PAPER Obesity is associated with impaired platelet-inhibitory ... - Nature

International Journal of Obesity (2003) 27, 907–911 & 2003 Nature Publishing Group All rights reserved 0307-0565/03 $25.00 www.nature.com/ijo

PAPER Obesity is associated with impaired platelet-inhibitory effect of acetylsalicylic acid in nondiabetic subjects M Tamminen1, R Lassila2,3, J Westerbacka1, S Vehkavaara1 and H Yki-Ja¨rvinen1* 1

Division of Diabetes; 2Department of Medicine, University of Helsinki, Helsinki, Finland; and 3Wihuri Research Institute, Helsinki, Finland

OBJECTIVE: Platelet aggregation responses to acetylsalicylic acid (ASA) show considerable interindividual variation, the causes of which are largely unknown. We determined whether variation in insulin action is associated with that of ASA on platelets. SUBJECTS: In all, 10 nonobese (age 5073 y, BMI 2571 kg/m2) and 11 obese (age 5272 y, BMI 3271 kg/m2) subjects. MEASUREMENTS: Insulin sensitivity of glucose uptake was determined by the euglycemic insulin clamp technique. Platelet aggregation responses to four doses of arachidonic acid (AA) and adenosine diphosphate (ADP) were assessed in platelet-rich plasma before and 1 h after ingestion of 50 mg ASA using Born’s turbidometric aggregometer. RESULTS: Whole-body insulin sensitivity (M-value 0–180 min) was 36% lower in the obese (4.570.6) than the nonobese (7.170.6 mg/kg min, Po0.01) group. Before ASA, all doses of AA induced complete aggregation. After ASA ingestion, ASA inhibited maximal aggregation more in the nonobese than the obese group at AA concentrations of 0.75, 1 and 1.5 mmol/l (P ¼ 0.016 for ANOVA). ADP-induced aggregation at high doses (2 and 3 mmol/l) was also less inhibited in the obese group. In vivo insulin sensitivity (r ¼ 0.68, Po0.001 for 1 mmol/l AA) and BMI (r ¼ 0.58, Po0.01 for 1 mmol/l AA) were closely correlated with residual aggregation after ASA administration. CONCLUSION: These data demonstrate that obese insulin-resistant subjects have a blunted response to platelet-inhibitory effect of ASA. If this blunted effect is of a single dose of ASA preserved in continuous use, it could contribute to the increased risk of atherothrombosis in insulin-resistant individuals. International Journal of Obesity (2003) 27, 907–911. doi:10.1038/sj.ijo.0802312 Keywords: insulin resistance; acetylsalicylic acid; platelet aggregation; obesity; arachidonic acid

Introduction Increased platelet activity could contribute to the increased risk of atherothrombotic vascular disease in type II diabetes.1,2 Several abnormalities have been described in platelets of diabetic patients, although many of the early studies did not distinguish between type I and II diabetes.3–7 The abnormalities include increased thromboxane production3,8 and a lowered threshold for platelet activation by agonists such as adenosine diphosphate (ADP)6 and collagen, which release arachidonic acid (AA) from membrane phospholipids.4,6,8 Platelets from diabetic patients may also have a shortened lifespan3,5 and increased expression of

*Correspondence: H Yki-Ja¨rvinen, Division of Diabetes, Department of Medicine, University of Helsinki, Haartmaninkatu 4, PO Box 340, 00029 HUS Helsinki, Finland. E-mail: [email protected] Received 16 October 2002; revised 4 February 2003; accepted 16 February 2003

glycoprotein receptors,7 thrombin generation,9 calcium concentrations,10 plasminogen activator inhibitor 1 release,11 shear-induced adhesion and aggregation to subendothelium,12 and a decreased activity of nitric oxide synthase.13 Platelets have insulin receptors that autophosphorylate their b-subunit when stimulated with insulin.14 Insulin has been shown to inhibit platelet aggregation both in vitro15,16 and in vivo.17,18 This action of insulin is associated with increased platelet cyclic GMP and AMP, and can be overcome by inhibiting nitric oxide (NO) synthesis with L-N-monomethylarginine (L-NMMA).19 In in vitro studies, insulin has been shown to inhibit dose dependently platelet aggregation to ADP in healthy subjects and lean type II diabetic patients.16,20 This in vitro effect of insulin has been suggested to be impaired in obese subjects and in obese type II diabetic patients compared to lean subjects.15 Acetylsalicylic acid (ASA) at doses exceeding 30 mg inhibits platelet function by permanent acetylation of

Obesity and ASA M Tamminen et al

908 platelet cyclooxygenase (COX-1) at the functionally important amino acid serine 529. This prevents the access of AA to the catalytic site of COX-1 at tyrosine 385 and irreversibly inhibits platelet-dependent thromboxane formation.21 This occurs in the portal circulation, which is why 95% of platelet COX-1 is inhibited although very little drug is detected systemically and why blood levels of ASA do not correlate with its antiplatelet effect.22 Since 1997, the American Diabetes Association has recommended ASA use for all adults with diabetes who have cardiovascular disease or its risk factors.23 A significant proportion of ASA users are considered resistant to the antiaggregating effect of ASA,24–26 but it is unknown if this applies to patients with type II diabetes and whether there are diabetes-related causes of ASA resistance. This would seem of interest as insulin resistance precedes type II diabetes and is associated with an increased risk of coronary heart disease and stroke.27 Given that insulin normally counteracts agonist-stimulated platelet aggregation16 and inhibits platelet recruitment to collagen in a whole-blood perfusion system,17 it seems plausible that insulin resistance may impair the ability of ASA to inhibit platelet aggregation. This question has not been addressed in previous studies. We therefore determined whether obesityassociated insulin resistance is characterized by altered ASA sensitivity of platelet aggregation.

Subjects and study design A total of 21 nondiabetic subjects were recruited for the study by advertising in a local paper. The subjects were divided into an obese and a nonobese group by their median (29.0 kg/m2) body mass index (BMI). Physical and biochemical characteristics of the study groups are shown in Table 1. The subjects were healthy as judged by history, physical examination, ECG and routine laboratory tests, and were not

Table 1

Characteristics of the subjects

Number of the subjects (male/female) Age (y) Weight (kg) Body mass index (kg/m2) Waist/hip ratio Fasting plasma glucose (mmol/l) HbA1c (%) Fasting serum insulin (mU/l) Fasting serum C-peptide (nmol/l) Serum total cholesterol (mmol/l) Serum HDL cholesterol (mmol/l) Serum triglycerides (mmol/l) Systolic blood pressure (mmHg) Diastolic blood pressure (mmHg) Smoking (7)

Nonobese

Obese

10 (5/5) 5073 7474 24.970.6 0.9070.02 5.670.2 5.770.2 772 0.570.1 4.870.3 1.470.1 1.170.2 12477 7873 4/6

11 (8/3) 5272 9573** 31.770.8*** 0.9970.02** 5.770.2 5.670.1 1373 0.970.1 5.170.2 1.370.1 1.370.1 12574 8272 2/9

Data are mean7s.e.m. **Po0.01, ***Po0.001 for difference between the obese and nonobese subjects.

International Journal of Obesity

taking any regular medications, including use of antiinflammatory drugs, for 2 weeks prior to the study. Smoking was prohibited on the study day and exercise and alcohol for 2 days before the study. Each subject was studied twice after a 10–12 h overnight fast starting at 8.00–8.30 a.m. At the first visit, in vivo insulin action was measured by using the euglycemic insulin clamp technique. At the second visit, blood samples for aggregation tests were withdrawn before and 1 h after oral administration of 50 mg ASA, as described in detail below. Written informed consent was obtained after the purpose, nature and potential risks had been explained to the subjects and the experimental protocol was approved by the Ethical Committee of the Department of Medicine, Helsinki University Central Hospital.

Materials and methods Whole-body insulin sensitivity Whole-body insulin sensitivity was determined by using the euglycemic insulin clamp technique.28 Two 18-gauge catheters (Venflon; Viggo-spectramed, Helsingborg, Sweden) were inserted into the left antecubital vein for infusion of glucose and insulin, and in a heated dorsal hand vein for sampling of arterialized venous blood as previously described.29 Insulin was infused in a primed-continuous fashion for 180 min. The rate of the continuous insulin infusion was 2 mU/kg min. The amount of glucose infused to maintain normoglycemia during hyperinsulinemia (0–180 min), corrected for changes in the glucose space (the M-value, mg/kg min), was used as a measure of whole-body insulin sensitivity.

Platelet aggregation Blood for the initial aggregation test was collected from median antecubital vein via an 18-gauge catheter. The patient thereafter chewed and swallowed a 50 mg tablet of ASA (Disperin 50 mg, Orion, Finland) with some water. After 60 min, another catheter was inserted in an intact antecubital vein for sampling of blood for repeated aggregation measurements. Blood for platelet aggregation measurements was collected in tubes containing 0.129 M sodium citrate (9:1) and centrifuged (170 g, 12 min at 221C) to obtain platelet-rich plasma (PRP). Platelet-poor plasma (PPP) was prepared by centrifugation of PRP at 5000 g for 5 min. The concentration of platelets in PRP was determined by a cell counter (Sysmex K-1000, TOA Medical Electronics, Japan) and adjusted to 300 106/ml with autologous PPP. In vitro platelet aggregation in PRP was measured using a four-channel turbidometric aggregometer (PACKS-4, Helena Laboratories, Beaumont, TX, USA).30 The aggregating agents were ADP (final concentrations 1, 1.5, 2 and 3 mmol/l; Sigma Chemicals, St Louis, MI, USA) and AA (final concentrations 0.5, 0.75, 1 and 1.5 mmol/l; Sigma Chemicals, St Louis, MI, USA). The maximal aggregation (%) response at each concentration of the agonist was determined at baseline and after 50 mg oral ASA.


909 Other measurements Plasma glucose concentrations were measured in duplicate by the glucose oxidase method31 with the use of a Beckman Glucose analyzer II (Beckman Instruments, Fullerton, CA, USA). HbA1C was measured by high-performance liquid chromatography with the use of fully automated Glycosylated Hemoglobin Analyzer System (Bio-Rad, Richmond, CA, USA). Serum-free insulin concentrations were determined by double-antibody radioimmunoassay (Pharmacia Insulin RIA kit; Pharmacia, Uppsala, Sweden) after precipitation with polyethylene glycol.32

Statistical analysis Analysis of ASA effects at various agonist concentrations between lean and obese subjects was made using analysis of variance (ANOVA) for repeated measures using the SYSTAT statistical package (Evanston, IL, USA) followed by pairwise comparison using Fischer’s least significant difference multiple comparison test. Normally distributed variables measured only once were compared using the unpaired t-test. Correlation analyses were performed conservatively using Spearman’s nonparametric correlation coefficient for all variables. Multiple linear regression analyses were performed to identify the independent determinants of platelet aggregation. Data are expressed as mean7s.e.m. P-values less than 0.05 were considered statistically significant.

Results Whole-body insulin sensitivity During the insulin infusion, serum-free insulin concentrations averaged 182714 in the obese and 16478 mU/l in the nonobese group (NS). Plasma glucose averaged 5.170.1 mmol/l in both groups during hyperinsulinemia. Whole-body insulin sensitivity was 36% lower in the obese (4.570.6) than the nonobese (7.170.6 mg/kg min, Po0.01) group. Both BMI (r ¼ 0.73, Po0.001), body weight (r ¼ 0.60, Po0.01), the waist/hip ratio (r ¼ 0.43, Po0.05), and concentrations of fasting serum insulin (r ¼ 0.76, Po0.001), C-peptide (r ¼ 0.86, Po0.001), serum triglycerides (r ¼ 0.53, Po0.05) and HDL cholesterol (r ¼ 0.49, Po0.05) were significantly correlated with wholebody insulin sensitivity.

Platelet aggregation Before ASA administration already the lowest concentration of AA (0.5 mmol/l) induced maximal aggregation in each subject without any differences between the groups (9571 vs 9472%, obese vs nonobese, NS). In the blood sample taken 1 h after 50 mg ASA, AA-induced aggregation was inhibited by ASA in the obese group only at the lowest dose of AA, while ASA significantly inhibited aggregation at all doses of AA in the nonobese group (Figure 1). Maximal aggregation

Figure 1 Maximal aggregation at four concentrations of AA (a) and ADP (b) at baseline and after ASA 50 mg. *Po0.05, **Po0.01, ***Po0.001 for obese vs nonobese after ASA 50 mg. +Po0.05, ++Po0.01, +++Po0.001 for difference between aggregation at baseline and after ASA.

after ASA was significantly greater in the obese than the nonobese group (P ¼ 0.016 for ANOVA, Figure 1). Before ASA, when ADP was used as the agonist, aggregation in response to the two lowest concentrations of ADP was slightly but not significantly greater in platelets of the obese than those of the nonobese group (Figure 1). Aggregation was significantly more inhibited by ASA at ADP concentrations of 2 mmol/l (7476 vs 9171% in nonobese vs obese, Po0.01) and 3 mmol/l (7976 vs 9371%, respectively, Po0.05) in the nonobese than the obese group (Figure 1). Before ASA administration, whole-body insulin sensitivity in the whole study group was inversely correlated with aggregation induced by the two lowest doses of ADP (1 and 1.5 mmol/l, Table 2). After ASA ingestion, there was a strong negative correlation between insulin sensitivity and maximal aggregation induced by AA at concentrations of 0.75, 1, 1.5 mmol/l, and ADP at concentrations of 1.5 and 3 mmol/l (Table 2). Aggregation induced with 1 mmol/l AA after ASA also correlated with BMI (r ¼ 0.58, Po0.01), body weight (r ¼ 0.56, Po0.01), waist/hip ratio (r ¼ 0.54, Po0.05), serum International Journal of Obesity


910 Table 2 Correlations between insulin sensitivity (mg/kg min) determined by the euglycemic clamp technique and maximal aggregation with different concentrations of AA and ADP in the whole study group

AA 0.75 mmol/l after ASA AA 1 mmol/l after ASA AA 1.5 mmol/l after ASA ADP 1 mmol/l at baseline ADP 1.5 mmol/l at baseline ADP 1.5 mmol/l after ASA ADP 3 mmol/l after ASA

r

P

0.67 0.68 0.63 0.48 0.48 0.52 0.45

o0.001 o0.001 o0.001 o0.05 o0.05 o0.05 o0.05

triglycerides (r ¼ 0.51, Po0.05), C-peptide (r ¼ 0.86, Po0.001), and fasting serum insulin (r ¼ 0.53, Po0.05) as well as with ADP-induced aggregation both before and after ASA administration (data not shown). In multiple linear regression analysis where features of insulin resistance (body weight, whole-body insulin sensitivity, waist/hip ratio, HDL cholesterol, and systolic blood pressure) were entered in the model the R2 was 0.61 (P ¼ 0.009). Of all variables, only whole-body insulin sensitivity was significant (Po0.05).

Discussion The present study was undertaken to examine whether variation in insulin action is associated with variation in the ability of ASA to inhibit platelet aggregation. We found that the ASA inhibition of platelet aggregation induced by AA and ADP was significantly blunted in obese insulin-resistant subjects compared to age- and gender-matched normalweight subjects. In vivo insulin sensitivity was closely inversely correlated with aggregation responses induced by ADP before ASA administration, and by both AA and ADP after oral ASA. These cross-sectional data demonstrate that platelets of insulin-resistant obese subjects do not respond to ASA as well as those of lean insulin-sensitive subjects. For platelets to function normally, both proaggregatory and antiaggregatory mechanisms need to be intact.33 These mechanisms were not examined in detail in the present study, and therefore the causes underlying the observed defects in platelet function are speculative. Any of the previously described abnormalities in the platelets of type II diabetic patients could be involved. These include increased platelet activation due to increases in thromboxane A2 synthesis8 and calcium levels,10 or increased numbers or function of GPIIb/IIIa complexes on platelets.7 Isoprostanes, free-radical catalyzed products of arachidonic acid, may also be involved.34,35 Increased isoprostane production can activate platelets through the thromboxane receptor, even when thromboxane synthesis is inhibited by ASA.36 In the present study, AA-induced aggregation was more strongly inhibited by ASA than ADP-induced aggregation. ADP at higher concentrations (2 and 3 mmol/l) overcame the inhibiInternational Journal of Obesity

tion of COX1 in the obese subjects, whose platelet aggregation returned to baseline. The difference between obese and nonobese subjects could reflect the impact of ADP-induced release of AA. The obese may continue to activate thromboxane receptors and aggregate platelets in spite of ASA. Also, the platelets of the obese are known to have reduced levels of cAMP and cGMP, which may lower the threshold for activation.15,17 Regarding possible defects in antiaggregatory mechanisms, insulin inhibits platelet aggregation both in vitro and in vivo, by a mechanism which appears to involve stimulation of NO production as in blood vessels.19 Both peripheral resistance arteries37 and platelets20 in vitro have been reported to have diminished sensitivity to NO in obese as compared to normal-weight subjects. In the present study, we found highly significant inverse correlation between insulin sensitivity and aggregation after ASA in the entire study group, even in multiple linear regression analysis. This finding supports the hypothesis that insulin resistance affects platelet function but does not prove causality. It is important to emphasize that abnormalities in platelet function tests in vitro and impaired platelet-inhibitory effect of ASA may not translate into an increased risk of cardiovascular events. However, the data suggest that future or ongoing trials examining the impact of ASA or other antiplatelet agents on vascular events or efficacy of ASA in patients with diabetes might want to include relative body weight or some measure of insulin resistance as a potential confounding variable. This would seem important since both obesity and other features of insulin resistance are associated with increased risk of coronary heart disease and stroke.27 Possibly, insulin-resistant obese subjects might benefit from ASA at higher dose than insulin-sensitive subjects, or therapies inhibiting platelet aggregation by blocking thromboxane-independent pathways or thromboxane receptors.

Acknowledgements Supported by grants from the Academy of Finland (Hannele Yki-Ja¨rvinen, Jukka Westerbacka), the Finnish Diabetes Research Foundation (Jukka Westerbacka), Liv och Ha¨lsa (Hannele Yki-Ja¨rvinen), the Juselius (Hannele Yki-Ja¨rvinen) and Novo Nordisk (Hannele Yki-Ja¨rvinen), Foundations.

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