Abstract
Background: Cardiovascular diseases are a leading cause of mortality after kidney transplantation. According to guidelines, acetylsalicylic acid (ASA) must be given as preventive antiplatelet therapy, but resistance to this drug is also well-known. Patients and Methods: A total of 214 renal transplant patients were included in our study and took 100 mg of ASA q.d. Aggregometry was performed to determine resistance. Twenty-four variables were examined using logistic regression analysis as possible causes of resistance. Results: ASA resistance was observed in 40.18% of the patients. Resistance reduced concomitant statin therapy and significantly increased simultaneous cyclosporine therapy. Conclusion: Our study assessed the post-transplant ASA resistance in a large population. Clarification of this matter is crutial, since one of the major preventive pharmacological therapies of cardiovascular mortality is not effective in a significant number of patients.
- Aggregometry
- acetylsalicylic acid resistance
- cardiovascular complication
- platelet aggregation
- transplantation
The leading cause of death after kidney transplantation has a cardiovascular origin. Matas et al. examined 2202 kidney transplant recipients and followed them up for a period of 10 years. The main causes of death were cardiovascular diseases (35% among deceased donor and 38% for living donor kidney transplant recipients), cancer (22% and 18% respectively), and miscellaneous infections (13% and 9% respectively) (1). In light of these results, one of the main goals of the postoperative care of patients with transplant is preventing cardiovascular complications. There are multiple possibilities of prevention. In accordance with the 2012 guideline of the European Society of Cardiology the most significant are lifestyle (including a healthy diet and regular exercise), keeping blood pressure, blood sugar and lipids in target range, and maintaining effective inhibition of platelet aggregation (2). Inhibition of platelet aggregation can be accomplished through cyclo-oxygenase (COX) inhibitor acetylsalicylic acid (ASA), purinergic receptor P2Y12 antagonist clopidogrel, or prasugrel, either alone or in combination, according to level IA evidence (2). During the course of an extended analysis of 135,000 patients inhibition of platelet aggregation reduced the risk of stroke, acute myocardial infarction and other vascular deaths by 22%, and the risk of non-fatal stroke by 25% (3). Resistance to antiplatelet therapy is a well-known phenomenon. Data pertaining to ASA resistance was summarised by Mani et al. ASA was found to be ineffective in 5-55% of cases. One of the downsides of that study was the use of different methods to measure resistance, as well as the inclusion of differently medicated patient groups, thus making it difficult to draw comparisons (4). One of the oldest ways to assess platelet function is to perform a bleeding time test, this technique, however, cannot be standardized, and thus it is less recommended (5). In addition to the aforementioned method, platelet function analyzers (PFA-100), flow cytometry and thromboelastometry can be used as well, however the most popular clinical method is platelet aggregometry (6). Aggregometry was first devised in 1962 and is currently the gold standard test for assessing the efficacy of anti-platelet therapy (7). In this study, the incidence of ASA resistance was investigated in renal transplant recipients.
Following these measurements, we tried to determine the cause of resistance, resorting to statistical methods which were based upon previous studies available in medical literature that had already identified a few possible factors as causes of resistance.
Patients and Methods
This investigation was approved by the Regional Ethics Committee of the University of Pecs (approval number: 4816) and written informed consent was signed by all participants.
During the research process encompassing a period from March 2009 to December 2013, 214 recipients were selected by the Department of Surgery, University of Pecs, from a group consisting of patients with previous kidney transplantation history (119 male; 95 female; average age=53.62 years). Patients were routinely given 100 mg of ASA q.d..
Blood samples for platelet aggregation testing were obtained from the cubital vein and collected in 3.8% sodium-citrate tubes. Measurements were performed at the First Department of Medicine, University of Pecs. Following multiple stages of centrifugation, first platelet-rich plasma (PRP), then platelet-poor plasma (PPP) were obtained. Platelet aggregation was induced by epinephrine (10 mM). Measurements were made using a turbidimetric Carat TX-4 (Carat Diagnosztika Ltd., Budapest, Hungary) platelet aggregometer, which calculates the degree of aggregation based on the difference between the optical densitiy of PPP and PRP. In order to determine ASA resistance, epinephrine-induced platelet response was examined. A decrease in maximum aggregation values is considered a result of antiplatelet therapy, if the value has fallen outside of the reference range (mean±2×SD) typical for the normal (untreated) population; otherwise therapy is deemed ineffective. ASA therapy was considered effective as long as the degree of aggregation stayed below 40%.
Further laboratory tests were performed at the Department of Laboratory Medicine, University of Pecs, to determine serum creatinine, glucose, cholesterol and triglyceride levels of the study subjects. Haemoglobin (HGB), haematocrit (HTC) and platelet count (PLT) were measured using an automated analyser. Patients' weight, height and body mass index (BMI) derived from the previously mentioned two values were measured in an outpatient setting. Tobacco use, and the number of therapeutic drugs (lipid-lowering drugs, immunosuppressants, calcium-channel blockers, angiotensin-converting-enzyme (ACE) inhibitors) were assessed. The incidence of chronic allograft nephropathy and steroid-resistant acute rejection were investigated, as well as influencing factors.
All registered clinical and laboratory parameters of ASA resistant/ non-responder and ASA responder patients were compared by multivariate logistic regression analysis for searching factors associated with ASA resistance using the 21.0 version of IBM SPSS Statistics software (IBM Corporation, Armonk, New York, United States). Value of p≤0.05 was considered statistically significant and Odds ratio (OR) and confidential interval (CI) were calculated for the following factors: age >60 years, smoking, BMI >25 kg/m2, presence of each cardiovascular risk factor, each medication, HGB <11 g/dl, HGB >14 g/dl, HCT <35%, HCT >40%, PLT >300×103/μl, haemoglobin A1C >6%, total cholesterol >5.2 mmol/l, LDL >3.5 mmol/l, tryglyceride >1.7 mmol/l, glucose >7.0 mmol/l, creatinine >120 mmol/l.
Results
The following are the results obtained from the aggregometric tests: resistance to ASA was found in 40.19% (86 patients) of the 214 transplant recipients. Treatment was effective in 59.81% (128 patients).
The next steps in the research were performing a logistic regression analysis of resistance and various risk factors in order to establish the possible causes of resistance; 24 variables were examined, which were all listed as potential causes of resistance in medical literature: age, serum creatinine, serum glucose, haematocrit, haemoglobin, tryglyceride, LDL cholesterol, platelet count, mycophenolate mofetil, methylprednisolone, tacrolimus, cyclosporine, mycophenolic acid, everolimus, sirolimus, calcium channel blockers, ACE inhibitors, anti-lipid therapy, steroid resistant acute rejection (SRAR), chronic allograft nephropathy (CAN), type of transplantation, diabetes, BMI, and smoking. Resistance was seen in a lesser proportion of patients receiving anti-lipid (statin) therapy. Furthermore, patients on cyclosporine-containing medicines demonstrated a higher rate of resistance compared to those that did not take such substances (Table I).
Factors found to have positive association to ASA resitance by multivariate logistic regression analysis.
Discussion
The higher incidence of cardiovascular complications after kidney transplantation compared to the general population can be attributed to multiple factors. One is various vascular injuries caused by the pre-transplant years of uraemia. Of course other main factors, such as smoking, diabetes mellitus, hypertension, hyperlipidaemia, obesity and certain non-controllable factors (age, gender and genetic predisposition) also play an important role in the development of cardiovascular complications (8). Moreover, kidney disease and the effect of certain drugs used therapeutically for patients with transplant must also be taken into account. A previous study showed that 42% of patients suffering from chronic kidney disease died of cardiovascular conditions, which are also the leading cause of death (27%) in the first few post-transplant years. It is important to note that over half of these patients died with a functioning kidney graft. Some significant pillars in the prevention of cardiovascular diseases are a healthy lifestyle, wholesome eating and thus reaching an ideal weight. The majority of our patients lead a healthy lifestyle, partly due to the heavy emphasis that was placed on patient education during ambulatory check up visits. The next well-known method of prevention is aggregation inhibition. However, our research has proven that in many cases, it is ineffective because a number of patients are resistant to ASA. Possible causes of resistance to ASA have been thoroughly investigated. In vitro examination of the effect of various immunosuppressive drugs on platelet aggregation was also performed. Aggregation was significantly increased by cyclosporine, while FK506, azathioprin and mycophenolate-mofetil resulted in a decrease (9). These drugs can also increase the development of cardiovascular diseases in other ways. It has been observed that lipid levels are worsened by most of them (cyclosporine, sirolimus, prednisolone), and that disturbances of glucose metabolism may be caused by tacrolimus. (10) Aggregometric measurements may also be influenced by the patient's posture at the time of the venepuncture, their cholesterol levels and the time of the procedure (11). The prevalence of resistance is significantly greater in smokers as opposed to patients who do not smoke (12). Intensified aggregation was seen in patients with diabetes, whose platelets have a greater tendency toward degranulation, as well as thromboxane A2 synthesis. In addition, diabetes-related macro- and microangiopathies are also common (coronary diseases, cerebrovascular events, peripheral vasculopathies, and retinopathy) (13). Ibuprofen use led to statistically significant decreases in ASA efficacy (14). The same study showed that diclofenac-containing drugs had a minimal negative impact on antiaggregation therapy. In other research, the following factors were found to reduce aggregation inhibition: being female (the age and fertility status were not made public), low haemoglobin count, old age with accompanying decreased metabolism, the prevalence of cardiovascular diseases, type 2 diabetes mellitus, kidney impairment and smoking (15). Other influencing factors might be patient adherence and heart rate. In the study, patient education and counselling concerning antithrombotic therapy was considered of great importance. Due to its insidious and unapparent effect, many patients do not consider aggregation inhibitor therapy important. Increased sympathetic tone correlates with an elevation of heart rate, and boosts aggregating ability (16). Other genetic factors that increase the capability of aggregation are COX1 gene polymorphism (affecting the aspirin-binding site, serine residue at position 529), platelet surface glycoprotein gene polymorphism, overexpression of the COX2 gene (17), initiation of platelet activation and alternative pathways of aggregation and the simultaneous activation of various pathways (18).
Numerous studies address the problem of patient non-compliance, therefore, all patients involved in our research received both verbal and written education to bring the possibilities of cardiovascular prevention and the importance of medication adherence to their attention. We believe that questions of the exact cause of resistance and its possible remedies will be answered by genetic investigations.
Summary
High-level resistance to ASA of kidney transplant patients was first proven by our study in a large number of recipients. We also sought answers to the cause of resistance, however, in the same way as previous studies in the past, we were also unable to find a definite solution to the problem. It is important to find potential causes of resistance and to restore aggregation inhibition, a basic element of cardiovascular prevention. Prophylactic treatment of patients is essential; furthermore, controlling the efficacy of therapy along with the treatment of risk factors should also be kept in mind.
Footnotes
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Funding Sources
None.
- Received September 20, 2014.
- Revision received October 20, 2014.
- Accepted October 27, 2014.
- Copyright © 2015 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved
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