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Effect of atorvastatin upon platelet activation in hypercholesterolemia, evaluated by flow cymetry

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Abstract

Hyperlipidemia is a well established risk factor for cardiovascular disease and atherothrombotic events, in which platelet activation also plays a significant role. However, very few studies have addressed platelet activation in hypercholesterolemia, the potential effect of lipid lowering drugs upon platelet hyperfunction, and the question of whether changes in the latter are correlated to normalization of plasma lipids. This study used whole blood flow cytometry to assess in vivo and in vitro platelet activation in a group of 33 patients with hypercholesterolemia, and also the ex vivo effect of atorvastatin (20 mg/day) upon such activation. A control group of 40 normolipidemic volunteers matched in terms of age, sex and added risk factors to the patient group was used. The results showed that hypercholesterolemic patients had in vivo a significantly greater percentage of GPIIb/IIIa- and phosphatidylserine-positive platelets compared with the control group (4.62±3.51% and 2.58±1.19% versus 2.73±1.08% and 1.54±0.68%, respectively). In vitro response of CD62 expression to thrombin was also greater in the patients than in the controls (92.51±6.00% versus 89.63±10.72%, p<0.05). Atorvastatin therapy normalized platelet hyperfunction in the patients studied and reduced GPIIb/IIIa response to ADP (from 82.65±6.43% to 75.84±4.89%, p<0.01). A significant correlation can be seen between such normalization and the decrease in plasma levels of total and LDL cholesterol.

Introduction

Platelet hyperaggregability is associated with risk factors for atherothrombotic events and diseases such as hypertension and hypercholesterolemia [1], [2], [3], [4], [5]. Therefore, drugs for the treatment of such conditions should have beneficial effects upon platelet hyperfunction. Changes in plasma lipoproteins affecting platelet function have been found in hyperlipidemia [6], [7]. Various research groups previously reported an increased in vitro platelet aggregation in the presence of low density lipoproteins cholesterol (LDL-C) [8], showing that high LDL-C levels could trigger spontaneous platelet aggregation in the absence of agonists [9], and that LDL-C, even at low concentrations, stimulated intraplatelet calcium mobilization [10], [11], promoting cell activation, and increasing platelet function and sensitivity to agonists [12], [13].

As to the effect of high density lipoproteins cholesterol (HDL-C), the results published in the literature are conflicting [8]. Some authors suggest that normal HDL-C levels inhibit platelet aggregation [14], while high levels stimulate aggregation [15], whereas other authors have seen no effects [16].

With the methods used in the mentioned studies it is not possible to assess the potential existence in vivo of activated platelet subpopulations that may be of great importance in the atherothrombotic process, and the presence of specific platelet activation markers is not directly measured. The reviewed studies on the effect of lipoproteins upon platelet function, were usually conducted using isolated and washed platelets, or platelet-rich plasma. In both cases the functional response is poorly physiological, since the platelets are removed from their natural environment, without the other blood cells [17]. Moreover, the platelets can be traumatized and artificially activated in vitro as a result of the manipulation required by these techniques. At present, platelet hyperfunction can be assessed in vivo and in vitro using whole blood flow cytometry. Flow cytometry also allows to study the ex vivo effect that a drug can have upon platelet activation. It is currently recognized that flow cytometry is an adequate method for detecting activated platelets in whole blood samples.

Regardless of the mechanisms involved in the process, the final effect of lipoprotein-platelet interactions may be the exposure of GPII/IIIa in its active form (GPIIb/IIIa*), the expression of P-selectin (CD62), and the expression of phosphatidylserine (PS), activation markers that are more physiological in vivo or in vitro, and more rapid, sensitive and relevant than the assessment of platelet aggregation. These platelet activation markers are interesting because integrin GPIIb/IIIa, a membrane glycoprotein, plays an essential role in aggregation through interaction with plasma fibrinogen [18]. Resting circulating platelets do not adhere to vascular endothelium [19] or leukocytes [20], but adhesion to both increases significantly after platelet activation. P-selectin in turn regulates adhesion of activated platelets to neutrophils and monocytes [21] and also to the endothelium [22], [23], and stabilizes the initial GPIIb/IIIa*-fibrinogen interaction, allowing the formation of large, stable platelet aggregates [24]. Thus, CD62 may play an important role in the pathogenesis of atherosclerosis, and is considered to be one of the most sensitive indicators for assessing platelet function status. This selectin is only expressed on the surface of activated platelets, and is contained in the alpha granules when the platelet is at rest. Phosphatidylserine (PS) is located in the internal hemilayer of the resting platelet membrane. When the platelet is activated, a transmembrane movement leading to PS exposure at the external hemilayer is started [25]. This loss of membrane phospholipid asymmetry leads to formation of the prothrombinase complex, a procoagulant surface that can therefore promote thrombus generation [26], [27]. Thus, exposure of this aminophospholipid is a good marker of platelet activation and can contribute to increase the prothrombotic status.

Despite the interest of the study of platelet activation in hyperlipidemia, the available information on these aspects is scarce and often conflicting. As an example, it has been reported that patients with high LDL-C levels do not have greater percentages of circulating platelets with GPIIb/IIIa in its active form as compared to control patients. However, platelets from such patients are more sensitive to the action of ADP [28]. A correlation appears to exist between decreased plasma cholesterol levels and CD62 expression [29]. Diet-induced lipidemia significantly increases during the postprandial period in the percentage of platelets expressing CD62 in their membranes, both in vivo and after ADP stimulation [30].

Based on the foregoing, it seems reasonable to think that the lipoprotein imbalance seen in hyperlipidemia may affect some of the platelet activation pathways and therefore platelet function, which could explain the changes seen in relation to the increased platelet aggregability in hypercholesterolemic patients. Statins (3-hydroxy-3-methylglutaryl-coenzyme A (HMG-COA) reductase inhibitors) have a broad range of properties that contribute to their capacity to reduce cardiovascular risk, including atheroma plaque stabilization, improved endothelial function, inhibition of smooth muscle cell proliferation and migration, reduced expression of adhesion molecules, prevention of cholesterol esterification and accumulation, reduced thrombogenic factor production, and reduced platelet activity. In this context, atorvastatin (Pfizer, NY, USA) is the most effective drug for reducing total cholesterol and LDL-C levels [31], and the study of its action upon platelet activation may therefore be highly relevant.

Since hypercholesterolemic patients have an increased risk of experiencing atherothrombotic events and platelets play a significant role in the development of such events, this study was undertaken to investigate whether these patients have a greater platelet activation than normolipidemic subjects. A study has also been made of whether these patients have hypercoagulability, assessed by PS exposure at the platelet surface, associated to the prothrombinase complex, and of the potential correlations between the plasma markers of atherothrombotic risk and the mentioned platelet markers. The main objective of the study was to assess the effect of atorvastatin upon the above mentioned platelet hyperfunction markers, as a new and interesting aspect of its antiatherogenic potential in hypercholesterolemic patients.

Section snippets

Material and methods

The study comprised 33 patients with primary hypercholesterolemia and/or mixed hyperlipidemia (21 women and 12 men), The demographic characteristics were: age 55.2±12.9 years, height 1.63±0.11 m, weight 68.7±13.9 kg (body mass index, BMI=25.8±3.5 kg/m2). They all had a negative carotid echo-Doppler study. Eighty-eight percent of the patients were non-alcohol drinkers, while the rest were moderate drinkers of alcohol (less than 30 g alcohol/day). Twenty-eight patients were non-smokers or smoked

Results

The side effects attributable to treatment with atorvastatin were poorly significant (one case each of headache, diarrhea and meteorism). These side effects occurred at the start of treatment, and were transient and of mild severity.

Table 1 gives the most relevant biochemical and hematological results. After 1 month of treatment, a significant decrease was seen in total cholesterol (TC) and LDL-C compared to baseline values (178±28 vs. 265±33 mg/dl, p<0.001 and 98±27 vs. 175±36 mg/dl, p<0.001,

Discussion

As expected, atorvastatin significantly reduced total cholesterol and LDL-C levels, in agreement with the results of other authors [33]. This lipid reduction involves a decreased risk of atherosclerotic disease [34], [35]. No significant changes were found in the other biochemical and hematological parameters routinely measured. Moreover, the absence of untoward side effects supports the efficacy and safety of this drug [36].

Although in vitro hypersensitivity of platelets to agonists has been

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