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Homocysteine levels and platelet reactivity in coronary artery disease patients treated with ticagrelor
Journal Pre-proof Homocysteine levels and platelet reactivity in coronary artery disease patients treated with Ticagrelor Monica Verdoia, MD, Roberta Rolla, MD, Federica Negro, MD, Francesco Tonon, MD, Patrizia Pergolini, MD, Matteo Nardin, MD, Marco Marcolongo, MD, Giuseppe De Luca, MD, PhD, on behalf of the Novara Atherosclerosis Study Group (NAS) PII:
S0939-4753(19)30357-6
DOI:
https://doi.org/10.1016/j.numecd.2019.09.018
Reference:
NUMECD 2151
To appear in:
Nutrition, Metabolism and Cardiovascular Diseases
Received Date: 6 June 2019 Revised Date:
19 August 2019
Accepted Date: 17 September 2019
Please cite this article as: Verdoia M, Rolla R, Negro F, Tonon F, Pergolini P, Nardin M, Marcolongo M, De Luca G, on behalf of the Novara Atherosclerosis Study Group (NAS), Homocysteine levels and platelet reactivity in coronary artery disease patients treated with Ticagrelor, Nutrition, Metabolism and Cardiovascular Diseases, https://doi.org/10.1016/j.numecd.2019.09.018. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier B.V. on behalf of The Italian Society of Diabetology, the Italian Society for the Study of Atherosclerosis, the Italian Society of Human Nutrition, and the Department of Clinical Medicine and Surgery, Federico II University.
Homocysteine levels and platelet reactivity in coronary artery disease patients treated with Ticagrelor
Monica Verdoia, MD, Roberta Rolla, MD; Federica Negro, MD, Francesco Tonon, MD, Patrizia Pergolini, MD, Matteo Nardin, MD; Marco Marcolongo, MD, Giuseppe De Luca, MD, PhD, on behalf of the Novara Atherosclerosis Study Group (NAS) Division of Cardiology (MV, FN, FT, GDL), Clinical Chemistry (PP, RR), Azienda OspedalieraUniversitaria “Maggiore della Carità”, Eastern Piedmont University, Novara, Italy; Division of Cardiology, Ospedale degli Infermi, ASL Biella, Italy (MV, MM); Department of Internal Medicine, ASST Spedali Civili, Brescia, Italy (MN) Running Title: Homocysteine, Ticagrelor and platelet reactivity
Keywords: Homocysteine, Ticagrelor, platelet reactivity, impedance aggregometry, coronary artery disease
Correspondence:
Giuseppe De Luca, MD, PhD. Associate Professor of Cardiology Ospedale “Maggiore della Carità”, Eastern Piedmont University, C.so Mazzini, 18 28100 Novara, Italy E-mail: [email protected] Tel:
+39-0321-3733141
Fax:
+39-0321-3733407
1
Abstract
Background. Recurrent atherothrombotic events have been reported in certain higher risk subsets of patients even with ticagrelor, a potent first-line antiplatelet agent for the management of patients with acute coronary syndrome (ACS). Hyperhomocysteinemia is a known determinant of platelet function abnormalities. Therefore, the aim of our study was to evaluate the impact of homocysteine (Hcy) levels on platelet reactivity in patients receiving Ticagrelor. Methods. Patients with ACS undergoing percutaneous coronary revascularization and on dual antiplatelet therapy with ASA + Ticagrelor (90mg/twice a day) were scheduled for platelet function assessment 30-90 days post-discharge. Aggregation tests were performed by Multiple Electrode Aggregometry (MEA). Suboptimal platelet inhibition (HRPR-high residual platelet reactivity was defined if above the lower limit of normality (417 AU*min). Results. We included 432 patients, divided according to Hcy tertiles. Higher Hcy levels were associated with age, renal failure, creatinine levels and use diuretics (p<0.001). Patients with higher Hcy levels displayed a higher platelet reactivity at COL test (p=0.002), and ADP test (p=0.04), with a linear relationship between Hcy and platelet aggregation after stimulation with collagen (r=0.202, p<0.001), thrombin receptor peptide (r=0.104, p=0.05) and ADP (r=0.145, p=0.006). However, Hcy levels did not significantly affect the rate of HRPR with Ticagrelor (9.9% vs 13.7% vs 10.7%, p=0.89; adjusted OR[95%CI] = [0.616-1.51], p=0.99). Conclusions. Among patients with ACS, despite the elevated platelet reactivity associated to hyperhomocysteinemia, the DAPT with ticagrelor could overcome such phenomenon, achieving an adequate platelet inhibition in the majority of the patients.
2
Background Antiplatelet strategies have dramatically reduced the rate of acute and long-term recurrent cardiovascular events among patients with coronary artery disease (CAD) (1-3). A dual antiplatelet therapy (DAPT), with Acetylsalicylic acid (ASA) and an adjunctive P2Y12adenosine diphosphate (ADP) antagonist, has further reduced the risk of ischemic events after percutaneous coronary revascularization (PCI; 4,5), with ticagrelor being preferred over clopidogrel in the context of acute coronary syndrome (ACS) patients (6). In fact, Ticagrelor represent a powerful and reversible, direct-acting P2Y12 blocker, that has demonstrated to improve the survival in ACS patients, both as an acute treatment and at long-term, even beyond the traditional 12 months treatment (7,8). However, a residual high on-treatment platelet reactivity (HRPR) despite DAPT has been documented especially in certain higher risk subsets of patients and even with the newer antiplatelet agents, conditioning the occurrence of thrombotic events and therefore the outcomes (9,10). In particular, an inadequate effectiveness of ticagrelor has been reported in over 10% of ACS patients. Therefore, large efforts have been invested in identifying the predictors of suboptimal response to new antiplatelet agents, with a particular focus the role of genetics and biomarkers (11,12). Homocysteine (Hcy) is a metabolite of methionine degradation, that has been linked in epidemiological studies with atherosclerosis progression, increased platelet hyperreactivity and thrombogenicity
(13,
14).
In
addition,
previous
reports
have
suggested
that
hyperhomocysteinemia could impair the effectiveness of antiplatelet strategies in patients with coronary artery disease (15). However, the hypothesis that Hcy levels could modulate platelet reactivity among patients treated with Ticagrelor has not been tested, so far. 3
Therefore, the aim of the present study is to evaluate the relationship between Hcy levels and platelet aggregation or the rate of HRPR among ACS patients receiving on DAPT with Ticagrelor. Methods We included patients admitted at the Division of Cardiology, “Maggiore della Carità” Hospital, Eastern Piedmont University in Novara, Italy, from September 2013 to May 2015 and requiring dual antiplatelet therapy for an acute coronary syndrome (ACS) and PCI. Main demographic, clinical and angiographic data, together with the indication to dual antiplatelet therapy were recorded at admission and included in a dedicated database. The study was approved by our local Ethical Committee and informed consent was obtained by all patients. 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 thrombocytopenia, thrombocytosis, anemia, and polycythemia with impaired hematocrit levels or refusal of providing written informed consent. Biochemical measurements Blood samples at admission and re-assessment were drawn following a fasting period of 12 h. Glucose, creatinine, glycosylated haemoglobin and lipid profile were determined as previously described (16). Blood cells count was performed in a blood sample collected in tripotassium EDTA (7.2 mg) tubes. These blood samples were analyzed within 2 h of venipuncture by automatic blood counter (A Sysmex XE-2100). Plasma homocysteine was measured using a competitive immunoassay with direct chemiluminescence detection. All the assays were 4
performed on Siemens ADVIA Centaur using ADVIA Centaur HCY Ready Pack. The lower detection limit was 0,5 µmol/L. The upper limit for method linearity without dilution was 65 µmol/L. The analytical variability was 7 %. Platelet aggregation Platelet reactivity assessment was scheduled after 30-90 days from discharge after the ACS event. For each patient platelet aggregation was determined by Multiplate- Multiple electrode aggregometry (MEA, 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. Platelets’ function was assessed after stimulation with collagen (3.2 µg/ml) (COL test), ADP (6.4 µM) with prostaglandin E1 and thrombin receptor activating peptide, (TRAP-6; 30 µM). Results were expressed as arbitrary Aggregation Units (AU) and plotted against time, defining platelet function as the area under curve (AUC or AU*min). Suboptimal response to ADP-antagonists was defined for ADP test AU*min higher than the lowerlimit of normality (LLN) (normal range: 417-1030) according to literature (18). Statistical analysis All statistical analyses were performed by SPSS Statistics Software 17.0. (SPSS Inc., Chicago, Illinois) Continues variables were represented as mean ± SD, while categorical variables as percentage. Chi-Square and the ANOVA test were used to compare clinical and chemistry features according to homocysteine levels. Linear regression analysis was used to evaluate the association between aggregation tests results and homocysteine levels. Multiple logistic regression analysis was performed to evaluate
5
independent predictors of HRPR, after correction for baseline differences that were entered in the model in block. A p value < 0.05 was considered statistically significant. Results Our population is represented by 432 patients, that were divided in three groups according to tertiles values of Hcy (<15.7, 15.7–21, >21 nmol/ml). Median distance from baseline to platelet reactivity assessment was 88.7 [60.9-97.4] days. Main demographic and clinical features of the enrolled population are displayed in Table 1. As shown, higher Hcy levels were associated with age, renal failure, creatinine levels and use diuretics (p<0.001). Patients with higher Hcy levels displayed a higher platelet reactivity at COL test (p=0.002), and ADP test (p=0.04) (Table 2), with a non-significant trend for enhanced mean platelet reactivity at TRAP test (p=0.33). At linear regression analysis, in fact, we observed a direct relationship between Hcy and platelet aggregation after stimulation with collagen (COL test r=0.202, p<0.001), thrombin receptor peptide (r=0.104, p=0.05) and ADP (r=0.145, p=0.006), Figure 1. The 11.4% of the patients displayed a HRPR on DAPT. However, Hcy levels did not significantly affect either the rate of HRPR with Ticagrelor (9.9% vs 13.7% vs 10.7%, p=0.89) (Figure 2). This findings were confirmed after correction for baseline confounding factors (adjusted OR[95%CI] = 1 [0.66-1.51], p=0.99).
6
Similar data were confirmed at subgroup analysis in special higher risk subsets of patients, with no significant interaction between the level of homocysteine (upper tertiles value, Hcy ≥21 nmol/ml) and age, gender, diabetes or renal failure on platelet reactivity (Figure 3).
Discussion The present study is the first large report evaluating the relationship between homocysteine levels
and
the
effect
of
DAPT
comprising
ticagrelor.
We
documented
that
hyperhomocysteinemia is associated with an enhanced platelet reactivity, despite not affecting the rate of HRPR with ticagrelor. The improvements in mechanical revascularization and pharmacological adjuncts have completely reversed the prognostic expectations of patients with ACS, increasing the survival and lowering the risk or recurrent ischemic events (19-21). Antithrombotic strategies certainly represent a pillar point in providing such outcome benefits, and especially the use of a dual antiplatelet therapy (DAPT) combining ASA with the new ADP-antagonists (22-24). In particular, ticagrelor, has emerged in several trials and meta-analyses (7,8,25) as a safe and effective antiplatelet agent, even in complex subsets of patients, therefore representing currently the most commonly indicated therapy in real-life ACS patients. However, a suboptimal response to ticagrelor, or HRPR still represents an important issue for patients with cardiovascular disease, more than doubling the risk of recurrent ischemia (18,26). Whilst metabolic factors and genetic polymorphisms have been identified as accounting for the large part of inadequate response to Clopidogrel (27,28), controversies still exist of the determinants of HRPR with ticagrelor (29) 7
Homocysteine is an intermediate amino acid derived from the methionine metabolism, whose plasmatic elevation is quite frequent, being dependent from diet, drugs, genetic asset and clinical status, and mainly from renal function (30). Previous studies have reported that even a mild hyperhomocysteinemia could increase the cardiovascular risk, by modulating both the progression of atherosclerosis and its thrombotic complications (31,32). In fact, Hcy has been reported to influence the normal coagulation balance, with an activation of factor V (33) and suppression of thrombomodulin and protein C (34). 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 (35,36). In addition, further studies have suggested that the pro-thrombotic 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 promote platelets’ activation. In fact, Spencer et al. documented a correlation between Hcy and P-selectin, an indicator of platelet activation, among 165 hypertensive patients (37). Similar results were reported in vitro in a Russian study, where platelet incubation with Hcy enhanced the adhesion of thrombin- activated platelets to collagen and fibrinogen (38), while Signorello et al. identified a higher production of tromboxanes in platelets incubated with homocysteine (39). However, inconclusive results have been reported so far in clinical practice, with even less data addressing patients receiving antiplatelet therapies. In effect, an experimental study in rats, showed that Hcy could modulate adenosine-metabolism, thus conditioning platelet reactivity (40). However, in a previous study, we demonstrated that among patients with coronary artery disease, elevated homocysteine was independently associated with suboptimal response to ASA, 8
but not to ADP-antagonists (15,41). However, in that study, the majority of patients was receiving Clopidogrel, a drug whose effectiveness is severely conditioned by metabolic factors, requiring a complex hepatic biotransformation (28). On the contrary different results could be expected with ticagrelor, offering a more potent and predictable direct platelet inhibition, although this matter has not been addressed so far in literature. The present study shows, in a large scale cohort of patients on DAPT, that Hcy elevation can increase platelet reactivity, in response to different activating stimuli. However, Ticagrelor could overcome such higher platelet activation, resulting in the absence of any difference in the rate of poor responders to Ticagrelor according to Hcy levels. Similarly, Karolczak K et al (42) in a study enrolling 126 patients with CAD, showed that higher plasma Hcy appeared a significant risk factor for blood platelet refractoriness to low ASA dose (OR=1.11; ±95%CI: 1.02-1.20, p<0.02, adjusted to age, sex and CAD risk factors). However, such observation could be prevented and even reversed by increasing the dose of ASA to 150 mg, suggesting a potential benefit of a more aggressive antiplatelet strategy. Moreover, the same authors (43) also reported in another study that in vitro aldosterone stimulates homocysteine production, in addition to displaying a direct effect on platelet reactivity. Therefore, it could be argued that the larger use of ACE-inhibitors of ARBs in the majority of ACS patients could have further contributed to reduce the levels of Hcy and the rate of HRPR in our ACS patients treated with DAPT. Nevertheless, the pathophysiological mechanisms linking hyperhomocysteinemia to platelet function, as much as its impact on the outcomes and the risk of thrombotic events, certainly require a better definition in larger dedicated studies, and especially for ACS patients treated 9
with DAPT. Nevertheless, until new data become available, elevated Hcy should be accounted as a marker of thrombotic risk, although not significantly conditioning the effectiveness of a DAPT comprising ticagrelor. Limitations The first limitations of our study is certainly represented by the cohort design of our study, comprising a heterogeneous population of all-comers ACS patients. However, such recruitment was made on purpose, considering the widespread use of Ticagrelor even in complex subsets of patients as among the elderly or renal failure patients, since we aimed at the evaluation of the actual prevalence and clinical impact of Hcy in a real-life population of patients treated with DAPT. However, our results were confirmed after adjustment for baseline confounding factors. A priori sample size analysis was not performed, in absence of a reference from literature providing data on the potential impact of Hcy on the prevalence of high-platelet reactivity with ticagrelor. Nevertheless, based on a superiority design, based on the observed rates of resistance in patients according to HCY upper tertile (HCY > 21 nmol/ml), and alfa error of 0.05, a total of 87073 patients would have been needed to show a statistically significant difference in HRPR between the two groups. Moreover, we did not define the levels of folic acid, despite its deficiency, due to an inadequacy of the dietary supply or to an increased requirement, is a known cause for hyperhomocysteinaemia and could have influenced our final results (44). Either, we did not investigate the role the genetic variant of methylenetetrahydrofolate reductase 677 C > T in our study population, as this mutation, has been associated with platelet dysfunction (45) and neither we excluded or accounted for the use of folates 10
supplementation. Indeed, despite the role of MTHFR and folates on the risk of arterial acute thrombosis is still debated, however, the evaluation of these additional factors in our study could have certainly further contributed to shed light on the role of these metabolic parameters on platelet reactivity in ACS patients. Furthermore, we did not perform light transmission aggregometry (LTA) tests, that still remains the gold standard for platelet function assessment, but rather we preferred bed-side whole blood aggregation tests, being more applicable in everyday clinical practice, whose clinical impact in patients on antiplatelet treatment has been previously validated. (46). In addition, we did not provide a control group for our analysis. However, the vast majority of ACS patients in our center are treated with ticagrelor according to guidelines, therefore not allowing to recruit a control group among the ACS population. Nevertheless, we previously reported similar data among elective patients receiving mainly clopidogrel (41). Finally, the absence of a long-term follow-up, did not allow to evaluate the clinical impact of Hcy elevation or HRPR on the occurrence of thrombotic events and survival. Conclusion. Among patients with ACS, elevated homocysteine is associated with an enhanced platelet reactivity. However, the DAPT with ticagrelor could overcome such phenomenon, achieving an adequate platelet inhibition in the majority of the patients, since the rate of HRPR with ticagrelor was low and not conditioned by Hcy levels.
Disclosures 11
The Authors declare no conflict of interest or funding source to disclose
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Table I. Clinical and demographical characteristics according to homocysteine tertiles
Baseline Clinical Characteristics
Hcy< 15.7 nmol/ml
15.7≥Hcy < 21 Hcy ≥21 nmol/ml nmol/ml
P value
(n = 140)
(n = 142)
(n = 150)
63.4±10.4
65.2±10.1
69.1±11.2
<0.001
78.6
72.5
82
0.46
26.7±4.1
27.2±4.9
27.2±4.9
0.64
Dyslipidemia(%)
55.4
59.9
56.8
0.83
Diabetes mellitus (%)
41.4
33.1
36
0.35
Renal failure (%)
7.2
16.9
29.5
<0.001
Active smokers (%)
35
25.4
35.8
0.36
Hypertension (%)
65
75.4
73
0.09
History of MI (%)
20
20.6
22.3
0.63
Age (mean±SD) Male Sex (%) Body mass Index (mean±SD)
16
Previous PCI (%)
25
31.7
30.4
0.32
Previous CABG (%)
8.6
8.5
9.5
0.80
Platelets Count (10^5/µl)
242±62.6
247.5±73.5
257.3±87.4
0.21
Haemoglobin( g/dl)
13.5±1.7
13.6±1.7
13.4±1.7
0.43
White blood cells(10^3/µl)
8.3±2.5
7.8±2.1
8.2±2.1
0.15
Creatinine (mg/dl)
0.9±0.25
1±0.3
1.2±.0.8
<0.001
Glycaemia (mg/dl)
121.3±44.3
118.6±36.8
117.6±36.4
0.71
Total cholesterol (mg/dL)
137.5±38.4
138.3±31
141.3±33.9
0.62
HDL cholesterol
40.2±11.4
41.9±11.9
42.2±12.5
0.29
Triglycerides (mg/dL)
123.2±71.4
117.7±51
127.9±75.2
0.43
HbA1c (%)
6.4±1.4
6.2±1
6.3±1.2
0.49
C reactive protein (mg/dl)
0.6±1.3
0.5±1.3
0.8±1.5
0.16
Biochemistry
0.26
Indication to angiography NSTE-ACS (%)
75
73.9
80.5
STEMI (%)
25
26.1
19.5
51.8±8.9
53.2±9
51.1±9.7
0.14
55.7
66.7
71.7
0.04
ACE inhibitors (%)
61.1
57.7
57.3
0.41
ARB (%)
11.4
21.1
18.7
0.11
Beta blockers (%)
88.6
90.8
86.7
0.60
Calcium antagonists (%)
18.6
21.8
22.7
0.40
Nitrates (%)
33.6
39.4
42
0.14
Diuretics (%)
17.1
27.5
44.7
<0.001
Statins (%)
88.6
92.3
90
0.70
Ejection fraction (mean±SD) Multivessel CAD (%) Therapy at admission
17
Table 2.Platelet function test according to homocysteine tertiles
Platelet function tests
Hcy< 15.7 nmol/ml
15.7≥Hcy < 21 nmol/ml
Hcy ≥21 nmol/ml
(n = 140)
(n = 142)
(n = 150)
COL test (mean±SD)
397.7±105
430.6±151
462.7±156
0.002
TRAP test (mean±SD)
1136.6±280
1174.4±305.5
1185.6±292
0.33
ADP test (mean±SD)
259.8±119.2
280.9±137.2
297.4±196
0.04
18
P value
Figures legend
Figure 1. Linear regression analysis relating homocysteine levels to platelet aggregation (Area Under the Curve, AUC) after stimulation with collagen (COL, fig. 1A), thrombin-activating peptide (TRAP, Fig. 1B) and Adenosine Diphosphate (ADP, Fig.1C).
Figure 2. Prevalence of high-on treatment platelet reactivity with ticagrelor based on homocysteine tertiles
Figure 3. Forrest plot displaying the impact of Homocysteine (as above the 3rd tertile) on HRPR in special subsets of patients
19
20
Figure 2
HRPR at ADP test (%)
20
p=0.89
15
10
5
0
I tert n=140
II tert n=142 Homocysteine (nmol/ml) 21
III tert n=150
22
Conflict of Interest Form Manuscript Name:
Homocysteine levels and platelet reactivity in coronary artery disease patients treated with Ticagrelor
Authors: Monica Verdoia, MD, PhD Roberta Rolla, MD; Federica Negro, MD, Francesco Tonon, MD, Patrizia Pergolini, MD, Matteo Nardin, MD; Marco Marcolongo, MD, Giuseppe De Luca, MD, PhD
Corresponding Author: Giuseppe De Luca Corresponding Author’s address: C.so Mazzini, 18 28100 Novara, Italy Corresponding Author telephone number: 0039 0321 3733294 Corresponding Author e-mail: [email protected] Details of nature of conflict of interest: none
What is known about the topic? -
Ticagrelor represent a powerful antiplatelet agent, that has demonstrated to improve the survival in ACS patients Inadequate effectiveness of ticagrelor has been reported in over 10% of ACS patients Hyperhomocysteinemia is a known determinant of platelet function abnormalities even among patients on DAPT
What does the study add? -
Among 432 patients with ACS, high-on treatment platelet reactivity was found in 11.4% of the patients, with no difference according to homocysteine levels
-
A dual antiplatelet therapy with ticagrelor could achieve an adequate platelet inhibition in the majority of patients.
S0939-4753(19)30357-6
DOI:
https://doi.org/10.1016/j.numecd.2019.09.018
Reference:
NUMECD 2151
To appear in:
Nutrition, Metabolism and Cardiovascular Diseases
Received Date: 6 June 2019 Revised Date:
19 August 2019
Accepted Date: 17 September 2019
Please cite this article as: Verdoia M, Rolla R, Negro F, Tonon F, Pergolini P, Nardin M, Marcolongo M, De Luca G, on behalf of the Novara Atherosclerosis Study Group (NAS), Homocysteine levels and platelet reactivity in coronary artery disease patients treated with Ticagrelor, Nutrition, Metabolism and Cardiovascular Diseases, https://doi.org/10.1016/j.numecd.2019.09.018. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier B.V. on behalf of The Italian Society of Diabetology, the Italian Society for the Study of Atherosclerosis, the Italian Society of Human Nutrition, and the Department of Clinical Medicine and Surgery, Federico II University.
Homocysteine levels and platelet reactivity in coronary artery disease patients treated with Ticagrelor
Monica Verdoia, MD, Roberta Rolla, MD; Federica Negro, MD, Francesco Tonon, MD, Patrizia Pergolini, MD, Matteo Nardin, MD; Marco Marcolongo, MD, Giuseppe De Luca, MD, PhD, on behalf of the Novara Atherosclerosis Study Group (NAS) Division of Cardiology (MV, FN, FT, GDL), Clinical Chemistry (PP, RR), Azienda OspedalieraUniversitaria “Maggiore della Carità”, Eastern Piedmont University, Novara, Italy; Division of Cardiology, Ospedale degli Infermi, ASL Biella, Italy (MV, MM); Department of Internal Medicine, ASST Spedali Civili, Brescia, Italy (MN) Running Title: Homocysteine, Ticagrelor and platelet reactivity
Keywords: Homocysteine, Ticagrelor, platelet reactivity, impedance aggregometry, coronary artery disease
Correspondence:
Giuseppe De Luca, MD, PhD. Associate Professor of Cardiology Ospedale “Maggiore della Carità”, Eastern Piedmont University, C.so Mazzini, 18 28100 Novara, Italy E-mail: [email protected] Tel:
+39-0321-3733141
Fax:
+39-0321-3733407
1
Abstract
Background. Recurrent atherothrombotic events have been reported in certain higher risk subsets of patients even with ticagrelor, a potent first-line antiplatelet agent for the management of patients with acute coronary syndrome (ACS). Hyperhomocysteinemia is a known determinant of platelet function abnormalities. Therefore, the aim of our study was to evaluate the impact of homocysteine (Hcy) levels on platelet reactivity in patients receiving Ticagrelor. Methods. Patients with ACS undergoing percutaneous coronary revascularization and on dual antiplatelet therapy with ASA + Ticagrelor (90mg/twice a day) were scheduled for platelet function assessment 30-90 days post-discharge. Aggregation tests were performed by Multiple Electrode Aggregometry (MEA). Suboptimal platelet inhibition (HRPR-high residual platelet reactivity was defined if above the lower limit of normality (417 AU*min). Results. We included 432 patients, divided according to Hcy tertiles. Higher Hcy levels were associated with age, renal failure, creatinine levels and use diuretics (p<0.001). Patients with higher Hcy levels displayed a higher platelet reactivity at COL test (p=0.002), and ADP test (p=0.04), with a linear relationship between Hcy and platelet aggregation after stimulation with collagen (r=0.202, p<0.001), thrombin receptor peptide (r=0.104, p=0.05) and ADP (r=0.145, p=0.006). However, Hcy levels did not significantly affect the rate of HRPR with Ticagrelor (9.9% vs 13.7% vs 10.7%, p=0.89; adjusted OR[95%CI] = [0.616-1.51], p=0.99). Conclusions. Among patients with ACS, despite the elevated platelet reactivity associated to hyperhomocysteinemia, the DAPT with ticagrelor could overcome such phenomenon, achieving an adequate platelet inhibition in the majority of the patients.
2
Background Antiplatelet strategies have dramatically reduced the rate of acute and long-term recurrent cardiovascular events among patients with coronary artery disease (CAD) (1-3). A dual antiplatelet therapy (DAPT), with Acetylsalicylic acid (ASA) and an adjunctive P2Y12adenosine diphosphate (ADP) antagonist, has further reduced the risk of ischemic events after percutaneous coronary revascularization (PCI; 4,5), with ticagrelor being preferred over clopidogrel in the context of acute coronary syndrome (ACS) patients (6). In fact, Ticagrelor represent a powerful and reversible, direct-acting P2Y12 blocker, that has demonstrated to improve the survival in ACS patients, both as an acute treatment and at long-term, even beyond the traditional 12 months treatment (7,8). However, a residual high on-treatment platelet reactivity (HRPR) despite DAPT has been documented especially in certain higher risk subsets of patients and even with the newer antiplatelet agents, conditioning the occurrence of thrombotic events and therefore the outcomes (9,10). In particular, an inadequate effectiveness of ticagrelor has been reported in over 10% of ACS patients. Therefore, large efforts have been invested in identifying the predictors of suboptimal response to new antiplatelet agents, with a particular focus the role of genetics and biomarkers (11,12). Homocysteine (Hcy) is a metabolite of methionine degradation, that has been linked in epidemiological studies with atherosclerosis progression, increased platelet hyperreactivity and thrombogenicity
(13,
14).
In
addition,
previous
reports
have
suggested
that
hyperhomocysteinemia could impair the effectiveness of antiplatelet strategies in patients with coronary artery disease (15). However, the hypothesis that Hcy levels could modulate platelet reactivity among patients treated with Ticagrelor has not been tested, so far. 3
Therefore, the aim of the present study is to evaluate the relationship between Hcy levels and platelet aggregation or the rate of HRPR among ACS patients receiving on DAPT with Ticagrelor. Methods We included patients admitted at the Division of Cardiology, “Maggiore della Carità” Hospital, Eastern Piedmont University in Novara, Italy, from September 2013 to May 2015 and requiring dual antiplatelet therapy for an acute coronary syndrome (ACS) and PCI. Main demographic, clinical and angiographic data, together with the indication to dual antiplatelet therapy were recorded at admission and included in a dedicated database. The study was approved by our local Ethical Committee and informed consent was obtained by all patients. 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 thrombocytopenia, thrombocytosis, anemia, and polycythemia with impaired hematocrit levels or refusal of providing written informed consent. Biochemical measurements Blood samples at admission and re-assessment were drawn following a fasting period of 12 h. Glucose, creatinine, glycosylated haemoglobin and lipid profile were determined as previously described (16). Blood cells count was performed in a blood sample collected in tripotassium EDTA (7.2 mg) tubes. These blood samples were analyzed within 2 h of venipuncture by automatic blood counter (A Sysmex XE-2100). Plasma homocysteine was measured using a competitive immunoassay with direct chemiluminescence detection. All the assays were 4
performed on Siemens ADVIA Centaur using ADVIA Centaur HCY Ready Pack. The lower detection limit was 0,5 µmol/L. The upper limit for method linearity without dilution was 65 µmol/L. The analytical variability was 7 %. Platelet aggregation Platelet reactivity assessment was scheduled after 30-90 days from discharge after the ACS event. For each patient platelet aggregation was determined by Multiplate- Multiple electrode aggregometry (MEA, 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. Platelets’ function was assessed after stimulation with collagen (3.2 µg/ml) (COL test), ADP (6.4 µM) with prostaglandin E1 and thrombin receptor activating peptide, (TRAP-6; 30 µM). Results were expressed as arbitrary Aggregation Units (AU) and plotted against time, defining platelet function as the area under curve (AUC or AU*min). Suboptimal response to ADP-antagonists was defined for ADP test AU*min higher than the lowerlimit of normality (LLN) (normal range: 417-1030) according to literature (18). Statistical analysis All statistical analyses were performed by SPSS Statistics Software 17.0. (SPSS Inc., Chicago, Illinois) Continues variables were represented as mean ± SD, while categorical variables as percentage. Chi-Square and the ANOVA test were used to compare clinical and chemistry features according to homocysteine levels. Linear regression analysis was used to evaluate the association between aggregation tests results and homocysteine levels. Multiple logistic regression analysis was performed to evaluate
5
independent predictors of HRPR, after correction for baseline differences that were entered in the model in block. A p value < 0.05 was considered statistically significant. Results Our population is represented by 432 patients, that were divided in three groups according to tertiles values of Hcy (<15.7, 15.7–21, >21 nmol/ml). Median distance from baseline to platelet reactivity assessment was 88.7 [60.9-97.4] days. Main demographic and clinical features of the enrolled population are displayed in Table 1. As shown, higher Hcy levels were associated with age, renal failure, creatinine levels and use diuretics (p<0.001). Patients with higher Hcy levels displayed a higher platelet reactivity at COL test (p=0.002), and ADP test (p=0.04) (Table 2), with a non-significant trend for enhanced mean platelet reactivity at TRAP test (p=0.33). At linear regression analysis, in fact, we observed a direct relationship between Hcy and platelet aggregation after stimulation with collagen (COL test r=0.202, p<0.001), thrombin receptor peptide (r=0.104, p=0.05) and ADP (r=0.145, p=0.006), Figure 1. The 11.4% of the patients displayed a HRPR on DAPT. However, Hcy levels did not significantly affect either the rate of HRPR with Ticagrelor (9.9% vs 13.7% vs 10.7%, p=0.89) (Figure 2). This findings were confirmed after correction for baseline confounding factors (adjusted OR[95%CI] = 1 [0.66-1.51], p=0.99).
6
Similar data were confirmed at subgroup analysis in special higher risk subsets of patients, with no significant interaction between the level of homocysteine (upper tertiles value, Hcy ≥21 nmol/ml) and age, gender, diabetes or renal failure on platelet reactivity (Figure 3).
Discussion The present study is the first large report evaluating the relationship between homocysteine levels
and
the
effect
of
DAPT
comprising
ticagrelor.
We
documented
that
hyperhomocysteinemia is associated with an enhanced platelet reactivity, despite not affecting the rate of HRPR with ticagrelor. The improvements in mechanical revascularization and pharmacological adjuncts have completely reversed the prognostic expectations of patients with ACS, increasing the survival and lowering the risk or recurrent ischemic events (19-21). Antithrombotic strategies certainly represent a pillar point in providing such outcome benefits, and especially the use of a dual antiplatelet therapy (DAPT) combining ASA with the new ADP-antagonists (22-24). In particular, ticagrelor, has emerged in several trials and meta-analyses (7,8,25) as a safe and effective antiplatelet agent, even in complex subsets of patients, therefore representing currently the most commonly indicated therapy in real-life ACS patients. However, a suboptimal response to ticagrelor, or HRPR still represents an important issue for patients with cardiovascular disease, more than doubling the risk of recurrent ischemia (18,26). Whilst metabolic factors and genetic polymorphisms have been identified as accounting for the large part of inadequate response to Clopidogrel (27,28), controversies still exist of the determinants of HRPR with ticagrelor (29) 7
Homocysteine is an intermediate amino acid derived from the methionine metabolism, whose plasmatic elevation is quite frequent, being dependent from diet, drugs, genetic asset and clinical status, and mainly from renal function (30). Previous studies have reported that even a mild hyperhomocysteinemia could increase the cardiovascular risk, by modulating both the progression of atherosclerosis and its thrombotic complications (31,32). In fact, Hcy has been reported to influence the normal coagulation balance, with an activation of factor V (33) and suppression of thrombomodulin and protein C (34). 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 (35,36). In addition, further studies have suggested that the pro-thrombotic 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 promote platelets’ activation. In fact, Spencer et al. documented a correlation between Hcy and P-selectin, an indicator of platelet activation, among 165 hypertensive patients (37). Similar results were reported in vitro in a Russian study, where platelet incubation with Hcy enhanced the adhesion of thrombin- activated platelets to collagen and fibrinogen (38), while Signorello et al. identified a higher production of tromboxanes in platelets incubated with homocysteine (39). However, inconclusive results have been reported so far in clinical practice, with even less data addressing patients receiving antiplatelet therapies. In effect, an experimental study in rats, showed that Hcy could modulate adenosine-metabolism, thus conditioning platelet reactivity (40). However, in a previous study, we demonstrated that among patients with coronary artery disease, elevated homocysteine was independently associated with suboptimal response to ASA, 8
but not to ADP-antagonists (15,41). However, in that study, the majority of patients was receiving Clopidogrel, a drug whose effectiveness is severely conditioned by metabolic factors, requiring a complex hepatic biotransformation (28). On the contrary different results could be expected with ticagrelor, offering a more potent and predictable direct platelet inhibition, although this matter has not been addressed so far in literature. The present study shows, in a large scale cohort of patients on DAPT, that Hcy elevation can increase platelet reactivity, in response to different activating stimuli. However, Ticagrelor could overcome such higher platelet activation, resulting in the absence of any difference in the rate of poor responders to Ticagrelor according to Hcy levels. Similarly, Karolczak K et al (42) in a study enrolling 126 patients with CAD, showed that higher plasma Hcy appeared a significant risk factor for blood platelet refractoriness to low ASA dose (OR=1.11; ±95%CI: 1.02-1.20, p<0.02, adjusted to age, sex and CAD risk factors). However, such observation could be prevented and even reversed by increasing the dose of ASA to 150 mg, suggesting a potential benefit of a more aggressive antiplatelet strategy. Moreover, the same authors (43) also reported in another study that in vitro aldosterone stimulates homocysteine production, in addition to displaying a direct effect on platelet reactivity. Therefore, it could be argued that the larger use of ACE-inhibitors of ARBs in the majority of ACS patients could have further contributed to reduce the levels of Hcy and the rate of HRPR in our ACS patients treated with DAPT. Nevertheless, the pathophysiological mechanisms linking hyperhomocysteinemia to platelet function, as much as its impact on the outcomes and the risk of thrombotic events, certainly require a better definition in larger dedicated studies, and especially for ACS patients treated 9
with DAPT. Nevertheless, until new data become available, elevated Hcy should be accounted as a marker of thrombotic risk, although not significantly conditioning the effectiveness of a DAPT comprising ticagrelor. Limitations The first limitations of our study is certainly represented by the cohort design of our study, comprising a heterogeneous population of all-comers ACS patients. However, such recruitment was made on purpose, considering the widespread use of Ticagrelor even in complex subsets of patients as among the elderly or renal failure patients, since we aimed at the evaluation of the actual prevalence and clinical impact of Hcy in a real-life population of patients treated with DAPT. However, our results were confirmed after adjustment for baseline confounding factors. A priori sample size analysis was not performed, in absence of a reference from literature providing data on the potential impact of Hcy on the prevalence of high-platelet reactivity with ticagrelor. Nevertheless, based on a superiority design, based on the observed rates of resistance in patients according to HCY upper tertile (HCY > 21 nmol/ml), and alfa error of 0.05, a total of 87073 patients would have been needed to show a statistically significant difference in HRPR between the two groups. Moreover, we did not define the levels of folic acid, despite its deficiency, due to an inadequacy of the dietary supply or to an increased requirement, is a known cause for hyperhomocysteinaemia and could have influenced our final results (44). Either, we did not investigate the role the genetic variant of methylenetetrahydrofolate reductase 677 C > T in our study population, as this mutation, has been associated with platelet dysfunction (45) and neither we excluded or accounted for the use of folates 10
supplementation. Indeed, despite the role of MTHFR and folates on the risk of arterial acute thrombosis is still debated, however, the evaluation of these additional factors in our study could have certainly further contributed to shed light on the role of these metabolic parameters on platelet reactivity in ACS patients. Furthermore, we did not perform light transmission aggregometry (LTA) tests, that still remains the gold standard for platelet function assessment, but rather we preferred bed-side whole blood aggregation tests, being more applicable in everyday clinical practice, whose clinical impact in patients on antiplatelet treatment has been previously validated. (46). In addition, we did not provide a control group for our analysis. However, the vast majority of ACS patients in our center are treated with ticagrelor according to guidelines, therefore not allowing to recruit a control group among the ACS population. Nevertheless, we previously reported similar data among elective patients receiving mainly clopidogrel (41). Finally, the absence of a long-term follow-up, did not allow to evaluate the clinical impact of Hcy elevation or HRPR on the occurrence of thrombotic events and survival. Conclusion. Among patients with ACS, elevated homocysteine is associated with an enhanced platelet reactivity. However, the DAPT with ticagrelor could overcome such phenomenon, achieving an adequate platelet inhibition in the majority of the patients, since the rate of HRPR with ticagrelor was low and not conditioned by Hcy levels.
Disclosures 11
The Authors declare no conflict of interest or funding source to disclose
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Table I. Clinical and demographical characteristics according to homocysteine tertiles
Baseline Clinical Characteristics
Hcy< 15.7 nmol/ml
15.7≥Hcy < 21 Hcy ≥21 nmol/ml nmol/ml
P value
(n = 140)
(n = 142)
(n = 150)
63.4±10.4
65.2±10.1
69.1±11.2
<0.001
78.6
72.5
82
0.46
26.7±4.1
27.2±4.9
27.2±4.9
0.64
Dyslipidemia(%)
55.4
59.9
56.8
0.83
Diabetes mellitus (%)
41.4
33.1
36
0.35
Renal failure (%)
7.2
16.9
29.5
<0.001
Active smokers (%)
35
25.4
35.8
0.36
Hypertension (%)
65
75.4
73
0.09
History of MI (%)
20
20.6
22.3
0.63
Age (mean±SD) Male Sex (%) Body mass Index (mean±SD)
16
Previous PCI (%)
25
31.7
30.4
0.32
Previous CABG (%)
8.6
8.5
9.5
0.80
Platelets Count (10^5/µl)
242±62.6
247.5±73.5
257.3±87.4
0.21
Haemoglobin( g/dl)
13.5±1.7
13.6±1.7
13.4±1.7
0.43
White blood cells(10^3/µl)
8.3±2.5
7.8±2.1
8.2±2.1
0.15
Creatinine (mg/dl)
0.9±0.25
1±0.3
1.2±.0.8
<0.001
Glycaemia (mg/dl)
121.3±44.3
118.6±36.8
117.6±36.4
0.71
Total cholesterol (mg/dL)
137.5±38.4
138.3±31
141.3±33.9
0.62
HDL cholesterol
40.2±11.4
41.9±11.9
42.2±12.5
0.29
Triglycerides (mg/dL)
123.2±71.4
117.7±51
127.9±75.2
0.43
HbA1c (%)
6.4±1.4
6.2±1
6.3±1.2
0.49
C reactive protein (mg/dl)
0.6±1.3
0.5±1.3
0.8±1.5
0.16
Biochemistry
0.26
Indication to angiography NSTE-ACS (%)
75
73.9
80.5
STEMI (%)
25
26.1
19.5
51.8±8.9
53.2±9
51.1±9.7
0.14
55.7
66.7
71.7
0.04
ACE inhibitors (%)
61.1
57.7
57.3
0.41
ARB (%)
11.4
21.1
18.7
0.11
Beta blockers (%)
88.6
90.8
86.7
0.60
Calcium antagonists (%)
18.6
21.8
22.7
0.40
Nitrates (%)
33.6
39.4
42
0.14
Diuretics (%)
17.1
27.5
44.7
<0.001
Statins (%)
88.6
92.3
90
0.70
Ejection fraction (mean±SD) Multivessel CAD (%) Therapy at admission
17
Table 2.Platelet function test according to homocysteine tertiles
Platelet function tests
Hcy< 15.7 nmol/ml
15.7≥Hcy < 21 nmol/ml
Hcy ≥21 nmol/ml
(n = 140)
(n = 142)
(n = 150)
COL test (mean±SD)
397.7±105
430.6±151
462.7±156
0.002
TRAP test (mean±SD)
1136.6±280
1174.4±305.5
1185.6±292
0.33
ADP test (mean±SD)
259.8±119.2
280.9±137.2
297.4±196
0.04
18
P value
Figures legend
Figure 1. Linear regression analysis relating homocysteine levels to platelet aggregation (Area Under the Curve, AUC) after stimulation with collagen (COL, fig. 1A), thrombin-activating peptide (TRAP, Fig. 1B) and Adenosine Diphosphate (ADP, Fig.1C).
Figure 2. Prevalence of high-on treatment platelet reactivity with ticagrelor based on homocysteine tertiles
Figure 3. Forrest plot displaying the impact of Homocysteine (as above the 3rd tertile) on HRPR in special subsets of patients
19
20
Figure 2
HRPR at ADP test (%)
20
p=0.89
15
10
5
0
I tert n=140
II tert n=142 Homocysteine (nmol/ml) 21
III tert n=150
22
Conflict of Interest Form Manuscript Name:
Homocysteine levels and platelet reactivity in coronary artery disease patients treated with Ticagrelor
Authors: Monica Verdoia, MD, PhD Roberta Rolla, MD; Federica Negro, MD, Francesco Tonon, MD, Patrizia Pergolini, MD, Matteo Nardin, MD; Marco Marcolongo, MD, Giuseppe De Luca, MD, PhD
Corresponding Author: Giuseppe De Luca Corresponding Author’s address: C.so Mazzini, 18 28100 Novara, Italy Corresponding Author telephone number: 0039 0321 3733294 Corresponding Author e-mail: [email protected] Details of nature of conflict of interest: none
What is known about the topic? -
Ticagrelor represent a powerful antiplatelet agent, that has demonstrated to improve the survival in ACS patients Inadequate effectiveness of ticagrelor has been reported in over 10% of ACS patients Hyperhomocysteinemia is a known determinant of platelet function abnormalities even among patients on DAPT
What does the study add? -
Among 432 patients with ACS, high-on treatment platelet reactivity was found in 11.4% of the patients, with no difference according to homocysteine levels
-
A dual antiplatelet therapy with ticagrelor could achieve an adequate platelet inhibition in the majority of patients.