Elevated levels of platelet microparticles in carotid atherosclerosis and during the postprandial state

Thrombosis Research 123 (2009) 881–886

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Regular Article

Elevated levels of platelet microparticles in carotid atherosclerosis and during the postprandial state Annika E. Michelsen a,⁎, Ann–Trude Notø b, Ellen Brodin b, Ellisiv B. Mathiesen c, Frank Brosstad a, John–Bjarne Hansen b a b c

Research Institute for Internal Medicine, University of Oslo, Oslo, Norway Center for Atherothrombotic Research in Tromsø (CART), Department of Medicine, Norway Department of Neurology, Institute of Clinical Medicine, University of Tromsø, Tromsø, Norway

a r t i c l e

i n f o

Article history: Received 9 May 2008 Received in revised form 12 September 2008 Accepted 30 October 2008 Available online 14 December 2008 Keywords: Platelets Microparticles Postprandial lipemia Carotid atherosclerosis

a b s t r a c t Background: Platelet microparticles (PMPs) possess proatherogenic and procoagulant properties which may play a role in atherogenesis and subsequent thromboembolic complications. The present study was conducted to investigate the possible relationship between carotid atherosclerosis and plasma concentrations of PMPs, and elucidate if plasma levels of PMPs were affected by postprandial hypertriglyceridemia. Methods and results: Subjects with ultrasound-assessed carotid atherosclerotic plaques (echogenic; n = 20 and echolucent; n = 20), assessed by ultrasonography, and subjects without carotid plaques (n = 20) were recruited from a population-based study and underwent a standard fat tolerance test. Subjects with carotid plaques had significantly higher levels of large PMPs than subjects without carotid atherosclerotic plaques (96.7 ± 50.4 µg/l versus 56.1 ± 34.9 µg/l), after adjustments for traditional cardiovascular risk factors and use cardiovascular drugs (p = 0.021). Plasma PMPs were not associated with plaque echogenicity. Postprandial hypertriglyceridemia induced a similar increase in plasma PMPs within all groups. Significant correlations were found between an increase in plasma triglycerides and percent elevation in total PMPs (r = 0.29, p b 0.05) and large PMPs (r = 0.34, p b 0.01) in the postprandial phase. Conclusions: Individuals with echogenic and echolucent carotid atherosclerotic plaques have statistically significant elevation of large plasma PMPs compared to age/sex-matched normal controls. Postprandial hypertriglyceridemia induces a significant, similar increase in plasma PMPs in individuals with and without carotid atherosclerotic plaques which could be of pathophysiological importance in atherogenesis. © 2008 Elsevier Ltd. All rights reserved.

Introduction Platelets play a crucial role in the initiation and progression of atherosclerosis. By adhering to sites with dysfunctional endothelium they release proinflammatory and procoagulant mediators, contributing to plaque formation and atherothrombosis [1–6]. When activated, platelets release membrane particles called platelet microparticles (PMPs). PMPs are procoagulant as they expose negatively charged phospholipids, contain activated coagulation factors Va [7,8] , Xa [9] and tissue factor [10]. Moreover, PMPs stimulate inflammation and atherosclerosis by expressing P-selectin on their membrane [11] and through release of platelet activating factor [12] and RANTES [13].

Abbreviations: PMP, platelet microparticle; TRL, triglyceride-rich lipoprotein; CVE, cerebrovascular events; CAD, coronary artery disease; GSM, grey scale median; CM, chylomicrons; LDL, low-density lipoprotein; HDL, high-density lipoprotein; MPV, mean platelet volume. ⁎ Corresponding author. Tel.: + 47 23 073616; fax: +47 23 07 36 30. E-mail address: [email protected] (A.E. Michelsen). 0049-3848/$ – see front matter © 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.thromres.2008.10.016

Postprandial hypertriglyceridemia is a physiological phenomenon occurring several times a day. Elevated postprandial levels of triglyceriderich lipoproteins (TRLs) have been associated with both coronary [14] and carotid artery atherosclerosis [15]. In fact, Zilversmit suggested atherosclerosis, at least in part, to be a postprandial disease, and hypothesized that accumulation of dietary TRLs promoted formation of atherosclerosis [16]. More recent studies provide a new perspective to this hypothesis by reporting a particular efficient penetration and selective retention of chylomicron remnants in vessel wall lesions [17]. Furthermore, previous studies have shown elevation of total [18] and endothelial-derived [19,20] microparticles in plasma, associated with impaired flow-mediated dilatation of the brachial artery [18,19], during the postprandial state, indicating a direct deteriorating impact of postprandial TRLs on endothelial function. Postprandial elevation of TRLs has been shown to activate platelets [21] and the coagulation system [22]. Carotid atherosclerosis is responsible for 20–30% of ischemic strokes and considered a major risk factor for cerebrovascular events (CVE) [23]. The carotid arteries are readily accessible to ultrasound imaging and evaluation of atherosclerotic plaque morphology. Plaques that appear echolucent are lipid-rich, covered by a thinner fibrous cap, whereas

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echogenic plaques have higher content of fibrous tissue and calcification [24]. Studies have shown that echolucent carotid plaques are associated with a high risk for ischemic CVE independent of the degree of stenosis [25,26]. The purpose of the present study was to investigate if subjects with carotid atherosclerotic plaques had elevated plasma concentrations of PMPs, and whether plasma PMPs were related to grade of plaque echogenicity. Furthermore, we wanted to study whether plasma levels of PMPs are influenced by postprandial lipemia.

re-calculated. In subjects with more than one plaque, the standardized GSM of the total plaque area was estimated as a weighted mean of the GSM value of each single plaque. The area of each plaque was divided by the total area of plaques in each subject, and this fraction was multiplied with standardized GSM value of each plaque. All scores were added to calculate the total standardized GSM score for each subject. Blood sampling

Material and methods Study participants The participants were recruited from a population health study database (the fifth survey of The Tromsø Study in 2001), which included ultrasound examination of the carotid arteries. Subjects were eligible for the plaque groups if they: 1) were aged 56–80 years, 2) had at least one plaque or more in the carotid bifurcation or internal carotid artery at the screening, 3) had a plaque thickness of ≥2.5 mm and plaque morphology classified as echolucent (grade 1) or echogenic (grade 4) according to the Gray-Weale criteria [27]. Subjects at a similar age and gender, but without plaques in their carotid arteries were used as controls. Exclusion criteria included the following conditions: regular use of lipid-lowering drugs or oral anticoagulants, cancer or other serious life- threatening medical conditions, hypothyroidism, renal, hepatic, or psychiatric disease, and current abuse of alcohol or drugs. Coronary artery disease (CAD) was defined as prior or present angina pectoris, myocardial infarction and peripheral vascular disease. CVE was defined as previous transient ischemic attacks, amaurosis fugax or stroke. First, subjects with and without carotid atherosclerotic plaques in the fifth survey of the Tromso study were invited to a screening visit for a standard questionnaire, physical examination, a second ultrasound examination and routine laboratory tests with special focus on exclusion criteria. The subjects archives at the University hospital of North Norway was also checked for exclusion criteria. Second, 40 subjects with and 20 subjects without carotid plaques who fulfilled the inclusion and exclusion criteria and wanted to participate were invited to a fat tolerance test. Informed written consent was obtained from the participants, and the study was approved by the regional ethical committee. The study was performed at the Clinical Research Centre at the University Hospital of North Norway in Tromsø. Ultrasound examination The ultrasound examination was performed as described previously [28,29]. Plaque morphology in terms of echogenicity was assessed by analysis of grey scale content of the plaques and calculation of grey scale median (GSM). Assessment of plaque morphology was made in all plaques present in the near and far walls of the common carotid, the bifurcation, and the internal carotid arteries on both sides (12 locations). All examinations and measurements of all plaques were recorded on videotape. Stored B-mode images were subsequently transferred to a personal computer and digitized into frames of 768 × 576 pixels of 256 grey levels each (0= black and 256 = white) with the use of a commercially available video grabber card (meteor II/Matrox Intellicam). Measurements of plaque area were made with the use of the Adobe Photoshop image-processing program (version 7.0.1), by encircling each plaque perimeter with a cursor. The grey-level distribution and GSM of each plaque was calculated. Standardization of GSM was performed according to the method by El-Barghouty [30]. A fixed area of the lumen of the carotid artery (300 pixels), the brightest area of the innermost adventitia adjacent to the plaque (150 pixels), and the plaque were marked. The plaque image was standardized by linearly adjusting the grey tonal range so that the lumen was assigned a GSM of 1 and the adventitia-media interface 200 [31]. After standardization, the GSM of the plaques were

After 12 hours overnight fasting and 48 hours refrain from physical exercise, blood samples were drawn from an antecubital vein on the right arm into vacutainers using a 19-gauge needle (Becton Dickinson, Meylan Cedex, France) without any additives (serum) or with 1/10th volume of 0.129 mol/l sodium citrate as anticoagulant (plasma). Blood was centrifuged immediately (plasma) or allowed to clot before centrifugation (2000× g for 15 minutes at 22 °C). Serum and plasma were stored in aliquots of 1 ml at −70 °C until analyzed. Fat tolerance test A fat-tolerance test was conducted using a test meal prepared from standard sour cream porridge containing 70% of calories from fat (66% saturated fat, 32% monounsaturated fat and 2% polyunsaturated fat). A weight-adjusted meal (1 gram fat per kg bodyweight) was served at 08:00 a.m. and consumed within 15 minutes. Blood samples for isolation of chylomicrons (CM), and serum- and plasma preparations were collected before the meal and every second hour during the next 8 hrs. Isolation of chylomicrons and measurement of lipids CM were isolated by over-layering 8 ml EDTA plasma with 5 ml of NaCl solution (density 1.006 kg/l NaCl solution with 0.02% sodium azide and 0.01% EDTA) in a cellulose nitrate tube (Beckman Instruments Inc, CA, USA) and centrifuged in a Beckman SW40 Ti swinging bucket rotor at 20.000 rpm for 1 hr at 4 oC. The CM, with Svedberg flotation (Sf) rates N 400, were carefully removed by aspiration from the top of the tube, divided into three aliquots in cryovials, and frozen at –70 oC until further analysis. Serum lipids were analysed on a Cobas Mira S (Roche Diagnostics, F. Hoffmann-La Roche Ltd, Basel, Switzerland) with reagents from ABX Diagnostics (Montpellier, France). Total cholesterol (CHOD-PAP) and triglycerides (GPO-PAP) were measured with enzymatic colorimetric methods. Low-density lipoprotein (LDL) and high-density lipoprotein cholesterol (HDL) were measured by selective inhibition colorimetric assays (LDL cholesterol direct and HDL cholesterol direct, respectively, ABX Diagnostics). Hematology and measurement of platelet microparticles Fasting blood samples were drawn into vacutainer tubes containing EDTA as anticoagulant (K3-EDTA 40 µl, 0.37 M per tube) and hematological variables was measured within two hours on an automated blood cell counter (Coulter Counter®, Coulter Electronics, Luton, UK). Citrated plasma samples were thawed and centrifuged at 11000× g for 4 minutes to eliminate trace amounts of platelets and platelet fragments. The supernatants were carefully removed and divided in two parts. One part of each sample was immediately mixed with an equal volume of assay buffer (Perkin-Elmer Life Science, Boston, MA, USA) supplied with a non-ionic detergent (lgepal CA-630, SigmaAldrich, St. Louis, MO, USA) to lyse microparticles. The other part was passed through a low-protein binding 0.1 µm filter (Millipore, Billerica, MA, USA). Filtration was performed to differentiate between small PMPs or small exosomes with specific properties and large PMPs. The filtrate (containing only PMPs smaller than 0.1 μm in diameter) was then mixed with an equal volume detergent-containing assay buffer.

A.E. Michelsen et al. / Thrombosis Research 123 (2009) 881–886 Table 1 Baseline characteristics of subjects with echolucent (n = 20) and echogenic (n = 20) carotid plaques and subjects without carotid artery plaques (n = 20). Variables Sex (% men) Age (yrs) Smoking status (%) BMI (kg/m2) Systolic BP (mm Hg) Diastolic BP (mm Hg) Total cholesterol (mmol/l) LDL cholesterol (mmol/l) HDL cholesterol (mmol/l) Triglycerides (mmol/l) Hypertension (%) Diabetes (%) CAD (%) CVE (%) β-blockers (%) Platelet inhibitors (%) Omega-3 FA (%) Degree of stenosis (%) Number of plaques Total plaque area (mm2) GSM (pixels)

Carotid plaques

Controls

F-test (p-value)

Echolucent

Echogenic

55 (11) 68.8 (65.4 – 72.2) 10 (2)

40 (8) 70.1 (67.3 – 72.8) 30 (6)

60 (12) 66.1 (66.6–70.1) 35 (7)

0.43 0.16 0.19

26.4 (24.6 – 28.1) 126 (117 – 134)

26.7 (24.6 – 28.8) 26.6 (24.8–28.4) 125 (116 – 134) 126 (118–133)

0.97 0.99

74 (68 – 79)

73 (69 – 77)

74 (69–79)

0.97

883

checked for frequency distribution. Variables not normally distributed were logarithmically transformed. Continuous variables are presented as mean (95% confidence interval (CI)), and categorical data as proportions (n). Serum triglycerides was not normally distributed and consequently logarithmically transformed. Differences in continuous and categorical variables between groups were analyzed by ANOVA and the Х2 -test, respectively. When indicated by an F-value below 0.05, a post-hoc test using the Bonferroni method was performed to identify significant group differences. General linear models for univariate analysis of variance were used for adjustments. Pearson's correlation coefficients were used to examine correlations between continuous variables. All analyses were performed using SPSS (SPSS Inc. Chicago, Illinois, USA) for Windows software, version 15.0.

6.15 (5.66 – 6.65) 6.55 (5.95 – 7.16)

6.04 (5.45–6.63)

0.38

4.06 (3.66 – 4.46)

4.11 (3.61 – 4.61)

3.75 (3.35–4.15)

0.42

Results

1.40 (1.14 – 1.66)

1.87 (1.61 – 2.14)

1.74 (1.52–1.95)

0.02

1.19 (0.90 – 1.48)

1.05 (0.75 – 1.34)

1.16 (0.90–1.41)

0.73

Characteristics of the study participants, including carotid plaque features, are shown in Table 1. There were no statistically significant differences between groups with regard to age, gender and traditional risk factors such as body mass index, blood pressure, LDL cholesterol, triglycerides, and the presence of hypertension, diabetes mellitus and previous cardiovascular and cerebrovascular diseases. The regular use of β-blockers and platelet inhibitors, and dietary supplementation with omega-3 fatty acids were similar among groups. Subjects with echolucent plaques had lower HDL cholesterol than subjects with echogenic plaques (p=0.08) and controls (p=0.016) (p for F-test=0.018). The difference in serum HDL cholesterol between groups remained significant even after adjustment for age, gender and body mass index (p=0.026). Hematological variables and plasma concentrations of PMPs measured from blood collected under fasting conditions and are shown in Table 2. Subjects with carotid plaques (echolucent and echogenic, n=40) had significantly higher levels of large PMPs than subjects without carotid atherosclerotic plaques (n=20) (96.7±50.4 µg/l versus 56.1±34.9 µg/l, p=0.018). The difference remained significant after adjustment for platelet count, mean platelet volume (MPV) and traditional coronary risk factors such as age, gender, body mass index, blood pressure, serum lipids, diabetes mellitus, hypertension and use of cardiovascular drugs (p=0.021). Plasma concentrations of large PMPs were significantly higher in subjects with echolucent carotid plaques than controls (p=0.02) and tended to be higher in subjects with echogenic carotid plaques compared to controls (p=0.11). There were no significant differences in levels of large PMPs and hematological variables between echogenicity groups. In particular, platelet count and MPV were similar in all groups. Plasma concentrations of total and small PMPs were not different between groups. Plasma concentration of total triglycerides (upper panel) and chylomicron triglycerides (lower panel) after ingestion of a standard fatenriched meal are shown in Fig.1. Postprandial triglyceride concentrations peaked 4 hrs after the meal in all groups. Subjects with echolucent plaques

20 (4) 0 (0) 20 (4) 0 (0) 20 (4) 10 (2)

25 (5) 5 (1) 25 (5) 0(0) 15 (3) 30 (6)

20 0 20 5 5 10

50 (10) 38 (32 – 43)

55 (11) 37 (31 – 43)

60 (12)

2.1 (1.5–2.6)

2.2 (1.6 – 3.0)

0.67

6.1 (3.0–9.3)

5.3 (3.2 – 7.0)

0.46

95.3 (87.3–103.2)

b0.001

35.8 (32.1 – 39.6)

(4) (0) (4) (1) (1) (2)

0.47 0.37 0.47 0.37 0.38 0.15 0.83 0.80

Values are means with 95% confidence intervals or percentages with numbers in brackets.

The two preparations, reflecting the total content of PMPs and the content of small PMPs, were then applied to the assay. PMPs in plasma were determined by a recently developed immunometric method reporting linear relation to PMPs measured by flow cytometry [32]. In short, the new method determines the concentration of GPIIb/IIIa in plasma after lysing PMPs in a non-ionic detergent. Intra-assay CV was 4.8%, while inter-assay CV was determined to be 12%. Statistics According to the morphological appearance of plaques subjects with carotid plaques were allocated to one echolucent group (grade 1 according to Gray-Weale criteria) (n = 20) and one echogenic group (grade 4 according to Gray-Weale criteria) (n = 20). All variables were

Table 2 Hematological variables and plasma concentrations of platelet-derived microparticles (PMPs) in subjects with echolucent (n = 20) and echogenic (n = 20) carotid plaques and subjects without carotid artery plaques (n = 20). Variables Hemoglobin (g/dl) White blood cells (× 109/l) Neutrophiles (× 109/l) Lymphocytes (×109/l) Monocytes (×109/l) Platelets (× 109/l) MPV (fl) Total PMPs (µg/l) Small PMPs (µg/l) Large PMPs (µg/l)

Carotid plaques Echolucent

Echogenic

14.1 (13.5 – 14.6) 6.29 (5.33 – 7.25) 3.67 (2.86 – 4.49) 1.76 (1.52 – 2.00) 0.52 (0.43 – 0.60) 270 (247 – 293) 8.15 (7.68 – 8.61) 184.2 (129.1 – 239.2) 74.0 (48.8 – 99.2) 110.2 (74.4 – 145.9)

14.2 (13.7 – 14.6) 5.64 (4.81 – 6.47) 3.10 (2.49 – 3.71) 1.80 (1.54 – 2.05) 0.49 (0.43 – 0.56) 257 (234 – 280) 7.97 (7.66 – 8.28) 157.0 (100.8 – 213.2) 73.8 (42.2 – 105.3) 83.3 (53.8 – 112.7)

Values are means with 95% confidence intervals in brackets. ⁎ p-values are adjusted for age, gender and body mass index.

Controls

F-test⁎ (p-value)

14.0 (13.5 – 14.4) 5.89 (4.87– 6.91) 3.48 (2.60 – 4.37) 1.69 (1.40 –1.98) 0.49 (0.41 – 0.58) 255 (230 – 279) 8.07 (7.73 – 8.40) 149.3 (46.6 – 251.9) 93.2 (46.2 – 162.7) 56.1 (39.8 – 72.4)

0.39 0.66 0.68 0.92 0.84 0.47 0.81 0.51 0.93 0.03

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Fig. 1. Plasma concentrations of total triglycerides (upper panel) and chylomicron triglycerides (lower panel) after intake of a standard fat-rich meal. Values are means ± SEM. ⁎ Subjects with echolucent plaques had significantly higher levels at 6 and 8 hours compared to controls (p b 0.05).

subjects with echogenic carotid atherosclerosis, suggesting pronounced platelet activation in this condition. Except for statistical lowered HDL cholesterol in subjects with echolucent carotid atherosclerosis, traditional cardiovascular risk factors were equally distributed between subjects with and without carotid atherosclerosis. Higher levels of PMPs have been reported in conditions associated with increased risk for cardiovascular diseases such as hypertriglyceridemia [35,36], hypertension [37] and diabetes mellitus [35,38]. Cardiovascular drugs such as aspirin [39], β-blockers [40] and statins [41,42] are known to dampen platelet function. The elevated levels of large PMPs in carotid atherosclerosis remained statistically significant even after adjustments for platelet count, mean platelet volume and traditional coronary risk factors such as age, gender, body mass index, blood pressure, serum lipids, diabetes mellitus, and hypertension. None of the subjects in our study used statins. Regular use of cardiovascular drugs in our study population was low, but neither use of aspirin nor β-blockers affected plasma levels of PMPs in adjusted analysis. These findings support actual higher levels of PMPs in subjects with carotid atherosclerosis compared to subjects without carotid atherosclerosis, independent of confounders associated with increased risk for cardiovascular diseases and medical treatment. Previous studies have shown increased platelet activation in carotid atherosclerosis [43] and detection of platelet-derived chemokines and growth factors in atherosclerotic plaques [44,45]. PMPs possess proatherogenic and procoagulant properties [7–13]. It is, therefore, tempting to speculate that PMPs play an important role in atherogenesis and the subsequent thromboembolic event in carotid atherosclerosis. However, the latter inference is complicated by a recent study by Leroyer et al. [5] reporting a selective accumulation of microparticles with high thrombogenicity in human atherosclerotic plaques obtained from patients undergoing carotid endarterectomy. Even though PMPs were abundant in plasma, PMPs were absent in atherosclerotic plaques. The mechanism beyond and pathophyciological consequences of the latter

had delayed clearance of triglyceride-rich lipoproteins reflected by significantly higher (pb 0.05) levels of total and chylomicron-related triglycerides 6 and 8 hours after ingestion of the meal compared to controls even after adjusting for age, gender, body mass index, smoking status, use of β-blockers, and fasting triglyceride levels. To see if postprandial triglyceridemia would affect plasma levels of PMPs, the plasma concentration of PMPs was measured at peak concentrations of postprandial triglycerides, 4 hours after ingestion of the test meal. The percent change in plasma PMPs (plasma concentrations at 4 hours minus baseline values) within each group are shown in Fig. 2. The percent change in total PMPs and large PMPs increased significantly within each group, but not between groups (p-values for F-tests were 0.35 and 0.31, respectively). For the whole study population, the postprandial increase in total triglycerides (plasma concentrations at 4 hours minus baseline values) was correlated with percent increase in total PMPs (r = 0.29, p b 0.05) and large PMPs (r = 0.34, p b 0.01). Discussion In the present study, we found that the plasma concentration of large PMPs was significantly higher in subjects with carotid atherosclerosis, independent of traditional cardiovascular risk factors and medical treatment. Plasma concentrations of large PMPs were not related to plaque echogenicity. A fat-rich meal was accompanied by a similar increase in plasma PMPs at peak levels of postprandial triglycerides in subjects with and without carotid atherosclerosis [33,34]. Plasma levels of PMPs were not significantly related to plaque morphology neither under fasting nor postprandial conditions. The present study is, to the best of our knowledge, the first study to report higher plasma concentrations of large PMPs in plasma from subjects with carotid atherosclerosis. Subjects with echolucent carotid atherosclerosis tended to have higher plasma levels of large PMPs than

Fig. 2. Bar graphs showing percent changes in total PMPs (upper) and large PMPs (lower) in plasma four hours after ingestion of a fat-rich meal (four hour concentration minus fasting concentrations). Values are means ± SEM. ⁎ p b 0.05, ⁎⁎ p b 0.01, refers to statistical changes within each group.

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observation is not fully understood, but may involve a selective removal of platelets by phagocytes [46]. Recently, we reported higher plasma levels of small and large PMPs in survivors of myocardial infarction, and a strong association between large PMPs and thrombin generation in plasma from patients with a previous myocardial infarction [47]. Small microparticles termed exosomes, released from platelets are enriched in CD63 important for adhesion and intercellular crosstalk, but they do not support coagulation [48]. These findings suggest differential pathophysiological impact of small and large PMPs. Hypertriglyceridemia has been shown to be an independent risk factor for CAD [49,50], and increasing evidence suggests that postprandial hyperlipidemia contributes to the development of atherosclerosis, and coronary and cerebrovascular diseases [14–17]. Previous studies have shown that postprandial hypertriglyceridemia induced by a fat-rich meal promoted elevation of total [18] and endothelial-derived [19,20] microparticles in plasma and attenuated flow-mediated dilatation of the vascular system [18,19] in healthy subjects, indicating a direct impact of postprandial TRLs on endothelial function. The latter assumption is supported by a significant correlation between the postprandial increase in endothelial-derived microparticles and the elevation in serum triglycerides [19,20]. In our study, a fat-rich meal was accompanied by a similar increase in plasma PMPs at peak levels of postprandial triglycerides in subjects with echolucent and echogenic carotid plaques as well as in subjects without carotid plaques. In agreement with studies on endothelial-derived microparticles in healthy individuals, we observed a significant correlation between peak levels of triglycerides and plasma concentrations of PMPs. However, recently Tushuizen et al [19] failed to show a postprandial elevation of total microparticles in patients with type 2 diabetes, and argued that excessive metabolic stress may have impaired the cells to respond properly to TRLs in the postprandial state. Even though plasma levels of large PMPs were higher under fasting conditions in subjects with carotid atherosclerosis in our study, we found similar elevation in plasma PMPs in subjects with and without carotid atherosclerosis. These observations suggest that the presence of carotid atherosclerosis did not attenuate shedding of microparticles from platelets as a response to postprandial TRLs. In conclusion, our findings provide evidence for increased platelet activation, assessed by PMPs, in carotid atherosclerosis and during postprandial hypertriglyceridemia. These findings may be of pathophysiological importance for the progression of atherosclerosis and thromboembolic complications. Conflict of interests None Acknowledgement Center for Atherothrombotic Research in Tromsø (CART) is supported by an independent grant from Pfizer AS. References [1] Hansson GK. Inflammation, atherosclerosis, and coronary artery disease. N Engl J Med 2005;352(16):1685–95. [2] Davi G, Patrono C. Platelet activation and atherothrombosis. N Engl J Med 2007;357 (24):2482–94. [3] Gawaz M. Role of platelets in coronary thrombosis and reperfusion of ischemic myocardium. Cardiovasc Res 2004;61(3):498–511. [4] Kockx MM. Apoptosis in the atherosclerotic plaque: quantitative and qualitative aspects. Arterioscler Thromb Vasc Biol 1998;18(10):1519–22. [5] Leroyer AS, Isobe H, Lesèche G, Castier Y, Wassef M, Mallat Z, et al. Cellular origins and thrombogenic activity of microparticles isolated from human atherosclerotic plaques. J Am Coll Cardiol 2007;49(7):772–7. [6] Mallat Z, Hugel B, Ohan J, Lesèche G, Freyssinet JM, Tedgui A. Shed membrane microparticles with procoagulant potential in human atherosclerotic plaques: a role for apoptosis in plaque thrombogenicity. Circulation 1999;99(3):348–53.

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