Biochimica et BiophysicaActa, 1097(1991) 123-127 © 1991 ElsevierSciencePublishers B.V. All rights reserved 0925-4439/91/$03.50 ADONIS 092544399100108W

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BBADIS 61058

Epinephrine potentiates activation of human platelets by low density lipoproteins Valery N. Bochkov, Tatyana A. Voino-Yasenetskaya and Vsevolod A. Tkachuk Institute of Experimental Cardiology, Cardiology,Research Center, Moscow (U.S.S.R.)

(Received 16 October 1990) (Revised manuscriptreceived 12 April 1991)

Keywords: Plateletactivation;LDL;Epinephrine;Atherosclerosis Low density lipoproteins activate phosphoinositide turnover, increase free cytoplasmic calcium concentration and stimulate phosphorylation of 20- and 47-kDa proteins in blood platelets. All these effects are substantially potentiated by epinephrine.

Introduction The mechanisms by which low density lipoproteins promote the development of atherosclerosis are not well understood. Recent evidence suggests that this phenomenon can be partly explained by the direct activating effect of LDL on blood platelets, which can specifically bind and possibly catabolise LDL particles [1,2]. Activation of platelets leads to their increased adhesion to the vascular wall, formation of thrombi and secretion of growth factors. All these events are known to participate in atherosclerotic plaque formation [3]. It was shown that the treatment of platelets with LDL leads to the activation of phospholipase C and the increase in cytoplasmic calcium concentration ([Ca2+]i) [2,4], as well as the phosphorylation of 47-kDa protein, catalysed by protein kinase C [5]. These events are thought to be a prerequisite for aggregation response [6]. In this work we report that LDL-dependent stimulation of blood platelets is potentiated by epinephrine. Our data provide further support to the relation of high levels of LDL and catecholamines to the increased risk of atherosclerosis. Materials and Methods Isolation of low density lipoproteins LDL (density 1.019-1.063 g/ml) were isolated from plasma of normocholesterolemic subjects by ultracen-

Correspondence: V.A. Tkachuk, Institute of Experimental Cardiology, CardiologyResearch Center, Moscow,U.S.S.R.

trifugation according to the procedure of Havel et al. [7], dialysed against a 0.15 M NaC1/1 mM EDTA, pH 7.4 solution, filtered (Millipore, 0.22 p~m pore size) and analysed by SDS-polyacrylamide gel electrophoresis. LDL were stored at 4°C and used within 3 weeks. We did not notice any change of LDL activity during this period. LDL concentration was expressed as their protein content. All experiments were repeated using LDL preparations obtained from different normocholesterolemic donors and gave similar results. The methylation of LDL was performed according to Ref. 8. Isolation of platelets Human blood was taken by venipuncture into 1/6 volume of acid citrate-dextrose anticoagulant (85 mM trisodium citrate, 71 mM citric acid, 9 mM D-glucose). Platelet-rich plasma (PRP) was obtained by centrifugation (200 × g, 15 min). PRP was centrifuged (1200 × g, 10 min) and pelleted platelets resuspended in equal volume of buffer, containing 150 mM NaC1, 1 mM MgC12, 2.7 mM KCI, 0.37 mM NaH2PO4, 1 mM CaC12, 5 mM D-glucose, 10 mM Hepes-NaOH (pH 6.55), 0.1 mg/ml of apyrase, 0.35% bovine serum albumin fraction V (BSA) and 50 units/ml of heparin (buffer A). Measurement of phosphoinositide metabolism Platelets, obtained by centrifugation of PRP were suspended to 10 9 cells/ml in buffer A, containing 100 ~.Ci/ml of myo-[2-3H]inositol (20 Ci/mmol, Amersham, U.K.). After incubation for 3 h at 37°C the cells were pelleted and resuspended (2-108 cells/ml) in buffer, containing 150 mM NaCI, 1 mM MgC12, 2.7 mM KC1, 0.37 mM NaH2PO4, 1 mM CaC12, 5 mM D-glucose, 10 mM Hepes-NaOH (pH 7.4), 0.1 mg/ml

124 of apyrase and 100 ~ g / m l of BSA (buffer B). 10 mM LiCI was then added and the platelets were incubated for 20 min at 37°C. 1 ml of cell suspension was mixed with 10 #1 of activator solution (physiological saline or 10 m g / m l of LDL or 1 - 10 4 M epinephrine or their combination) and incubated 5 rain at 37°C. Incubation was terminated by the addition of a hot (90°C) solution containing 1%, SDS and 30 mM EDTA. The platelet lysate was then diluted with H 2 0 ( x 4 ) and chromatographed on 0.5 ml column of Dowex AG, 1 x 4, formate form (Bio-Rad, U.S.A.). The column was washed with water (12 ml) followed by 6 ml of 60 mM ammonium formate. [3H]inositol phosphates were eluted with 6 ml of 1 M ammonium formate solution containing (1.1 M formic acid [9]. Using this procedure the recovery of [3H]inositol mono-, bis- and trisphosphates was in excess of 90 percent.

Measurement of [Ca: +]i Indo-1 pentaacetoxymethyl ester (Sigma, U.S.A.)was added to platelet preparation in buffer A to the final concentration of 5 /xM. After incubation for 1 h at room temperature, the cells were pelleted (1200 x g, 10 rain) and resuspended in equal volume of buffer B. The measurements of lndo-1 fluorescence were performed on a Jasco FP-770 spectrofluorimeter at 330 nm (excitation) and 400 nm (emission) wavelengths. The suspension was not stirred. The temperature was maintained at 35°C. Immediately before measurements an aliquot of cell suspension was diluted 1:10 with buffer B, without albumin and apyrase. Calibration of fluorescent signal and calculations of [Ca 2+ ]i were performed as described [10]. Basal [Ca2+]i level was 97 _+ 18 nM (n = 4).

Measurement of protein phosphorylation Platelets were isolated in buffer A containing no Nail 2PO 4, pH 6.55. After cell counting the suspension was centrifuged and resuspended to 109 cells/ml in the same buffer, containing 0.5 m C i / m l [32p]HsPO 4 (Amersham, U.K.). After 2 h at 37°C 4 volumes of phosphate-free solution A were added and cells pelleted. The pellet was finally resuspended to 2 × 10 ~ cells/ml in phosphate-free buffer B, pH 7.4. The incubation was started by mixing prewarmed (37°C) cell suspension with agonist solution at final agonist concentration given in the legends to Figs. 3 and 4. At indicated times 50 ~1 aliquots were mixed with 50 /xl of solution, containing 2% SDS, 10% 2mercaptoethanol and traces of bromophenol blue, briefly vortexed and frozen in liquid nitrogen. Immediately before electrophoresis the samples were thawed in a boiling water bath and additionally boiled for 2 rain. Discontinious SDS-polyacrylamide gel electrophoresis was performed in vertical gradient (1(I-15%) gel

slabs according to Ref. 11 in Protean Dual Slab Cell (Bio-Rad, U.S.A.). After electrophoresis the gels were fixed, stained with Coomassie R-250, dried and autoradiographed. The pattern of phosphorylation was quantitated using Beckman CDS-200 scanning densitometer at 600 nm. Molecular weights of polypeptides were assessed using molecular weight markers (Dalton Mark VII, Sigma, U.S.A.). Two-dimensional gel-electrophoresis was performed according to Ref. 12 with some modifications. Protein samples, containing 100 /xg of protein were prcpared exactly as described above. Solubilisation of samples with SDS prior to isoelectrofocusing (IEF) was described before [13]. IEF was performed in 4 × 130 mm tubes, containing 5% polyacrylamide gel, 9 M urea, 2% CHAPS (3-[(3-cholamidopropyl)-dimethylammonio]- lpropanesulfonate) (Sigma, U.S.A.), 1.6% Ampholine 5-8 and 0.4% Bio-Lyte 3/10. The separation was performed in Pharmacia G E - 2 / 4 L S Electrophoresis Apparatus. The upper chamber was filled with 20 mM NaOH and the lower chamber contained 6 mM H3PO 4. IEF was performed overnight to achieve 400(I volt/h. After IEF tubes were processed as in Ref. 14. Briefly, the gels were stained, incubated in water, then in 63 mM Tris-HCl (pH 6.8) and finally in the same buffer, containing 2.3% SDS. The gel rod was placed on top of discontinious gradient slab gel, fixed with 1% agarose in 63 mM Tris-HCl (pH 6.8) and further processed as described for one-dimensional electrophoresis.

Protein determinations Protein concentrations of LDL preparations and platelet samples were determined according to Lowry [15]. Results

Actiuation of phospholipase C Data, presented in Fig. 1, indicate that incubation of [3H]inositol-labelled platelets with 1 0 0 / x g / m l of LDL (concentration known to saturate specific binding sites on these cells [1]) increased the concentration of inositol phosphates in these cells. This effect is thought to reflect activation of phosphoinositide-specific phospholipase C [16]. It can also be seen that LDL-induced response was substantially potentiated by epinephrine, w h i l e e p i n e p h r i n e alone had no effect on phosphoinositide methabolism. Similar effect was described previously for other platelet agonists [17].

Increase in [Ca 2 +]i We have found that the addition of LDL to the suspension of platelets, labelled with indo-1 induced rapid increase in [Ca2+]i. The elevation of Ca~ + was reversible, reached maximum in about 30 s and decrcased to basal level in 2-3 rain (data not shown). The

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Fig. 1. The effects of LDL and epinephrine on the levels of inositol phosphates in human blood platelets. [3H]inositol-labelled platelets were incubated with LDL (100 p,g/ml), epinephrine (1.10 ~ M) or their combination for 5 rain at 37°C. The incubation was terminated and inositol phosphates were extracted and analysed as described in Materials and Methods. Each point represents radioactivity in l0 s cells. Significance of differences: basal-epinephrine, not significant; basaI-LDL, p < 0.01: LDL-(LDL+epinephrine), P < 0.005. Similar results were obtained in two independent experiments.

action of LDL was concentration-dependent (ECs0 = 16 +_ 9 / z g / m l , n = 4). It was more pronounced in the presence of 10 -6 M epinephrine. Epinephrine alone did not alter Ca~ + level (Fig. 2). These effects of LDL were similar to those described for a known platelet agonist, thrombin [17].

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ILOL], g/ma Fig. 2. The effect of epinephrine on LDL-induced [Ca 2+ ]i increase. Epinephrine (1.10 6 M) was added to the suspension of lndo-1loaded platelets 15 s prior to the addition of LDL. The results are expressed as the difference between the maximum level achieved during the stimulation and the basal level (97_+ 18 nM, n = 4). The data are typical of four experiments.

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Fig. 3. The effect of LDL on phosphorylation of platelet proteins. 32p-labelled platelets were incubated for 60 s at 37°C without (a) or with (b) 200 g g / m l of LDL and phosphoproteins were analysed by two-dimensional gel-electrophoresis with subsequent autoradiography. Spots, incorporating highest radioactivity are indicated by the arrows. The same changes were observed in three independent experiments.

LDL-induced protein phosphorylation Ca 2+ ions are known to activate in platelets a specific protein kinase, which phosphorylates myosin light chain, having a molecular mass of about 20 kDa [6]. Activation of phosphoinositide turnover also leads to phosphorylation of several proteins, catalyzed by diacylglycerol-activated protein kinase C [18]. In platelets treated with physiologic stimuli the main substrate of protein kinase C is a cytoplasmic 47-kDa protein, which is known to exist in multiple phosphorylated forms with slightly different p I values [19]. We used two-dimensional electrophoresis to study the influence of LDL on the state of phosphorylation of platelet proteins. We observed that LDL induced a rather selective phosphorylation of an acidic 20-kDa protein and of a group of basic proteins with the molecular mass about 47 kDa (Fig. 3). Similar, although more prominent changes were observed after addition of platelet activating factor and adenosine diphosphate (data not shown). We studied the time-course of 47-kDa protein phosphorylation. It was reversible (Fig. 4a) and proceeded similarly to that induced by ADP (Fig. 4b). Addition of epinephrine alone did not induce changes in protein phosphorylation, but LDL- and ADP-induced phosphorylation of p47 was markedly increased in the pres-

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