Apo A-I Binding to Platelets Detected by Flow Cytometry

Apo A-I Binding to Platelets Detected by Flow Cytometry

Thrombosis Research 103 (2001) 117 – 122 REGULAR ARTICLE Apo A-I Binding to Platelets Detected by Flow Cytometry$ Derya Ozsavcı1, Turay Yardimci1, ...

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Thrombosis Research 103 (2001) 117 – 122

REGULAR ARTICLE

Apo A-I Binding to Platelets Detected by Flow Cytometry$

Derya Ozsavcı1, Turay Yardimci1, Gulderen Yanıkkaya Demirel2, Fikriye Uras1, Nezih Hekim2 and Orhan Nuri Ulutin3 1 Department of Biochemistry, Marmara University Faculty of Pharmacy, Istanbul, Turkey; 2Dr. Pakize I. Tarzi Laboratory, Istanbul, Turkey; and 3Thrombosis Research Laboratory, Cerrahpasa School of Medicine, Istanbul University, Istanbul, Turkey (Received 3 February 2001 by Editor N. Akar; revised/accepted 14 April 2001)

Abstract Lipoprotein–platelet interactions are very important in atherosclerosis and thrombosis. Several studies have been carried out on specific binding of various lipoproteins to platelets. But there is considerable disagreement about the details of these binding sites. Although low-density lipoprotein (LDL) receptors of several cells have been studied extensively, there is little datum about high-density lipoprotein (HDL) receptors. Apolipoprotein (apo) A-I may play a major role in the determination of the specificity of HDL receptors. In this study, binding of apo A-I to platelets was investigated by using a flow cytometric method. Citrated blood samples were obtained from five healthy and seven hypercholesterolemic subjects. Apo A-I antibody was incubated with the citrated whole blood before and after activation with ADP or thrombin

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This article has been presented at 16th International Congress on Thrombosis, May 5 – 8, 2000, Porto, Portugal.

Abbreviations: TRAP, thrombin receptor agonist peptide; Apo, apolipoprotein; HDL, LDL, VLDL, high-, low- and very-lowdensity lipoproteins; FITC, fluorescein isothiocyanate; MFI, mean fluorescence intensity; PBS, phosphate-buffered saline; ADP, adenosine diphosphate; MoAb, monoclonal antibody; Gp, glycoprotein. Corresponding author: Derya Ozsavcı, Department of Biochemistry, Marmara University Faculty of Pharmacy, Tibbiye Caddesi, No. 49, Haydarpasa, Kadikoy 81010, Istanbul, Turkey. Tel: +90 (216) 449 2303; Fax: +90 (216) 449 2303; E-mail: .

receptor agonist peptide (TRAP). Then fluorescein isothiocyanate (FITC)-labeled secondary antibodies were added and analyzed on a Becton-Dickinson FACSort flow cytometer. In the hypercholesterolemic group, apo A-I binding to platelets was found to be significantly decreased after activation with TRAP ( P < .05), but not after activation with ADP. In the control group, after platelet activation with ADP or TRAP, the apo A-I MFI values were not found to be significantly different from the values of resting platelets ( P > .05). In this study, we demonstrated that apo A-I can bind to platelets, and this supports the hypothesis that apo A-I may play a major role in HDL binding to platelets. D 2001 Elsevier Science Ltd. All rights reserved. Key Words: Apolipoprotein A-1; Platelet HDL receptor; Hypercholesterolemia; Flow cytometry; GpIIb/IIIa receptor

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lasma lipoproteins have been shown to affect platelet activation [1 – 3]. Aviram and Brook [4] and Hassall et al. [5] found that low-density lipoprotein (LDL), and in some cases very-low-density lipoprotein (VLDL), could sensitize platelets in vitro against various agonists if these lipoproteins were added to PRP or they were directly interacted with isolated, washed platelets. The same effect was observed when LDL was added to whole blood and the platelet function was determined using filtrag-

0049-3848/01/$ – see front matter D 2001 Elsevier Science Ltd. All rights reserved. PII S0049-3848(01)00279-1

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ometry [6]. On the other hand, it has been observed that isolated high-density lipoprotein (HDL) has opposing effects on platelet aggregation and, in some cases, shows a mild inhibitory effect or antagonizes the effects of LDL [5]. HDL particles have been shown to inhibit [7], stimulate or not change platelet aggregation [4,8,9]. In recent years, the use of fluorescently labeled antibodies and flow cytometry has permitted the detection of activation antigens on individual platelets [10–12]. Several studies have been reported regarding specific binding of various lipoproteins to human platelets. However, there is considerable disagreement about the details of these binding sites [4,13]. LDL receptors are present in hepatocytes, lymphocytes, fibroblasts and platelets. Independent binding sites for HDL have been reported in hepatic tissue in addition to those found in testes, ovaries and adrenals [14]. With these receptors, they regulate both their intracellular cholesterol concentration and plasma cholesterol concentration. However, we do not have enough data about the HDL binding sites on platelets. Several investigators have reported that gel-filtered platelets from normal subjects bind both to LDL and HDL and they have found cross-reactions between the binding sites for these ligands [13]. Recently, GpIIb/IIIa has been proposed to be the platelet receptor for HDLs. However, it is not obvious at present if binding of HDL3 to GpIIb/ IIIa is related to any physiological response [15]. Although it is not definitely established, it is suggested that apolipoprotein (apo) A-I has a role in determining the specificity of the HDL receptors [14,16]. It seems likely that HDL particles can bind to platelet GpIIb/IIIa receptors via apo A-I. In this way, we have analyzed the apo A-I binding to platelets, and the effects of hypercholesterolemia and platelet activation on this binding using a flow cytometric method.

1. Materials and Methods 1.1. Materials Mouse anti-human apo A-I monoclonal antibody (MoAb) was obtained in purified form from

Calbiochem, Novabiochem (La Jolla, CA, USA). As secondary antibody, F (ab)2 polyclonal antimouse fluorescein isothiocyanate (FITC) from Beckman Coulter (Brea, CA, USA) was used. Thrombin receptor agonist peptide (TRAP) and adenosine diphosphate (ADP), both from Stago (Asnieres, France), were used as activators of platelets. 1.2. Subjects and Blood Collection Five normal and seven hypercholesterolemic subjects were used for this study. These patients had normal platelet counts and none of them was receiving medication such as acetylsalicylic acid or other drugs. Venipuncture of subjects was performed without stasis using a 21-gauge butterfly needle. After discarding initial 2 ml of blood, whole blood was collected into 3.8% sodium citrated vacutainer tubes (Becton Dickinson, San Jose, California, USA). 1.3. Analysis of Apo A-I Binding to Platelets by Flow Cytometric Method Titration of MoAb binding was performed with serial dilutions in phosphate-buffered saline (PBS), and all the tests were done in duplicate. Optimal MoAb concentration was determined after several experiments and 1/100 concentration was determined to be the optimum. Fifty microliters of citrated whole blood was added into three tubes, containing 50 ml 0.1 mmol ADP, 50 ml TRAP or 50 ml PBS, respectively. The tubes were mixed and incubated for 5 min at room temperature. Twenty microliters of samples from each of these tubes was added into both control and test-labeled tubes. Five microliters of diluted apo A-I monoclonal antibody was added to all of the test tubes, but not to the control tubes. After 15 min of incubation at room temperature in dark conditions, 5 ml of FITC-labeled secondary antibody was added to all the tubes. Then, the tubes were mixed and incubated again for 15 min at room temperature in the dark. The samples were diluted to 2 ml with PBS and stored at 2–8C. Flow cytometric analysis was performed within 2 h. The analyses of all the samples were carried out on a FACSort flow cytometer (Becton Dickinson, USA). The instrument was equipped with

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a 488-nm argon ion laser. The alignment of the instrument was controlled daily by calibrator beads. The platelet population was identified by means of light scattering characteristics and enclosed in an electronic gate. Fifty thousand platelets from each sample were analyzed. A discriminator was set on the forward scatter to eliminate the possible contamination of debris on the scattergram. A listmode gate was set around the platelets. All of the test samples, which were incubated with secondary antibody, were used as non-specific binding controls. The test samples were evaluated according to the cursor, and were set on these non-specific binding controls. 1.4. Biochemical Parameters Hitachi 917 analyzer was used for the analyses of biochemical parameters. 1.5. Statistical Methods Statistical analyses were done by standard statistical tests, namely unpaired t test and paired t test.

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2. Results Apo A-I results were evaluated before and after activation with TRAP or ADP in terms of mean fluorescence intensity (MFI) values. Logarithmic forward and side scatter scattergrams were used to be able to observe platelets as a compact cell population. Examples of these scattergrams and histograms obtained from unstimulated samples and the ones activated by ADP or TRAP are shown in Fig. 1. MFI values obtained from the samples of hypercholesterolemic patients and normal subjects are shown in Fig. 2. In the hypercholesterolemic group, apo A-I binding to platelets was found to be decreased significantly after activation with TRAP ( P=.05), but not after activation with ADP ( P=.40, P>.05). In the control group, after platelet activation with ADP or TRAP, the apo A-I MFI values were not found to be significantly different from the values of resting platelets, respectively ( P=.65, P>.05) ( P=.37, P>.05). When we compared the results of apo A-I binding (MFI) to inactivated platelets of both normocholesterolemic and hypercholesterolemic

Fig. 1. Scattergrams and histograms of samples unstimulated and activated by ADP or TRAP. M1 shows the percentage of positive fluorescence of antigen; the marker is set according to values obtained with isotypic control.

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nogen (486 ± 29 mg/dl) values were higher than in the control group (Table 2). However, HDL cholesterol levels were not significantly different in either group.

3. Discussion

Fig. 2. MFI values obtained from the samples of hypercholesterolemic patients and normal subjects.

patients, there was no significant difference between them ( P=.18, P>.05). Apo A-I binding to platelets after activation with TRAP or ADP did not give any significant relationship between the control and hypercholesterolemic groups, respectively ( P=.08, P>.05) ( P=.73, P>.05). 2.1. Biochemical Parameters For the hypercholesterolemic group (Table 1), serum cholesterol (288 ± 17 mg/dl), LDL cholesterol (186 ± 23 mg/dl), VLDL cholesterol (32 ± 8 mg/dl), triglyceride (178 ± 27 mg/dl) and fibri-

Atherosclerosis is associated with the following risk factors: hypercholesterolemia, hypertension, male sex, obesity, diabetes mellitus, smoking and HDL cholesterol levels below 35 mg/dl [15, 17–19]. Platelets and lipoproteins have a role in atherosclerosis, thrombosis and acute coronary syndromes [3,4,14,18]. It has been reported in several studies that there is a direct relation between plasma lipoproteins and hemostatic function of platelets [4,14,18,20]. As it is known that platelet behavior varies in hypercholesterolemic subjects, further investigation has been carried out to understand the interaction of platelets with lipoproteins [21]. Increased activity of the coagulation mechanism and high levels of cholesterol and phospholipids in the platelets of hypercholesterolemic patients have been noted [13]. A membrane abnormality originating from the megakaryocyte in the bone marrow during platelet formation might induce this condition, or it could be a result of the platelet plasma–lipoprotein interaction. Platelets have been shown to possess specific LDL and HDL receptors. The apo B and apo E binding to specific LDL receptors has been described in various tissues and platelets, but there is limited datum concerning the apo A-specific HDL receptors [16]. Incuba-

Table 1. Biochemical parameters of the hypercholesterolemic group Cholesterol LDL HDL VLDL 10 3 Triglyceride Fibrinogen Age Sex (Platelet count/Ml) (mg/dl) cholesterol cholesterol cholesterol (mg/dl) (mg/dl) (mg/dl) (mg/dl) (mg/dl) Case 1 Case 2 Case 3 Case 4 Case 5 Case 6 Case 7 Mean S.D.

313 274 286 282 300 262 298 287.9 17.2

230 186 181 186 151 181 189 186.2 23.2

54 50 48 48 50 37 51 48.2 5.3

29 44 37 28 25 24 39 32.2 7.7

147 219 188 182 178 140 190 177.8 27

520 469 500 430 490 500 490 485.6 28.9

54 32 56 37 48 72 38 48 13.9

F M M F F M M

150 215 139 133 158 248 69 158.9 58.1

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Table 2. Biochemical parameters of the control group Cholesterol LDL HDL VLDL Triglyceride Fibrinogen Age Sex 10 3 (Platelet count/Ml) (mg/dl) cholesterol cholesterol cholesterol (mg/dl) (mg/dl) (mg/dl) (mg/dl) (mg/dl) Case 1 Case 2 Case 3 Case 4 Case 5 Mean S.D.

179 165 191 185 200 184 13.1

105 102 125 126 153 122.2 20.4

60 57 53 49 52 54.2 4.3

14 18 13 10 22 15.4 4.6

tion of lipoproteins with isolated platelets leads to high platelet activation by LDL and VLDL and depression of activity by HDL and chylomicrons [13]. It was reported that HDL only minimally inhibits LDL binding, VLDL is a more effective inhibitor and LDL inhibits HDL binding [4]. In this study, we have detected the binding of apo A-I (the major apoprotein of HDL) to platelets by using flow cytometric assay. Thus, MFI of apo A-I antibody binding to platelets was evaluated. Flow cytometric results obtained in studies performed with platelet receptor antibodies are expressed in terms of percentage of the cells recognizing the specific antibodies. These parameters do not give an exact picture of the minimal variations in the receptor expressions. In contrast, results expressed as MFI are much more sensitive. Our results, therefore, have been expressed accordingly. In the studies carried out by Curtiss and Plow, it was clarified that when LDL and HDL were radio-iodinated and their interaction with washed human platelets was evaluated, LDL was found to be a poor inhibitor of 125I-HDL binding to platelets, whereas HDL was found to be an effective inhibitor of 125I-LDL binding. It has also been reported that there are 7000 and 1500 binding sites for LDL and HDL, respectively, in unstimulated platelets [14]. In this study, apo A-I binding to resting platelets and platelets activated by ADP or TRAP in hypercholesterolemic and normal subjects was measured and the effect of platelet activation to this binding evaluated. Curtiss and Plow reported that binding of HDL and LDL was independent of platelet activation in a study carried out with radio-iodinated HDL and LDL [14]. However, in our study, it was observed that

71 68 63 50 112 72.8 23.3

276 290 290 310 360 305.2 32.9

32 29 28 38 50 35.4 9.0

F F F F M

217 202 176 170 200 193 19.5

apo A-I binding to platelets decreased significantly following activation with a strong agonist like TRAP especially in the hypercholesterolemic group, but it was not significant in the normal group. Koller et al. [22] have recently reported that lipoproteins bind to both components of GpIIb/IIIa complex in isolated membranes and intact platelets. In another study, it was reported that HDL3 binds to GpIIb/IIIa, which is a fibrinogen receptor in platelets, and this binding is associated with phospholipase D activation, and phosphatidic acid and diacylglycerol formation [15]. Our findings show that apo A-I, and thus HDL, is likely to bind to platelets. If the binding site of apo A-I to platelets is GpIIb/IIIa, then there might be suppression in the binding of apo A-I to platelets since fibrinogen also binds to these sites after activation with TRAP. Despite the fact that HDL levels in both hypercholesterolemic and normal subjects were similar, MFI of apo A-I binding to platelets in the hypercholesterolemic group was found to be higher than in the control group (not significant). This situation probably results from the antiatherogenic effect of HDL in hypercholesterolemia [15]. It might also be due to platelet activation in vivo independent of the increased levels of cholesterol, triglyceride and fibrinogen. In conclusion, in this preliminary study, we detected the binding of apo A-I to platelets by flow cytometry. Our findings suggest that HDL binds to platelets via apo A-I. In order to achieve more reliable results, it is possible to try out the flow cytometric study with a higher number of subjects in both groups and include patients with HDL levels below 35 mg/dl in the hypercholesterolemic group; in addition, other binding methods could be used.

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We would like to express our gratitude to Dr. Nural Bekiroglu for assistance in statistical analyses.

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