Clopidogrel resistance
Assistant Editor-in-Chief: Leah Biller
Synonyms and related keywords:  Clopidogrel non-responders,  clopidogrel hyporesponders, clopidogrel non-responsiveness,  clopidogrel hyporesponsiveness, clopidogrel failure

# Overview
Administration of the same dose of a drug to all patients has the advantages of simplicity and ease of use.  However, data regarding the variability in platelet inhibition across patients highlights the potential importance of tailoring the antiplatelet or dose of an antiplatelet to the pharmacodynamic response of the patient.  Patients who do not achieve adequate inhibition in response to a dose of clopidogrel are variably termed Clopidogrel non-responders or Clopidogrel hyporesponders.  A recent European Society of Cardiology working group has suggested the term "elevated platelet reactivity despite treatment".
This chapter reviews the underlying etiology and clinical relevance of clopidogrel non-responsiveness.

# Definitions of Clopidogrel Non Responsiveness
There are multiple definitions of clopidogrel non-responsiveness 
- Gurbel et al: Change in inhibition of platelet aggregation (IPA) of < 10% using light transmittance aggregometry (LTA)
- Angiolillo et al: IPA < 40% by LTA 
- Lau et al: Platelet aggregation >= to 70% by LTA 
It should also be noted that the degree of non-responsiveness will also vary depending upon the timing following clopidogrel administration that responsiveness is tested. For instance, Gurbel et al have shown that using the same assay and the same definition, at 2 hours following clopidogrel administration, the rate of non-responsiveness was 60%; by one day the number was 33%, and by one month the number was 15%.  Thus, non-responsiveness may vary depending upon the degree of activation of the platelets themselves.  As the platelets become less activated following an acute coronary syndrome episode, the rate of non-responsiveness may be lower.  This variability in platelet activation and variability in non-responsiveness raises important questions regarding the potential differences in the optimal acute dose and the optimal chronic dose of clopidogrel and other thienopyridines.

# Incidence of Clopidogrel Resistance
The incidence of clopidogrel resistance varies significantly from 5% to 44%.  The incidence varies depending upon
- The definition of clopidogrel resistance
- The timing of assessing clopidogrel resistance in relation to an acute coronary syndrome episode
- There may be circadian rhythm to platelet aggregation

# Association of Clopidogrel Non-Responsiveness with Adverse Clinical Outcomes
There are a large number of studies associating clopidogrel non-responsiveness with adverse outcomes:
Despite these associations of clopidogrel hyporesponsiveness with adverse outcomes, there is no large scale data suggesting that acting upon test results and modifying therapy based upon test results is associated with improved outcomes.  It is important to ascertain if the patient has been compliant with the medication before declaring that the patient is a clopidogrel non-responder.

# Is there a Threshold Effect to Efficacy or are Clinical Outcomes Improved with Higher and Higher Doses (a Continuous Relationship to Clinical Outcomes)
One unresolved question is whether there is a threshold effect  whereby clinical outcomes are not further improved above a certain level of platelet inhibition, or alternatively whether clinical outcomes are further  improved with higher and higher doses  in which case there is a continuous variable relationship between platelet inhibition and clinical outcomes.  Data supporting a potential threshold effect comes from Gurbel et al.  When data regarding the relationship between stent thrombosis and clinical outcomes was plotted as a cumulative distribution function rather than a bell curve, it was noted that stent thrombosis was infrequent above 40-50% inhibition.

# Mechanisms Underlying Clopidogrel Resistance
There are multpiple mechanisms underlying clopidogrel resistance: 

## Clinical Factors
- Poor patient compliance
- Under-dosing: Some patients may alter the dosing to take the drug every other day
- Poor absorption
- The presence of an acute coronary syndrome and increased platelet activation
- Co-morbidities such as diabetes mellitus that is known to be assoicated with heightened platelet activation 
- Elevated body mass index
- Elevated platelet count

## Cellular Factors
- Accelerated platelet turnover
- Reduced CYP3A metabolic activity
- Increased ADP exposure
- Up-regulation of the P2Y12 pathway
- Up-regulation of the P2Y1 pathway
- Up-regulation of the P2Yindependent pathways (collagen, epinephrine, thomboxane A2, thrombin)

## Genetic Basis
Clopidogrel is a pro-drug. When it appears in the bloodstream following absorption, it is not in the active form. This inactive metabolite or pro-drug must circulate to the liver to be metabolized and converted to the active metabolite (there appear to be 4 active isomers). Genetic polymorphisms that have been related to variability in clopidogrel metabolism include:
- Polymorphisms of CYP
- Polymorphisms of GPIa
- Polymorphisms of P2Y12
- Polymorphisms of GPIIIa
Variability in the function of the CYP 2C19 allele has been postulated to be related to the ability to metabolize clopidogrel.  
The three individual alleles and their relative ability to metabolize clopidogrel are as follows:
- *17 hypermetabolizer allele
- *1 normal metabolizer allele
- *2 poor metabolizer allele, genetic functional variant 681 G>A
Based upon the combinations (pairs) of these three alleles, four types of metabolizers have been identified based upon the ability of the patients to generate active metabolite and pharmacodynamics:
- Ultra-metabolizers (UM): (30% of patients)
- Extensive metabolizers (EM): (36% of patients)
- Intermediate metabolizers (IM): (29% of patients)
- Poor metabolizers (PM): (5% of patients)
It should be noted that the active metabolites of clopidogrel and prasugrel are equally potent,  and that differences in pharmacodynamic and clinical outcomes are due to differences in the generation of active metabolite rather than potency of the active metabolite.  Carriers of the allele (those patients with a least one copy of the *2 allele) had a higher 450 day event rate (12.1%) versus those patients with no copies of the allele (an 8.0% event rate, HR 1.53, p=0.014).
In a similar but slightly different finding, Simon et al have demonstrated that it was only those patients who carried two copies of the *2 allele (*2 / *2) and not just one copy (*1 / *2) who had a higher risk of adverse events (death, MI, stroke).
In a third study, Collet et al demonstrated that among 259 young survivors of a first myocardial infarction who were treated with chronic clopidogrel, death, MI, and urgent revascularization occurred more often in carriers  (*2 / *2 or *1 / *2) than in non-carriers (*1 / *1)(HR = 3.69 , p=0.0005), as did stent thrombosis (HR = 6.02 , p=0.0009).  These findings were true in a multivariate model of potential confounders.
Although both prasugrel and clopidogrel require cytochrome P450 (CYP) enzymes for activation, a substudy of 1,466 patients enrolled in the TRITON-TIMI 38 study found that  CYP variations did not affect:
- Levels of prasugrel's active metabolite
- Prasugrel's inhibition of platelet aggregation, or
- Clinical cardiovascular event rates in persons treated with prasugrel 

## Inhibition of Metabolism by Co-Ingestion of Other Drugs

### Statins
Statins have been found to interfere with the generation of clopidogrels active metabolite.     One statin that does not interfere with clopidogrel metabolism is pravastatin.  Non-randomized data from clinical trials have not confirmed a higher risk of adverse outcomes among patients co-ingesting statins in addition to clopidogrel versus those treated with clopidogrel alone.  It is possible that the higher loading dose of 600 mg used in current clinical pracitce overcomes this interference.

### Omeprazole and Proton Pump Inhibitors
Omeprazole induces a conformational change in the CYP enzyme system and may alter the metabolism of clopidogrel.  In a double-blind placebo-controlled trial, stented patients treated with clopidogrel were randomized to treatment with either omeprazole (20 mg/day) or placebo. Following 7 days of treatment, the residual platelet aggregation was significantly hgiher in the omeprazole group (p < 0.0001).  The clinical impact of this finding and whether this inhibition can be overcome with a higher dose of clopidogrel is not clear.
There have been non-randomized retrospective analyses of the clinical outcomes among patients treated with omeprazole vs no omeprazole.    However, these non-randomized analyses are very confounded by the fact that patients treated with omeprazole are more often diabetics, had undergone CABG, had a history of cerebrovascular disease and peripheral arterial disease, had previously been on clopidogrel, and more often had renal disease.   Indeed, it is notable that among patients not treated with clopidogrel, treatment with a proton pump inhibitor (PPI) was associated with a 1.6 fold higher event rate in CREDO despite multivariate adjustment for confounders  This points to the potential role of unidentified confounders in the association of PPIs with clinical outcomes.
Pantoprazole and esomeprazole are not associated with a phramcodynamic or clinical effect in non-randomized analyses 

# Gold Standard Tests of Clopidogrel Responsiveness
Light transmittance aggregometry (LTA): This is a laboratory based study that evaluates the aggregation or clumping of platelets in response to aggregating stimuli. For historical reasons, it is broadly accepted as the gold standard in-vitro test of platelet function.  The most immediate information for basic diagnostic considerations is obtained by using agents such as adenosine diphosphate (ADP), epinephrine and collagen. Both ADP and epinephrine are contained in storages organelles within the platelets and are released during formation of the primary hemostatic plug thus enhancing further platelet aggregation. Conversely, collagen is found in the supporting connective tissue of the vessels and is considered to be the first proagulant factor that the platelet encounters following vessels injury. Other reagents such as arachidonic acid, ristocetin, serotonin, calcium and Factor VIII are also used to study platelet response for more specific purposes. Different aggregating agents stimulate alternative pathways of activation in the platelets and different concentrations of the same agonist are often used to elicit dose-dependent response.
Aside from the detection and diagnosis of acquired or congenital qualitative platelet defects, LTA has an important role to reveal patterns of GPIIb/IIIa-dependent platelet-to-platelet aggregation in response to specific agonists (e.g. arachidonic acid to assess aspirin response; ADP to test thienopyridines response). 
The chambers of a typical aggregometer are designed so that a beam of infrared light shines through two cuvettes. One of these contains the sample, namely a suspension of platelet rich plasma (PRP) obtained by a relatively low centrifugal force centrifugation. The other cuvette contains a reference sample of platelet poor plasma (PPP) obtained by centrifuging the blood sample at a relatively high force. Silicon photodiodes detect the light able to pass through the samples, with PRP arbitrarily considered to be 0% light transmission (or 0% aggregation) and PPP considered to be 100% light transmission (or 100% aggregation). The optical aggregation output is proportional to the continuously measured difference in light transmission between the PRP and PPP samples. Following the addition of a stimulus to the cuvette containing PRP, changes in light transmission occur as a consequence of platelet response and are recorded over time. In fact, the larger size of activated platelet allows less light to pass through the PRP: this is recorded as less light transmission relative to the PPP. Conversely, when platelets form aggregates, more light is able to pass through the test sample.
Aggregation recordings are curves characterized by several features:
- Shape changes;
- A first wave of aggregation that may reverse (primary aggregation);
- A second wave of aggregation that occurs when the granule contents become the stimulus and lead to further aggregation;
- Maximum amount of change in light transmission caused by the stimulus (percent aggregation);
- Late amount of aggregation recorded after a certain timeframe;
- Slope of the aggregation, or percentage of aggregation per minute.
Designed as a measure of defective platelet function, the main disadvantage of LTA is that the test is time-consuming, expensive, weakly standardized and need to be performed in specialized lab by specialized personnel. Additionally, several procedural variables may account for poor reproducibility.
Vasodilator-Stimulated Phosphoprotein (VASP) The phosphorylation (P) of vasodilator-stimulated phosphoprotein (VASP) is a test based on flow cytometry which is very specific to the P2Y12 signaling pathway. It is commercially available as a kit marketed as PLT VASP/P2Y12 (BioCytex, Marseilles, France). 
VASP is an intracellular platelet protein that is not phosphorilated at baseline. VASP-P is mediated by the cAMP cascade, which is enhanced by prostaglandin E1 (PGE1) and inhibited by the link between ADP and P2Y12 receptors. Therefore, VASP-P is a marker of P2Y12 receptor inhibition, whereas its non-phosphorylated counterpart correlates with the non-inhibited form of the P2Y12 receptor. 
By using the PLT VASP/P2Y12 kit, the effect of clopidogrel can be demonstrated by the persistence of VASP in its phosphorylated state (VASP-P) induced by PGE1 despite the simultaneous addition of ADP.
More in detail, the blood sample is first incubated with PGE1 alone or PGE1 + ADP. Subsequently, after a cellular permeabilization, VASP-P is labeled by indirect no wash immunofluorescence using a specific monoclonal antibody. The two tested conditions are then evaluated by means of dual color flow cytometry analysis. Final results are usually expressed in terms of platelet reactivity index (PRI), which is calculated using corrected mean fluorescence intensities (MFIc) in the presence of PGE1 alone or PGE1 and ADP simultaneously, according to the following formula:
PRI = x100
Assessment of VASP-P requires a low sample volume and is performed on whole blood. Another advantage is the opportunity to ship blood samples at room temperature to a central core laboratory. Ultimately, it correlates well with light transmittance aggregometry and VerifyNow technologies. However, sample preparation is time consuming and the reliability of the results is highly dependent from the presence of a skilled technician. Also, a flow cytometer is required.

# Point of Care Devices

## VerifyNow
The VerifyNow system (Accumetrics, San Diego, Ca,  USA; ) is a bedside test that allows for monitoring of the efficacy of thienopyridines, aspirin, and glycoprotein IIbIIIa inhibitors.
Formerly known as the Ultegra rapid platelet function analyzer, the VerifyNow system is a turbidimetric based optical detection system which measures platelet induced aggregation as an increase in light transmittance.
This system is a point-of-care and consists of an instrument, a disposable assay device and controls. The assay device contains a lyophilized preparation of human fibrinogen-coated beads, platelet agonist, preservative and buffer.
Three assays are currently available, which differ according to the platelet agonist contained in the mixing chamber:
After activation, the GP IIb/IIIa receptors on platelets will bind to the fibrinogen-coated microbeads and cross link to other microbeads resulting in a clearing of the beads and platelets within the detection well. The instrument uses light transmittance to measure the rate at which this clearing occurs.
The main advantage of this test is that the patient sample is a low sample volume of 3.2% citrated whole blood, which is automatically dispensed from the blood collection tube into the assay device by the instrument, with no blood handling required by the user. Another advantage is that the instrument provides the results in minutes.

## Platelet Function Analyzer (PFA-100)
The Platelet Function Analyzer-100(PFA-100, Siemens Healthcare Diagnostics, Inc., Deerfield, IL) is a test based on the principle of shear-induced aggregation. Although it is not a COX-1specific assay, it allows monitoring of aspirin effects by providing a quantitative measure of platelet-related hemostasis in anticoagulated whole blood.
The system comprises a microprocessor-controlled tool and a disposable test cartridge containing a biologically active nitrocellulose membrane. To perform the test, 0.8 ml of citrated whole blood is transferred into the reservoir of the cartridge within 4 h of blood sampling. After warming the anticoagulated blood to 37 C, the instrument aspirates a blood sample under vacuum from the reservoir through a 200 m diameter stainless steel capillary and a 150 m aperture cut into the membrane, which is coated with collagen and epinephrine (CEPI) or collagen and ADP (CADP).
The presence of these biochemical stimuli, and the high shear rates of 50006000 s1 generated under the standardized flow conditions, result in platelet aggregate forms that block the aperture of the membrane. The time required to obtain full occlusion of the aperture is reported as the closure time (CT).
Prolonged CT with only the CEPI cartridge is observed with mild inherited platelet function disorders and with aspirin ingestion, while prolonged CTs with both CEPI and CADP cartridges are associated with more severe inherited platelet dysfunctions. 
An advantage of the PFA-100 application as a platelet function assay is that it is a rapid, accurate, simple, and reproducible test that requires only a small volume of blood. It cannot be considered as a point of care as minimal pipetting is required to use this test. One of its major disadvantages is that it is poorly sensitive in detecting effects of thienopyridines and therefore should not be used for this purpose. Newer generation assays are currently under development to assess thienopyridine effects.

# Clinical Utility of Point of Care Testing Versus Genetic Testing
In so far as point of care testing results are more readily available, these may be a more suitable choice for use in clinical practice as compared to genetic testing.  Furthermore, there may be mechanisms other than variability in metabolism that account for differences in response to clopidogrel which are assessed by point of care tests and not by genetic testing.

# Strategies to Overcome Clopidogrel Non-Responsiveness
Due to the severity of its consequences, how to manage suboptimal clopidogrel response is a major clinical problem. The most important aspect is to guarantee patient compliance. The second aspect to evaluate is any potential drug-drug interactions. Studies are currently ongoing with the goal to better elucidate the interaction between clopidogrel and PPIs. The following strategies can be proposed to overcome inadequate clopidogrel responsiveness:
- increase clopidogrel dosing
- triple antiplatelet therapy
- using a different P2Y12 antiplatelet agent.

## Increase clopidogrel dosing
Several studies have shown that a high clopidogrel loading dose regimen ( 600 mg) achieves more potent platelet inhibition when compared to a standard 300 mg loading dose.  This has also been associated with better clinical outcomes in patients undergoing PCI .  ]. A high maintenance dose (150 mg/day) dose regimen of clopidogrel has found to be associated with enhanced platelet inhibition compared to the currently recommended 75 mg/day , in particular in patients with high posttreatment platelet reactivity while on 75mg  .  The OPTIMUS (Optimizing antiPlatelet Therapy In diabetes MellitUS) study selectively evaluated type 2 diabetes mellitus patients with high platelet reactivity while on 75mg clopidogrel and showed that 150 mg clopidogrel maintenance dose induced greater platelet inhibition compared with 75 mg dosing . In a recently published observational study, Lemesle et al. showed better clinical outcomes in PCI patients treated with 600-mg loading dose followed by a high maintenance dose (150 mg/day) without a significant increase in bleeding events. The CURRENT OASIS 7 TRIAL (Clopidogrel optimal loading dose Usage to Reduce recurrent EveNTs/Optimal Antiplatelet Strategy for InterventionS; NCT00335452) showed that doubling the loading and maintenance dose of clopidogrel is associated with improved outcomes in cardiovascular mortality, MI and stroke at 30 days in patients undergoing PCI. This trial enrolled 25087 patients which were then divided into PCI and no PCI cohots. Patients were then also randomized in 2X2 factorial distribution to double dose clopidogrel and standard dose clopidogrel, as well as low dose aspirin(75=100mg) and high dose aspirin(300-325mg). Patients were followed at day 30 for primary end points which were cardiovascular death, MI and stroke and safety outcome which was major bleeding at day 30. Results of this study showed that among the PCI patients, the risk of stent thrombosis was reduced by 30% and the risk of MI was reduced by 22% in the group that received the high dose Clopidogrel compared to the group that received the standard dose. Thsi was statistically significant. The high dose group had more major bleeding, but there was no increase in intracerebral or fatal bleeds. No benefit of the higher dose was found in the group of patients who did not have PCI. 
Several currently ongoing clinical trials are evaluating safety and/or efficacy of a tailored treatment with high clopidogrel maintenance dose in patients with inadequate response to clopidogrel. These include GRAVITAS (Gauging Responsiveness with a VerifyNow Assay: Impact on Thrombosis And Safety; NCT00645918), ARCTIC (Double Randomization of a Monitoring Adjusted Antiplatelet Treatment Versus a Common Antiplatelet Treatment for DES Implantation, and Interruption Versus Continuation of Double Antiplatelet Therapy; NCT00827411), and DANTE (Dual Antiplatelet Therapy Tailored on the Extent of Platelet Inhibition, NCT00774475).

## Triple Antiplatelet Therapy
In the acute phase of therapy, adding a glycoprotein IIb/IIIa inhibitor may be considered as this leads to more potent platelet inhibition. Recently, Cuisset et al. showed that the rate of cardiovascular events at 1 month was significantly lower when abciximab was added compared to conventional dual antiplatelet therapy in clopidogrel nonresponders (n=149) referred for elective PCI . The 3T/2R trial showed that better clinical outcomes in aspirin or clopidogrel non responders undergoing elective PCI treated with tirofiban.  In the maintenance phase of therapy, triple antiplatelet therapy achieved with the adjunctive use of cilostazol, a phosphodiesterase III inhibitor, is another option. The OPTIMUS-2 study showed that in a diabetic population cilostazol markedly enhances P2Y12 inhibitionThis may explain the reduced stent thrombosis rates observed with this triple antiplatelet treatment regimen compared to standard dual antiplatelet therapy and reduced target lesion revascularization rates in patients treated with both bare-metal and drug-eluting stents with greater effects among diabetics. All the above strategies have not been associated with increased bleeding.

## Using a different P2Y12 receptor antagonists
Although clopidogrel has largely replace ticlopidine due to its better safety profile, it has been shown that ticlopidine may improve platelet inhibition among suboptimal responders.
However, the future likely resides with the use of newer agents. New ]s are currently under different phases of clinical development (e.g. prasugrel, cangrelor, ticagrelor, elinogrel). These agents have more potent and less variable inhibitory effects than clopidogrel. Prasugrel, a third generation thienopyridine, has already completed its phase III investigation and received approval for clinical use in Europe . If prasugrel yields better clinical outcomes without increasing bleeding hazards in clopidogrel non-responders is under investigation.