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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.
Keywords: clopidogrel, HPLC, platelet function assays, pharmacogenetic assaysThis review article discuss the principles of pharmacokinetic, pharmacodynamic and pharmacogenetic assays used for monitoring clopidogrel therapy and comparatively discuss their advantages and disadvantages.
ADP adenosine diphosphate APTT activated partial thromboplastin time AU aggregation units CAD coronary artery disease CADP collagen‐ADP cAMP cyclic adenosine monophosphate CFT clot formation time CLPM clopidogrel metabolite CTM converted to thiol metabolite of clopidogrel EDTA‐K ethylenediaminetetraacetic potassium salt HPLC high‐performance liquid chromatography HTPR high on‐treatment platelet reactivity INR international normalized ratio LTA light transmission aggregometry MCF maximum clot firmness MFI mean fluorescence intensity ML maximum lysis MPV mean platelet volume MS mass spectrometry NSAID nonsteroidal anti‐inflammatory drug PCI percutaneous coronary intervention PFA platelet function assay PGE1 prostaglandin E1 POCT point of care test PRI platelet reactivity index PRP platelet‐rich plasma PRU platelet reactivity units PT prothrombin time ROTEM rotational thromboelastometry STEMI ST‐elevated myocardial infarction TEG thromboelastography UHPLC ultra‐high‐performance liquid chromatography VASP vasodilator‐stimulated phosphoprotein vWF von Willebrand factor
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.
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
Metabolic activation of clopidogrel prodrug in patients. CLPM, clopidogrel metabolite; CTM, converted to thiol metabolite of clopidogrel
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
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 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 , 22 , 23 A study on clopidogrel resistance in India using optical aggregometry has shown that, in the population, 2.54% were resistant, 12.7% were semi‐responders, and 84.7% were responders to clopidogrel. 24 There are several underlying causes for clopidogrel resistance. Age, gender, obesity, long‐term medications, and stress, such as individual factors, may contribute to clopidogrel resistance. Mainly, genetic factors such as mutations in P2Y12 gene, P450 CYP3A gene, and COX1 gene and polymorphism in platelet glycoprotein receptors affect the resistance. Further, overproduction of platelets by bone marrow, transfusion of platelets, failure in compliance, insufficient dose, and absorption by hepatic cells and certain drug interactions, such as nonsteroidal anti‐inflammatory drugs (NSAIDs), will also result in clopidogrel resistance. 25 , 26 , 27
Platelet function tests play a major role in determining the effect of antiplatelet drugs, such as clopidogrel, on inhibiting platelet aggregation. Although light transmission aggregometry (LTA) is considered as gold standard, it has many limitations. PFTs should possess several features for them to be ideal in use. They should be cost‐effective, less labor expertise, high sensitivity and specificity, produce quick results, and define the clinical situation more precisely. Further, they should have standardized test procedure with proper quality control, reference ranges to quantify the antiplatelet effect, and measure nonresponsiveness to antiplatelet drug, specific for the platelet receptor to measure the drug and its active metabolites. Importantly, they should have the capacity to monitor platelet activation‐induced physiologically relevant agonists and to detect high‐risk and low‐risk patients for antiplatelet drug resistance. 28
However, when most PFTs are considered they are less sensitive and cannot be performed in relation to normal physiological state. The reason is that, PFTs are performed on anticoagulated whole blood with relatively high concentrations of agonists including platelet aggregation. Further, the platelets will form more stabilized thrombus at high shear rate, which is difficult to be achieved by PFTs. Normal pathological shear rate is >10 000 s −1 . 29 At low shear rate of PFT, the effect of clopidogrel inhibitor is overrated, as the inhibitor fails to ban the platelet deposition on reactive surfaces due to shear gradient. 30 In addition, PFTs have different cut‐off values to define the clopidogrel nonresponsiveness, hence it has become problematic to build up better correlation of results between different PFTs with different principles. 31
Most basic laboratory test is full blood count, where the mean platelet volume (MPV) parameter implies the platelet activation and clopidogrel resistance. 32 ADP is the commonly used agonist for monitoring clopidogrel action by PFTs. There are two ADP receptors on the surface of platelets which involve in platelet aggregation. P2Y1 receptor stimulates ADP‐induced platelet aggregation via phospholipase C and phosphatidylinositol pathways. Second receptor is P2Y12, which stimulates platelet activation by inhibiting adenylyl cyclase signaling pathway. P2Y12 receptor essentially plays a role in producing more stabilized ADP‐induced platelet aggregation. Hence, the clopidogrel therapy mainly targets on blocking this P2Y12 receptors. 33
Platelet function tests are affected by many pre‐analytical variables, such as operator errors and phlebotomy errors, which may cause damage to the vessel walls inducing platelet coagulation. Other factors which affect the PFT results are age, gender, and clinical conditions (acquired platelet disorders, vWF, Hermansky‐Pudlak syndrome, and Chediak‐Higashi syndrome), certain foods—garlic, turmeric, and cumin seeds—and drugs such as aspirin, aspirin‐containing drugs, NSAIDs, tricyclic antidepressants, some antibiotics, and antihistamines, and other long‐term medications. 34
Prior to the PFT, the anticoagulated whole blood sample of the patient should be tested for full blood count/platelet count, to exclude pseudothrombocytopenia due to cold platelet agglutinin, and MPV, to identify high MPV which indicates platelet turnover and low values indicate bone marrow failure. Blood film and morphology will be used to confirm the conditions, such as primary platelet disorders (Gray platelet syndrome), presence of platelet clumps (pseudothrombocytopenia), and thrombocytopenia. 35 , 36 , 37
Plasma sample should be tested for prothrombin time (PT), international normalized ratio (INR), activated partial thromboplastin time (APTT), and fibrinogen levels to check the fibrinogen levels as they are important for platelet aggregation. 36 , 37 Collected samples should be performed within 30‐90 minutes, which is known as the turnaround time, and be controlled by minimizing the sample transportation time, centrifugation time, and increasing the calibration ranges. It is vital to perform PFT immediately in the case of serious bleeding, >6 units of red cells in 12 hours. 35
Except for high‐performance liquid chromatography (HPLC), which requires whole blood collected in ethylenediaminetetraacetic potassium salt (EDTA‐K), other PFA could be performed on blood collected in 3.2% trisodium citrate anticoagulant at room temperature and physiological pH. Agonists play an important role by increasing the intracellular/cytosolic Ca 2+ , thereby inducing the platelet activation. Sodium citrate has the ability to chelate the extracellular calcium (Ca 2+ ). Hence, citrate will have a slight chance to chelate the intracellular Ca 2+ as well as causing decreased platelet aggregation when the samples are taken for analysis 4 hours after collection. 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.
PFA available to monitor the effect of clopidogrel in patients with CAD. HPLC‐MS, high performance liquid chromatography‐mass spectrometry; LTA, light transmission aggregometry; PFA, platelet function assay; ROTEM, rotational thromboelastometry; TEG, thromboelastography; VASP, vasodilator‐stimulated phosphoprotein
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 measuring the change in optical density when light passes through the PRP after the addition of the agonists, commonly used as ADP, arachidonic acid, collagen, epinephrine, and thrombin. ADP is regarded as the most common agonist to determine the effect of clopidogrel on inhibiting platelet aggregation. Low‐dose ADP (1, 2.5, or 5 µm) will initially bind to P2Y1 receptors on the platelet, inducing intracellular calcium efflux. Hence, results in altering the shape of platelets causing primary wave of platelet aggregation. Secondary wave is formed due to the release of ADP from platelet storage granules. Low‐dose ADP induces only the reversible primary wave. ADP has the capacity to bind for P2Y12 receptors. When high concentration of ADP agonist (10 and 20 µm) is added, it will bind to P2Y12 receptors, initiating the platelet aggregation, without changing the shape. P2Y12 receptor is the major receptor for ADP and is responsible for complete platelet aggregation. However, if the patient is a better clopidogrel responder, clopidogrel will block the binding of ADP to P2Y12 receptors, thus preventing the second wave of platelet aggregation. Such that, the percentage of light transmission in PRP will provide the percentage of maximum platelet 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. If high, adjust with PPP. Counts less than the range will reduce the aggregation responses. 22 , 44 , 45 , 46 , 47 , 48 Cut‐off value for suboptimal clopidogrel response in terms of percentage aggregation is ≥ 70% for 10µM ADP and ≥ 50% for 5µM ADP 49
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 reactivity index (PRI) derived from MFI [PRI(%) = 100 × (MFI(PGE1) − MFI(PGE1 + ADP))/MFI(PGE1)]. The main advantage associated with this assay is that it is highly specific for the P2Y12 receptors. 44
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
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 computation is expressed via thromboelastometric curve and numerical parameters. ROTEM delta has the ability to identify hypo‐ and hyperfunctional stages of clotting process. ROTEM can measure the parameters: clotting time, clot formation time (CFT), maximum clot firmness (MCF), and alpha angle, which use the angle between middle axis and tangent to the clotting curve through 2‐mm amplitude point. ROTEM has special techniques to monitor the coagulation pathway via intrinsic and extrinsic pathways, heparin action, and clot firmness after blocking hyperfibrinolysis by aprotinin. There are many variables that can be measured by ROTEM analysis. Commonly used parameters are coagulation time, CFT, α‐angle, amplitude at 10 minutes after CT, MCF, maximum lysis (ML), EXTEM (measures extrinsic pathway), APTEM (measure clot firmness after blocking hyperfibrinolysis), FIBTEM (measure clot firmness after blocking the platelets), INTEM (measures intrinsic pathway), and HEPTEM (same as INTEM, but inhibits heparin). 55 , 56 , 57 , 58 , 59 Assay should solely depend on the instructions provided by the manufacturer as the results may vary depending on the temperature, reagent status, sample stability, and instrument status. 57 In monitoring the effect of clopidogrel, ROTEM analysis could be performed using EXTEM and FIBTEM reagents. Thus, produces the results CT, MCF, and CFT of EXTEM and FIBTEM, respectively. 56
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 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 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 is another point of care assay to monitor the action of clopidogrel. This can be performed on less volume of citrated whole blood. This assay monitors the platelet aggregation and effect of antiplatelet drugs under higher shear stress. Hence, this assay can be performed rapidly in less time using less labor which is an added advantage when compared with conventional aggregation assays. PFA‐100 has cartridges coated with collagen and epinephrine or ADP. Blood drawn from the patient who is under clopidogrel therapy will flow under higher shear rate through the capillary and a small aperture of PFA‐100 analyzer toward the coated cartridge. Platelets will aggregate and form the ADP‐induced platelet plug by blocking the aperture. The time taken for complete occlusion of the aperture is recorded as closure time (CT). Collagen‐ADP (CADP) cartridge measures the platelet dysfunction due to clopidogrel. Prolonged CT indicates the better response to clopidogrel. 64 , 65 , 66 , 67 CT for CADP cartridge is 55‐137 seconds. 68
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 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 exceed the threshold limit for platelet size, such platelets are not considered for the resultant platelet count. Percentage inhibition of platelet aggregation provides the degree of platelet aggregation in the presence of clopidogrel. Percentage of inhibition is expressed as the ratio between platelet counts before and after exposure to ADP. Plateletworks is widely used for monitoring the effectiveness of antiplatelet drugs such as clopidogrel and aspirin. 53 , 73 , 74 , 75 Blood samples should be analyzed within 10 minutes to obtain accurate results. There are studies to prove the significant correlation between Plateletworks and LTA. However LTA determines only macroaggregation while Plateletworks is sensitive for microaggregation. 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 is generally used for identification and quantification of chemical compounds. HPLC consists of a stationary phase which is packed either with a resin or silica gel beads and mobile phase. Methanol and acetonitrile are used as solvents for the separation of analyte. Molecular interaction between stationary phase, analyte, and solvent decides the retention time. Separation of analytes in normal HPLC depends on the polarity where stationary phase is polar and mobile phase is nonpolar. However, in reverse phase HPLC, the separation is based on hydrophobic interactions between polar mobile phase and nonpolar stationary phase. During the separation and quantification of active metabolites, the internal diameter of the HPLC column, particle size, and pore size of the stationary phase and pump pressure should be considered for optimum performance. 77
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
CYPA1, CYP2B6, and CYP2C19 are CYP450 enzymes that participate in the first step of converting clopidogrel prodrug to 2‐oxo‐clopidogrel. The second step which forms the thiol metabolite is catalyzed by the enzymes CYP2B6, CYP2C9, CYP3A4/5, and CYP2C19. CYP2C19 is considered as the main enzyme involved in the whole process as it provides 44.9% to the first step and 20.6% to the second step. In addition, CYP3A4 too contributes 39.8% to the second step. 84 , 85 Hence, bioactivation process of clopidogrel could be altered due to genetic polymorphisms of the genes CYP2C19, CYP3A4/5, CYP2C9, and CYP2B6 that code for their respective enzymes. Out of these genes, CYP2C19 gene polymorphism is studied mainly due its association with both the steps. CYP2C19 is highly polymorphic, where *1/*1 allele is considered as normal and two loss of functional (LoF) alleles due to single nucleotide polymorphism are (*2) and (*3). These LoF alleles result in poor clopidogrel therapeutic outcome. Although CYP2C19 and CYP2C9 LoF cause poor clopidogrel outcome, CYP1A2,CYP2B6, and CYP3A4/5 alleles have not shown any considerable effect on clopidogrel effect. The Clinical Pharmacogenetic Consortium has developed a guide to interpret CYP2C19 genetic test. It explained that *1/*17, *1/*17, and *17/*17 genotypes show normal enzyme activity and clopidogrel 75 mg daily dose could be administered. Genotypes *1/*2, *1/*3, and *2/*17 indicate intermediate enzyme activity and genotypes *2/*2, *2/*3, and *3/*3 show absence of enzyme activity. Patients with these genotypes should be treated with either Prasugrel 10 mg daily or Ticagrelor 90 mg twice a day. 86 Prasugrel and Ticagrelor are used for clopidogrel poor responders as these are not affected by CYP2C19 LoF allele. 87 It was found that CYP2C19*2 resulted in poor platelet inhibition by clopidogrel and hence causes poor outcomes in PCI patients. 88 CYP2C19*17 gene polymorphism too can be studied which results in enhanced enzyme activity due to increased gene transcription. 89 Clopidogrel absorption in the intestine is controlled by a transporter protein called P‐glycoprotein multidrug resistance‐1, which is coded by the gene ABCB1. Polymorphism of ABCB1 gene has shown poor absorption of clopidogrel by intestines. 90 CES1 is another genetic polymorphism which could be studied as the CES1 enzyme synthesized by the gene results in the metabolism of clopidogrel. LoF of this allele causes increased concentration of active thiol metabolite, 91 whereas polymorphism of PON1 gene reduces the enzyme activity of PON1 esterase and results in reduction of active thiol metabolite concentration. 92 Gain of functional haplotypes H2 of P2Y12 gene increases atherothrombotic events and affects the action of clopidogrel. 93 However, due to the difficulty in predicting the outcome of every genetic variation associated with clopidogrel, Clinical Pharmacogenetics Implementation Consortium guidelines for CYP2C19 genotype and clopidogrel therapy have not endorsed to follow CYP2C19‐guided therapy.
Several pharmacogenetic tests are available to determine clopidogrel response by analyzing different CYP2C19 variants. Whole blood is used for polymerase chain reaction and primer extension reaction, which is the underlying technique of the tests AccuType ™ CP and Cytochrome P450 2C19 10 mutations assay (ARUP Laboratories, Salt Lake City, UT, USA). This technique is used by clopidogrel efficacy test (by 23andMe Company, Sunnyvale, CA, USA); however, it is performed for salivary samples. Medication panel (Navigenics, Inc, Foster City, CA, USA) and drug response test (Pathway Genomics, San Diego, CA, USA) use salivary samples on two different techniques: TaqMan SNP genotype assay and DNA testing, respectively. DNA testing is an underlying principle in many other tests such as Clopidogrel genetic test (TheranostiCs Lab, Auckland, New Zealand), CYP450 2C19 (Plavix ™ ) gene test for buccal swabs and Verigene CYP2C19 XP/CYP2C19 nucleic acid test. PlaVitest by Genelec 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
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
Reference ranges for clopidogrel responsiveness
