is used, blood sample becomes highly nonphysiological and has reduced the accuracy of results obtained via thromboelastogram (TEG). 38 When citrated blood is used, the degree of platelet aggregation will not be changed up to 2 hours; however, the aggregation gradually decreases thereafter. Even small rise in quantity of sodium citrate will show significant inhibition of platelet aggregation along with ADP, and collagen as sodium citrate acts as the major agent associated with ADP-induced aggregation. At low plasma Ca 2+ (0.1 mmol/L), citrated blood fails to generate thrombin, thus reduces the growth and stability of thrombus. Generally, 0.25 ± 0.05 mmol/L free calcium is needed for thrombin generation.
Thereby, citrated blood seem to affect the reproducibility and sensitivity of the assay to monitor the ADP-induced platelet aggregation in patients under clopidogrel therapy. 39,40 In order to overcome the faults, hirudin anticoagulant was introduced, which is extracted from leeches. Hirudin can directly inhibit the thrombin without changing the Ca 2+ levels in the sample. Since thrombin is inhibited, coagulation of the blood sample was also inhibited as a result of blocking the conversion of fibrinogen to fibrin.
Further, hirudin can maintain the ADP levels in the samples for more than 4 hours. In addition, the poor platelet impedance observed in citrated blood due to the effect from collagen and ADP was resolved when using hirudin anticoagulant. Moreover, Multiplate assay requires tight platelet aggregation for a remarkable change in impedance, which could not be strongly detected by citrated samples. Hence, hirudin is highly recommended for use while lepirudin, phenylalanyl-L-prolyl-L-arginine chloromethyl ketone also can be used. [41][42][43] The assays ( Figure 2) that are available to monitor the effect of clopidogrel on platelet activity are discussed comparatively under this review.

Light transmission aggregometry
Light transmission aggregometry is considered as the gold standard test performed on either whole blood or platelet-rich plasma (PRP) and is used worldwide. The platelet function is measured by measur- aggregation (% MPA) and percentage inhibition of platelet aggregation (% IPA). When light passes through PRP, initially considered platelet aggregation is 0% (no aggregation), then the light transmission through the PRP is 0% (hence, 100% inhibition by clopidogrel). If aggregation initiates upon the addition of agonist, light transmission too increases.
At platelet poor plasma (PPP), it is considered that 100% light transmission for 100% platelet aggregation (hence, 0% inhibition by clopidogrel). It could be interpreted that the decreased light transmission indicates the better clopidogrel responders. In order to overcome the limitations of above conventional method, a new method was developed where PRP was incubated with prostaglandin E1 (PGE1). Once pretreated with PGE1, prior to addition of ADP, the effect of P2Y1 receptors will be completely inhibited. Common ADP agonist doses are 5, 10, or 20 µmol/L. LTA is affected by pre-analytical variables, such as anti-inflammatory drugs, food stuff like garlic, turmeric, and caffeine, and high fat diet, and should adjust the count within 200-400 × 109/L.

Vasodilator-stimulated phosphoprotein assay
Vasodilator-stimulated phosphoprotein is an actin regulatory protein and a substrate for both cAMP and cGMP (cyclic guanosine monophosphate)-dependent protein kinases and involves in the filopodia formation and adhesion of platelets. When P2Y12 receptors are blocked by clopidogrel and PGE1 is stimulated, activated adenylyl cyclase will initiate the VASP phosphorylation. Hence, VASP phosphorylation will indicate the P2Y12 inhibition by clopidogrel. 50,51 However, still the direct relationship between the VASP phosphorylation and ADP-induced in vivo platelet aggregation has not yet described. 52 Citrated anticoagulant blood samples are used along with the commercially available kit included with ADP and PGE1. Assay is based on the flow cytometric technique. Upon incubation of platelets with PGE1 and ADP, platelets will be reacted with CD61 phycoerythrin-labeled platelet-specific antibody and FITC-labeled phosphorylated VASP-specific mouse monoclonal antibody. The used antibody is specific for the phosphorylated form of VASP.
Likely, pretreated samples will be analyzed via flow cytometer which produces results as geometric mean fluorescence intensity (MFI).
The degree of phosphorylation of VASP will be directly proportional to the platelet inhibition by clopidogrel and is expressed as platelet The main advantage associated with this assay is that it is highly specific for the P2Y12 receptors. 44

| Point of care tests
Point of care tests (POCTs) are tests that can be performed on whole blood at or near the bedside of the patient, more easily, such that rapid results can be obtained without pipetting and pretreatments to the sample. Frequently, VerifyNow P2Y12 assay, Multiplate assay, PFA-100, Plateletworks, TEG, and ROTEM are designed as POCTs and used to monitor the effect of clopidogrel. POCTs are developed to overcome the limitations associated with LTA, such as labor intensiveness, cost, and time. POCTs are generally at high cost than normal LTA, but they have the ability to provide clot quality and monitors clot formation and progression even after the point of clot formation. 35,53,54

| Rotational thromboelastometry: ROTEM delta and ROTEM platelet
Blood collected within 4 hours into 3.2% sodium citrate is needed to be incubated at 37°C before examination. ROTEM delta uses viscoelastic measurement. Blood is filled into the cuvette and a cylindrical pin, known as the sensor, is immersed into the cuvette so that there is 1-mm gap between pin and cuvette wall, bridge by the blood. Sensor is rotated sideways by a spring. The pin rotates till the blood is in liquid form; however, when blood starts clotting, rotation of the pin slows down. Kinetic motion of the pin is detected mechanically and com- However, ROTEM delta lacks the ability to monitor antiplatelet drug therapy due to increased production of thrombin during the assay. Thrombin thus produced stimulates platelets, preventing the detection of platelet inhibition by antiplatelet drugs. In order to overcome the above limitation, ROTEM platelet was combined to ROTEM delta such that single sample can be analyzed simultaneously with two techniques. ROTEM platelet consists of two channels which allow whole blood analysis via impedance principle. Sample cuvette is inserted into temperature-controlled channel and then two wires of the electrodes are placed inside the cuvette. When the activated platelets aggregate around the surface of the electrode wires, impedance between wires increases producing an impedance curve. The magnetic stirrer inside the cuvette prevents the deposition of blood cells at the incubation. If the patient is effectively responding to clopidogrel therapy, the curve lies below the reference curve. Impedance curve provides three parameters, where area under curve (Ω × min) gives overall platelet aggregation, amplitude at 6 minutes (Ω/min) gives degree of platelet aggregation after activation, and maximum slope (Ω) indicates time taken for platelet aggregation. ROTEM platelet was specifically designed to monitor antiplatelet therapy by three assays. ARATEM assay monitors aspirin therapy by activator arachidonic acid, ADPTEM assay monitors clopidogrel using ADP, and TRAPTEM monitors thrombin by thrombin-receptor activating peptide-6. 56,60

| Thromboelastography
Thromboelastography is a rapid POCT performed on citrated whole blood. This technique quantitatively measures the viscoelastic properties of the platelets in forming the platelet plug. The principle will be same as that of ROTEM platelet, which has a disposable cup with a detection pin fixed at the center. Difference is that the TEG rotates the cup and ROTEM rotates the pin first when the clot forms. Prior to clotting, blood has the minimum viscosity. Therefore, the waves of the cup cannot induce the movement of the pin. When blood starts to coagulate, blood viscosity increases, and the clot gets attached to both the cup and pin causing the movement of the pin upon induced by the cup. When the viscosity increases gradually, amplitude of the pin movement also increases. Once the fibrinolysis starts, platelet clot dissolves decreasing the blood viscosity. Change of amplitude is expressed graphically against time. 61 Conventional TEG lacks the ability to determine the ADP receptor inhibition for the reason that the excess production of thrombin through the technique. Conventional TEG will show normal maximum amplitude for a patient under clopidogrel therapy who was shown to have an ultimate platelet inhibition via LTA, which was considered as a major problem associated with conventional TEG.
ROTEM expresses CT, α-angle, CFT, MCF, and clot lysis, whereas those parameters are expressed as reaction value (R value), α-angle, K-value, maximum amplitude, and amplitude at 30 minutes (LY30) in TEG. However, by overcoming the above problem, modified TEG has the capability to monitor the clopidogrel action, without thrombin generation. ADP is used as the agonist to measure the degree of platelet aggregation of patients under clopidogrel therapy. 58

| VerifyNow P2Y12 assay
VerifyNow assay is performed as a POCT on citrated whole blood as a turbidometry assay. When PGE1 is introduced, ADP induces platelet coagulation and aggregates with the help of fibrinogen-coated beads. Most important fact is that, the assay is sensitive and specifically targets the P2Y12 receptors while inhibiting the action of P2Y1.
As in LTA, the platelet aggregation is determined by the percentage of the light transmission and expressed in PRU (P2Y12 reaction units). Low PRU indicates the high P2Y12 receptor inhibition and better response to clopidogrel. Measurement of inhibition of P2Y12 receptor inhibition as a result of thrombin receptor-activating peptide-induced platelet aggregation has become an added advantage.

Percentage inhibition is reported as [Base PRU − Posttreatment
PRU]/[Baseline PRU] × 100. VerifyNow assay is a rapid test which can be performed even at bedside within 5 minutes, which has been an advantage when compared with LTA and VASP phosphorylation assays. VerifyNow assay also has the capability to monitor the effect of clopidogrel on P2Y12 receptors directly thereby helps to determine the adequacy of the loading dose of clopidogrel in patients who will be subjected to coronary stenting. Further, the assay has a simple technique and interpretation of results can be done easily. 51,62,63 Cut-off value for the suboptimal clopidogrel response in terms of percentage aggregation is ≥70% for 10µM ADP and ≥50% for 5µM ADP. 49

| Platelet function assay-100
Platelet function assay-100 is another point of care assay to monitor

| Multiplate analyzer
Multiplate analyzer is a sensitive, novel, point of care platelet function analyzer which can be performed on citrated whole blood using electrical impedance aggregometry principle. The main aim of the assay was to monitor the platelet function inhibitors. When the samples obtained from the patients who are under clopidogrel therapy are exposed to ADP agonist, platelets will get stimulated and results in ADP-induced platelet aggregation. Once the aggregated platelets attach tightly to the sensor wires in the Multiplate device, an electrical resistance will be created between the wires. ADP with 200-μmol/L concentration interacts with P2Y1 and P2Y12 receptors inducing irreversible aggregation. However, ADP + PGE1: 200 μmol/L + 300 nmol/L inhibits aggregation by P2Y1 receptor, thus increasing specificity for P2Y12 receptors. Change in the impedance can be shown by the graph; arbitrary aggregation units (AU) against time. In addition, the three parameters, area under the curve, height of the curve, and maximum slope, express the complete platelet reactivity, platelet aggregation, and velocity, respectively. 69,70

| Plateletworks analyzer
Plateletworks is a POCT device performed on whole blood.
Plateletworks analyzer measures the platelet count of the sample before the addition of the ADP agonist and then after platelet aggregation upon addition of ADP. Usually, reference platelet count taken from the K3-EDTA anticoagulant blood without ADP and other count is taken from citrated anticoagulant sample with ADP agonist. 71 In normal patients, the platelets get aggregated in the presence of agonist and the resultant platelet count is considered to be zero.
When the blood flows through the aperture, the constant electric current will develop an electrical pulse which is amplified and converted to obtain the platelet count. 72 When the platelet aggregates Minimum studies have been conducted to show the prediction of outcomes of cardiovascular diseases. 71,73,76 In the determination of the cut-off values for Plateletworks assay, it had shown 63% sensitivity and 58.5% specificity using ROC curve analysis. 74

| High-performance liquid chromatography
High-performance liquid chromatography is generally used for identification and quantification of chemical compounds. HPLC Around 5%-40% of patients who are under clopidogrel therapy may show resistance to clopidogrel and the cause may be due to impaired drug absorption by the hepatic cells or impaired drug metabolic activity. However, the cause can be clearly explained via analysis of drug metabolite levels in the patient's blood via HPLC method. However, due to low levels of the prodrug in plasma after ingestion and instability of thiol derivatives, it has become difficult to use HPLC method for determination of drug levels in patient plasma. 78 Hence, most of the studies were conducted using plasma of healthy volunteers and spiking the samples with prepared concentrations of clopidogrel. 79,80 Thereby, those studies were only able to determine either the prodrug only or one of the derivates from CTM or CLPM. One such study was able to analyze the pharmacokinetic properties of only CTM isomers, but not the isomers or prodrug or CLPM. They have analyzed the H1-H4 isomers using a reverse-phase ultra-high-performance liquid chromatography and tandem mass spectrometry (MS) with a concentration accuracy for a range of 0.5-250 ng/mL. 81 Once the HPLC technique is validated by the means of the parameters linearity, stability, precision, and accuracy, it was identified as the most sensitive, specific analysis method to monitor the effect of clopidogrel on patient responsiveness. 82,83 Two studies have been found to analyze the clopidogrel and its metabolites in patients with myocardial infarction and PCI. One study has determined the plasma levels of clopidogrel, inactive carboxyl metabolite, and active H4 thiol metabolite in the plasma of ST-elevated myocardial infarction patients. 11 The second study studied the plasma levels of clopidogrel, CLPM, active H4, and inactive H3 of patients under PCI. The study has found that the maximum absorption of 75 mg of drug was 2 ng/mL in 1.4 hours and 300 mg of drug was 4.5 ng/mL in 1.2 hours by intestines. Platelet aggregation with 75 mg dose was found to be between 37 and 747 AU/min. The significant correlation between maximum concentration of H4 isomer and platelet aggregation depicts the importance of use of both the above parameters in the detection of response to clopidogrel therapy. 78  Corporation can be performed on either whole blood or buccal swabs using extended CYP2C19 DNA mutation panel. 94 CYP2C19*2 genetic test produced by Spartan Biosciences is a rapid test which could be performed within 1 hour on buccal swab. 87 Clopidogrel antiplatelet effect was studied by using a rapid test called Rapid Infinity analyzer. DNA extracted from the whole blood collected in EDTA was hybridized to BioFilmChip microarray in the analyzer, hence can be used to determine CYP19*2, *4 and CYP2C9*2, *3 polymorphisms. 95 Rapid Verigene and classical TaqMan assays have been compared by analyzing CYP2C19*2, *3, *4, *5 and *17 polymorphisms. Study have found that turnaround time, labor requirement, and relative cost is less in the Verigene assay than TaqMan assay, although reagent cost of Verigene assay is considerably high. Hence, Verigene is considered having better performance. 96 F I G U R E 3 Differentiation of results obtained from conventional and modifies light transmission aggregometry performed on clopidogrel responder and non-responder patients (Bagoly et al, 2013). 44 Conventional adenosine diphosphate (ADP) aggregation (without prostaglandin E1 [PGE1]) is shown in the left side and P2Y12 specific ADP aggregation with PGE1 is shown in right side. Top graphs shows the samples obtained from clopidogrel responder patients and below shows the samples obtained from clopidogrel resistant patient

| COMPARISON OF THE PF TS
Light transmission aggregometry is the most acceptable method to determine the validity of other novel PFTs. In contrast to these advantages, the major disadvantage is that the higher concentrations of ADP agonist have the ability to induce the platelet aggregation without altering the shape of platelets. Other drawbacks of this assay are listed in Table 2. 25,44,97 A study conducted has shown that the traditional method of LTA cannot be used as a test to monitor the effect of clopidogrel.
Conventional ADP represents platelet aggregation due to both P2Y1 and P2Y12 receptors, whereas modified method with PGE1 shows the platelet aggregation specific to PGE1 (Figure 3). Further, when comparing the VerifyNow P2Y12 assay, Modified ADP/PGE1 assay, and VASP phosphorylation assay, there was a 12%-54% variation in the cut-off values for clopidogrel nonresponsiveness between the above assays. Percentage of clopidogrel nonresponders detected by modified AD/PGE1 aggregation method and VerifyNow P2Y12 assay was much similar. When monitoring the clopidogrel active metabolite of clopidogrel in the plasma of patients, VASP assay was identified as more sensitive as the assay is more specific for P2Y12 assay. This study has concluded that the highest correlation was shown between the ADP/PGE1 aggregation assay and VASP phosphorylation assay and less correlation between conventional LTA and VerifyNow assay and VASP phosphorylation assay in monitoring the efficiency of action of clopidogrel. 44 POCTs, such as VerifyNow P2Y12 assay, PFA-100, Impact cone and Platelet analyzer, and Thromboelastograph Platelet Mapping System, were developed to overcome the limitations of the LTA technique although it is considered as the gold standard test.
A study conducted to compare the effectiveness of LTA, modified TEG, and PFA-100 has recruited 28 patients on clopidogrel therapy. The correlation between the LTA and TEG results was considerably acceptable (κ = 0.81). PFA-100 had shown variable results for only two individuals who have been detected as resistant to clopidogrel by LTA and hence no relation was shown with either TEG or LTA. However, PFA-100 was not recommended to demonstrate considerable difference in clopidogrel responses, but may be useful to demonstrate aspirin responses. 64 As defined by the upper curve, Figure 4  respectively. Points C and D reflect the late aggregation before and after clopidogrel administration, respectively. 46,98 Both TEG and ROTEM have the capability to analyze the physical properties of the clot including the clot stability and strength, fibrin formation, clot formation, and clot lysis, but not thrombolysis or fibrinolysis. Moreover, both platelet function and defects associated with coagulation cascade are also monitored. Controversial result was obtained from the studies, 100-102 that TEG has a very low ability in predicting the hemorrhage and guiding the transfusion of blood products, whereas. However, TEG platelet mapping can predict the excess hemorrhage in patients undergoing coronary artery bypass and under clopidogrel and aspirin therapy. 103 The patients who were under clopidogrel therapy for 5 days prior to bypass were shown 70% cut-off value for platelet responsiveness for clopidogrel and had the capacity to determine the hemorrhage level at the surgery and transfusion guide. 104,105 Vasodilator-stimulated phosphoprotein phosphorylation assay has a higher ability to determine thienopyridine-induced inhibition of platelets when compared with turbidimetric platelet aggregation assays. Flow cytometric analysis was able to define the associated clinical situation in detail. However, epinephrine action on α2A receptors can initiate the dephosphorylation of VASP, and nitric oxide donors can induce the VSP phosphorylation through cGMP causing false results in the analysis of the clopidogrel action. [106][107][108] GRAVITAS trial (Gauging Responsiveness with a VerifyNow P2Y12 Assay: Impact on Thrombosis and Safety) has setup the cut-off value for clopidogrel high on-treatment platelet reactivity (HTPR) as ≥208 P2Y12 PRU, which has been found in more than half of the selected subjects and has shown a >5-fold increased risk of cardiovascular death, myocardial infarction, and stent thrombosis at 60 days. 109 Significant correlations were found between multiplate analyzer, PFA-100, and LTA after ADP-induced platelet aggregation in samples with clopidogrel therapy. However, sensitivity (78%), specificity (95%), accuracy (92%), positive predictive value (80%), and negative predictive value (95%) of Multiplate assay are significantly higher (<0.0001) than LTA. 110 Multiplate assay keeps the cellular environment unchanged, hence the assay becomes more rapid. 111 Many studies have observed that PFA-100 is more sensitive in determining the effect of clopidogrel rather than aspirin. 112 However, a study has shown that PFA-100 cannot be used in the determination of residual antiplatelet activity of clopidogrel. The actual platelet resistance in clopidogrel-related HTPR can be detected by PFA-100 when compared to aspirin. 113 Light transmission aggregometry had shown 60% sensitivity and specificity in HTPR once compared with VASP assay. Although both the assays have a high negative predictive value (94%), they lack a proper standardization technique. 114 The cut-off value for clopidogrel responsiveness when measured with Multiplate analyzer was found to be >416 AU/min under 84% and 70% sensitivity and specificity, respectively. 115 Although there are many platelet function assays, none of the tests have been optimized or fully standardized to study the overall effect of clopidogrel and patient responsiveness to the drug. Reason is that, PFTs have their own pros and cons (Table 1). 36,36,49,67,76,97,99,[116][117][118][119][120][121][122] By comparing the results obtained from the study, it was concluded that VASP phosphorylation assay, VerifyNow P2Y12 assay, and LTA with 20 µmol/L ADP are ideal PFTs to monitor clopidogrel responsiveness. 54,66,70,117,124 When compared with LTA, VASP-P assay was more suitable 125 and out of LTA, Multiplate assay, Verify Now assay, and TEG, Multiplate assay was found to be ideal to monitor the clopidogrel. 126 Limitations of LTA has paved the way for the development of standardized PFTs such as PFA-100, Multiplate, and VerifyNow P2Y12 assays. 36

| SUG G E S TED IMPROVEMENTS FOR PL ATELE T FUN C TI ON TE S TING
An anticoagulant, such as hirudin, should be used instead of the 3.2% trisodium citrate when possible. The anticoagulant used must not interfere with the normal physiology of the sample. 54,130 An advanced principle with a simple technique which can be used globally for different agonists without altering the physiological environment of the sample would be ideal. 67 Further, PFT should have the capacity to determine not only platelet aggregation, but also to measure the procoagulant activity of platelets. Most importantly, reference ranges need to be established to differentiate the normal from different clinical conditions and also identify low-/high-risk patients for clopidogrel and other antiplatelet drug nonresponsiveness. When the current clinical requirements are considered, the need for the PFT to be used as a screening test has increased, in order to obtain results and quick decisions. 131 It would be better to develop more standardized and quality-controlled PFT, minimizing the effect from pre-analytical errors to the final result of the assay in addition to being cost-effective.
Assays more specifically assess the metabolism of clopidogrel and its thiol metabolites will help to study the increasing patterns of patient nonresponsiveness to clopidogrel. Performing large randomized clinical trials in different populations will benefit to identify the clinical would provide a better understanding regarding clopidogrel therapy.

D I SCLOS U R E S
None declared.

AUTH O R S' CO NTR I B UTI O N S
All the authors have made substantial contributions to conception and design, or acquisition of data, or analysis and interpretation of data. Involved in drafting the manuscript and revising it critically for important intellectual content. All authors gave the final approval of the version to be published. Each author have participated sufficiently in the work to take public responsibility for appropriate portions of the content, and agreed to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

DATA AVA I L A B I L I T Y S TAT E M E N T
Data sharing is not applicable to this article as no new data were created or analyzed in this study.