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Relation of Hypoalbuminemia to Response to Aspirin in Patients With Stable Coronary Artery Disease
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Relation of Hypoalbuminemia to Response to Aspirin in Patients with Stable Coronary Artery Disease Arthur Shiyovich MD , Liat Sasson MD , Eli Lev MD , Alejandro Solodky MD , Ran Kornowski MD , Leor Perl MD PII: DOI: Reference:
S0002-9149(19)31248-2 https://doi.org/10.1016/j.amjcard.2019.10.055 AJC 24285
To appear in:
The American Journal of Cardiology
Received date: Revised date: Accepted date:
9 September 2019 26 October 2019 30 October 2019
Please cite this article as: Arthur Shiyovich MD , Liat Sasson MD , Eli Lev MD , Alejandro Solodky MD , Ran Kornowski MD , Leor Perl MD , Relation of Hypoalbuminemia to Response to Aspirin in Patients with Stable Coronary Artery Disease, The American Journal of Cardiology (2019), doi: https://doi.org/10.1016/j.amjcard.2019.10.055
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 Inc.
Relation of Hypoalbuminemia to Response to Aspirin in Patients with Stable Coronary Artery Disease Arthur Shiyovich MD*, Liat Sasson MD *, Eli Lev MD, Alejandro Solodky MD, Ran Kornowski MD, Leor Perl MD
Department of Cardiology, Rabin Medical Center, Beilinson Hospital, Petah-Tikva, and "Sackler" Faculty of Medicine, Tel-Aviv University Israel. *Equal contribution Corresponding author: Shiyovich Arthur MD. Beilinson Hospital, Rabin Medical Center, 39 Jabotinski Street, Petah Tikva, Israel, 49100. email: [email protected] Tel: +972-39377100 Fax: +97239377101 Running title: hypoalbuminemia is associated with aspirin resistance
1
Abstract
Serum albumin (SA) level is a powerful cardiovascular prognostic marker, suggested to be involved in regulation of platelet function. High on-aspirin platelet reactivity (HAPR) is associated with increased risk for deleterious cardiovascular events. The aim of the current study was to evaluate the association between HAPR and albumin levels in patients with stable coronary artery disease (CAD) treated with aspirin. Patients with known stable CAD, who were taking aspirin (75-100 mg qd) regularly for at least one month, were screened for the current study. Exclusion criteria: cancer, sepsis or acute infection, active inflammatory/rheumatic disease, recent major surgery, chronic liver failure, the administration of other anti-platelet drugs, non-adherence with aspirin and thrombocytopenia. Blood was drawn from the participants and sent for SA level and platelet function test (VerifyNow). HAPR was defined as aspirin reaction units (ARU) >550. Overall 116 patients were analyzed; age 69 ±10, 28% women. Twenty (17%) were hypoalbuminemic (≤3.5 g/dL). Hypoalbuminemic patients had similar characteristics to the normal albumin group except mildly higher creatinine in the former. SA levels were significantly lower in the hypoalbuminemic group (3.2±0.2 g/dL vs. 4.2±0.4 g/dL, respectively, p<0.001) while mean ARU was significantly higher compared to the normal albumin group (548±45 vs. 444±66 ARU, respectively, p<0.001). A significant inverse association was observed between SA and ARU with (R2=0.67, p<0.001). Multivariate analysis adjusted for potential confounders found that albumin ≤3.5 is the strongest predictor of HAPR among patients with stable CAD (HR=4.9, 95%CI=2.2-32, p=0.002). In conclusion, hypoalbuminemia is strongly
associated with HAPR among patients with stable CAD.
Key words: serum albumin level; High on-aspirin platelet reactivity (HAPR); coronary artery disease
2
Introduction
Albumin is a pivotal protein in human plasma with a great variety of physiological roles that include: maintaining capillary permeability and plasma osmotic pressure, transporting various substances affecting their metabolism and activity, anti-oxidative and anti-inflammatory functions, as well as significant relations with coagulation pathways and platelet function 1, 2. Hypoalbuminemia is emerging as a powerful cardiovascular prognostic marker 2-5. Thrombotic complications were also found to be related with hypoalbuminemia 6, 7. One of the suggested mechanisms for the increased cardiovascular morbidity and
worse outcomes in patients with hypoalbuminemia is that albumin interferes with mechanisms supporting platelet aggregation 6, 8. However, empiric data to support such mechanisms are scarce. Aspirin is the most commonly used antiplatelet agent in patients with cardiovascular diseases. An inadequate response to prescribed aspirin in these patients is associated with increased risk for subsequent deleterious events 9. The aim of the current study was to evaluate the association between platelet function (i.e. platelet aggregation) and albumin levels in patients with coronary artery disease (CAD) treated with aspirin.
Methods The current study included two prospective subsets of patients as following; the first group comprised a historic prospective cohort of patients (n=66) while the second group included contemporary patients that were enrolled ad-hoc (n=50). All patients had known stable CAD defined as patients with previously proven CAD (a stenosis of at least 50% in one or more epicardial coronary arteries by coronary angiography) that were free from acute coronary syndrome, percutaneous coronary intervention or coronary artery bypass graft surgery within 6 months prior to inclusion in the study, were regularly taking Aspirin (75-100 mg qd) for at least a month and were either evaluated in the outpatient clinics (e.g. routine follow-up) or hospitalized in our institution, a tertiary medical center, for evaluation of symptoms other than those classified as exclusion criteria. Exclusion criteria: acute coronary syndrome, known malignancy (patients with remission >2 year were eligible), sepsis or acute infection, active inflammatory/rheumatic disease, major surgery in the last 6 months, chronic liver failure, treatment with anti-platelet drugs other than Aspirin ( i.e Clopidogrel, Ticagrelor or Prasugrel), treatment with
3
oral anticoagulation, allergy to- or non-adherence with Aspirin and thrombocytopenia (<100K/micl). The institutional review board approved the study, which was performed in accordance with the Helsinki declaration.
Following informed consent and patients’ verbal confirmation of adherence with Aspirin within the last month and in the day of the test, blood was drawn from the antecubital vein and split in two sets of experimental batches; one sent to the biochemical laboratory for CBC and biochemistry including serum albumin level while the second was used for the platelet function test (VerifyNow) as elaborated below. Since in the first (historic) cohort serum albumin level measurement was not performed upon enrollment, as was done in the second cohort, we included only patients who had albumin results ≤1-month from enrollment and the VerifyNow test (see below). The VerifyNow system (Accumetrics, San Diego, CA) is a whole-blood assay based on light transmission measurements. The Aspirin-specific cartridge is used to assess platelet dysfunction caused by Aspirin. In the Aspirin-specific assay, arachidonic acid activates platelets, and the activated platelets bind to fibrinogen-coated beads to form an aggregate. The degree of aggregation is quantified by a corresponding increase in light transmission and is reported in Aspirin reaction units (ARU). High on-aspirin platelet reactivity (HAPR) was defined as ARU >550 in consistency with previous studies 10, 11. Data were obtained from the hospital’s computerized medical records and included demographic and clinical characteristics, cardiovascular risk factors and co-morbidity, procedural characteristics and workup (e.g. blood tests, echocardiography). Patients were divided into two groups based in the serum albumin level: hypoalbuminemic; SA≤3.5 g/dL and normoalbuminemic SA>3.5 g/dL (normal range 3.5-5.5 g/dL) as this cutoff was also shown to be of prognostic value in patients with coronary artery disease 12. Glomerular filtration rate was calculated by the Modification of Diet in Renal Disease formula. Patient characteristics were presented as mean and standard deviation (SD) for continuous variables and n and percent for the categorical data. Chi-square or Fischer exact tests were used for analysis of categorical variables when appropriate. The unpaired student t-test was used for analysis of continuous variables. The Kolmogorov–Smirnov test was performed to assess the normal distribution of the data in the sample. Spearman’ correlation was used to assess differences in the rates of ARU with the change in the levels of SA. A multivariate analysis study was performed
4
using binary hierarchical logistic regression, to assess the independent factors associated with HAPR. The following potential confounders that were selected were: age, sex, BMI, diabetes mellitus, chronic kidney disease (glomerular filtration rate, using the Modification of Diet in Renal Disease formula, below 50 mL/min/1.73 m2), left ventricular ejection fraction, anemia (hemoglobin<10g/dL), creatinine levels, prior acute MI, past CABG, use of PPI and albumin below or equal to 3.5g/dL. In the first model, all confounders were considered in a single block. However, due to the strong correlation of chronic kidney disease with HAPR, a hierarchical regression was performed, where the effect of CKD was first examined, controlled and followed by a second stage where the independent impact of all other confounders, including levels of albumin, were assessed. Statistical analysis was performed using IBM SPSS Statistics 25 software. For each test, P< 0.05 was considered as statistically significant.
Results Overall 116 patients were included in the current study mean age. 69 ±10, 28% women. A total of 20 patients (17%) were hypoalbuminemic as per the previously mentioned definitions. Baseline characteristics of the study cohort; comparison between hypoalbuminemic and patients with normal albumin level is presented in Tables 1 and 2. Overall the baseline characteristics of the two groups were well balanced except statistically significant worse renal function in the hypoalbuminemic group. Mean serum albumin levels was significantly lower in the hypoalbuminemic group (3.2±0.2 g/dL vs. 4.2±0.4 g/dL, respectively p<0.001) while mean ARU was significantly higher in the hypoalbuminemic compared with the normal albumin group (548±45 vs. 444±66 ARU, respectively, p<0.001). Figure 2 displays ARU according to serum albumin levels of all patients. A strong and statistically significant inverse relationship exists between these two parameters as can be observed in the graph (Spearmans’ correlation coefficient of -0.81, R2=0.67, p<0.001). Moreover, 83% (10/12) of patients with abnormal ARU were hypoalbuminemic and 100% had serum albumin lower than 4 g/dL. Among the hypoalbuminemic patients 55% (11/20) had an abnormal ARU.
5
Multivariate analysis adjusted for multiple potential confounders (Table 3) found that albumin ≤3.5 is the strongest predictor of HAPR in these patients (HR=4.9, 95%CI=2.2-32, p=0.002). Another statistically significant independent predictor of inadequate platelets response was CKD (HR=1.86, 95%CI-1.15-11.6, p=0.04). To analyze the factors associated with the occurrence of HAPR, due to the fact that CKD was more prevalent in the hypoalbuminemic group, we used hierarchical logistic regression to control for the strong and well-established impact of CKD on HAPR. We have organized the independent variables into two hierarchical blocks: CKD as a first block, and the rest of the factors in the second. In this model, the second block demonstrated a strong independent, although attenuated, impact of albumin< 3.5 on HAPR (supplementary table 1). The model presented good fit, according to the Hosmer-Lemeshow statistics (p = 0.948).
Discussion The current study found a strong inverse association between SA levels and ARU in patients with stable coronary artery disease who were treated with aspirin pharmacotherapy. More specifically, hypoalbuminemic patients were found to be at almost 5-fold greater risk for aspirin resistance. This unique association remains strong and statistically significant following adjustment to potential confounders, which are considered to be linked with abnormal ARU. To the best of our knowledge, this is the first investigation to evaluate and describe such an association. Furthermore, such a finding substantiates one of the more prominent potential explanations for the strong association between hypoalbuminemia and worse cardiovascular outcomes shown in recent studies 2-4, 12, 13
.
The findings in this study are consistent with those of Paar et al. 8 that recently assessed platelet aggregation according to low, normal and high levels of albumin (by adding albumin in vitro) in whole blood samples of healthy volunteers and found that it was significantly higher in the presence of low concentrations of albumin compared with physiological concentration. Our findings are also consistent with those of Basili et al. 6 that showed in a combination of in vivo and in vitro studies that reduction in albumin is progressively associated with increased platelet aggregation and increased risk for portal vein thrombosis in patients with liver cirrhosis. Furthermore, markers of increased platelet aggregation improved (decreased) following albumin infusion administered to hypoalbuminemic patients.
6
The pathophysiological mechanisms explaining the observed association between albumin and inadequate response to aspirin have not been fully elucidated. However, based on previous studies using in vitro and in vivo (mostly animal studies) models, the following explanations can be suggested: first, albumin binds and thus prevents the degradation of the platelet aggregation inhibitor prostacyclin (PGI2) as well as increases its availability for its receptor on platelets 14, 15. Furthermore, it has been reported that albumin has high affinity for binding platelet-activating factor (PAF) and as a result suppressing its pro-aggregation activity in a doseresponse manner 16, 17. Albumin has also been shown to prevent the formation of, as well as inactivate, the platelet agonist thromboxane A2, the first through binding of arachidonic acid (the substrate of platelet-derived cyclooxygenases) and the second by direct binding 18. Additionally, albumin can promote the activity of inducible nitric oxide synthase in macrophages which leads to enhanced formation of the potent platelet inhibitor nitric oxide (NO) 19, 20. Furthermore, albumin has been reported to exert an antioxidant effect by binding and inactivating free metal (i.e. copper or iron) which catalyze reactive oxidant species (ROS), by quenching ROS 7, 21
and inactivation of Nox2, an important cellular producer of ROS, and a plausible platelet activator
6, 22, 23
. In
fact, genetic deficiency of Nox2 has been reported in association with impaired platelet activation and increased thrombosis 23, 24. Aspirin is extensively bound to albumin (approximately 90%) in a saturated and complex manner, which changes with changing concentrations of both, hence affecting the pharmacokinetics and possibly its inhibitory effect over platelet aggregation 25, 26. Furthermore, the binding of aspirin to albumin has been shown to result in acetylation of albumin which resulted in altered affinity for binding other substances (e.g. reduced affinity for prostaglandins) 27, 28. This could compromise the direct anti-thrombotic effects of albumin as above-mentioned, especially when albumin levels are reduced, however additional studies are needed to substantiate this hypothesis 27. Remarkably, except albumin levels, only CKD independently predicted inadequate response to aspirin in the current study. Previous studies reported that the following parameters were associated with increased risk for aspirin resistance; CKD, prior myocardial infarction, diabetes mellitus, high platelet count and low hemoglobin 29
. The discrepancy with our findings could stem from the relatively moderate sample size, and the use of a
7
laboratory test rather than clinical outcome as the definition of inadequate response. Nevertheless, in great consistency with our findings, Larsen et al. 30 recently found that the only conventional risk factor (albumin was not evaluated) independently predicting clinical outcomes in patients with stable CAD taking aspirin, was renal insufficiency, hence emphasizing the importance of this risk factor. The limitations, as well as the implications of the current study, should be discussed. First, this is cross-sectional and observational study from a single center, with a relatively small sample size and scarce female patients, thus generalizability of the findings is limited and causality between the hypoalbuminemia and platelet aggregation cannot be firmly substantiated. Nevertheless, the strong and gradual association between hypoalbuminemia and HAPR, as well as the vast amount of data showing that these patients are at increased risk for deleterious outcomes, imply that these patients should be monitored closely. The reasons for hypoalbuminemia should be investigated and interventions aimed at increasing albumin levels or altering the anti-thrombotic regimen to improve patient outcomes should be evaluated in future studies. Additionally, further investigation with better control and adjustment for renal function, a potential confounder, is in order. In order to improve the validity of the findings we enrolled ad-hoc contemporary patients, in addition to our historic cohort. However, unaccounted differences between the time periods might have introduced some bias. Third, although we excluded known causes for acute reduction in levels of albumin (e.g. acute infection) the reasons for the low albumin were not evaluated in this study nor were clinical outcomes or the impact of changes over time in albumin values In conclusion, the current study reports, for the first time, the significant association between serum albumin level and HAPR among patients with stable coronary artery disease treated with aspirin. Hypoalbuminemia confers an almost 5-fold greater risk for HAPR compared with patients with normal albumin levels. These findings may support the hypothesis that increased platelet activation is one of the mechanisms mediating hypoalbuminemia and worse cardiovascular outcomes. Conflict of interest The authors declare no conflict of interest
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References
1.
Garcia-Martinez R, Caraceni P, Bernardi M, Gines P, Arroyo V, Jalan R. Albumin: Pathophysiologic basis of its role in the treatment of cirrhosis and its complications. Hepatology (Baltimore, Md.) 2013;58:1836-1846
2.
Arques S. Human serum albumin in cardiovascular diseases. Eur J Int Med 2018;52:8-12
3.
Chien SC, Chen CY, Lin CF, Yeh HI. Critical appraisal of the role of serum albumin in cardiovascular disease. Biomarker Research 2017;5:31
4.
Plakht Y, Gilutz H, Shiyovich A. Decreased admission serum albumin level is an independent predictor of long-term mortality in hospital survivors of acute myocardial infarction. Soroka acute myocardial infarction ii (sami-ii) project. Int J Cardiol 2016;219:20-24
5.
Shiyovich A, Bental T, Assali A, Vaknin-Assa H, Kornowski R, Perl L. Changes over time in serum albumin levels predict outcomes following percutaneous coronary intervention. J Cardiol 2019
6.
Basili S, Carnevale R, Nocella C, Bartimoccia S, Raparelli V, Talerico G, Stefanini L, Romiti GF, Perticone F, Corazza GR, Piscaglia F, Pietrangelo A, Violi F. Serum albumin is inversely associated with portal vein thrombosis in cirrhosis. Hepatology Comm 2019;3:504-512
7.
Folsom AR, Lutsey PL, Heckbert SR, Cushman M. Serum albumin and risk of venous thromboembolism. Thrombosis and Haemostasis 2010;104:100-104
8.
Paar M, Rossmann C, Nusshold C, Wagner T, Schlagenhauf A, Leschnik B, Oettl K, Koestenberger M, Cvirn G, Hallstrom S. Anticoagulant action of low, physiologic, and high albumin levels in whole blood. PloS one 2017;12:e0182997
9.
Schwartz KA. Aspirin resistance: A clinical review focused on the most common cause, noncompliance. The Neurohospitalist 2011;1:94-103
10.
Price MJ, Angiolillo DJ, Teirstein PS, Lillie E, Manoukian SV, Berger PB, Tanguay JF, Cannon CP, Topol EJ. Platelet reactivity and cardiovascular outcomes after percutaneous coronary intervention: A
9
time-dependent analysis of the gauging responsiveness with a verifynow p2y12 assay: Impact on thrombosis and safety (gravitas) trial. Circulation 2011;124:1132-1137 11.
Campo G, Fileti L, de Cesare N, Meliga E, Furgieri A, Russo F, Colangelo S, Brugaletta S, Ferrari R, Valgimigli M. Long-term clinical outcome based on aspirin and clopidogrel responsiveness status after elective percutaneous coronary intervention: A 3t/2r (tailoring treatment with tirofiban in patients showing resistance to aspirin and/or resistance to clopidogrel) trial substudy. J Am Coll Cardiol 2010;56:1447-1455
12.
Oduncu V, Erkol A, Karabay CY, Kurt M, Akgun T, Bulut M, Pala S, Kirma C. The prognostic value of serum albumin levels on admission in patients with acute st-segment elevation myocardial infarction undergoing a primary percutaneous coronary intervention. Coronary Artery Disease 2013;24:88-94
13.
Kurtul A, Ocek AH, Murat SN, Yarlioglues M, Demircelik MB, Duran M, Ergun G, Cay S. Serum albumin levels on admission are associated with angiographic no-reflow after primary percutaneous coronary intervention in patients with st-segment elevation myocardial infarction. Angiology 2015;66:278-285
14.
Weiss HJ, Turitto VT. Prostacyclin (prostaglandin i2, pgi2) inhibits platelet adhesion and thrombus formation on subendothelium. Blood 1979;53:244-250
15.
Tsai AL, Hsu MJ, Patsch W, Wu KK. Regulation of pgi2 activity by serum proteins: Serum albumin but not high density lipoprotein is the pgi2 binding and stabilizing protein in human blood. Biochimica et biophysica acta 1991;1115:131-140
16.
Grigoriadis G, Stewart AG. Albumin inhibits platelet-activating factor (paf)-induced responses in platelets and macrophages: Implications for the biologically active form of paf. Brit J Bharmacol 1992;107:73-77
17.
Clay KL, Johnson C, Henson P. Binding of platelet activating factor to albumin. Biochimica et biophysica acta 1990;1046:309-314
18.
Maclouf J, Kindahl H, Granstrom E, Samuelsson B. Interactions of prostaglandin h2 and thromboxane a2 with human serum albumin. Eur J Biochem 1980;109:561-566
10
19.
Poteser M, Wakabayashi I. Serum albumin induces inos expression and no production in raw 267.4 macrophages. Brit J Pharmacol 2004;143:143-151
20.
Podesser BK, Hallstrom S. Nitric oxide homeostasis as a target for drug additives to cardioplegia. Brit J Pharmacol 2007;151:930-940
21.
Pratico D, Pasin M, Barry OP, Ghiselli A, Sabatino G, Iuliano L, FitzGerald GA, Violi F. Iron-dependent human platelet activation and hydroxyl radical formation: Involvement of protein kinase c. Circulation 1999;99:3118-3124
22.
Violi F. Editorial commentary: Nox2: A new challenge for antiplatelet treatment? Trends in Cardiovascular Medicine 2018;28:435-436
23.
Violi F, Carnevale R, Loffredo L, Pignatelli P, Gallin JI. Nadph oxidase-2 and atherothrombosis: Insight from chronic granulomatous disease. Arteriosclerosis, Thrombosis, and Vascular Biology 2017;37:218225
24.
Carnevale R, Loffredo L, Sanguigni V, Plebani A, Rossi P, Pignata C, Martire B, Finocchi A, Pietrogrande MC, Azzari C, Soresina AR, Martino S, Cirillo E, Martino F, Pignatelli P, Violi F. Different degrees of nadph oxidase 2 regulation and in vivo platelet activation: Lesson from chronic granulomatous disease. J Am Heart Assoc 2014;3:e000920
25.
Juurlink DN, Gosselin S, Kielstein JT, Ghannoum M, Lavergne V, Nolin TD, Hoffman RS. Extracorporeal treatment for salicylate poisoning: Systematic review and recommendations from the extrip workgroup. Ann Em Med 2015;66:165-181
26.
Lee S, Johnson D, Klein J, Eppler J. Protein binding of acetylsalicylic acid and salicylic acid in porcine and human serum. Veterinary and Human Toxicology 1995;37:224-225
27.
Liyasova MS, Schopfer LM, Lockridge O. Reaction of human albumin with aspirin in vitro: Mass spectrometric identification of acetylated lysines 199, 402, 519, and 545. Biochem Pharmacol 2010;79:784-791
28.
Attallah AA, Lee JB. Indomethacin, salicylates and prostaglandin binding. Prostaglandins 1980;19:311
11
29.
Larsen SB, Grove EL, Neergaard-Petersen S, Wurtz M, Hvas AM, Kristensen SD. Determinants of reduced antiplatelet effect of aspirin in patients with stable coronary artery disease. PloS one 2015;10:e0126767
30.
Larsen SB, Grove EL, Neergaard-Petersen S, Wurtz M, Hvas AM, Kristensen SD. Reduced antiplatelet effect of aspirin does not predict cardiovascular events in patients with stable coronary artery disease. J Am Heart Assoc 2017;6
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Figure 1. Aspirin reactivity units (ARU) by albumin levels
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Relation of Hypoalbuminemia to Response to Aspirin in Patients with Stable Coronary Artery Disease Arthur Shiyovich MD , Liat Sasson MD , Eli Lev MD , Alejandro Solodky MD , Ran Kornowski MD , Leor Perl MD PII: DOI: Reference:
S0002-9149(19)31248-2 https://doi.org/10.1016/j.amjcard.2019.10.055 AJC 24285
To appear in:
The American Journal of Cardiology
Received date: Revised date: Accepted date:
9 September 2019 26 October 2019 30 October 2019
Please cite this article as: Arthur Shiyovich MD , Liat Sasson MD , Eli Lev MD , Alejandro Solodky MD , Ran Kornowski MD , Leor Perl MD , Relation of Hypoalbuminemia to Response to Aspirin in Patients with Stable Coronary Artery Disease, The American Journal of Cardiology (2019), doi: https://doi.org/10.1016/j.amjcard.2019.10.055
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 Inc.
Relation of Hypoalbuminemia to Response to Aspirin in Patients with Stable Coronary Artery Disease Arthur Shiyovich MD*, Liat Sasson MD *, Eli Lev MD, Alejandro Solodky MD, Ran Kornowski MD, Leor Perl MD
Department of Cardiology, Rabin Medical Center, Beilinson Hospital, Petah-Tikva, and "Sackler" Faculty of Medicine, Tel-Aviv University Israel. *Equal contribution Corresponding author: Shiyovich Arthur MD. Beilinson Hospital, Rabin Medical Center, 39 Jabotinski Street, Petah Tikva, Israel, 49100. email: [email protected] Tel: +972-39377100 Fax: +97239377101 Running title: hypoalbuminemia is associated with aspirin resistance
1
Abstract
Serum albumin (SA) level is a powerful cardiovascular prognostic marker, suggested to be involved in regulation of platelet function. High on-aspirin platelet reactivity (HAPR) is associated with increased risk for deleterious cardiovascular events. The aim of the current study was to evaluate the association between HAPR and albumin levels in patients with stable coronary artery disease (CAD) treated with aspirin. Patients with known stable CAD, who were taking aspirin (75-100 mg qd) regularly for at least one month, were screened for the current study. Exclusion criteria: cancer, sepsis or acute infection, active inflammatory/rheumatic disease, recent major surgery, chronic liver failure, the administration of other anti-platelet drugs, non-adherence with aspirin and thrombocytopenia. Blood was drawn from the participants and sent for SA level and platelet function test (VerifyNow). HAPR was defined as aspirin reaction units (ARU) >550. Overall 116 patients were analyzed; age 69 ±10, 28% women. Twenty (17%) were hypoalbuminemic (≤3.5 g/dL). Hypoalbuminemic patients had similar characteristics to the normal albumin group except mildly higher creatinine in the former. SA levels were significantly lower in the hypoalbuminemic group (3.2±0.2 g/dL vs. 4.2±0.4 g/dL, respectively, p<0.001) while mean ARU was significantly higher compared to the normal albumin group (548±45 vs. 444±66 ARU, respectively, p<0.001). A significant inverse association was observed between SA and ARU with (R2=0.67, p<0.001). Multivariate analysis adjusted for potential confounders found that albumin ≤3.5 is the strongest predictor of HAPR among patients with stable CAD (HR=4.9, 95%CI=2.2-32, p=0.002). In conclusion, hypoalbuminemia is strongly
associated with HAPR among patients with stable CAD.
Key words: serum albumin level; High on-aspirin platelet reactivity (HAPR); coronary artery disease
2
Introduction
Albumin is a pivotal protein in human plasma with a great variety of physiological roles that include: maintaining capillary permeability and plasma osmotic pressure, transporting various substances affecting their metabolism and activity, anti-oxidative and anti-inflammatory functions, as well as significant relations with coagulation pathways and platelet function 1, 2. Hypoalbuminemia is emerging as a powerful cardiovascular prognostic marker 2-5. Thrombotic complications were also found to be related with hypoalbuminemia 6, 7. One of the suggested mechanisms for the increased cardiovascular morbidity and
worse outcomes in patients with hypoalbuminemia is that albumin interferes with mechanisms supporting platelet aggregation 6, 8. However, empiric data to support such mechanisms are scarce. Aspirin is the most commonly used antiplatelet agent in patients with cardiovascular diseases. An inadequate response to prescribed aspirin in these patients is associated with increased risk for subsequent deleterious events 9. The aim of the current study was to evaluate the association between platelet function (i.e. platelet aggregation) and albumin levels in patients with coronary artery disease (CAD) treated with aspirin.
Methods The current study included two prospective subsets of patients as following; the first group comprised a historic prospective cohort of patients (n=66) while the second group included contemporary patients that were enrolled ad-hoc (n=50). All patients had known stable CAD defined as patients with previously proven CAD (a stenosis of at least 50% in one or more epicardial coronary arteries by coronary angiography) that were free from acute coronary syndrome, percutaneous coronary intervention or coronary artery bypass graft surgery within 6 months prior to inclusion in the study, were regularly taking Aspirin (75-100 mg qd) for at least a month and were either evaluated in the outpatient clinics (e.g. routine follow-up) or hospitalized in our institution, a tertiary medical center, for evaluation of symptoms other than those classified as exclusion criteria. Exclusion criteria: acute coronary syndrome, known malignancy (patients with remission >2 year were eligible), sepsis or acute infection, active inflammatory/rheumatic disease, major surgery in the last 6 months, chronic liver failure, treatment with anti-platelet drugs other than Aspirin ( i.e Clopidogrel, Ticagrelor or Prasugrel), treatment with
3
oral anticoagulation, allergy to- or non-adherence with Aspirin and thrombocytopenia (<100K/micl). The institutional review board approved the study, which was performed in accordance with the Helsinki declaration.
Following informed consent and patients’ verbal confirmation of adherence with Aspirin within the last month and in the day of the test, blood was drawn from the antecubital vein and split in two sets of experimental batches; one sent to the biochemical laboratory for CBC and biochemistry including serum albumin level while the second was used for the platelet function test (VerifyNow) as elaborated below. Since in the first (historic) cohort serum albumin level measurement was not performed upon enrollment, as was done in the second cohort, we included only patients who had albumin results ≤1-month from enrollment and the VerifyNow test (see below). The VerifyNow system (Accumetrics, San Diego, CA) is a whole-blood assay based on light transmission measurements. The Aspirin-specific cartridge is used to assess platelet dysfunction caused by Aspirin. In the Aspirin-specific assay, arachidonic acid activates platelets, and the activated platelets bind to fibrinogen-coated beads to form an aggregate. The degree of aggregation is quantified by a corresponding increase in light transmission and is reported in Aspirin reaction units (ARU). High on-aspirin platelet reactivity (HAPR) was defined as ARU >550 in consistency with previous studies 10, 11. Data were obtained from the hospital’s computerized medical records and included demographic and clinical characteristics, cardiovascular risk factors and co-morbidity, procedural characteristics and workup (e.g. blood tests, echocardiography). Patients were divided into two groups based in the serum albumin level: hypoalbuminemic; SA≤3.5 g/dL and normoalbuminemic SA>3.5 g/dL (normal range 3.5-5.5 g/dL) as this cutoff was also shown to be of prognostic value in patients with coronary artery disease 12. Glomerular filtration rate was calculated by the Modification of Diet in Renal Disease formula. Patient characteristics were presented as mean and standard deviation (SD) for continuous variables and n and percent for the categorical data. Chi-square or Fischer exact tests were used for analysis of categorical variables when appropriate. The unpaired student t-test was used for analysis of continuous variables. The Kolmogorov–Smirnov test was performed to assess the normal distribution of the data in the sample. Spearman’ correlation was used to assess differences in the rates of ARU with the change in the levels of SA. A multivariate analysis study was performed
4
using binary hierarchical logistic regression, to assess the independent factors associated with HAPR. The following potential confounders that were selected were: age, sex, BMI, diabetes mellitus, chronic kidney disease (glomerular filtration rate, using the Modification of Diet in Renal Disease formula, below 50 mL/min/1.73 m2), left ventricular ejection fraction, anemia (hemoglobin<10g/dL), creatinine levels, prior acute MI, past CABG, use of PPI and albumin below or equal to 3.5g/dL. In the first model, all confounders were considered in a single block. However, due to the strong correlation of chronic kidney disease with HAPR, a hierarchical regression was performed, where the effect of CKD was first examined, controlled and followed by a second stage where the independent impact of all other confounders, including levels of albumin, were assessed. Statistical analysis was performed using IBM SPSS Statistics 25 software. For each test, P< 0.05 was considered as statistically significant.
Results Overall 116 patients were included in the current study mean age. 69 ±10, 28% women. A total of 20 patients (17%) were hypoalbuminemic as per the previously mentioned definitions. Baseline characteristics of the study cohort; comparison between hypoalbuminemic and patients with normal albumin level is presented in Tables 1 and 2. Overall the baseline characteristics of the two groups were well balanced except statistically significant worse renal function in the hypoalbuminemic group. Mean serum albumin levels was significantly lower in the hypoalbuminemic group (3.2±0.2 g/dL vs. 4.2±0.4 g/dL, respectively p<0.001) while mean ARU was significantly higher in the hypoalbuminemic compared with the normal albumin group (548±45 vs. 444±66 ARU, respectively, p<0.001). Figure 2 displays ARU according to serum albumin levels of all patients. A strong and statistically significant inverse relationship exists between these two parameters as can be observed in the graph (Spearmans’ correlation coefficient of -0.81, R2=0.67, p<0.001). Moreover, 83% (10/12) of patients with abnormal ARU were hypoalbuminemic and 100% had serum albumin lower than 4 g/dL. Among the hypoalbuminemic patients 55% (11/20) had an abnormal ARU.
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Multivariate analysis adjusted for multiple potential confounders (Table 3) found that albumin ≤3.5 is the strongest predictor of HAPR in these patients (HR=4.9, 95%CI=2.2-32, p=0.002). Another statistically significant independent predictor of inadequate platelets response was CKD (HR=1.86, 95%CI-1.15-11.6, p=0.04). To analyze the factors associated with the occurrence of HAPR, due to the fact that CKD was more prevalent in the hypoalbuminemic group, we used hierarchical logistic regression to control for the strong and well-established impact of CKD on HAPR. We have organized the independent variables into two hierarchical blocks: CKD as a first block, and the rest of the factors in the second. In this model, the second block demonstrated a strong independent, although attenuated, impact of albumin< 3.5 on HAPR (supplementary table 1). The model presented good fit, according to the Hosmer-Lemeshow statistics (p = 0.948).
Discussion The current study found a strong inverse association between SA levels and ARU in patients with stable coronary artery disease who were treated with aspirin pharmacotherapy. More specifically, hypoalbuminemic patients were found to be at almost 5-fold greater risk for aspirin resistance. This unique association remains strong and statistically significant following adjustment to potential confounders, which are considered to be linked with abnormal ARU. To the best of our knowledge, this is the first investigation to evaluate and describe such an association. Furthermore, such a finding substantiates one of the more prominent potential explanations for the strong association between hypoalbuminemia and worse cardiovascular outcomes shown in recent studies 2-4, 12, 13
.
The findings in this study are consistent with those of Paar et al. 8 that recently assessed platelet aggregation according to low, normal and high levels of albumin (by adding albumin in vitro) in whole blood samples of healthy volunteers and found that it was significantly higher in the presence of low concentrations of albumin compared with physiological concentration. Our findings are also consistent with those of Basili et al. 6 that showed in a combination of in vivo and in vitro studies that reduction in albumin is progressively associated with increased platelet aggregation and increased risk for portal vein thrombosis in patients with liver cirrhosis. Furthermore, markers of increased platelet aggregation improved (decreased) following albumin infusion administered to hypoalbuminemic patients.
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The pathophysiological mechanisms explaining the observed association between albumin and inadequate response to aspirin have not been fully elucidated. However, based on previous studies using in vitro and in vivo (mostly animal studies) models, the following explanations can be suggested: first, albumin binds and thus prevents the degradation of the platelet aggregation inhibitor prostacyclin (PGI2) as well as increases its availability for its receptor on platelets 14, 15. Furthermore, it has been reported that albumin has high affinity for binding platelet-activating factor (PAF) and as a result suppressing its pro-aggregation activity in a doseresponse manner 16, 17. Albumin has also been shown to prevent the formation of, as well as inactivate, the platelet agonist thromboxane A2, the first through binding of arachidonic acid (the substrate of platelet-derived cyclooxygenases) and the second by direct binding 18. Additionally, albumin can promote the activity of inducible nitric oxide synthase in macrophages which leads to enhanced formation of the potent platelet inhibitor nitric oxide (NO) 19, 20. Furthermore, albumin has been reported to exert an antioxidant effect by binding and inactivating free metal (i.e. copper or iron) which catalyze reactive oxidant species (ROS), by quenching ROS 7, 21
and inactivation of Nox2, an important cellular producer of ROS, and a plausible platelet activator
6, 22, 23
. In
fact, genetic deficiency of Nox2 has been reported in association with impaired platelet activation and increased thrombosis 23, 24. Aspirin is extensively bound to albumin (approximately 90%) in a saturated and complex manner, which changes with changing concentrations of both, hence affecting the pharmacokinetics and possibly its inhibitory effect over platelet aggregation 25, 26. Furthermore, the binding of aspirin to albumin has been shown to result in acetylation of albumin which resulted in altered affinity for binding other substances (e.g. reduced affinity for prostaglandins) 27, 28. This could compromise the direct anti-thrombotic effects of albumin as above-mentioned, especially when albumin levels are reduced, however additional studies are needed to substantiate this hypothesis 27. Remarkably, except albumin levels, only CKD independently predicted inadequate response to aspirin in the current study. Previous studies reported that the following parameters were associated with increased risk for aspirin resistance; CKD, prior myocardial infarction, diabetes mellitus, high platelet count and low hemoglobin 29
. The discrepancy with our findings could stem from the relatively moderate sample size, and the use of a
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laboratory test rather than clinical outcome as the definition of inadequate response. Nevertheless, in great consistency with our findings, Larsen et al. 30 recently found that the only conventional risk factor (albumin was not evaluated) independently predicting clinical outcomes in patients with stable CAD taking aspirin, was renal insufficiency, hence emphasizing the importance of this risk factor. The limitations, as well as the implications of the current study, should be discussed. First, this is cross-sectional and observational study from a single center, with a relatively small sample size and scarce female patients, thus generalizability of the findings is limited and causality between the hypoalbuminemia and platelet aggregation cannot be firmly substantiated. Nevertheless, the strong and gradual association between hypoalbuminemia and HAPR, as well as the vast amount of data showing that these patients are at increased risk for deleterious outcomes, imply that these patients should be monitored closely. The reasons for hypoalbuminemia should be investigated and interventions aimed at increasing albumin levels or altering the anti-thrombotic regimen to improve patient outcomes should be evaluated in future studies. Additionally, further investigation with better control and adjustment for renal function, a potential confounder, is in order. In order to improve the validity of the findings we enrolled ad-hoc contemporary patients, in addition to our historic cohort. However, unaccounted differences between the time periods might have introduced some bias. Third, although we excluded known causes for acute reduction in levels of albumin (e.g. acute infection) the reasons for the low albumin were not evaluated in this study nor were clinical outcomes or the impact of changes over time in albumin values In conclusion, the current study reports, for the first time, the significant association between serum albumin level and HAPR among patients with stable coronary artery disease treated with aspirin. Hypoalbuminemia confers an almost 5-fold greater risk for HAPR compared with patients with normal albumin levels. These findings may support the hypothesis that increased platelet activation is one of the mechanisms mediating hypoalbuminemia and worse cardiovascular outcomes. Conflict of interest The authors declare no conflict of interest
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References
1.
Garcia-Martinez R, Caraceni P, Bernardi M, Gines P, Arroyo V, Jalan R. Albumin: Pathophysiologic basis of its role in the treatment of cirrhosis and its complications. Hepatology (Baltimore, Md.) 2013;58:1836-1846
2.
Arques S. Human serum albumin in cardiovascular diseases. Eur J Int Med 2018;52:8-12
3.
Chien SC, Chen CY, Lin CF, Yeh HI. Critical appraisal of the role of serum albumin in cardiovascular disease. Biomarker Research 2017;5:31
4.
Plakht Y, Gilutz H, Shiyovich A. Decreased admission serum albumin level is an independent predictor of long-term mortality in hospital survivors of acute myocardial infarction. Soroka acute myocardial infarction ii (sami-ii) project. Int J Cardiol 2016;219:20-24
5.
Shiyovich A, Bental T, Assali A, Vaknin-Assa H, Kornowski R, Perl L. Changes over time in serum albumin levels predict outcomes following percutaneous coronary intervention. J Cardiol 2019
6.
Basili S, Carnevale R, Nocella C, Bartimoccia S, Raparelli V, Talerico G, Stefanini L, Romiti GF, Perticone F, Corazza GR, Piscaglia F, Pietrangelo A, Violi F. Serum albumin is inversely associated with portal vein thrombosis in cirrhosis. Hepatology Comm 2019;3:504-512
7.
Folsom AR, Lutsey PL, Heckbert SR, Cushman M. Serum albumin and risk of venous thromboembolism. Thrombosis and Haemostasis 2010;104:100-104
8.
Paar M, Rossmann C, Nusshold C, Wagner T, Schlagenhauf A, Leschnik B, Oettl K, Koestenberger M, Cvirn G, Hallstrom S. Anticoagulant action of low, physiologic, and high albumin levels in whole blood. PloS one 2017;12:e0182997
9.
Schwartz KA. Aspirin resistance: A clinical review focused on the most common cause, noncompliance. The Neurohospitalist 2011;1:94-103
10.
Price MJ, Angiolillo DJ, Teirstein PS, Lillie E, Manoukian SV, Berger PB, Tanguay JF, Cannon CP, Topol EJ. Platelet reactivity and cardiovascular outcomes after percutaneous coronary intervention: A
9
time-dependent analysis of the gauging responsiveness with a verifynow p2y12 assay: Impact on thrombosis and safety (gravitas) trial. Circulation 2011;124:1132-1137 11.
Campo G, Fileti L, de Cesare N, Meliga E, Furgieri A, Russo F, Colangelo S, Brugaletta S, Ferrari R, Valgimigli M. Long-term clinical outcome based on aspirin and clopidogrel responsiveness status after elective percutaneous coronary intervention: A 3t/2r (tailoring treatment with tirofiban in patients showing resistance to aspirin and/or resistance to clopidogrel) trial substudy. J Am Coll Cardiol 2010;56:1447-1455
12.
Oduncu V, Erkol A, Karabay CY, Kurt M, Akgun T, Bulut M, Pala S, Kirma C. The prognostic value of serum albumin levels on admission in patients with acute st-segment elevation myocardial infarction undergoing a primary percutaneous coronary intervention. Coronary Artery Disease 2013;24:88-94
13.
Kurtul A, Ocek AH, Murat SN, Yarlioglues M, Demircelik MB, Duran M, Ergun G, Cay S. Serum albumin levels on admission are associated with angiographic no-reflow after primary percutaneous coronary intervention in patients with st-segment elevation myocardial infarction. Angiology 2015;66:278-285
14.
Weiss HJ, Turitto VT. Prostacyclin (prostaglandin i2, pgi2) inhibits platelet adhesion and thrombus formation on subendothelium. Blood 1979;53:244-250
15.
Tsai AL, Hsu MJ, Patsch W, Wu KK. Regulation of pgi2 activity by serum proteins: Serum albumin but not high density lipoprotein is the pgi2 binding and stabilizing protein in human blood. Biochimica et biophysica acta 1991;1115:131-140
16.
Grigoriadis G, Stewart AG. Albumin inhibits platelet-activating factor (paf)-induced responses in platelets and macrophages: Implications for the biologically active form of paf. Brit J Bharmacol 1992;107:73-77
17.
Clay KL, Johnson C, Henson P. Binding of platelet activating factor to albumin. Biochimica et biophysica acta 1990;1046:309-314
18.
Maclouf J, Kindahl H, Granstrom E, Samuelsson B. Interactions of prostaglandin h2 and thromboxane a2 with human serum albumin. Eur J Biochem 1980;109:561-566
10
19.
Poteser M, Wakabayashi I. Serum albumin induces inos expression and no production in raw 267.4 macrophages. Brit J Pharmacol 2004;143:143-151
20.
Podesser BK, Hallstrom S. Nitric oxide homeostasis as a target for drug additives to cardioplegia. Brit J Pharmacol 2007;151:930-940
21.
Pratico D, Pasin M, Barry OP, Ghiselli A, Sabatino G, Iuliano L, FitzGerald GA, Violi F. Iron-dependent human platelet activation and hydroxyl radical formation: Involvement of protein kinase c. Circulation 1999;99:3118-3124
22.
Violi F. Editorial commentary: Nox2: A new challenge for antiplatelet treatment? Trends in Cardiovascular Medicine 2018;28:435-436
23.
Violi F, Carnevale R, Loffredo L, Pignatelli P, Gallin JI. Nadph oxidase-2 and atherothrombosis: Insight from chronic granulomatous disease. Arteriosclerosis, Thrombosis, and Vascular Biology 2017;37:218225
24.
Carnevale R, Loffredo L, Sanguigni V, Plebani A, Rossi P, Pignata C, Martire B, Finocchi A, Pietrogrande MC, Azzari C, Soresina AR, Martino S, Cirillo E, Martino F, Pignatelli P, Violi F. Different degrees of nadph oxidase 2 regulation and in vivo platelet activation: Lesson from chronic granulomatous disease. J Am Heart Assoc 2014;3:e000920
25.
Juurlink DN, Gosselin S, Kielstein JT, Ghannoum M, Lavergne V, Nolin TD, Hoffman RS. Extracorporeal treatment for salicylate poisoning: Systematic review and recommendations from the extrip workgroup. Ann Em Med 2015;66:165-181
26.
Lee S, Johnson D, Klein J, Eppler J. Protein binding of acetylsalicylic acid and salicylic acid in porcine and human serum. Veterinary and Human Toxicology 1995;37:224-225
27.
Liyasova MS, Schopfer LM, Lockridge O. Reaction of human albumin with aspirin in vitro: Mass spectrometric identification of acetylated lysines 199, 402, 519, and 545. Biochem Pharmacol 2010;79:784-791
28.
Attallah AA, Lee JB. Indomethacin, salicylates and prostaglandin binding. Prostaglandins 1980;19:311
11
29.
Larsen SB, Grove EL, Neergaard-Petersen S, Wurtz M, Hvas AM, Kristensen SD. Determinants of reduced antiplatelet effect of aspirin in patients with stable coronary artery disease. PloS one 2015;10:e0126767
30.
Larsen SB, Grove EL, Neergaard-Petersen S, Wurtz M, Hvas AM, Kristensen SD. Reduced antiplatelet effect of aspirin does not predict cardiovascular events in patients with stable coronary artery disease. J Am Heart Assoc 2017;6
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Figure 1. Aspirin reactivity units (ARU) by albumin levels
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