Introduction

Cardiovascular disease is the leading cause of mortality in patients with type 2 diabetes mellitus, accounting for an estimated 65–80 % of deaths in these patients [1]. Although there have been substantial reductions in cardiovascular related morbidity and mortality in the general population over the last 40 years attributed to improved treatment of cardiovascular risk factors and disease, the same benefit has not been observed in those with diabetes mellitus [2, 3].

A landmark observational study suggested that patients with diabetes without prior myocardial infarction had a similar risk of coronary heart disease to patients with prior myocardial infarction without diabetes [4].

Numerous pathophysiological mechanisms have been proposed to explain the exceptionally poor outcome of patients with diabetes and coronary artery disease including aggressive atherosclerosis [5], abnormal endothelial function [6, 7], impaired fibrinolysis, platelet hyperactivity [8]. Increased platelet activity plays a major role in the pathogenesis of thrombo-ischaemic complications in diabetic patients.

Significant interest emerged for the widespread implementation of interventions for lowering cardiovascular risk in patients with diabetes, such as the use of aspirin therapy.

Several studies have demonstrated a beneficial role of antiplatelet therapy with aspirin in primary and secondary prevention of coronary heart disease in patients with diabetes [9].

Subgroup analysis of a diabetic cohort of the US Physicians Health Study demonstrated a reduction in the myocardial infarction rate ranging from 10.2 % in the placebo treated group to 4.0 % for the aspirin-treated group [10].

However, aspirin’s antiplatelet effect is not uniform in all patients and reduced antiplatelet activity has been reported between 20 and 30 % of patients [11, 12]. This group seems to have a greater than threefold increase in the risk of major adverse events in stable coronary artery disease [13, 14].

The frequency of this phenomenon varies significantly, depending on the applied detection method and the investigated group of patients. Various study have investigated the effectiveness of antiplatelet therapy and, in their conclusion, several acquired conditions are supposed to play a role in the aspirin responsiveness [15], such as glycaemic control [16–18], hypertension, hypercholesterolemia, smoking or drugs [19–21], age [22] as well as genetic polymorphisms [23, 24].

Beside these factors, different modes of ASA administration might affect the rate of ASA absorption and, consequently, platelet response [25, 26] in fact a reduced absorption of acetyl salicylic acid could compromise the response to anti-aggregation activity. Especially in diabetic patients, in which the peripheral circulation, even in the splanchnic area, is frequently affected, but also in patients with chronic gastroenteric disorders, on gastroprotective therapy or simply with diffuse vascular disease, an insufficient adsorption can be the main factor that makes antiaggregation inefficient.

The aim of our study was to investigate in a cohort of diabetic patients, with and without known cerebral or cardiovascular disease, the prevalence of aspirin resistance and whether this resistance can be reversed by using a more bioavailable formulation of salicylic acid such as the intravenous via and or soluble salt.

Methods

We prospectively enrolled 163 diabetic patients between November 2011 and December 2012. The patients were recruited from consecutive patients presenting to the outpatient clinic for follow-up treatment of type 2 diabetes who were chronically taking acetyl salicylic acid 100 mg daily. We enrolled both male and female over 18 years old with type 2 diabetes diagnosed at least 12 months earlier on pharmacological or insulin therapy and that were taking ASA 100 mg at least for 30 days before the enrolment. Compliance on aspirin was determined by patient interview both at study enrolment and follow-up. Patients taking oral anticoagulation therapy, with a personal history of haemorrhagic disorder, on chronic treatment with non-steroid anti-inflammatory drugs, under administration of heparin or low-molecular-weight heparin within 24 h before enrolment, acute coronary syndrome (ACS) or major cerebral vascular event or hospitalization for any cause within the 30 days before, excessive low or high platelet count (<130,000/μl or >450.000/μl) and history of haematological disorder have been excluded. The assumption of other anti aggregation therapy acting on P2Y12, such as clopidogrel, was not considered an exclusion criterion since the tests used to evaluate the aggregation status are specific for COX1 inhibition. Enrolled patients were taking ASA both in primary prevention, in order to reduce the risk of cardiovascular event in a high risk population, and in secondary prevention after ACS or transient ischaemic attack (TIA)/stroke. We also enrolled those patients with coronary artery disease (CAD) diagnosed by Treadmill test, myocardial single-photon emission computed tomography (SPECT) or coronary angiography.

This study complied with the Declaration of Helsinki and was approved by the Ethical Committee of the San Giovanni Battista Hospital (Turin, Italy); all patients signed an informed consent.

In all enrolled patients, platelets aggregation was assessed a preliminary test using PFA100 and VerifyNow between 8.00 and 10.00 am and the last assumption of enteric coated ASA was between 12.00 am and 2.00 pm of the previous day. Patients found resistant by at least one of the tests were called to receive and infusion of 288 mg of lysine acetylsalicylate (Flectadol®) corresponding to 160 mg of acetylsalicylic acid in 100 ml of saline solution between 10.00 and 12.00 am before the daily dose administration. All patients were instructed not to take aspirin for the following 24 h. Blood samples both for PFA100 and for VerifyNow were collected 1 and 24 h after the end of the infusion. Patients in whom the resistance have been reversed received 288 mg of soluble salt of lysine acetylsalicylate (Cardirene 160®) corresponding to 160 mg of acetylsalicylic acid instead of aspirin as their chronic antiaggregation treatment. In our study the dosage was chosen based on the availability in Italy, at the time of enrolment, of the only dosages of 160 mg and 300 mg of oral lysine acetylsalicylate.

The platelet function of these patients was re-tested 1 month after the beginning of oral lysine acetylsalicylate (LAS).

Efficacy of anti-aggregation therapy was assessed using the platelet function analyser system (PFA 100) and the VerifyNow aspirin test.

In the PFA-100 device (Siemens, Marburg, Germany) the citrate whole blood is sucked at high shear rates in a capillary closed by a membrane with a micro opening. Some platelet agonists, collagen/epinephrine (first reaction cartridge) and collagen/ADP (second cartridge), trigger platelet activation and aggregation on the membrane’s opening. This gradually slows down the blood’s flow until the complete occlusion of the opening. PFA-100 measures the closure time (CT) from the start test [27, 28]. Aspirin usually prolongs only the collagen/epinephrine-induced CT [29]. Resistance to aspirin was defined as a normal collagen/epinephrine CT. In our laboratory, normal ranges for the first cartridge CT are 85–160 s. Patients with CTs >160 were defined as aspirin responders; patients with CTs <160 were defined as aspirin resistant.

Studies using PFA100 show that the resistance to the ASA stands in a range between 10 and 15 % [19, 30]. In the subgroup of aspirin resistant it has been detected a higher rate of adverse cardiovascular events [14].

In VerifyNow device (Accumetrics, San Diego, CA) the citrate whole blood is put in contact with fibrinogen-coated beads tied to the receptors on the platelet surface. Arachidonic acid used as agonist, induces platelet aggregation on the beads. The test measures the aggregation with a optical turbidimetric method [31]: the increase of the light transmittances is directly proportional to the degree of platelet aggregation. VerifyNow aspirin test measures platelet aggregation as aspirin reaction unit (ARU) with a cutoff for aspirin resistance at 550 ARU. Patients with ARU < 550 were defined as aspirin responders; patients with ARU > 550 were defined as aspirin resistant.

This test is highly specific for aspirin action on COX1, since there is a different cartridge for testing P2Y12 inhibitors, such as clopidogrel. Using the VerifyNow test the resistance rate to the aspirin range from 6 to 20 % [32, 33].

We considered to be resistant to ASA those patients who resulted resistant in at least one of the two tests used.

Statistics

Statistical significance was considered at the 5 % level (p = 0.05). Continuous variables are presented as mean ± standard deviation and categorical variables are presented as frequencies and percentages. Differences between groups were evaluated by using independent samples 2-sided Student’s t-test for continuous variables and Fisher’s exact test for categorical variables. The agreement between the aspirin resistance status assessed by the various platelet function tests was evaluated with the use of the Cohen’s k coefficient.

Results

One hundred sixty-three patients with diabetes mellitus were enrolled; 60 female (37 %) and 103 male (63 %) with a mean age of 68 ± 7 years. All patients were taking aspirin at least for 30 days before the enrolment, 89 patients (55 %) in primary prevention while the remaining 74 patients (45 %) as secondary prevention (34 % for CAD and 11 % for stroke/TIA); the mean duration of diabetes mellitus was 18 ± 9 years with a mediocre metabolic control (HbA1c 62 ± 11,9 mmol/mol), the mean BMI was 29,01 ± 6,01 which shows poor control of body weight.

Assessment of platelet function in basal sample showed a prevalence of aspirin resistance of 18,4 % (30 patients) in at least one of the two tests used; among these subjects 10 were resistant in both tests, whereas 12 only with PFA100 and 8 only with VerifyNow, showing a moderate concordance between assays (k = 0,5).

There were no statistically significant differences in clinical characteristics between aspirin resistant and aspirin sensitive patients (Table 1), apart from BMI, higher in resistant population (in PFA100 and VerifyNow resistant group respectively 32,5 ± 6,3 and 31,0 ± 9,8, and 29,1 ± 3,8 in the sensitive group; p = 0,008) with no difference between those resistant to VerifyNow and PFA100, and a slightly higher prevalence of clopidogrel assumption between resistant patients.

Table 1 Clinical characteristics compared between resistant and sensitive patients
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No adverse reactions occurred during or following the infusion.

In 27 out of 30 patients (90 %) the aspirin resistance was reversed within 24 h from the infusion (in 22 at 1 h and in 5 at 24 h) using both platelets function tests. All the values of PFA100 and VerifyNow recorded at each point are listed in Tables 2–3; the trends of aggregation tests for each resistant patients are shown in Figs. 1 and 2.

Table 2 VerifyNow values for resistant patients
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Table 3 PFA 100 values for resistant patients
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Fig. 1
figure 1

VerifyNow values trends. Cut off value for resistance is over 550 ARU

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Fig. 2
figure 2

PFA 100 values trends. Cut off value for resistance is under 160 s

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Three patients (one resistant with PFA100 and two with VerifyNow) were found resistant with that assay after the infusion at 1 and 24 h. All patients whose aspirin resistance had been reversed were treated with LAS 288 mg (corresponding to ASA 160 mg) daily for 1 month.

All 27 patients were tested 1 month after the infusion on daily therapy with Cardirene and 25 patients have been found fully aspirin-sensitive, while two patients, both found resistant with PFA100, have been found with borderline value (150 and 153 s).

Discussion

In our study the prevalence of aspirin resistance among diabetic patients was 18 %, as reported in previous studies [12]. In our cohort it does not seem influenced by age, sex, duration of diabetes or metabolic control, smoking, hypertension, dyslipidaemia, creatinine level, or by the presence of a previous ischaemic event [30]; also the therapy associated with aspirin was found to be irrelevant. In previous studies some of these characteristics were reported to be related with a higher prevalence of aspirin resistance, particularly metabolic control [18, 19], but data are still controversial.

In our population we found the resistant group to have higher BMI than sensitive group. As described in previous papers (17, 34) a greater volume of distribution could influence the efficacy, and increasing the dose could have played a role in reversing ASA resistant in this group. Nevertheless also in the sensitive group there are obese patients and in the resistant group there are patients with BMI <25, showing as there is not a direct correlation between BMI and ASA responsiveness and increasing the dose can not be considered the only explanation to the reversibility obtained.

The relative higher prevalence of patients assuming clopidogrel in the resistant group is probably an incidental finding considering the very small overall amount of patient assuming thienopyridines in our population, since patients with very recent ACS were excluded. Alternatively the use of clopidogrel could define a group of patients with higher risk for cardiovascular events.

The high prevalence of aspirin resistance in a population already with higher risk than general population could have important clinical implication [13, 14, 35]. Abnormal platelet function may be related to several mechanisms, including metabolic alterations, oxidative stress and endothelial dysfunction. Platelets in diabetic subjects are characterized by enhanced thromboxane synthesis; furthermore hyperglycaemia induces reactive oxygen species (ROS) inducing oxidative stress. ROS lead to activation of transcription factors, such as NF-kB and activator protein-1, and subsequent production of pro-inflammatory cytokines and adhesion molecules and to reduction of synthesis of nitric oxide [36]. These mechanisms are implicated in a reduced response to aspirin activity, but also other mechanisms have been proposed for aspirin resistance [37, 38].

In our study we provide a support for the assumption that delayed or impaired intestinal absorption plays and important role in the absence of laboratory platelet response to aspirin in diabetic patients. In particular we focused on the pharmaceutical formulation since the absorption can be deeply altered by different formulations [26, 39]. This is also underlined by the persistence of the reversion of aspirin resistance not only after the infusion but also after 1 month of oral assumption of soluble salt at the same dose of the infusion instead of enteric-coated pills, in most of the patients who had their resistance reversed. This formulation has shown not to increase the adverse events or gastric intolerance or compliance [40, 41]. This observation could provide a further option for improving the antiaggregation therapy in high-risk patients, as diabetic ones, tailoring the therapy on the basis of quick and easy test available as point of care in many centres.

Nevertheless there is still a small percentage (10 %) of patients that are non-responsive to antiaggregation therapy even after endovenous administration confirming the important role of the others mechanisms previously described.

One contributing cause for the efficiency of intravenous aspirin may be the, even slightly, higher dose, chosen based on the commercial availability. Several studies demonstrated that the inhibition of thromboxane formation was dose-dependent [42, 43], but other studies were unable to reveal any dose-effect relationship using aspirin doses between 30 and 325 mg per day [44]. Some authors have also hypothesized that an accelerated platelet turnover or the interindividual variability in the recovery of platelet cyclooxygenase activity during the dosing interval may limit the duration of the antiplatelet effect of low-dose aspirin in patients with diabetes mellitus and it might explain an incomplete laboratory response to aspirin therapy. Following these assumptions, they have demonstrated improved platelet antiaggregation with an administration twice daily at low dose (75 mg) compared to single daily administration at high dose (320 mg) [45, 46].

Our study, in agreement with previous findings, suggests that different platelet function assays that are suitable for detecting aspirin resistance cannot be used interchangeably since there is only a moderate concordance between the tests used (K = 0,5) [47–49]. Furthermore the prevalence of aspirin resistance might have been overestimated by the use of tests on whole blood due to enhanced interaction, in diabetic patients, between platelets and red blood cells, which release large contents of adenosine diphosphate (ADP) y. The tests used have been chosen on the basis of accessibility, economic sustainability, ease and rapidity in their execution among tests validated.

This study has several important limitations; we did not measure the blood salicylate or serum/plasma thromboxane A2 metabolite levels, number of cases was small, the laboratory follow up was at short time and the assessment of compliance only by interview.

Despite such limitations our study highlight a new promising approach to reduce the prevalence of laboratory aspirin resistance.

In addition, our study is an observational study, for this reason no formal sample size determination was performed and all patients belong to a sequential single-arm cohort,-depending on their presentation at our outpatient clinic.

As this study was planned as proof of concept, a further prospective controlled clinical trial with two arms for comparison between the standard therapy and the “reversed resistance” with adequate sample size needs to be performed.

In conclusion, diabetic patients show markedly augmented platelet function despite regular oral intake of 100 mg of aspirin daily. A single intravenous dose of lysine acetylsalicylate, corresponding to 160 mg of acetyl salicylic acid, instead of the standard oral 100 mg of the enteric-coated formulation, can reverse the augmented platelet aggregability and laboratory aspirin resistance in the large majority of patients found resistant with standard therapy. The chronic therapy with an oral drug with a more favourable pharmacokinetic profile, such as the soluble salt, has shown to maintain the efficacy of antiaggregation in short follow-up in those who had their resistance reversed. Additional clinical studies are needed to find the optimal dosing and pharmaceutical formulation and to confirm the clinical significance of reversion of laboratory aspirin resistance in diabetic patients.