Interindividual variability in the response to clopidogrel has been shown to be related to the clinical ischemic outcomes. Although testing of platelet function or genetic profile is recommended to evaluate the response to clopidogrel, standardized testing and definitive antiplatelet therapy after testing need to be established.
by Yusuke Yamaguchi and Professor Mitsuru Murata
Clinical background
Platelet activation and aggregation play a pivotal role in arterial thrombosis formation; therefore, antiplatelet therapy to inhibit platelet function is considered effective for preventing and treating atherothrombosis. The combination of aspirin and clopidogrel has been shown to be more effective than aspirin alone for improving clinical ischemic outcomes in patients with coronary artery disease (CAD). This dual antiplatelet therapy contributes substantially to prevent the occurrence of cardiovascular events in patients with acute coronary syndrome (ACS) or percutaneous coronary intervention (PCI). Current guidelines recommend aspirin and clopidogrel for these patients; however, some patients still develop cardiovascular events despite dual therapy. It has been shown in the last decade that the responsiveness to clopidogrel is highly variable in individuals and that a suboptimal response to clopidogrel is a risk factor for cardiovascular events. The interindividual variability in the effect of clopidogrel is due to multiple factors [Table 1].
Effects of CYP2C19 on clopidogrel
Clopidogrel, a second generation thienopyridine, is an inactive prodrug that requires a 2-step metabolic conversion to an active metabolite. This active metabolite inhibits adenosine diphosphate (ADP)-induced platelet aggregation by selectively and irreversibly binding P2Y12 receptors on the platelet membrane. Several isoforms of cytochrome P450 (CYP), including CYP2C19, CYP3A4, CYP1A2, CYP2B6, and CYP2C9, have been shown to be involved in the metabolic pathway. Of these enzymes, CYP2C19 is considered to be the main determinant of clopidogrel metabolism that produces the active form.
It is known that CYP2C19 has numerous single nucleotide polymorphisms (SNPs), of which CYP2C19*2 (681G>A, located in exon 5) has been studied extensively and shown to be associated with a loss of function of the enzyme. CYP2C19*2 clearly associates with both the pharmacokinetics (i.e., area under the concentration curve and maximal plasma concentration of clopidogrel active metabolite) and the pharmacodynamics (i.e., inhibition of ADP-induced platelet aggregation) of clopidogrel. CYP2C19*2 is detected more frequently in Asians than in Caucasians, with approximately 40–50% and 30% having at least one CYP2C19*2 allele, respectively. In addition to CYP2C19*2, CYP2C19*3, *4, *5, *6, *7, and *8 have been identified as loss-of-function alleles.
Methods to evaluate the effect of clopidogrel on platelet inhibition
Different laboratory tests [Table 2] can be used to assess platelet function in patients treated with clopidogrel. ADP-induced platelet aggregation in platelet-rich plasma measured by light transmission aggregometry is used most commonly, with numerous published studies using this method to measure platelet function. The majority of these studies measured platelet function as maximal platelet aggregation rate induced by 5, 10, or 20 µmol/l ADP. The platelet aggregation rate 5–8 min after the addition of ADP has also been reported. The POPULAR study [1] on clopidogrel-treated patients following elective PCI showed that 42.9% maximal platelet aggregation rate induced by 5 µmol/l ADP or 64.5% induced by 20 µmol/l ADP correlated with the 1-year mortality rate, myocardial infarction (MI), stent thrombosis, and stroke.
The VerifyNow P2Y12 test (Accumetrics Inc, SanDiego, CA) has been developed as a point-of-care device to quickly and accurately assess platelet function in patients. This test is a whole-blood, light transmission-based optical detection assay that measures the light transmittance of ADP-induced platelet aggregation in a cartridge containing fibrinogen-coated beads and is able to specifically evaluate P2Y12 receptor inhibition. The results are reported as P2Y12 reaction units (PRU), with a lower PRU value being associated with higher P2Y12 inhibition. A meta-analysis of individual patient data in six observational studies [2] revealed that a PRU value of 230 at PCI is the best cut-off value for predicting the occurrence of cardiovascular events, including death, MI, and stent thrombosis, in patients with stable CAD or non-ST elevated ACS undergoing PCI over 1 year.
The effect of clopidogrel on platelet function can be also evaluated by detecting vasodilator-stimulated phosphoprotein (VASP). VASP is phosphorylated by cyclic adenosine monophosphate (cAMP) produced in the adenylate cyclase cascade downstream of the P2Y12 receptor. By binding to the P2Y12 receptor and suppressing the cascade, ADP leads to an increase in VASP dephosphorylation, whereas inhibition of the receptor by clopidogrel active metabolite leads to an increase in VASP phosphorylation. This test measures VASP phosphorylation in a flow cytometric assay with the result expressed as platelet reactivity index (PRI) that represents the ratio of the phosphorylated and dephosphorylated VASP. A lower PRI value reflects higher P2Y12 inhibition.
Clinical utility of laboratory testing
Numerous studies, including our meta-analysis [3], have reported that patients with a suboptimal response to antiplatelet therapy have increased cardiovascular events [Figure 1A], and data have been accumulated on testing of platelet function to establish a reliable cut-off value for clinical risk. However, it remains unclear how to monitor suboptimal responses in daily clinical practice due to the lack of a standardised method to measure and interpret the results of platelet function. Furthermore, there is no guideline for alternative treatment strategies to the “one-size-fits-all” 75 mg/day clopidogrel regime because conclusive evidence that personalised antiplatelet therapy improves patient outcomes has not been established from large-scale randomised trials. However, a meta-analysis [4] recently reported the evaluation of the clinical efficacy and safety of intensified antiplatelet therapy involving reloading clopidogrel, using glycoprotein IIb/IIIa inhibitors periprocedural PCI, increasing the maintenance dose of clopidogrel, or switching to prasugrel. Although there were several limitations, this meta-analysis showed that intensified antiplatelet therapy reduces cardiovascular death and stent thrombosis without increasing major bleeding.
Meanwhile, CYP2C19 genotype does not always seem to predict cardiovascular events, although it is a major predictor for suboptimal response to clopidogrel. To date, many large-scale clinical trials, including the recent Genotype Information and Functional Testing (GIFT) trial [5], which investigate an association of CYP2C19 genotype with cardiovascular events, have been performed. However, the results of these trials were inconsistent. Indeed, we showed heterogeneity in the odds ratio of the cardiovascular events between the carriers and non-carriers of CYP2C19*2 allele in our meta-analysis [Figure 1B]. Considering that CYP2C19*2 contributes to only about 5% of the variability in response to clopidogrel [6], many other genetic factors may contribute to the variability apart from CYP2C19. Therefore, genetic testing including additional factors such as SNPs in other CYPs or ABCB1 (encoding p-glycoprotein) would be expected to improve identification of patients with a suboptimal response.
Current status and future prospects
In 2009, the U.S. Food and Drug Administration (FDA) released a black box warning that significant attention needs to be paid to clopidogrel pharmacogenomics. Similarly, the American and European guidelines published in 2011 gave a Class IIb recommendation for testing of platelet function or genetic profile in patients treated with clopidogrel and for consideration of the use of an alternate P2Y12 inhibitor in patients with inadequate platelet inhibition.
The primary goal of testing of platelet function and genetic profile is to identify patients with a suboptimal response to antiplatelet therapy and provide them with a tailor therapy to improve the clinical ischemic outcomes without an
associated bleeding risk. Although these laboratory tests provide sufficient evidence to predict outcomes, personalised antiplatelet therapy on the basis of these tests has not been established in the guidelines. Currently, several clinical trials are ongoing that evaluate the effect of personalised antiplatelet therapy on the basis of laboratory tests. These trials will hopefully provide important data to establish guidelines, to allow clinicians to properly select laboratory tests, and to plan personalised antiplatelet therapy in patients with a suboptimal response.
References
1. Breet NJ, van Werkum JW, Bouman HJ, Kelder JC, Ruven HJ, Bal ET, et al. JAMA 2010; 303: 754–762.
2. Brar SS, ten Berg J, Marcucci R, Price MJ, Valgimigli M, Kim HS, et al. J Am Coll Cardiol 2011; 58: 1945–1954.
3. Yamaguchi Y, Abe T, Sato Y, Matsubara Y, Moriki T, Murata M. Platelets. Epub 2012 Jul 3, doi: 10.3109/09537104.2012.700969
4. Aradi D, Komócsi A, Price MJ, Cuisset T, Ari H, Hazarbasanov D, et al. Int J Cardiol. Epub 2012 Jun 15, doi: 10.1016/j.ijcard.2012.05.100
5. Price MJ, Murray SS, Angiolillo DJ, Lillie E, Smith EN, Tisch RL, et al. J Am Coll Cardiol 2012; 59: 1928–1937.
6. Hochholzer W, Trenk D, Fromm MF, Valina CM, Stratz C, Bestehorn HP, et al. J Am Coll Cardiol 2010; 55: 2427–2434.
The authors
Yusuke Yamaguchi and Mitsuru Murata MD, PhD
Dept of Laboratory Medicine, Keio University School of Medicine,
35 Shinanomachi, Shinjuku-ku, Tokyo, 160-8582, Japan
E-mail: yusukeyamaguchi@z8.keio.jp
25-Hydroxyvitamin D Total ELISA
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, /in Featured Articles /by 3wmediaAD: a serious set-back
, /in Featured Articles /by 3wmediaAlzheimer’s disease (AD) is now the fifth leading cause of death in people over 65 years old. The prevalence of AD is increasing rapidly as the world population ages; data show that the incidence increases exponentially after the age of 65, with more than 40% of those aged over 85 now affected. According to a 2012 WHO report, nearly 36 million people globally are living with dementia, around two thirds of whom have AD, and this number is predicted to triple by 2050. The 18th World Alzheimer’s day on the 21st of September emphasized the need to reduce the stigma of dementia and make communities ‘dementia-friendly’. While these aims are laudable, the pressing need is for very early diagnosis and timely effective treatment if health services are not to be totally overwhelmed by the escalating numbers of AD patients needing care.
Two major abnormalities, clearly visible at autopsy, are present in abundance in the brains of AD patients, namely beta-amyloid plaques (Aβ) and neurofibrillary tangles (tau protein). However these lesions are not very evident using even advanced neuroimaging techniques, and the disease is most frequently diagnosed by psychological tests and rule-out of other causes of neurodegeneration, so that many early cases remain undiagnosed. Clinical research to allow early diagnosis has mainly focused on fluid biomarkers, and genetic risk factors and markers. Stanford University School of Medicine, USA, has been concentrating on the former approach with the aim of eventually developing a simple blood test that would confirm the onset of AD several years before clinical symptoms were apparent. Initially researchers compared signalling proteins from the blood of patients with and without AD. Their more recent work uses animal models to compare neurons from the hippocampal formation, which are very vulnerable and die in the early stages of AD, with neurons which are not affected until the late stages of the disease. Several labs based in Europe are concentrating on finding cerebrospinal fluid markers present in the early stages of AD, such as total tau, phosphorylated tau and the 42 amino acid form of Aβ, which would allow early specific and sensitive diagnosis. The search for genetic markers has demonstrated that the genes APOE and PICALM consistently affect Aβ.
Early diagnosis, however, must be followed by effective treatment. Currently cholinesterase inhibitors and NMDA receptor antagonists are used to alleviate symptoms but are not curative. Sadly just before this year’s World Alzheimer’s day it was announced that two antibody drugs targetting Aβ, namely Bapineuzumab from Pfizer and Solanezumab from Eli Lilly, had proved to be no better than placebo in Phase III clinical trials. Last year the European Parliament called for dedicated plans to reduce the burden of AD; a new funding model to ensure that big pharma doesn’t withdraw from the AD challenge could be the most valuable strategy.
Heart fatty acid binding protein and troponin: a match made in heaven?
, /in Featured Articles /by 3wmediaPlasma levels of heart-type fatty acid binding protein (H-FABP) have been shown to rise early after the onset of acute myocardial infarction (AMI). Recent evidence suggests combining H-FABP with troponin gives superior diagnostic accuracy compared to the alternative ‘early markers’ of myocardial necrosis, creatine kinase-MB (CK-MB) and myoglobin. However, using a single measurement at the time of presentation to the Emergency Department (ED), H-FABP is unlikely to have sufficient sensitivity to safely ‘rule out’ AMI, even when combined with a standard troponin assay. With the advent of high sensitivity troponin assays which have higher diagnostic sensitivity at the time of presentation, it is possible that H-FABP could be combined with levels of high sensitivity troponin and potentially with other clinical information to enable safe ‘rule out’ of AMI using a single blood test at the time of presentation. Further work in this area is needed.
by Dr Richard Body
Background
Suspected cardiac chest pain accounts for approximately one quarter of acute medical admissions, although only a minority of the patients admitted will ultimately be diagnosed with an acute coronary syndrome [1]. Meanwhile, up to 2% of patients with acute myocardial infarction (AMI) have that diagnosis missed and are inadvertently discharged, leading to a worse prognosis [2]. There is therefore tremendous potential to reduce unnecessary hospital admissions in this patient group, although advances in diagnostic technology are clearly necessary in order to do so.
High sensitivity troponin
Cardiac troponins are regulatory proteins contained within the myofibrillar apparatus of cardiac myocytes. They are released into the bloodstream following myocardial necrosis and their detection allows highly sensitive and specific diagnosis of AMI. Indeed, the detection of a rise and/or fall of cardiac troponin in serum or plasma is integral to the diagnosis of AMI. With the advent of high sensitivity troponin (hs-cTn) assays, which have greater analytical and diagnostic sensitivity than standard assays, it is tempting to believe that the hunt for an ‘early rule out’ strategy for acute coronary syndromes is over. Standard troponin assays lack the diagnostic sensitivity to enable safe exclusion of acute myocardial infarction (AMI) when measured at the time of presentation. This creates a period of ‘troponin blindness’, when patients with AMI still have low circulating troponin levels prior to the development of a late troponin rise. Hs-cTn assays have been shown to improve diagnostic sensitivity at the time of initial presentation to the Emergency Department (ED). While this reduces the magnitude of our problem with ‘troponin blindness’, it does not overcome the problem completely. Even hs-cTn assays fail to identify approximately 10% of patients with AMI at the time of presentation [3, 4]. With hs-cTn assays it may be possible to reduce the time taken to confidently ‘rule out’ AMI with serial sampling from 6 to 9 hours after arrival (or 10–12 hours from symptom onset) to as little as 3 hours after arrival [4, 5]. This approach still needs to be validated against a hs-cTn reference standard, however, and there are a few other reasons to be cautious. The sensitivity of the Siemens troponin I Ultra assay (a sensitive assay but not high sensitivity), which had a diagnostic sensitivity of 100% at 3 hours after presentation in Keller et al.’s original study (evaluated against the reference standard of testing 6 hours after arrival), was actually only 94.5% at 6 to 12 hours from symptom onset [4]. Further, high sensitivity troponin T (hs-cTnT) has been shown to have a sensitivity of only 92.2% when measured 2 hours after presentation, which is still some way from a satisfactory rule out strategy [6]. Using the new Abbott Architect high sensitivity troponin I assay, sensitivity for AMI is 98.2% (with 95% confidence intervals extending down to 96.9%), again using a standard troponin assay as the reference standard [5]. Even if we accept that no rule out strategy will be 100% sensitive and consider this 3-hour troponin to be a satisfactory rule out strategy, that still means an anxious wait for patients and would still, in health systems like the United Kingdom, necessitate admission to an inpatient ward for investigation.
Interest in ‘early markers’ of myocardial necrosis
There has been interest in the role of ‘early markers’ of myocardial necrosis for many years. As troponin is predominantly an intracellular constituent and levels do not peak for 12 to 24 hours after the onset of infarction [7], many have investigated the value of biomarkers with release kinetics suggesting that they may enable earlier identification of AMI. Thus, the measurement of creatine kinase-MB (CK-MB) and myoglobin levels in combination with troponin were shown to improve early diagnosis of AMI as early as 2001 [8]. More recently, the ASPECT study from 14 countries in the Asia-Pacific region examined the value of CK-MB, myoglobin and troponin I (using assays from Alere, San Diego, CA, USA) measured at presentation and 120 minutes later in patients with a Thrombolysis In Myocardial Infarction (TIMI) score of 0/7. The authors found that 9.8% of patients could be discharged using this strategy with a 0.9% incidence of adverse cardiac events within 30 days [9]. Around the same time, the Randomised Assessment Using Panel Assay of Cardiac Biomarkers (RATPAC) study demonstrated that serial evaluation of CK-MB, myoglobin and troponin I over 90 minutes led to an increase in the proportion of patients successfully discharged from the ED, although this came at a cost of rebound-overuse of Coronary Care resources, perhaps as a function of the lack of specificity of myoglobin and CK-MB. The strategy was found to be not cost effective [10].
Heart-type fatty acid binding protein
Heart-type fatty acid-binding protein (H-FABP) is a cytosolic protein that is abundantly expressed in human myocardial cells, where it facilitates intracellular fatty acid transport within cardiac myocytes [11]. Plasma H-FABP levels rise early after the onset of AMI. McCann et al. evaluated H-FABP (Hycult Biotechnology ELISA) and troponin T (cTnT; Roche Elecsys, 4th generation) in 415 patients who were admitted to an acute cardiology unit on suspicion of an acute coronary syndrome. They demonstrated that H-FABP had superior sensitivity to troponin in patients who presented early (<4h) after symptom onset [Figure 1] [12]. A meta-analysis of 16 studies including 3,709 patients with suspected AMI demonstrated a pooled sensitivity of 84% [95% confidence intervals (CI) 76–90%] and a pooled specificity of 84% (95% CI 76–89%), although there was significant heterogeneity between studies [13]. It is clear that measurement of H-FABP alone cannot enable safe ‘rule out’ of AMI. Combining H-FABP with troponin will, however, yield a higher diagnostic sensitivity. Body et al. [14] demonstrated that the combination of H-FABP and troponin I offers both superior sensitivity and superior specificity to the combination of CK-MB, myoglobin and troponin I [Figure 2].
A systematic review by Carroll et al. demonstrated that, in 4 studies, the combination of H-FABP and troponin had an overall sensitivity of between 76 and 97% [15]. Two of these studies did, however, use insensitive troponin assays with diagnostic sensitivities of 42% and 55% respectively. The use of more sensitive troponin assays may be expected to yield higher diagnostic performance. Indeed, in the study by Body et al., the sensitivity of the combination of H-FABP and troponin increased from 82% to 87% when a more sensitive troponin assay was used [14, 16]. If only low risk patients (using the modified Goldman risk stratification tool) who had normal H-FABP and normal cTnT were considered for early discharge, a sensitivity and negative predictive value of 99% could be achieved, although this strategy may have a specificity as low as 19%, meaning that only a minority of patients would be eligible for early discharge while 1% of AMIs would still be missed [16].
H-FABP and high sensitivity troponin
It is clear that neither H-FABP nor troponin (even using a high sensitivity assay) can be used to safely exclude a diagnosis of AMI when measured at the time of presentation to the ED. The combination of H-FABP and standard troponin assays improves overall diagnostic sensitivity but is still unable to ‘rule out’ this important diagnosis. By combining H-FABP with high sensitivity troponin assays, it may be possible to further increase sensitivity and thus achieve an effective early rule out strategy. Evidence in this area is still limited. However, Aldous et al. did evaluate the combination of H-FABP (Hycult Biotech) and hs-cTnT in a cohort of 384 patients presenting to the ED with suspected acute coronary syndromes. This combination had a sensitivity of 90.0% for AMI and a specificity of 73.5%. Notably, the sensitivity of the H-FABP assay alone was particularly low in this study (50.0%), which may be a function of the high diagnostic cut-off employed (60ng/ml) when compared to the cut-off employed by McCann et al. using the same assay (5ng/ml) [12, 17]. Using this high diagnostic cut-off, however, the combination of H-FABP and hs-cTnT measured at the time of presentation may help to ‘rule in’ the diagnosis of AMI, with a specificity of 99.4% (95% CI 97.9–99.9%) [17].
Inoue et al. also evaluated both hs-cTnT and H-FABP (DS Pharma Biomedical, Osaka) in 432 ED patients with suspected acute coronary syndromes. In this study, H-FABP had a similar area under the receiver operating characteristic (ROC) curve (AUC) to hs-cTnT (0.83 versus 0.82), although hs-cTnT had a higher sensitivity at the diagnostic cut-off (87.9% vs. 78.5%) [18]. The authors do not report the diagnostic value of the combination of both biomarkers. Meanwhile, in 1,818 patients with suspected acute coronary syndromes, Keller et al. reported that H-FABP had an AUC of 0.89, which rose to 0.97 when combined with high sensitivity troponin I (Abbott Architect STAT high sensitive troponin) [5]. This implies that the combination has high diagnostic accuracy, although the sensitivity and negative predictive value of the strategy were not reported.
H-FABP and prognosis
H-FABP levels may also have prognostic value in patients with suspected acute coronary syndromes. Viswanathan et al. studied 1,080 consecutive patients presenting with suspected acute coronary syndromes [19]. They measured both H-FABP (Randox Evidence Biochip) and troponin I using a sensitive assay (Siemens Advia troponin I Ultra) and followed patients for a median of 18 months. H-FABP predicted death or AMI occurring during follow up, even in troponin negative patients and after adjustment for age and serum creatinine. For predicting death or AMI, H-FABP had an AUC of 0.79 (95% CI 0.74–0.84)
compared to 0.77 (95% CI 0.72–0.82) for troponin I.
Future work
Further work is still needed to determine whether the combination of H-FABP and high sensitivity troponin will enable safe rule out of acute coronary syndromes in the ED. Combination with other clinical information available from risk stratification tools (such as the modified Goldman or TIMI scores) or the ECG may further increase sensitivity, enabling AMI to be safely excluded in a proportion of patients presenting to the ED. Further, with the increase in false positive results given by high sensitivity troponin assays, H-FABP may help to ‘rule in’ the diagnosis of AMI in patients with troponin elevations at the time of presentation, before the results of serial testing are available. This will facilitate early treatment and triage to an appropriate level of care in the hospital, while avoiding the risks of unnecessary treatment for those patients with false positive elevations.
Conclusions
H-FABP is a promising biomarker for use in patients with suspected acute coronary syndromes. Used alone or in combination with a standard troponin assay, sensitivity will be insufficient to safely ‘rule out’ AMI. Further work is needed to determine whether combination with a high sensitivity assay can enable safe ‘rule out’ for a proportion of patients, and to evaluate whether H-FABP may have a role in the differentiation between ‘true positive’ and ‘false positive’ troponin elevations at the time of initial presentation.
References
1. Goodacre S, et al. The health care burden of acute chest pain. Heart 2005; 91: 229–230.
2. Pope JH, et al. Missed diagnoses of acute cardiac ischaemia in the Emergency Department. N Engl J Med 2000; 342: 1163–1170.
3. Reichlin T, et al. Early diagnosis of myocardial infarction with sensitive cardiac troponin assays. N Engl J Med 2009; 361: 858–867.
4. Keller T, et al. Sensitive troponin I assay in early diagnosis of acute myocardial infarction. N Engl J Med 2009; 361(9): 868–877.
5. Keller T, et al. Serial changes in highly sensitive troonin I assay and early diagnosis of myocardial infarction. JAMA 2011; 306(24): 2684–2693.
6. Aldous SJ, et al. Diagnostic and prognostic utility of early measurement with high-sensitivity troponin T assay in patients presenting with chest pain. CMAJ 2012; 184: E260-E268.
7. Tucker JF, et al. Early diagnostic efficiency of cardiac troponin I and troponin T for acute myocardial infarction. Acad Emerg Med 1997; 4(1): 13–21.
8. McCord J, et al. Ninety-minute exclusion of acute myocardial infarcdtion by use of quantitative point-of-care testing of myoglobin and troponin I. Circulation 2001; 104: 1483–1488.
9. Than M, et al. A 2-h diagnostic protocol to assess patients with chest pain symptoms in the Asia-Pacific region (ASPECT): a prospective observational validation study. Lancet 2011; 377(9771): 1077–1084.
10. Fitzgerald P, et al, on behalf of the RATPAC investigators. Cost-effectiveness of point-of-care biomarker assessment for suspected myocardial infarction: The RATPAC trial (Randomised Assessment of Treatment Using Panel Assay of Cardiac markers). Acad Emerg Med 2011; 18(5): 488–495.
11. Schaap FG, et al. Impaired Long-Chain Fatty Acid Utilization by Cardiac Myocytes Isolated From Mice Lacking the Heart-Type Fatty Acid Binding Protein Gene. Circ Res 1999; 85(4): 329–337.
12. McCann C, et al. Novel biomarkers in early diagnosis of acute myocardial infarction compared with cardiac troponin T. Eur Heart J 2008; 29(23): 2843–2850.
13. Bruins Slot MH, et al. Heart-type fatty acid-binding protein in the early diagnosis of acute myocardial infarction: a systematic review and meta-analysis. Heart 2010; 96(24): 1957–1963.
14. Body R, et al. A FABP-ulous ‘rule out’ strategy? Heart fatty acid binding protein and troponin for rapid exclusion of acute myocardial infarction. Resuscitation 2011; 82(8): 1041–1046.
15. Carroll C, et al. Heart-type fatty acid binding protein as an early marker for myocardial infarction: systematic review and meta-analysis. Emerg Med J 2012.
16. Body R, et al. Reply to Letter: Still FABP-ulous even with a more sensitive troponin assay. Resuscitation 2012; 83(2): e29–e30.
17. Aldous S, et al. Heart fatty acid binding protein and myoglobin do not improve early rule out of acute myocardial infarction when highly sensitive troponin assays are used. Resuscitation 2012; 83(2): e27–e28.
18. Inoue K, et al. Heart fatty acid-binding protein offers similar diagnostic performance to high-sensitivity troponin T in Emergency Room patients presenting with chest pain. Circulation 2011; 75: 2813–2820.
19. Viswanathan K, et al. Heart-Type Fatty Acid-Binding Protein Predicts Long-Term Mortality and Re-Infarction in Consecutive Patients With Suspected Acute Coronary Syndrome Who Are Troponin-Negative. J Am Coll Cardiol 2010; 55(23): 2590–2598.
The author
Richard Body, MB ChB MRCSEd(A&E) FCEM PhD
Emergency Department,
Manchester Royal Infirmary,
Oxford Road, Manchester, M13 9WL, UK
e-mail: richard.body@manchester.ac.uk
Variability of the response to clopidogrel: mechanisms, availability of testing, and relation to clinical outcomes
, /in Featured Articles /by 3wmediaInterindividual variability in the response to clopidogrel has been shown to be related to the clinical ischemic outcomes. Although testing of platelet function or genetic profile is recommended to evaluate the response to clopidogrel, standardized testing and definitive antiplatelet therapy after testing need to be established.
by Yusuke Yamaguchi and Professor Mitsuru Murata
Clinical background
Platelet activation and aggregation play a pivotal role in arterial thrombosis formation; therefore, antiplatelet therapy to inhibit platelet function is considered effective for preventing and treating atherothrombosis. The combination of aspirin and clopidogrel has been shown to be more effective than aspirin alone for improving clinical ischemic outcomes in patients with coronary artery disease (CAD). This dual antiplatelet therapy contributes substantially to prevent the occurrence of cardiovascular events in patients with acute coronary syndrome (ACS) or percutaneous coronary intervention (PCI). Current guidelines recommend aspirin and clopidogrel for these patients; however, some patients still develop cardiovascular events despite dual therapy. It has been shown in the last decade that the responsiveness to clopidogrel is highly variable in individuals and that a suboptimal response to clopidogrel is a risk factor for cardiovascular events. The interindividual variability in the effect of clopidogrel is due to multiple factors [Table 1].
Effects of CYP2C19 on clopidogrel
Clopidogrel, a second generation thienopyridine, is an inactive prodrug that requires a 2-step metabolic conversion to an active metabolite. This active metabolite inhibits adenosine diphosphate (ADP)-induced platelet aggregation by selectively and irreversibly binding P2Y12 receptors on the platelet membrane. Several isoforms of cytochrome P450 (CYP), including CYP2C19, CYP3A4, CYP1A2, CYP2B6, and CYP2C9, have been shown to be involved in the metabolic pathway. Of these enzymes, CYP2C19 is considered to be the main determinant of clopidogrel metabolism that produces the active form.
It is known that CYP2C19 has numerous single nucleotide polymorphisms (SNPs), of which CYP2C19*2 (681G>A, located in exon 5) has been studied extensively and shown to be associated with a loss of function of the enzyme. CYP2C19*2 clearly associates with both the pharmacokinetics (i.e., area under the concentration curve and maximal plasma concentration of clopidogrel active metabolite) and the pharmacodynamics (i.e., inhibition of ADP-induced platelet aggregation) of clopidogrel. CYP2C19*2 is detected more frequently in Asians than in Caucasians, with approximately 40–50% and 30% having at least one CYP2C19*2 allele, respectively. In addition to CYP2C19*2, CYP2C19*3, *4, *5, *6, *7, and *8 have been identified as loss-of-function alleles.
Methods to evaluate the effect of clopidogrel on platelet inhibition
Different laboratory tests [Table 2] can be used to assess platelet function in patients treated with clopidogrel. ADP-induced platelet aggregation in platelet-rich plasma measured by light transmission aggregometry is used most commonly, with numerous published studies using this method to measure platelet function. The majority of these studies measured platelet function as maximal platelet aggregation rate induced by 5, 10, or 20 µmol/l ADP. The platelet aggregation rate 5–8 min after the addition of ADP has also been reported. The POPULAR study [1] on clopidogrel-treated patients following elective PCI showed that 42.9% maximal platelet aggregation rate induced by 5 µmol/l ADP or 64.5% induced by 20 µmol/l ADP correlated with the 1-year mortality rate, myocardial infarction (MI), stent thrombosis, and stroke.
The VerifyNow P2Y12 test (Accumetrics Inc, SanDiego, CA) has been developed as a point-of-care device to quickly and accurately assess platelet function in patients. This test is a whole-blood, light transmission-based optical detection assay that measures the light transmittance of ADP-induced platelet aggregation in a cartridge containing fibrinogen-coated beads and is able to specifically evaluate P2Y12 receptor inhibition. The results are reported as P2Y12 reaction units (PRU), with a lower PRU value being associated with higher P2Y12 inhibition. A meta-analysis of individual patient data in six observational studies [2] revealed that a PRU value of 230 at PCI is the best cut-off value for predicting the occurrence of cardiovascular events, including death, MI, and stent thrombosis, in patients with stable CAD or non-ST elevated ACS undergoing PCI over 1 year.
The effect of clopidogrel on platelet function can be also evaluated by detecting vasodilator-stimulated phosphoprotein (VASP). VASP is phosphorylated by cyclic adenosine monophosphate (cAMP) produced in the adenylate cyclase cascade downstream of the P2Y12 receptor. By binding to the P2Y12 receptor and suppressing the cascade, ADP leads to an increase in VASP dephosphorylation, whereas inhibition of the receptor by clopidogrel active metabolite leads to an increase in VASP phosphorylation. This test measures VASP phosphorylation in a flow cytometric assay with the result expressed as platelet reactivity index (PRI) that represents the ratio of the phosphorylated and dephosphorylated VASP. A lower PRI value reflects higher P2Y12 inhibition.
Clinical utility of laboratory testing
Numerous studies, including our meta-analysis [3], have reported that patients with a suboptimal response to antiplatelet therapy have increased cardiovascular events [Figure 1A], and data have been accumulated on testing of platelet function to establish a reliable cut-off value for clinical risk. However, it remains unclear how to monitor suboptimal responses in daily clinical practice due to the lack of a standardised method to measure and interpret the results of platelet function. Furthermore, there is no guideline for alternative treatment strategies to the “one-size-fits-all” 75 mg/day clopidogrel regime because conclusive evidence that personalised antiplatelet therapy improves patient outcomes has not been established from large-scale randomised trials. However, a meta-analysis [4] recently reported the evaluation of the clinical efficacy and safety of intensified antiplatelet therapy involving reloading clopidogrel, using glycoprotein IIb/IIIa inhibitors periprocedural PCI, increasing the maintenance dose of clopidogrel, or switching to prasugrel. Although there were several limitations, this meta-analysis showed that intensified antiplatelet therapy reduces cardiovascular death and stent thrombosis without increasing major bleeding.
Meanwhile, CYP2C19 genotype does not always seem to predict cardiovascular events, although it is a major predictor for suboptimal response to clopidogrel. To date, many large-scale clinical trials, including the recent Genotype Information and Functional Testing (GIFT) trial [5], which investigate an association of CYP2C19 genotype with cardiovascular events, have been performed. However, the results of these trials were inconsistent. Indeed, we showed heterogeneity in the odds ratio of the cardiovascular events between the carriers and non-carriers of CYP2C19*2 allele in our meta-analysis [Figure 1B]. Considering that CYP2C19*2 contributes to only about 5% of the variability in response to clopidogrel [6], many other genetic factors may contribute to the variability apart from CYP2C19. Therefore, genetic testing including additional factors such as SNPs in other CYPs or ABCB1 (encoding p-glycoprotein) would be expected to improve identification of patients with a suboptimal response.
Current status and future prospects
In 2009, the U.S. Food and Drug Administration (FDA) released a black box warning that significant attention needs to be paid to clopidogrel pharmacogenomics. Similarly, the American and European guidelines published in 2011 gave a Class IIb recommendation for testing of platelet function or genetic profile in patients treated with clopidogrel and for consideration of the use of an alternate P2Y12 inhibitor in patients with inadequate platelet inhibition.
The primary goal of testing of platelet function and genetic profile is to identify patients with a suboptimal response to antiplatelet therapy and provide them with a tailor therapy to improve the clinical ischemic outcomes without an
associated bleeding risk. Although these laboratory tests provide sufficient evidence to predict outcomes, personalised antiplatelet therapy on the basis of these tests has not been established in the guidelines. Currently, several clinical trials are ongoing that evaluate the effect of personalised antiplatelet therapy on the basis of laboratory tests. These trials will hopefully provide important data to establish guidelines, to allow clinicians to properly select laboratory tests, and to plan personalised antiplatelet therapy in patients with a suboptimal response.
References
1. Breet NJ, van Werkum JW, Bouman HJ, Kelder JC, Ruven HJ, Bal ET, et al. JAMA 2010; 303: 754–762.
2. Brar SS, ten Berg J, Marcucci R, Price MJ, Valgimigli M, Kim HS, et al. J Am Coll Cardiol 2011; 58: 1945–1954.
3. Yamaguchi Y, Abe T, Sato Y, Matsubara Y, Moriki T, Murata M. Platelets. Epub 2012 Jul 3, doi: 10.3109/09537104.2012.700969
4. Aradi D, Komócsi A, Price MJ, Cuisset T, Ari H, Hazarbasanov D, et al. Int J Cardiol. Epub 2012 Jun 15, doi: 10.1016/j.ijcard.2012.05.100
5. Price MJ, Murray SS, Angiolillo DJ, Lillie E, Smith EN, Tisch RL, et al. J Am Coll Cardiol 2012; 59: 1928–1937.
6. Hochholzer W, Trenk D, Fromm MF, Valina CM, Stratz C, Bestehorn HP, et al. J Am Coll Cardiol 2010; 55: 2427–2434.
The authors
Yusuke Yamaguchi and Mitsuru Murata MD, PhD
Dept of Laboratory Medicine, Keio University School of Medicine,
35 Shinanomachi, Shinjuku-ku, Tokyo, 160-8582, Japan
E-mail: yusukeyamaguchi@z8.keio.jp
Biomarkers of vascular calcification in patients with impaired kidney function
, /in Featured Articles /by 3wmediaChronic renal failure is a disease with a high and increasing prevalence. Currently about 10% of the population of Europe and North America are affected. The disease is associated with a high morbidity and mortality mainly attributed to cardiovascular diseases. In fact patients with more advanced stages of chronic renal failure have a greater risk of dying due to cardiovascular disease than of renal failure itself. Approximately 50% of these patients die from cardiovascular complications.
by Professor Berthold Hocher
Accelerated vascular calcification (VC) is but one of the important mechanisms of cardiovascular disease in dialysis patients. Under the setting of end-stage renal disease (ESRD), VC is more severe and develops in both the intima and the media of the blood vessels. VC is an active and regulated process mediated by vascular smooth muscle cells [Fig. 1], which undergo a phenotypic change to osteoblasts or chondrocytes, which, in turn, release promoters of VC and apoptosis. VC is markedly up regulated in dialysis patients, and this may be explained by the up-regulation of such promoters of VC as hyperphosphatemia, hypercalcemia, cholesterol, hyperleptinemia down-regulation of the inhibitors of VC such as matrix Gla protein, fetuin-A [1].
Fetuin-A
Fetuin-A is a 62-kilodalton glycoprotein, which belongs to the cystatin superfamily of proteins. In humans, the 349-amino acid protein, as secreted from the liver, consists of two chains: a heavy and a light chain joined by a connecting segment and linked by disulfide bonds. The N-terminus of the heavy chain consists of two cystatin domains, D1 and D2; the acidic amino acids in the D1 domain appear to account for fetuin’s ability to inhibit precipitation of calcium and phosphorus. Indeed, fetuin-A accounts for up to one-half of the in vitro capacity of the serum to prevent the precipitation of calcium and phosphorus. It is now recognised that fetuin-A can actively regulate the cell-mediated process of osteogenesis in the vessel wall, inhibits mineralisation in a concentration-dependent manner, enhances the phagocytosis of apoptotic bodies by vascular smooth muscle cells, limiting their ability to nucleate calcium phosphate. Finally, fetuin-A is an antagonist of bone morphogenetic protein-2, the promoter of VC in vascular cells.
A number of studies have demonstrated an association between serum fetuin-A levels and all-cause mortality of dialysis patients. This association of low fetuin-A levels and mortality was confirmed by clinical trial on 664 hemodialysis (HD) and 323 peritoneal dialysis (PD) patients during a median follow-up of 2.8 years. In this study, an increase in serum fetuin-A by 0.1 g per litre corresponded to a 9% lower death risk. The death predictable value of fetuin-A in this study was independent of serum C-reactive protein (CRP) levels. At the same time, in multivariate analysis of biomarkers of prediction of mortality dialysis patients where serum C-reactive protein was entered, fetuin-A lost its predictable value. The latter fact suggests further investigation of the role of fetuin-A in dialysis patients is needed to fully elucidate the pathomechanisms lowering serum fetuin-A levels in ESRD [1].
Fibroblast growth factor 23 (FGF-23)
FGF-23 is a hormone secreted by osteoblasts. It plays a role in the regulation of phosphorus and in the metabolism of vitamin D. Depletion of FGF-23 causes hyperphosphatemia, up-regulation of 1,25- dihydroxyvitamin D, ectopic calcification and early death. FGF-23 is involved in physiological maintenance of normal serum phosphate levels in the settings of variable dietary phosphorus intake. In the settings of impaired/reduced nephron mass, normal serum phosphate levels are maintained in part by reactive increase of FGF-23, which promotes excretion of phosphate via the remaining nephrons and decreases the absorption of dietary phosphorus by inhibiting the synthesis of 1,25-dihydroxyvitamin D. Depletion of FGF-23 with chronic kidney disease (CKD) progression leads to hyperphosphatemia, ectopic calcification and premature death. It was previously reported that increased serum phosphate levels and decreased 1,25-dihydroxyvitamin D levels are associated with increased mortality.
In the recent study by Gutiérrez et al., multivariable adjusted analyses showed that an increase in serum phosphate levels higher than 5.5 mg/dl and an increase of FGF-23 was associated with a 20% increase in the mortality risk, suggesting hyperphosphatemia and increased FGF-23 are sensitive biomarkers for assessment of the risk of death [reviewed in 1].
Receptor activator of NF-kB ligand–Osteoprotegerin System Osteoblasts regulate differentiation and activation of osteoclasts under conditions of normal bone turnover. Osteoblasts synthesise and secrete a protein called receptor activator of NF-κB ligand (RANKL). RANKL binds to its receptor on pre-osteoclasts and thus regulates bone turnover. Osteoprotegerin (OPG) is also secreted by osteoblasts and modulates the effects of RANKL by blocking osteoblast differentiation. These two key players are also involved in the transformation of vascular smooth muscle cells into bone formatting cells in blood vessels under the condition of chronic renal failure.
Several studies indicate a pathogenic role of OPG in the pathogenesis of cardiovascular diseases in uremic and also non-uremic patients. The OPG/RANKL system plays a key role in the pathogenesis of endothelial function. Tseng et al. suggest that an imbalance between bone formatting hormones and bone degrading hormones may play a key role in the pathogenesis of vascular calcification. High OPG might indicate a reduced degradation capacity of calcified arteries. These authors suggest that an induction of RANKL in the vessel walls might overcome this problem and thus offer even new therapeutic options for vascular calcification. However, this hypothesis needs for sure further investigations [2–4].
Vitamin D
Vitamin D is a multifunctional hormone that can affect many essential biological functions, ranging from immune regulation to mineral ion metabolism. A close association between altered activity of vitamin D and vascular calcification has been reported in various human diseases, including patients with atherosclerosis, osteoporosis and CKD. Experimental studies have shown that excessive vitamin D activities can induce vascular calcification, and such vascular pathology can be reversed by reducing vitamin D activities. The human relevance of these experimental studies is not clear, as vitamin D toxicity is relatively rare in the general population. Contrary to the relationship between vitamin D and vascular calcification, in experimental uremic models low levels of vitamin D were shown to be associated with extensive vascular calcification – a phenomenon that is very similar to the vascular pathology seen in patients with CKD. The current treatment approach of providing vitamin D analogues to patients with CKD often poses a dilemma, as studies linked vitamin D treatment to subsequent vascular calcification. In any case, a close monitoring of the vitamin D status in patients with CKD is indicated to ensure that these patients have vitamin D levels associated with the best survival likelihood [5, 6].
Osteopontin
Osteopontin (OPN) was initially identified in osteoblasts as a mineralisation-modulatory matrix protein. Recently, OPN has been studied as a multifunctional protein that is up regulated in a variety of acute and chronic inflammatory conditions, such as wound healing, fibrosis, autoimmune disease and atherosclerosis. OPN is highly expressed at sites with atherosclerotic plaques, especially those associated with macrophages and foam cells. In the context of atherosclerosis, OPN is generally regarded as a pro-inflammatory and pro-atherogenic molecule. The role of OPN in VC, which is closely related to chronic and active inflammation, is that of a negative regulator. It is an inhibitor of calcification and an active inducer of decalcification. OPN expression and its regulatory molecular mechanisms remain elusive during the process
of vascular calcification. Therefore, further research with regard to the role of OPN in diseases associated with VC is needed to identify potential OPN-related therapeutic targets [7].
References
1. Chaykovska L, Tsuprrykov O, Hocher B. Biomarkers for the prediction of mortality and morbidity in patients with renal replacement therapy. Clin Lab 2011; 57(7–8): 455–467.
2. Shin JY, Shin YG, Chung CH. Elevated serum osteoprotegerin levels are associated with vascular endothelial dysfunction in type 2 diabetes. Diabetes Care 2006; 29(7): 1664–1666.
3. Tseng W, Graham LS, Geng Y, Reddy A, Lu J, Effros RB, et al. PKA-induced receptor activator of NF-kappaB ligand (RANKL) expression in vascular cells mediates osteoclastogenesis but not matrix calcification. J Biol Chem 2010; 285(39): 29925–29931.
4. Ozkok A, Caliskan Y, Sakaci T, Erten G, Karahan G, Ozel A, et al. Osteoprotegerin/RANKL axis and progression of coronary artery calcification in hemodialysis patients. Clin J Am Soc Nephrol 2012; 7(6): 965–973.
5. Lieb W, Gona P, Larson MG, Massaro JM, Lipinska I, Keaney JF Jr, et al. Biomarkers of the osteoprotegerin pathway: clinical correlates, subclinical disease, incident cardiovascular disease, and mortality. Arterioscler Thromb Vasc Biol 2010; 30(9): 1849–1854.
6. Ellam TJ, Chico TJ. Phosphate: the new cholesterol? The role of the phosphate axis in non-uremic vascular disease. Atherosclerosis 2012; 220(2): 310–318.
7. Ketteler M, Rothe H, Krüger T, Biggar PH, Schlieper G. Mechanisms and treatment of extraosseous calcification in chronic kidney disease. Nat Rev Nephrol 2011; 7(9): 509–516.
The author
Berthold Hocher, M.D., Ph.D.
Institute of Nutritional Science, University of Potsdam,
D-14558 Nuthetal-Potsdam, Germany
E-mail: hocher@uni-potsdam.de
www.uni-potsdam.de/eem
Molecular allergology offers new opportunities – for the lab and the clinician
, /in Autoimmunity & Allergy, Featured Articles /by 3wmediaby Dr Magnus Borres Molecular allergology enables quantification of IgE antibodies to single allergen protein components at the molecular level. This helps the clinician establish the cause of allergic sensitisation, evaluate the risk for severe allergic reactions and improve patient management. New tests and technologies enable the laboratory to assist in an efficient manner.