ORIGINAL ARTICLE
Homocysteine Levels Influence Platelet Reactivity in Coronary Artery Disease Patients Treated With Acetylsalicylic Acid Monica Verdoia, MD,* Alon Schaffer, MD,* Patrizia Pergolini, MD,† Roberta Rolla, MD,† Lucia Barbieri, MD,* Giorgio Bellomo, MD,† Fabiola Sinigaglia, MD,‡ Paolo Marino, MD,* Harry Suryapranata, MD, PhD,§ and Giuseppe De Luca, MD, PhD*‡; on behalf of the Novara Atherosclerosis Study Group (NAS)
Background: Suboptimal platelet inhibition with antiplatelet treatments is associated with a severe prognosis in patients with coronary artery disease (CAD), and the identification of its determinants is still challenging. Homocysteine elevation has emerged as a prothrombotic factor, influencing coagulative status and endothelial function and potentially modulating platelet aggregation. We therefore aimed to evaluate the effects of homocysteine (Hcy) levels on platelet reactivity in patients receiving acetylsalicylic acid (ASA) with or without ADP antagonists.
Methods: Patients undergoing coronary angiography and receiving ASA (100–160 mg daily) for .7 days, with or without ADP antagonists, were included. Aggregation tests were performed by multiple electrode aggregometry. Suboptimal platelet inhibition was defined as on-treatment aggregation above the lower limit of normality. Results: Our population is represented by 508 ASA-treated patients, 406 (80.1%) of whom on dual antiplatelet therapy (ASA and ADP antagonists). Hcy levels above the median (15.1 nmol/mL) were associated with male gender (P = 0.04), hypertension (P = 0.004), hypercholesterolemia (P = 0.03), aging, renal failure (P , 0.001, respectively), previous coronary bypass grafting (P = 0.04), therapy with calcium antagonists (P = 0.04) and diuretics (P = 0.001), and multivessel CAD (P = 0.03). Higher Hcy is directly related with serum creatinine and uric acid (P , 0.001). Suboptimal platelet inhibition was found in 16 patients (3.2%) for ASA and for ADP antagonists in 80 patients (19.7%). Hcy levels significantly affected suboptimal response to ASA, but not to ADP-mediated aggregation. In fact, a linear relationship was found between homocysteine and platelet reactivity after stimulation with arachidonic acid (r = 0.14, P = 0.004) and collagen (r = 0.12, P = 0.02), but Received for publication November 9, 2014; accepted February 3, 2015. From the *Department of Cardiology, Azienda Ospedaliera-Universitaria Maggiore della Carità, Eastern Piedmont University, Novara, Italy; †Department of Clinical Chemistry, Azienda Ospedaliera-Universitaria Maggiore della Carità, Eastern Piedmont University, Novara, Italy; ‡Department of Translational Medicine, Centro di Biotecnologie per la Ricerca Medica Applicata (BRMA), Eastern Piedmont University, Novara, Italy; and §Department of Cardiology, UMC St Radboud, Nijmegen, the Netherlands. The authors report no conflicts of interest. Reprints: Giuseppe De Luca, MD, PhD, Ospedale “Maggiore della Carità,” Eastern Piedmont University, C.so Mazzini, 18, 28100 Novara, Italy (e-mail: [email protected]). Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.
J Cardiovasc Pharmacol ä Volume 66, Number 1, July 2015
not with ADP (r = 0.02, P = 0.77). Moreover, after correction for baseline differences, Hcy above the median was confirmed as an independent predictor of impaired ASA response [adjusted odds ratio (95% confidence interval) = 3.7 (1.08–12.4), P = 0.04].
Conclusions: Among patients with CAD, elevated homocysteine is an independent predictor of suboptimal response to ASA, but not to ADP antagonists. Key Words: homocysteine, acetylsalicylic acid, platelet reactivity, impedance aggregometry, coronary artery disease (J Cardiovasc Pharmacol Ô 2015;66:35–40)
BACKGROUND Antiplatelet strategies have dramatically improved the outcome and reduced the rate of acute cardiovascular events among patients with coronary artery disease (CAD).1–3 Acetylsalicylic acid (ASA) represents the pivotal antiplatelet agent in both primary and secondary prevention,4 while dual antiplatelet therapy (DAPT), with adjunctive ADP antagonists, has significantly reduced the recurrence of ischemic events in high-risk patients, as after an acute coronary syndrome or coronary stenting.5,6 However, several studies have raised the problem of a residual high on-treatment platelet reactivity (HRPR), significantly conditioning the prognosis and the occurrence of thrombotic events in up to 10%–15% of patients.7–9 Therefore, large efforts have been accomplished to identify the predictors of suboptimal response to antiplatelet agents, with a particular focus on the role of genetics and biomarkers.10,11 Homocysteine (Hcy) is a metabolite of methionine degradation, playing a manifold and partly undefined role in pro-oxidant, proatherogenic, and prothrombotic mechanisms. Indeed, even mild hyperhomocysteinemia has been claimed for not only inducing changes in coagulation process, fibrinolysis, or endothelial dysfunction but also impairing the function of blood platelets,12 therefore conditioning the severity and outcome of patients with CAD.13,14 In fact, Hcy has been reported to enhance the metabolism of eicosanoids, leading to the overproduction of platelet thromboxanes, and also to enhance in vitro platelet adhesion.15 However, the hypothesis that Hcy levels could modulate platelet reactivity even among those patients treated with antiplatelet agents has been seldom tested, with contrasting results. www.jcvp.org |
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Therefore, aim of this study was to evaluate the relationship between Hcy levels and platelet aggregation among patients with CAD receiving chronic therapy with ASA, with or without ADP antagonists.
Statistical Analysis
METHODS We included patients with a recent admission to the Division of Cardiology at “Maggiore della Carità” Hospital, Eastern Piedmont University in Novara, Italy, from January 2009 to April 2014, having undergone coronary angiography for documented or suspected CAD. All patients were scheduled for platelet aggregation tests after .7 days of antiplatelet therapy with ASA (100–160 mg daily) or DAPT associating ASA to an ADP antagonist (clopidogrel 75 mg, prasugrel 10 mg daily, or ticagrelor 90 mg twice a day). The study was approved by our local ethical committee. Main demographic, clinical, and angiographic data, together with the indication to DAPT, were recorded at admission and included in a dedicated database. All patients were required a written informed consent. Main cardiovascular risk factors were identified; hypertension was defined as systolic pressure .140 mm Hg and/or diastolic pressure was .90 mm Hg or if the individual was taking antihypertensive medications. The diagnosis of diabetes was based on previous history of diabetes treated with or without drug therapies, fasting glucose .126 g/dL, or HbA1c .6.5% at the moment of admission. Exclusion criteria were recent administration of GPIIb/IIIa inhibitors, thrombocytopenia, thrombocytosis, anemia, and polycythemia with impaired hematocrit levels or refusal of providing written informed consent.
All statistical analyses were performed by SPSS Statistics Software 17.0 (SPSS Inc, Chicago, IL). Continuous variables were represented as mean 6 SD, while categorical variables as percentage. Chi-square and analysis of variance tests were used to compare clinical and chemistry features according to homocysteine levels. Linear regression analysis was used to evaluate the association between aggregation test results and homocysteine levels. Multiple logistic regression analysis was performed to evaluate independent predictors of HRPR, after correction for baseline differences that were entered in the model in blocks. A P-value of ,0.05 was considered statistically significant.
RESULTS
For each patient, inhibition of platelet aggregation was determined by Multiplate multiple electrode aggregometry [Roche Diagnostics (Schweiz) AG].17 A whole-blood sample was obtained from every patient and stored in Vacutainer standard lithium heparin tubes. The aggregation tests were performed from 30 minutes to 2 hours from blood collection. Platelet aggregation was assessed after stimulation with arachidonic acid (AA) (0.5 mM) (ASPI test), collagen (3.2 mg/mL) (COL test), ADP (6.4 mM) with prostaglandin E1, and thrombin receptor–activating peptide (TRAP-6; 30 mM). Results were expressed as arbitrary aggregation units (AUs) and plotted against time, defining platelet function as area under the curve (AUC or AU*min). HRPR with ASA was considered for ASPI test AU*min above lower limit normal (normal range, 862–1344), while suboptimal response to ADP antagonists was defined for ADP test AU*min .lower limit normal (normal range, 417–1030).
Our population is represented by 508 ASA-treated patients. Among them, 406 (80.1%) were additionally treated with ADP antagonists (DAPT). Patients were divided according to Hcy median values (15.1 nmol/mL). Table 1 shows main clinical, demographic, and chemistry features of the included population. Higher Hcy levels were associated with male gender (P = 0.04), hypertension (P = 0.004), hypercholesterolemia (P = 0.03), aging, renal failure (P , 0.001, respectively), previous coronary bypass grafting (P = 0.04), therapy with calcium antagonists (P = 0.04) and diuretics (P = 0.001), and multivessel CAD (P = 0.03). Hcy elevation is directly related with serum creatinine and uric acid (P , 0.001). Suboptimal platelet inhibition with ASA was found in 16 patients (3.2%) at ASPI test, with a nonsignificant higher rate among patients with Hcy above the median {3.9% vs. 2.4%, P = 0.31; odds ratio [OR] [95% confidence interval (CI)] = 1.69 [0.60–4.7], P = 0.31}. Among the 406 patients receiving DAPT, 80 (19.7%) displayed HRPR at ADP test, with no relationship with Hcy elevation [18.2% vs. 21%, P = 0.43; OR (95% CI) = 0.82 (0.50–1.35), P = 0.43]. However, when evaluating aggregation results as continuous variables, a linear relationship was found between homocysteine and platelet reactivity after stimulation with AA (ASPI test: r = 0.14, P = 0.004), in overall population, as shown in Figure 1 and when evaluating separately patients receiving or not an ADP antagonist (DAPT: r = 0.1, P = 0.058; ASA monotherapy: r = 0.19, P = 0.08). Similar results were found also after platelet stimulation with collagen (COL test: r = 0.12, P = 0.02; Fig. 2). Hcy levels resulted slightly elevated in patients with HRPR at ASPI test (20.3 6 11.9 nmol/mL vs. 17.1 6 8.2 nmol/mL, P = 0.14), and after correction for baseline differences, Hcy above the median resulted as an independent predictor of HRPR with ASA [adjusted OR (95% CI) = 3.7 (1.08–12.4), P = 0.04]. On the contrary, ADP-mediated platelet aggregation was not related to Hcy levels, in both patients receiving ADP antagonists (r = 0.02, P = 0.77, Fig. 3A) and in patients not on DAPT (r = 0.03, P = 0.78, Fig. 3B), and results were confirmed at multivariate analysis [adjusted OR (95% CI) = 0.70 (0.40–1.24), P = 0.23].
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Biochemistry Analysis Blood samples were drawn after a fasting period of 12 hours. Glucose, creatinine, glycosylated hemoglobin, and lipid profile were determined as previously described.16 Blood cell count was performed in a blood sample collected in tripotassium EDTA (7.2 mg) tubes. These blood samples were analyzed within 2 hours of venipuncture by automatic blood counter (Sysmex XE-2100).
Platelet Aggregation
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TABLE 1. Clinical and Demographic Characteristics According to Homocysteine Median Values (15.1 nmol/mL) Baseline Clinical Characteristics
Hcy , 15.1 (n = 254)
Hcy ‡ 15.1 (n = 254)
Age (mean 6 SD) Male gender, % Dyslipidemia, % Diabetes mellitus, % Renal failure, % Active smokers, % Hypertension, % History of MI, % Previous PCI, % Previous CABG, % Biochemistry Platelet counts, 103/mL Hemoglobin, g/dL White blood cells, 103/mL Creatinine, mg/dL Glycemia, mg/dL Total cholesterol, mg/dL Triglycerides, mg/dL HbA1c, % C-reactive protein, mg/dL Uric acid, mg/dL Indication to angiography Stable angina/Silent ischemia, % Acute coronary syndrome, % Arrhythmias/ Valvulopathy/LV dysfunction, % Multivessel CAD Therapy at admission ACE inhibitors, % ARB, % Beta blockers, % Calcium antagonists, % Nitrates, % Diuretics, % Statins, % ADP antagonists Clopidogrel, % Ticagrelor/ Prasugrel, %
64.8 6 10.6 71.1 68 30.6 6.3 21.7 66 21.8 30 10.3
69.2 6 11 79.1 58.2 32.5 20.3 25.1 77.7 28.2 31.7 13.7
217.2 13.6 8.4 0.9 131.1 155.1 134 6.4 0.9 5.6
6 6 6 6 6 6 6 6 6 6
54.8 1.5 4.7 0.4 55.7 39.2 74.3 1.6 1.9 1.5
227.3 13.5 8.2 1.2 128.4 156.8 145.7 6.4 1.1 6.4
6 6 6 6 6 6 6 6 6 6
71.5 1.7 2.5 0.7 64.9 41.8 103.7 1.4 2.4 2
P ,0.001 0.04 0.03 0.51 ,0.001 0.65 0.004 0.12 0.70 0.04 0.07 0.74 0.59 ,0.001 0.62 0.64 0.15 0.72 0.13 ,0.001 0.35
26.3
30.6
66.3
62.4
7.4
6.9
50
59
0.03
35.6 20.9 51.8 17 37.5 21.3 54.9
36 26.5 58.1 24.9 38.3 34.4 53.8
0.99 0.17 0.18 0.04 0.93 0.001 0.86 0.09
60.2 21.3
57 28.2
ACE, angiotensin converting enzyme; ADP, adenosine diphosphate; ARB, angiotensin receptor blocker; CABG, coronary artery bypass graft; CAD, coronary artery disease; MI, myocardial infarction; PCI, percutaneous coronary intervention.
DISCUSSION This study is the largest report evaluating the relationship between homocysteine levels and the extent of platelet aggregation in a cohort of CAD patients treated with ASA or DAPT. Our main findings are that hyperhomocysteinemia is an independent predictor of suboptimal response to ASA, but it does not influence ADP-mediated platelet aggregation. High on-treatment platelet reactivity still represents one of the Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.
Homocysteine, ASA, and Platelet Reactivity
most intriguing challenges for patients with cardiovascular disease.18 In fact, despite a recent meta-analysis clearly demonstrating that reducing HRPR significantly decreases ischemic events,19 the great efforts for progressive dose adjustments,20 and the introduction of new more potent antiplatelet agents,21 still suboptimal platelet inhibition is observed in a large percentage of patients, thus influencing the outcomes. Several explanations have been proposed for the “resistance” to antiplatelet agents, and especially for clopidogrel, where genetic polymorphisms and variable absorption have been identified as conditioning its effectiveness.10,22 However, HRPR has been described in 0.5%–75% of patients with ASA, which represents the basis of every antiplatelet strategy, and the recent results of the ISAR-ASPI study have demonstrated in over 7000 post-PCI patients a significant impact of high on-aspirin treatment platelet reactivity, assessed by Multiplate impedance aggregometry, on the occurrence of death or stent thrombosis at 12 months.23 Indeed, metabolic disorders and circulating factors, such as in diabetes, have been suggested to increase platelet activity, thus potentially influencing the effectiveness of antiplatelet drugs.24,25 Homocysteine is an intermediate amino acid derived from methionine metabolism, whose plasmatic elevation is quite frequent, being dependent from diet, drugs, genetic asset, and clinical status, and mainly from renal excretion. Previous studies reported that even mild homocysteine elevation could increase the cardiovascular risk, by modulating both the progression of atherosclerosis and especially by favoring thrombotic complications.26–28 In fact, Hcy has been reported to influence the normal coagulation balance, with activation of factor V29 and suppression of thrombomodulin and protein C.30 Moreover, hyperhomocysteinemia can rapidly blunt endothelial function in both hypertensive and healthy individuals, by raising the levels of endothelin-1, von Willebrand factor, and other markers of endothelial activation.31,32 However, further studies have suggested that the prothrombotic effect of Hcy could depend on a direct effect on platelets, as Hcy enhances the production of pro-oxidant species that can induce inflammation and therefore favor platelet activation. Indeed, Spencer et al33 documented a correlation between Hcy and P-selectin, an indicator of platelet activation, among 165 hypertensive patients. Similar results were reported in vitro in a Russian study, where platelet incubation with Hcy enhanced adhesion of thrombin-activated platelets to collagen and fibrinogen,34 while Signorello et al15 identified a higher production of thromboxanes in platelets incubated with homocysteine. However, few studies have addressed so far the role of hyperhomocysteinemia in modulating the response to antiplatelet therapy. Zhang et al35 reported a higher rate of ASA “resistance” among hemodialysis patients, with Hcy emerging as an independent predictor. More recently, Karolczak et al36 included 126 patients with CAD, finding that the ASA-mediated reductions in platelet aggregation were significantly related with Hcy levels and that Hcy diminished in vitro antiplatelet effect of ASA and augmented platelet aggregation by up to 62% for collagen stimulation and 15% with AA. www.jcvp.org |
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FIGURE 1. Linear regression analysis relating homocysteine levels to platelet aggregation [area under the curve (AUC)] after AA stimulation (ASPI test).
On the contrary, a French study documented in patients with cerebrovascular ischemic disorders an elevation of Hcy levels and of the indicators of endothelial dysfunction, but not of platelet activation.32 This study confirms, in a large-scale population of patients with CAD, that hyperhomocysteinemia is significantly associated to platelet reactivity and suboptimal ASA response by the use of 2 different whole-blood tests. Indeed, Hcy elevation was associated to a worse cardiovascular risk profile, and especially to renal failure; however, Hcy $ 15.1 nmol/mL was confirmed as an independent predictor of HRPR with AA after correction for potential confounders. An experimental study in rats showed that Hcy modulated adenosine metabolism, thus conditioning platelet
reactivity.37 However, no study on ADP-mediated aggregation has been conducted so far in humans and especially not in patients receiving DAPT. Moreover, this is the first study investigating the impact of Hcy levels on response to ADP antagonists, showing no impact of Hcy levels on ADPmediated platelet aggregation. Present results may suggest the need of higher dosage of ASA, to overcome the phenomenon of HRPR in patients with Hcy elevation, as in fact, overall higher platelet aggregation, in response to collagen, was maintained even despite DAPT. In fact, we previously reported a lack of association between Hcy levels and periprocedural myocardial injury in patients undergoing PCI, where the use of glycoprotein IIb/IIIa inhibitors in the majority of patients, in
FIGURE 2. Linear regression analysis relating homocysteine levels to platelet aggregation [area under the curve (AUC)] after collagen stimulation (COL test).
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Homocysteine, ASA, and Platelet Reactivity
FIGURE 3. Linear regression analysis relating homocysteine levels to platelet aggregation [area under the curve (AUC)] after adenosine diphosphate stimulation in patients treated with (A) or without (B) ADP antagonists.
association with DAPT, could probably have prevented a large quote of events.38 In addition, Hcy-lowering strategies could be addressed to prevent HRPR. In a previous study, folate administration significantly reduced the urinary excretion of 11-dehydro-TXB2, a degradation product of platelet thromboxanes, among hyperhomocysteinemic patients.39 However, the clinical benefits of homocysteine-lowering therapies in cardiovascular disease are still debated.40,41 Therefore, future large-scale studies should be advised to evaluate whether folate supplementation can reduce platelet reactivity among patients with Hcy elevation.
Limitations Availability of long-term clinical follow-up data with the evaluation of the clinical impact elevated homocysteine and HRPR on the occurrence of thrombotic events would have certainly strengthened the conclusions of our study. This issue certainly needs investigations in future trials. Furthermore, although the population of ASA only–treated patients represented only about 20% of our patients, AAmediated platelet aggregation (as evaluated by the ASPI test) is strictly dependent on the metabolic activity of the cyclooxygenase and cannot be modified in the presence of an ADP antagonist. In fact, similar results were observed in patients with or without ADP antagonists. In addition, we did not consider high-dose ASA in patients displaying HRPR with standard therapy. In fact, high-dose ASA is not currently recommended and not used in clinical practice in the majority of countries, as previous large trials have reported no benefit of such strategy, with potential harmful effects of increased bleeding risk.42 Finally, we did not perform light transmission aggregometry tests, which still remain the gold standard for platelet function assessment, but rather, we preferred bedside wholeblood aggregation tests, being more applicable in everyday clinical practice, whose clinical impact in patients on antiplatelet treatment has been previously validated.43 Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.
CONCLUSIONS Among patients with CAD, elevated homocysteine is independently associated with suboptimal response to ASA, but not to ADP antagonists. REFERENCES 1. De Luca G, Suryapranata H, Marino P. Reperfusion strategies in acute ST-elevation myocardial infarction: an overview of current status. Prog Cardiovasc Dis. 2008;50:352–382. 2. De Luca G, Smit JJ, Ernst N, et al. Impact of adjunctive tirofiban administration on myocardial perfusion and mortality in patients undergoing primary angioplasty for ST-segment elevation myocardial infarction. Thromb Haemost. 2005;93:820–823. 3. De Luca G, Ucci G, Cassetti E, et al. Benefits from small molecule administration as compared with abciximab among patients with STsegment elevation myocardial infarction treated with primary angioplasty: a meta-analysis. J Am Coll Cardiol. 2009;53:1668–1673. 4. Antithrombotic Trialists’ Collaboration. Collaborative meta-analysis of randomised trials of antiplatelet therapy for prevention of death, myocardial infarction, and stroke in high risk patients. BMJ. 2002;324:71–86. 5. Brener SJ, Steinhubl SR, Berger PB, et al; CREDO Investigators. Prolonged dual antiplatelet therapy after percutaneous coronary intervention reduces ischemic events without affecting the need for repeat revascularization: insights from the CREDO trial. J Invasive Cardiol. 2007;19:287–290. 6. Schnorbus B, Daiber A, Jurk K, et al. Effects of clopidogrel, prasugrel and ticagrelor on endothelial function, inflammatory and oxidative stress parameters and platelet function in patients undergoing coronary artery stenting for an acute coronary syndrome. A randomised, prospective, controlled study. BMJ Open. 2014;4:e005268. 7. Steinhubl SR, Talley JD, Braden GA, et al. Point-of-care measured platelet inhibition correlates with a reduced risk of an adverse cardiac event after percutaneous coronary intervention: results of the GOLD (AU-Assessing Ultegra) multicenter study. Circulation. 2001;103:2572–2578. 8. Collet JP, Cuisset T, Rangé G, et al; ARCTIC Investigators. Bedside monitoring to adjust antiplatelet therapy for coronary stenting. N Engl J Med. 2012;367:2100–2109. 9. Pettersen AÅ, Seljeflot I, Abdelnoor M, et al. High on-aspirin platelet reactivity and clinical outcome in patients with stable coronary artery disease: results from ASCET (Aspirin Nonresponsiveness and Clopidogrel Endpoint trial). J Am Heart Assoc. 2012;1:e000703. 10. Laine M, Arméro S, Peyrol M, et al. Clinical impact of genetically determined platelet reactivity. J Cardiovasc Transl Res. 2013;6:398–403. 11. Bernlochner I, Steinhubl S, Braun S, et al. Association between inflammatory biomarkers and platelet aggregation in patients under chronic clopidogrel treatment. Thromb Haemost. 2010;104:1193–1200.
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Homocysteine Levels Influence Platelet Reactivity in Coronary Artery Disease Patients Treated With Acetylsalicylic Acid Monica Verdoia, MD,* Alon Schaffer, MD,* Patrizia Pergolini, MD,† Roberta Rolla, MD,† Lucia Barbieri, MD,* Giorgio Bellomo, MD,† Fabiola Sinigaglia, MD,‡ Paolo Marino, MD,* Harry Suryapranata, MD, PhD,§ and Giuseppe De Luca, MD, PhD*‡; on behalf of the Novara Atherosclerosis Study Group (NAS)
Background: Suboptimal platelet inhibition with antiplatelet treatments is associated with a severe prognosis in patients with coronary artery disease (CAD), and the identification of its determinants is still challenging. Homocysteine elevation has emerged as a prothrombotic factor, influencing coagulative status and endothelial function and potentially modulating platelet aggregation. We therefore aimed to evaluate the effects of homocysteine (Hcy) levels on platelet reactivity in patients receiving acetylsalicylic acid (ASA) with or without ADP antagonists.
Methods: Patients undergoing coronary angiography and receiving ASA (100–160 mg daily) for .7 days, with or without ADP antagonists, were included. Aggregation tests were performed by multiple electrode aggregometry. Suboptimal platelet inhibition was defined as on-treatment aggregation above the lower limit of normality. Results: Our population is represented by 508 ASA-treated patients, 406 (80.1%) of whom on dual antiplatelet therapy (ASA and ADP antagonists). Hcy levels above the median (15.1 nmol/mL) were associated with male gender (P = 0.04), hypertension (P = 0.004), hypercholesterolemia (P = 0.03), aging, renal failure (P , 0.001, respectively), previous coronary bypass grafting (P = 0.04), therapy with calcium antagonists (P = 0.04) and diuretics (P = 0.001), and multivessel CAD (P = 0.03). Higher Hcy is directly related with serum creatinine and uric acid (P , 0.001). Suboptimal platelet inhibition was found in 16 patients (3.2%) for ASA and for ADP antagonists in 80 patients (19.7%). Hcy levels significantly affected suboptimal response to ASA, but not to ADP-mediated aggregation. In fact, a linear relationship was found between homocysteine and platelet reactivity after stimulation with arachidonic acid (r = 0.14, P = 0.004) and collagen (r = 0.12, P = 0.02), but Received for publication November 9, 2014; accepted February 3, 2015. From the *Department of Cardiology, Azienda Ospedaliera-Universitaria Maggiore della Carità, Eastern Piedmont University, Novara, Italy; †Department of Clinical Chemistry, Azienda Ospedaliera-Universitaria Maggiore della Carità, Eastern Piedmont University, Novara, Italy; ‡Department of Translational Medicine, Centro di Biotecnologie per la Ricerca Medica Applicata (BRMA), Eastern Piedmont University, Novara, Italy; and §Department of Cardiology, UMC St Radboud, Nijmegen, the Netherlands. The authors report no conflicts of interest. Reprints: Giuseppe De Luca, MD, PhD, Ospedale “Maggiore della Carità,” Eastern Piedmont University, C.so Mazzini, 18, 28100 Novara, Italy (e-mail: [email protected]). Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.
J Cardiovasc Pharmacol ä Volume 66, Number 1, July 2015
not with ADP (r = 0.02, P = 0.77). Moreover, after correction for baseline differences, Hcy above the median was confirmed as an independent predictor of impaired ASA response [adjusted odds ratio (95% confidence interval) = 3.7 (1.08–12.4), P = 0.04].
Conclusions: Among patients with CAD, elevated homocysteine is an independent predictor of suboptimal response to ASA, but not to ADP antagonists. Key Words: homocysteine, acetylsalicylic acid, platelet reactivity, impedance aggregometry, coronary artery disease (J Cardiovasc Pharmacol Ô 2015;66:35–40)
BACKGROUND Antiplatelet strategies have dramatically improved the outcome and reduced the rate of acute cardiovascular events among patients with coronary artery disease (CAD).1–3 Acetylsalicylic acid (ASA) represents the pivotal antiplatelet agent in both primary and secondary prevention,4 while dual antiplatelet therapy (DAPT), with adjunctive ADP antagonists, has significantly reduced the recurrence of ischemic events in high-risk patients, as after an acute coronary syndrome or coronary stenting.5,6 However, several studies have raised the problem of a residual high on-treatment platelet reactivity (HRPR), significantly conditioning the prognosis and the occurrence of thrombotic events in up to 10%–15% of patients.7–9 Therefore, large efforts have been accomplished to identify the predictors of suboptimal response to antiplatelet agents, with a particular focus on the role of genetics and biomarkers.10,11 Homocysteine (Hcy) is a metabolite of methionine degradation, playing a manifold and partly undefined role in pro-oxidant, proatherogenic, and prothrombotic mechanisms. Indeed, even mild hyperhomocysteinemia has been claimed for not only inducing changes in coagulation process, fibrinolysis, or endothelial dysfunction but also impairing the function of blood platelets,12 therefore conditioning the severity and outcome of patients with CAD.13,14 In fact, Hcy has been reported to enhance the metabolism of eicosanoids, leading to the overproduction of platelet thromboxanes, and also to enhance in vitro platelet adhesion.15 However, the hypothesis that Hcy levels could modulate platelet reactivity even among those patients treated with antiplatelet agents has been seldom tested, with contrasting results. www.jcvp.org |
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Therefore, aim of this study was to evaluate the relationship between Hcy levels and platelet aggregation among patients with CAD receiving chronic therapy with ASA, with or without ADP antagonists.
Statistical Analysis
METHODS We included patients with a recent admission to the Division of Cardiology at “Maggiore della Carità” Hospital, Eastern Piedmont University in Novara, Italy, from January 2009 to April 2014, having undergone coronary angiography for documented or suspected CAD. All patients were scheduled for platelet aggregation tests after .7 days of antiplatelet therapy with ASA (100–160 mg daily) or DAPT associating ASA to an ADP antagonist (clopidogrel 75 mg, prasugrel 10 mg daily, or ticagrelor 90 mg twice a day). The study was approved by our local ethical committee. Main demographic, clinical, and angiographic data, together with the indication to DAPT, were recorded at admission and included in a dedicated database. All patients were required a written informed consent. Main cardiovascular risk factors were identified; hypertension was defined as systolic pressure .140 mm Hg and/or diastolic pressure was .90 mm Hg or if the individual was taking antihypertensive medications. The diagnosis of diabetes was based on previous history of diabetes treated with or without drug therapies, fasting glucose .126 g/dL, or HbA1c .6.5% at the moment of admission. Exclusion criteria were recent administration of GPIIb/IIIa inhibitors, thrombocytopenia, thrombocytosis, anemia, and polycythemia with impaired hematocrit levels or refusal of providing written informed consent.
All statistical analyses were performed by SPSS Statistics Software 17.0 (SPSS Inc, Chicago, IL). Continuous variables were represented as mean 6 SD, while categorical variables as percentage. Chi-square and analysis of variance tests were used to compare clinical and chemistry features according to homocysteine levels. Linear regression analysis was used to evaluate the association between aggregation test results and homocysteine levels. Multiple logistic regression analysis was performed to evaluate independent predictors of HRPR, after correction for baseline differences that were entered in the model in blocks. A P-value of ,0.05 was considered statistically significant.
RESULTS
For each patient, inhibition of platelet aggregation was determined by Multiplate multiple electrode aggregometry [Roche Diagnostics (Schweiz) AG].17 A whole-blood sample was obtained from every patient and stored in Vacutainer standard lithium heparin tubes. The aggregation tests were performed from 30 minutes to 2 hours from blood collection. Platelet aggregation was assessed after stimulation with arachidonic acid (AA) (0.5 mM) (ASPI test), collagen (3.2 mg/mL) (COL test), ADP (6.4 mM) with prostaglandin E1, and thrombin receptor–activating peptide (TRAP-6; 30 mM). Results were expressed as arbitrary aggregation units (AUs) and plotted against time, defining platelet function as area under the curve (AUC or AU*min). HRPR with ASA was considered for ASPI test AU*min above lower limit normal (normal range, 862–1344), while suboptimal response to ADP antagonists was defined for ADP test AU*min .lower limit normal (normal range, 417–1030).
Our population is represented by 508 ASA-treated patients. Among them, 406 (80.1%) were additionally treated with ADP antagonists (DAPT). Patients were divided according to Hcy median values (15.1 nmol/mL). Table 1 shows main clinical, demographic, and chemistry features of the included population. Higher Hcy levels were associated with male gender (P = 0.04), hypertension (P = 0.004), hypercholesterolemia (P = 0.03), aging, renal failure (P , 0.001, respectively), previous coronary bypass grafting (P = 0.04), therapy with calcium antagonists (P = 0.04) and diuretics (P = 0.001), and multivessel CAD (P = 0.03). Hcy elevation is directly related with serum creatinine and uric acid (P , 0.001). Suboptimal platelet inhibition with ASA was found in 16 patients (3.2%) at ASPI test, with a nonsignificant higher rate among patients with Hcy above the median {3.9% vs. 2.4%, P = 0.31; odds ratio [OR] [95% confidence interval (CI)] = 1.69 [0.60–4.7], P = 0.31}. Among the 406 patients receiving DAPT, 80 (19.7%) displayed HRPR at ADP test, with no relationship with Hcy elevation [18.2% vs. 21%, P = 0.43; OR (95% CI) = 0.82 (0.50–1.35), P = 0.43]. However, when evaluating aggregation results as continuous variables, a linear relationship was found between homocysteine and platelet reactivity after stimulation with AA (ASPI test: r = 0.14, P = 0.004), in overall population, as shown in Figure 1 and when evaluating separately patients receiving or not an ADP antagonist (DAPT: r = 0.1, P = 0.058; ASA monotherapy: r = 0.19, P = 0.08). Similar results were found also after platelet stimulation with collagen (COL test: r = 0.12, P = 0.02; Fig. 2). Hcy levels resulted slightly elevated in patients with HRPR at ASPI test (20.3 6 11.9 nmol/mL vs. 17.1 6 8.2 nmol/mL, P = 0.14), and after correction for baseline differences, Hcy above the median resulted as an independent predictor of HRPR with ASA [adjusted OR (95% CI) = 3.7 (1.08–12.4), P = 0.04]. On the contrary, ADP-mediated platelet aggregation was not related to Hcy levels, in both patients receiving ADP antagonists (r = 0.02, P = 0.77, Fig. 3A) and in patients not on DAPT (r = 0.03, P = 0.78, Fig. 3B), and results were confirmed at multivariate analysis [adjusted OR (95% CI) = 0.70 (0.40–1.24), P = 0.23].
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Biochemistry Analysis Blood samples were drawn after a fasting period of 12 hours. Glucose, creatinine, glycosylated hemoglobin, and lipid profile were determined as previously described.16 Blood cell count was performed in a blood sample collected in tripotassium EDTA (7.2 mg) tubes. These blood samples were analyzed within 2 hours of venipuncture by automatic blood counter (Sysmex XE-2100).
Platelet Aggregation
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TABLE 1. Clinical and Demographic Characteristics According to Homocysteine Median Values (15.1 nmol/mL) Baseline Clinical Characteristics
Hcy , 15.1 (n = 254)
Hcy ‡ 15.1 (n = 254)
Age (mean 6 SD) Male gender, % Dyslipidemia, % Diabetes mellitus, % Renal failure, % Active smokers, % Hypertension, % History of MI, % Previous PCI, % Previous CABG, % Biochemistry Platelet counts, 103/mL Hemoglobin, g/dL White blood cells, 103/mL Creatinine, mg/dL Glycemia, mg/dL Total cholesterol, mg/dL Triglycerides, mg/dL HbA1c, % C-reactive protein, mg/dL Uric acid, mg/dL Indication to angiography Stable angina/Silent ischemia, % Acute coronary syndrome, % Arrhythmias/ Valvulopathy/LV dysfunction, % Multivessel CAD Therapy at admission ACE inhibitors, % ARB, % Beta blockers, % Calcium antagonists, % Nitrates, % Diuretics, % Statins, % ADP antagonists Clopidogrel, % Ticagrelor/ Prasugrel, %
64.8 6 10.6 71.1 68 30.6 6.3 21.7 66 21.8 30 10.3
69.2 6 11 79.1 58.2 32.5 20.3 25.1 77.7 28.2 31.7 13.7
217.2 13.6 8.4 0.9 131.1 155.1 134 6.4 0.9 5.6
6 6 6 6 6 6 6 6 6 6
54.8 1.5 4.7 0.4 55.7 39.2 74.3 1.6 1.9 1.5
227.3 13.5 8.2 1.2 128.4 156.8 145.7 6.4 1.1 6.4
6 6 6 6 6 6 6 6 6 6
71.5 1.7 2.5 0.7 64.9 41.8 103.7 1.4 2.4 2
P ,0.001 0.04 0.03 0.51 ,0.001 0.65 0.004 0.12 0.70 0.04 0.07 0.74 0.59 ,0.001 0.62 0.64 0.15 0.72 0.13 ,0.001 0.35
26.3
30.6
66.3
62.4
7.4
6.9
50
59
0.03
35.6 20.9 51.8 17 37.5 21.3 54.9
36 26.5 58.1 24.9 38.3 34.4 53.8
0.99 0.17 0.18 0.04 0.93 0.001 0.86 0.09
60.2 21.3
57 28.2
ACE, angiotensin converting enzyme; ADP, adenosine diphosphate; ARB, angiotensin receptor blocker; CABG, coronary artery bypass graft; CAD, coronary artery disease; MI, myocardial infarction; PCI, percutaneous coronary intervention.
DISCUSSION This study is the largest report evaluating the relationship between homocysteine levels and the extent of platelet aggregation in a cohort of CAD patients treated with ASA or DAPT. Our main findings are that hyperhomocysteinemia is an independent predictor of suboptimal response to ASA, but it does not influence ADP-mediated platelet aggregation. High on-treatment platelet reactivity still represents one of the Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.
Homocysteine, ASA, and Platelet Reactivity
most intriguing challenges for patients with cardiovascular disease.18 In fact, despite a recent meta-analysis clearly demonstrating that reducing HRPR significantly decreases ischemic events,19 the great efforts for progressive dose adjustments,20 and the introduction of new more potent antiplatelet agents,21 still suboptimal platelet inhibition is observed in a large percentage of patients, thus influencing the outcomes. Several explanations have been proposed for the “resistance” to antiplatelet agents, and especially for clopidogrel, where genetic polymorphisms and variable absorption have been identified as conditioning its effectiveness.10,22 However, HRPR has been described in 0.5%–75% of patients with ASA, which represents the basis of every antiplatelet strategy, and the recent results of the ISAR-ASPI study have demonstrated in over 7000 post-PCI patients a significant impact of high on-aspirin treatment platelet reactivity, assessed by Multiplate impedance aggregometry, on the occurrence of death or stent thrombosis at 12 months.23 Indeed, metabolic disorders and circulating factors, such as in diabetes, have been suggested to increase platelet activity, thus potentially influencing the effectiveness of antiplatelet drugs.24,25 Homocysteine is an intermediate amino acid derived from methionine metabolism, whose plasmatic elevation is quite frequent, being dependent from diet, drugs, genetic asset, and clinical status, and mainly from renal excretion. Previous studies reported that even mild homocysteine elevation could increase the cardiovascular risk, by modulating both the progression of atherosclerosis and especially by favoring thrombotic complications.26–28 In fact, Hcy has been reported to influence the normal coagulation balance, with activation of factor V29 and suppression of thrombomodulin and protein C.30 Moreover, hyperhomocysteinemia can rapidly blunt endothelial function in both hypertensive and healthy individuals, by raising the levels of endothelin-1, von Willebrand factor, and other markers of endothelial activation.31,32 However, further studies have suggested that the prothrombotic effect of Hcy could depend on a direct effect on platelets, as Hcy enhances the production of pro-oxidant species that can induce inflammation and therefore favor platelet activation. Indeed, Spencer et al33 documented a correlation between Hcy and P-selectin, an indicator of platelet activation, among 165 hypertensive patients. Similar results were reported in vitro in a Russian study, where platelet incubation with Hcy enhanced adhesion of thrombin-activated platelets to collagen and fibrinogen,34 while Signorello et al15 identified a higher production of thromboxanes in platelets incubated with homocysteine. However, few studies have addressed so far the role of hyperhomocysteinemia in modulating the response to antiplatelet therapy. Zhang et al35 reported a higher rate of ASA “resistance” among hemodialysis patients, with Hcy emerging as an independent predictor. More recently, Karolczak et al36 included 126 patients with CAD, finding that the ASA-mediated reductions in platelet aggregation were significantly related with Hcy levels and that Hcy diminished in vitro antiplatelet effect of ASA and augmented platelet aggregation by up to 62% for collagen stimulation and 15% with AA. www.jcvp.org |
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FIGURE 1. Linear regression analysis relating homocysteine levels to platelet aggregation [area under the curve (AUC)] after AA stimulation (ASPI test).
On the contrary, a French study documented in patients with cerebrovascular ischemic disorders an elevation of Hcy levels and of the indicators of endothelial dysfunction, but not of platelet activation.32 This study confirms, in a large-scale population of patients with CAD, that hyperhomocysteinemia is significantly associated to platelet reactivity and suboptimal ASA response by the use of 2 different whole-blood tests. Indeed, Hcy elevation was associated to a worse cardiovascular risk profile, and especially to renal failure; however, Hcy $ 15.1 nmol/mL was confirmed as an independent predictor of HRPR with AA after correction for potential confounders. An experimental study in rats showed that Hcy modulated adenosine metabolism, thus conditioning platelet
reactivity.37 However, no study on ADP-mediated aggregation has been conducted so far in humans and especially not in patients receiving DAPT. Moreover, this is the first study investigating the impact of Hcy levels on response to ADP antagonists, showing no impact of Hcy levels on ADPmediated platelet aggregation. Present results may suggest the need of higher dosage of ASA, to overcome the phenomenon of HRPR in patients with Hcy elevation, as in fact, overall higher platelet aggregation, in response to collagen, was maintained even despite DAPT. In fact, we previously reported a lack of association between Hcy levels and periprocedural myocardial injury in patients undergoing PCI, where the use of glycoprotein IIb/IIIa inhibitors in the majority of patients, in
FIGURE 2. Linear regression analysis relating homocysteine levels to platelet aggregation [area under the curve (AUC)] after collagen stimulation (COL test).
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Homocysteine, ASA, and Platelet Reactivity
FIGURE 3. Linear regression analysis relating homocysteine levels to platelet aggregation [area under the curve (AUC)] after adenosine diphosphate stimulation in patients treated with (A) or without (B) ADP antagonists.
association with DAPT, could probably have prevented a large quote of events.38 In addition, Hcy-lowering strategies could be addressed to prevent HRPR. In a previous study, folate administration significantly reduced the urinary excretion of 11-dehydro-TXB2, a degradation product of platelet thromboxanes, among hyperhomocysteinemic patients.39 However, the clinical benefits of homocysteine-lowering therapies in cardiovascular disease are still debated.40,41 Therefore, future large-scale studies should be advised to evaluate whether folate supplementation can reduce platelet reactivity among patients with Hcy elevation.
Limitations Availability of long-term clinical follow-up data with the evaluation of the clinical impact elevated homocysteine and HRPR on the occurrence of thrombotic events would have certainly strengthened the conclusions of our study. This issue certainly needs investigations in future trials. Furthermore, although the population of ASA only–treated patients represented only about 20% of our patients, AAmediated platelet aggregation (as evaluated by the ASPI test) is strictly dependent on the metabolic activity of the cyclooxygenase and cannot be modified in the presence of an ADP antagonist. In fact, similar results were observed in patients with or without ADP antagonists. In addition, we did not consider high-dose ASA in patients displaying HRPR with standard therapy. In fact, high-dose ASA is not currently recommended and not used in clinical practice in the majority of countries, as previous large trials have reported no benefit of such strategy, with potential harmful effects of increased bleeding risk.42 Finally, we did not perform light transmission aggregometry tests, which still remain the gold standard for platelet function assessment, but rather, we preferred bedside wholeblood aggregation tests, being more applicable in everyday clinical practice, whose clinical impact in patients on antiplatelet treatment has been previously validated.43 Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.
CONCLUSIONS Among patients with CAD, elevated homocysteine is independently associated with suboptimal response to ASA, but not to ADP antagonists. REFERENCES 1. De Luca G, Suryapranata H, Marino P. Reperfusion strategies in acute ST-elevation myocardial infarction: an overview of current status. Prog Cardiovasc Dis. 2008;50:352–382. 2. De Luca G, Smit JJ, Ernst N, et al. Impact of adjunctive tirofiban administration on myocardial perfusion and mortality in patients undergoing primary angioplasty for ST-segment elevation myocardial infarction. Thromb Haemost. 2005;93:820–823. 3. De Luca G, Ucci G, Cassetti E, et al. Benefits from small molecule administration as compared with abciximab among patients with STsegment elevation myocardial infarction treated with primary angioplasty: a meta-analysis. J Am Coll Cardiol. 2009;53:1668–1673. 4. Antithrombotic Trialists’ Collaboration. Collaborative meta-analysis of randomised trials of antiplatelet therapy for prevention of death, myocardial infarction, and stroke in high risk patients. BMJ. 2002;324:71–86. 5. Brener SJ, Steinhubl SR, Berger PB, et al; CREDO Investigators. Prolonged dual antiplatelet therapy after percutaneous coronary intervention reduces ischemic events without affecting the need for repeat revascularization: insights from the CREDO trial. J Invasive Cardiol. 2007;19:287–290. 6. Schnorbus B, Daiber A, Jurk K, et al. Effects of clopidogrel, prasugrel and ticagrelor on endothelial function, inflammatory and oxidative stress parameters and platelet function in patients undergoing coronary artery stenting for an acute coronary syndrome. A randomised, prospective, controlled study. BMJ Open. 2014;4:e005268. 7. Steinhubl SR, Talley JD, Braden GA, et al. Point-of-care measured platelet inhibition correlates with a reduced risk of an adverse cardiac event after percutaneous coronary intervention: results of the GOLD (AU-Assessing Ultegra) multicenter study. Circulation. 2001;103:2572–2578. 8. Collet JP, Cuisset T, Rangé G, et al; ARCTIC Investigators. Bedside monitoring to adjust antiplatelet therapy for coronary stenting. N Engl J Med. 2012;367:2100–2109. 9. Pettersen AÅ, Seljeflot I, Abdelnoor M, et al. High on-aspirin platelet reactivity and clinical outcome in patients with stable coronary artery disease: results from ASCET (Aspirin Nonresponsiveness and Clopidogrel Endpoint trial). J Am Heart Assoc. 2012;1:e000703. 10. Laine M, Arméro S, Peyrol M, et al. Clinical impact of genetically determined platelet reactivity. J Cardiovasc Transl Res. 2013;6:398–403. 11. Bernlochner I, Steinhubl S, Braun S, et al. Association between inflammatory biomarkers and platelet aggregation in patients under chronic clopidogrel treatment. Thromb Haemost. 2010;104:1193–1200.
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