Arachidonic acid of platelet phospholipids is decreased after extracorporeal removal of plasma low density lipoproteins in patients with familial hypercholesterolemia

Atherosclerosis 131 (1997) 97 – 106

Arachidonic acid of platelet phospholipids is decreased after extracorporeal removal of plasma low density lipoproteins in patients with familial hypercholesterolemia Robert Bra¨utigam a, Carola Bra¨utigam a, Reinhard Lorenz b, Werner O. Richter c, Bernd Engelmann a,* a

Physiologisches Institut der Uni6ersita¨t Mu¨nchen, Pettenkoferstr. 12, D-80 336 Mu¨nchen, Germany b Institut fu¨r Prophylaxe der Kreislaufkrankheiten, Uni6ersita¨t Mu¨nchen, Mu¨nchen, Germany c II. Medizinische Klinik, Klinikum Großhadern, Uni6ersita¨t Mu¨nchen, Mu¨nchen, Germany

Received 14 August 1996; received in revised form 17 December 1996; accepted 21 January 1997

Abstract Platelet phospholipid composition was analyzed before and after extracorporeal removal of low density lipoproteins (LDL) by LDL apheresis in six patients with familial hypercholesterolemia. Elevated levels of total plasma cholesterol and the portion of plasma cholesterol carried by LDL were reduced by 56 and 66% after LDL apheresis. Platelet cholesterol contents remained unaffected. While the phosphatidylcholine (PC):sphingomyelin (SM) ratio in plasma lipoproteins was increased by 22% following apheresis, the same parameter was lowered by 14% in platelets. LDL apheresis induced decreases in the percentages of distinct molecular species containing arachidonic acid in platelet diacyl subgroups of PC, phosphatidylinositol (PI) and phosphatidylserine (PS) as well as in alkenylacyl (plasmalogen) phosphatidylethanolamine (PE). Directly after apheresis, the percentages of molecular species with arachidonic acid of diacyl PC, diacyl PI and alkenylacyl PE were reduced by 20, 23 and 8%, respectively. Two days after the procedure, total arachidonic acid of diacyl PC, diacyl PS and alkenylacyl PE was lowered by 11, 20 and 8%. Overall, the amount of phospholipid bound arachidonic acid was reduced by 16% after apheresis (from 79.1 to 66.4 nmol/108 platelets). The results are thus in agreement with previous data indicating decreased phospholipid bound arachidonic acid in red blood cells after apheresis (Engelman B, Bra¨utigam C, Kulschar R et al. Biochim Biophys Acta 1994;1196:154). Urinary 2,3-dinor thromboxane B2, an estimate of platelet thromboxane A2 (TXA2) production, tended to be decreased following the procedure. The percentage change in the TXA2 metabolite was positively related to the magnitude of change induced by apheresis in phospholipid bound arachidonic acid. In summary, the results suggest that in patients with hypercholesterolemia, the level of plasma LDL is an important determinant of the arachidonic acid content of several platelet phospholipids. © 1997 Elsevier Science Ireland Ltd. Keywords: Alkenylacyl (plasmalogen) phosphatidylcholine; Diacyl phosphatidylethanolamine; Oleic acid; Linoleic acid; Immunoabsorption

Abbre6iations: LDL, low density lipoproteins; HDL, high density lipoproteins; VLDL, very low density lipoproteins; PC, phosphatidylcholine; LPC, lyso phosphatidylcholine; PE, phosphatidylethanolamine; PS, phosphatidylserine; PI, phosphatidylinositol; SM, sphingomyelin; C:P, total cholesterol:total phospholipid; Diacyl-, phospholipid molecule with ester bonds at the C1- and the C2-atom of the glycerol backbone; Alkenylacyl-(plasmalogen), phospholipid molecule with an enolether bond at the C1-atom and an ester bond at the C2-atom of the glycerol backbone; TXA2, thromboxane A2; TXB2, thromboxane B2; 16:0, Hydrocarbon chain with 16 C atoms (palmitic acid); 18:0, Hydrocarbon chain with 18 C atoms (stearic acid); 18:1 (n-9), Hydrocarbon chain with 18 C atoms and one double bond (oleic acid); 18:2 (n-6), Linoleic acid; 20:0, Arachidic acid; 20:3 (n-6), Eicosatrienoic acid; 20:4 (n-6), Arachidonic acid; 20:5 (n-3), Eicosapentaenoic acid; 22:4 (n-6), Docosatetraenoic acid; 22:6 (n-3), Docosahexaenoic acid; 16:0/20:4, Molecular species containing 16:0 at the C1-atom and esterified with 20:4 at the C2-atom of the glycerol backbone. * Corresponding author. Tel.: + 49 89 5996394; fax: + 49 89 5996378; e-mail: [email protected] 0021-9150/97/$17.00 © 1997 Elsevier Science Ireland Ltd. All rights reserved. PII S 0 0 2 1 - 9 1 5 0 ( 9 7 ) 0 6 0 8 7 - 5

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1. Introduction Profound changes in phospholipid composition are elicited by activation of platelets in response to platelet agonists (e.g. thrombin or collagen). Due to the action of phospholipases A2 and C, plasma membrane phospholipids such as diacyl phosphatidylcholine (PC)1, diacyl phosphatidylinositol (PI) as well as other phospholipids are degraded to a substantial extent [1]. Plasma low density lipoproteins (LDL) may also exert agonist-like effects on platelet phospholipids, although somewhat differing results have been obtained [2]. However, lipoproteins (LDL and high density lipoproteins (HDL)) could also alter platelet lipid composition by delivering or extracting lipid molecules from platelets. Indeed, rapid exchange of lipid molecules such as free fatty acids and phospholipids between both compartments has been described [3–6]. As the above mentioned investigations were performed under in vitro conditions, it is largely unknown whether the platelet lipid composition is influenced by plasma lipoproteins in vivo. Studies performed on patients with elevated plasma LDL contents indicate several alterations in platelet phospholipid composition. These include increases in platelet cholesterol levels [7] and enhanced percentages of PI associated arachidonic acid [8,9]. Both disturbances of platelet lipid composition have been proposed to be responsible for the increased production of thromboxane A2 (TXA2) in platelets of patients with type IIa hypercholesterolemia [10]. To directly assess the influence of lipoproteins on platelet lipids in patients with hypercholesterolemia under in vivo conditions, we analyzed, in this study, whether the substantial and rapid reduction of plasma LDL content by extracorporeal LDL apheresis affected the composition of phospholipid and cholesterol within platelet membranes. As physiological and pathophysiological changes in platelet function are related to alterations in specific phospholipids, the molecular species composition of major platelet glycerophospholipids (diacyl and alkenylacyl phosphatidylcholine, diacyl and alkenylacyl phosphatidylethanolamine (PE), diacyl phosphatidylserine (PS), diacyl PI) was assessed before and after LDL apheresis.

2. Materials and methods

2.1. Subjects Six male patients (donors 1 – 6 in Table 6 and in Figs. 1–3) with heterozygous familial hypercholesterolemia, in whom regular LDL apheresis (by immunoadsorption, see below) was performed weekly,

participated in the study. Their mean age was 5296 years. (9 S.D.; range 42–60 years.) The treatment had been started 6–28 months before the analyses. All patients presented angiographically verified coronary heart disease. In three patients, in whom myocardial infarction had been previously diagnosed, a coronary bypass operation had been performed. All patients received a daily dose of 40 mg of the hydroxymethylglutaryl reductase inhibitor simvastatin. While being treated by LDL apheresis and simvastatin, the patients received, in addition, b adrenoreceptor antagonists, allopurinol, calcium entry-blockers, nitrates and mexiletine. Aspirin medication had been stopped at least three weeks before the analyses. None of the patients was treated with vitamin E. The patients were instructed to follow a cholesterol-lowering diet (300 mg cholesterol/day, 30% of daily energy intake as fat, with 10% as saturated, monounsaturated, and polyunsaturated fatty acids, respectively). Informed consent was obtained from all patients and the study was carried out according to the principles of the Helsinki declaration.

2.2. Procedure of the LDL apheresis In the patients with familial hypercholesterolemia, plasma LDL levels were lowered by immunoabsorption every week [11]. Briefly, blood cells and plasma were separated and the plasma run over a column made from polyclonal anti apo B antibodies coupled to Sepharose AB gel. After passage through the column, plasma depleted of LDL was reinjected into the patient’s forearm vein. The column was regenerated as described in [11]. The treatment lasted 3–4 h. During 2/3 of the time of the procedure heparin (379 10 I.U./kg body weight as bolus and 2597 I.U./kg body weight per h as infusion) and dextrose solution (2.5–3 ml/min) were used as anticoagulants.

2.3. Clinical laboratory parameters Total plasma cholesterol and triglycerides were measured enzymatically using an automatic clinical chemistry analyzer. Separation of lipoprotein fractions was performed by ultra centrifugation for 24 h using a Beckman TI 50 rotor (d=1.006 g/ml; 50.000 rpm at 4°C). After isolation of very low density lipoproteins (VLDL), present in the upper fraction), cholesterol was determined in the high density lipoproteins (obtained after precipitation of the bottom fraction with heparin and MgCl2) and in low density lipoproteins (by subtracting HDL cholesterol from total bottom cholesterol).

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2.4. Determination of 2,3 -dinor-TXB2 contents in 24 h urine The patients collected their urine into polypropylene bottles over the 24 h period before the start of apheresis (pre), during the 24 h period immediately following apheresis (24 h post) as well as between the first and second day after apheresis (starting exactly one day after the end of apheresis; 48 h post). The determination of urinary 2,3-dinor-TXB2 content was performed as previously described [12]. Only urine samples negative for microhematuria and proteinuria were used for the analyses. 25 ml of urine were added to 200 ml of an assay buffer containing an antibody against TXB2 as well as a tritium labeled TXB2 tracer. The values were compared to calibration curves generated using standard 2,3-dinor-TXB2, tritium labeled TXB2 and the antibody.

2.5. Assessment of platelet total phospholipid and cholesterol contents Venous blood (anticoagulated with 0.38% citrate) was drawn immediately before (pre), directly after (post), as well as two days after the end of LDL apheresis (48 h post). The blood was centrifuged at 180×g for 20 min, the supernatant recovered and again centrifuged under the same conditions. Plateletrich plasma was subsequently centrifuged at 3000× g for 3 min and the pellet washed two times at room temperature with a buffer composed of (mM): 138 NaCl, 3 KCl, 1 MgCl2, 15 Hepes, 9 citrate, 5 EDTA, 5 glucose, containing, in addition, 350 mg/100 ml albumin and 5 mg/100 ml apyrase (pH 6.3). After the second washing, the supernatant was removed and the cells suspended in the same buffer. The cells were counted in a coulter counter. Platelet lipids were extracted according to Bligh and Dyer [13] using chloroform containing 50 mg/l butylated hydroxytoluene. The amount of total platelet phospholipids was estimated by measuring the total phosphate content in the lipid extracts [14]. Total cholesterol contents were assessed by using a commercially available kit (Boehringer Mannheim).

2.6. Analysis of plasma and platelet phospholipid fractions Lipids were extracted [13] from plasma and washed platelets (isolated as detailed above) and phospholipids separated and quantitated as described [15]. Silica G60 plates (Merck, Darmstadt, Germany) were sprayed with K + oxalate (1% in methanol – H2O (2:3)) and activated for 30 min at 80°C. The plates were developed for the first dimension with the solvent CHCl3 – CH3OH– NH3 –H2O (45:37:6:4), dried and for the second dimen-

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sion with CHCl3 –CH3OH–CH3COCH3 –CH3COOH– H2O (40:15:15:12:8). The spots corresponding to the different phospholipids were visualized using iodine vapour and the amounts of phospholipids quantitated by phosphate analysis [14]. Duplicate determinations taken from the same donor but treated independently during lipid extraction and phospholipid separation agreed within 6.1% for all phospholipids with percentages \ 2%. For analysis of plasma and platelet plasmalogen phospholipids (alkenylacyl PC (plasmenylcholine) and alkenylacyl PE (plasmenylethanolamine)), the following procedure was employed [16]. Phospholipids were separated by two-dimensional thin layer chromatography (TLC) on DC 60 plates (Merck), using for the first dimension the solvent CHCl3 –CH3OH– NH3 –H2O (90:54:5.5:5.5) and for the second dimension the solvent CHCl3 –CH3OH–CH3COOH–H2O (90:40:12:2). After visualisation with diphenylhexatriene spray, the spots corresponding to PC and PE were scraped from the plates and eluted from the silica by addition of chloroform–methanol (1:4), the extraction being repeated twice. The phospholipids were applied to small TLC plates (10 cm × 10 cm) and the plates put onto glass tanks that contained fresh HCl (37% in H2O). The distance between the silica layer of the plates and the upper margin of the acid was 5.5 cm. The plates were exposed for 3 min to HCl fumes in order to cleave selectively the acid labile enol ether bond of plasmalogens. After drying the plates were developed in CHCl3 – CH3OH–CH3COOH–H2O (90:40:12:2) in order to separate PC from LPC and PE from lyso PE. The phosphate content of the spots containing the lyso phospholipids reflected the amount of plasmenylcholine and plasmenylethanolamine present in plasma lipoproteins and platelets. During the whole procedure, lipids were protected against oxidation by addition of 50 mg/l butylated hydroxytoluene to the solvents.

2.7. Molecular species analysis of platelet glycerophospholipids Molecular species analysis of platelet glycerophospholipids was performed as previously described for red blood cell phospholipids [17,18] with some modifications. Washed platelets (from : 50 ml blood) were isolated as detailed above, and lipids extracted [13] using chloroform containing 50 mg/l butylated hydroxytoluene. Platelet PC, PE, PS and PI were isolated by two-dimensional TLC as described above for analysis of platelet and plasma phospholipid fractions. The spots corresponding to the different phospholipids were visualized using diphenylhexatriene spray. The phospholipids were extracted from the silica according to Arvidson [19] and, subsequently, dispersed

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by sonication in 2 ml of the following media. For PC: 50 mM tris(hydroxymethyl)aminomethane, 30 mM boric acid, 5 mM CaCl2 and 8 U of phospholipase C from Chlostridium welchii, pH 7.4. For PE and PS: 30 mM K2HPO4, 30 mM boric acid and 60 – 100 U of phospholipase C from Bacillus cereus (pH 7.0). For PI: 30 mM tris(hydroxymethyl)aminomethane, 30 mM boric acid and 0.5 U of PI-specific phospholipase C (from Bacillus cereus), pH 7.4. All phospholipases were obtained from Sigma (Deisenhofen, Germany). After addition of 4 ml of diethylether and a 12–16 h incubation under argon one-dimensional TLC (diethylether–hexane, 3:2) was used to check for the completeness of the formation of diacylglycerol. Subsequently, 25 mg of 3,5-dinitrobenzoylchloride and 1 ml of dry pyridine were added to the dried neutral lipids. The mixture was heated for 10 min at 65°C and then immersed in an ice-bath for 15 s. 3 ml of ice-cold H2O and 2 ml of hexane were added. Thereafter, 2 ml of 1 M NaCl was added to the hexane phase, the upper phase sucked off and 2 ml of H2O added. The hexane phase was recovered and the H2O phase reextracted twice with hexane. The hexane phases were combined. In order to separate the different diradylglycerol subclasses (diacyl, alkenylacyl and alkylacyl), the samples were applied to HPTLC plates (Merck, Darmstadt, Germany) and developed in hexane – diethylether (7:3). The spots corresponding to diacyl- and alkenylacylglycerol were scraped off and the silica extracted with diethylether. Subsequently, the samples were dissolved in acetonitrile– isopropanol (8:2). The samples were separated into the different molecular species using an ODS Hypersil column (200 mm ×2.1 mm, Hewlett Packard, Bo¨blingen, Germany) coupled to an HPLC pump (Gilson, obtained from Abimed, Langenfeld, Germany). For detection of the absorption of the peaks at 254 nm a UV detector was used (Hewlett Packard 1050). The flow rate was 0.25 ml/min. The different molecular species were identified by gas chromatographic analyses of fatty acid methyl esters and dimethylacetals obtained after hydrolyzing the diradylglycerols of the collected peaks with 14% borontrifluoride in methanol. In addition, derivatization of single molecular species (either obtained from Sigma or synthesized) was used to confirm the identity of the peaks. The individual molecular species thus separated in six different subgroups of platelet glycerophospholipids are shown in Tables 3 – 5.

The calculations were performed using the SigmaStat program. Mean values9 S.D. unless otherwise indicated. PB 0.05 were considered to be significant.

3. Results In six patients with familial hypercholesterolemia, a single treatment with LDL apheresis (by immunoabsorption, Section 2) reduced total plasma cholesterol contents by 56 (post) and 32% (48 h post) (from 7.4790.79 (pre) to 3.259 0.34 (post) (P B0.05 post versus pre) and 5.109 0.89 mmol/l plasma (48 h post) (PB 0.05 versus pre and post, mean values9S.D.)). Total triglyceride levels were lowered by 50% at post and reached preapheresis values at 48 h post. The procedure caused a 66% decrease in plasma LDL cholesterol, the values for this parameter still being lowered by 34% at 48 h post compared to pre (from 5.739 1.21 (pre) to 1.93 9 0.70 (post) (PB0.05 versus pre) and 3.789 1.17 mmol/l plasma (48 h post) (PB

2.8. Statistics The data were analyzed by paired Student’s t-test (comparisons between values obtained before (pre) and after apheresis (post)) or by repeated measures ANOVA (comparisons between values at pre, post and 48 h post) followed by the Student-Newman-Keuls test.

Fig. 1. Sum of percentages of molecular species with arachidonic acid of platelet diacyl phospholipids as determined before (pre, empty columns), directly after (post, columns with vertical lines), and 2 days after LDL apheresis (48 h post, columns with horizontal lines) in six patients with hypercholesterolemia. Numbers given on x-axis refer to different patients. Mean9 S.E.M. * P B0.05; a vs. pre

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Fig. 2. Sum of percentages of molecular species with arachidonic acid of platelet alkenylacyl (plasmalogen) phospholipids as determined before (pre, empty columns), directly after (post, columns with vertical lines) and two days after LDL apheresis (48 h post, columns with horizontal lines) in six patients with hypercholesterolemia. Numbers given on x-axis refer to different patients. Mean 9S.E.M. * P B 0.05; a vs. pre; b vs. post

0.05 versus pre and post, mean values 9S.D.)). Plasma contents of HDL cholesterol were not altered after LDL apheresis (not shown). LDL apheresis induced a 39% reduction in plasma C:P ratio (at post, Table 1). At 48 h post, plasma C:P was still lowered by 27% (compared to pre). The procedure did not alter the C:P ratio of the LDL and HDL particles themselves (data not shown). Similarly, C:P of the platelets was unchanged at post and 48 h post (Table 1). In particular, no change in the contents of total platelet cholesterol was noticed. The percentages of major plasma and platelet glycerophospholipids and of SM, as determined before and after apheresis are

Fig. 3. Content of the TXA2 metabolite 2,3-dinor TXB2 in 24 h urine collected by the patients over the 24 h interval immediately before apheresis (pre, empty columns), over the 24 h period directly after apheresis (24 h post, columns with vertical lines) as well as over the time interval between 24 h and 48 h after the procedure (48 h post, columns with horizontal lines). Numbers given on x-axis refer to different patients. The mean values ( 9S.E.M., given in pg/mg creatinine) of the urinary contents of 2,3-dinor-TXB2 in the six patients were as follows. Pre: 2689 58; 24 h post: 233950; 48 h post: 234 9 56.

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given in Table 2. In plasma, the procedure induced a 18% reduction in the percentage of SM. The percentages of glycerophospholipids containing a choline head group (diacyl and alkenylacyl PC) tended to be elevated at post. Accordingly, the ratio of total PC:SM was raised by 22% immediately after apheresis. In addition, a 40% increase in plasma LPC levels was observed at post. The percentage of platelet diacyl PC was lowered by 7% after apheresis. Concomitantly, the percentage of SM was increased by 8% (Table 2). The ratio of total PC:SM was thus reduced by 14% following the procedure. The percentages of the other platelet phospholipids shown in Table 2 were not altered by apheresis. The analyses of individual molecular species of platelet diacyl phospholipids (PC, PE, PS, PI) and of platelet plasmalogen phospholipids (alkenylacyl PC and PE) as determined before, directly and two days after apheresis are given in Tables 3–5. Table 3 shows data gained from analyses of platelet diacyl PC and PE. A major difference in species composition between the two phospholipids is related to their contents of arachidonic acid (20:4), linoleic (18:2) and oleic acid (18:1). While diacyl PC was found to contain mainly species with 18:2 and 18:1 at pre, in diacyl PE species with 20:4 predominated. Essentially similar results have been obtained in studies on platelets from healthy donors [20]. In diacyl PC, LDL apheresis caused a 24% decrease in the percentage of the arachidonic acid containing species 16:0/20:4 (Table 3). Also the other species with 20:4, namely 18:0/20:4, tended to be reduced at post. In contrast, the level of the species 16:0/22:6 was increased directly after apheresis. The procedure did not induce significant alterations in the molecular species composition of diacyl PE (Table 3). Molecular species analysis of the two anionic phospholipids diacyl PS and diacyl PI also revealed differences in particular in species containing 18:1, 18:2 and 20:4 (Table 4). The percentages of the two species with arachidonic acid (16:0/20:4 and 18:0/20:4) were considerably higher in diacyl PI compared to diacyl PS at pre. Following LDL apheresis the percentage of 18:0/20:4 was lowered by 32 (post) and 28% (48 h post). Concomitantly the levels of the two species with oleic and linoleic acid (16:0/18:1 and 18:0/18:2) were elevated at the two time points (by 37 (post) and 32% (48 h post)). Similarly, the species 18:0/18:1 of diacyl PS tended to be increased after apheresis. A 29% fall in the percentage of 16:0/20:4 in diacyl PI was observed at post (Table 4). Two days later, an even stronger reduction in the level of this species was noticed (by 35% compared to pre). The percentage of 18:0/18:1 of diacyl PI showed a tendency towards an elevation after apheresis. LDL apheresis did not elicit significant changes in the species composition of alkenylacyl PC. The major species of plasmenylcholine, namely 16:0/20:4, tended

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Table 1 Cholesterol:phospholipid ratios of plasma and platelets as well as platelet total cholesterol and phospholipid contents in six patients with hypercholesterolemia before and after LDL apheresis

Plasma Cholesterol:phospholipid Platelets Total cholesterol (mmol/2×108 cells) Total phospholipids (mmol/2×108 cells) Cholesterol:phospholipid

Pre

Post

48 h Post

2.28 9 0.69

1.38 90.35*

1.66 90.55*

0.129 0.03 0.24 90.05 0.51 90.12

0.12 90.03 0.23 90.04 0.52 90.12

0.12 9 0.04 0.25 9 0.08 0.50 9 0.09

Means 9S.D. * PB0.05 (vs. pre).

to be increased after apheresis (Table 5). In contrast, the same species in alkenylacyl PE was reduced by 25 and 19% at post and 48 h post, respectively. Immediately after apheresis an 80% increase in the species 16:0/20:3 of alkenylacyl PE was observed. Two days after apheresis, the sum of 16:0/18:1 and 18:0/18:2 was raised by 52% compared to pre in the same phospholipid fraction (Table 5). The data given in Tables 3 – 5 indicate that the extracorporal reduction of apo B containing lipoproteins by LDL apheresis is in particular accompanied by changes in molecular species of platelet phospholipids containing arachidonic acid. Accordingly, the effect of the procedure on the levels of the sum of species with arachidonic acid in the different glycerophospholipids was evaluated. In Figs. 1 and 2 the values for the Table 2 Levels of individual phospholipids in plasma and platelets of six patients with hypercholesterolemia as determined before and after LDL apheresis Percentages of total phospholipids

Plasma Diacyl PC Alkenylacyl SM Diacyl PE Alkenylacyl Diacyl PI LPC PC:SM Platelets Diacyl PC Alkenylacyl SM Diacyl PE Alkenylacyl Diacyl PS Diacyl PI PC:SM

PC

PE

PC

PE

Mean 9S.D. * PB0.05 (vs. pre).

Pre

Post

66.69 6.20 3.069 0.53 17.99 5.64 2.309 0.47 1.809 0.23 1.879 0.51 4.87 91.38 4.41 91.92

68.59 2.85 3.469 1.07 14.693.50* 2.0290.29 1.7390.44 1.6790.65 6.8490.80* 5.389 1.66*

39.294.42 2.0790.80 18.492.67 13.89 3.37 13.49 2.80 9.369 1.03 3.8090.74 2.309 0.42

36.49 2.49* 2.329 0.66 19.99 2.42* 13.19 3.37 14.49 2.09 10.19 1.53 4.019 0.37 1.9790.28*

arachidonic acid contents are indicated separately for the six different patients. The percentage of species of diacyl PC containing arachidonic acid was reduced by 20 (post) and 11% (48 h post) after apheresis, while the level of the polyunsaturated fatty acid was unaltered by the procedure in diacyl PE (Fig. 1). The arachidonic acid content of diacyl PS was lowered in five of the six patients at post and reduced by 20% at 48 h post. LDL apheresis induced a 23% reduction in the level of PI associated 20:4 directly after apheresis (Fig. 1). In alkenylacyl PC, the percentage of species with 20:4 was unaltered at post (Fig. 2). However, at 48 h post this parameter was increased in all donors as compared to pre and post. In alkenylacyl PE, which carries the major proportion of phospholipid bound arachidonic acid in most of the patients (see below), the amount of arachidonic acid was lowered by 8% after apheresis (Fig. 2). Table 6 summarizes the effect of LDL apheresis on the total amount of phospholipid bound arachidonic acid as determined in platelets of the six patients with hypercholesterolemia. In three of the six patients (donors 2, 3 and 5) alkenylacyl PE contained the major part of total phospholipid bound arachidonic acid while in two further patients (donors 1 and 4) diacyl PE carried most of the phospholipid associated arachidonic acid. In platelets of donor 6, arachidonic acid was predominantly contained in diacyl PC. Overall, LDL apheresis induced a 16% fall in platelet phospholipid bound arachidonic acid. The percentage change in phospholipid bound arachidonic acid was not related to the decrease in plasma total cholesterol or LDL cholesterol levels induced by LDL apheresis at post (not shown). In order to evaluate a possible contribution of heparin to the observed changes in molecular species of platelet phospholipids after apheresis, 50 I.U. of (unfractionated) heparin/kg body weight was given intravenously to two hypercholesterolemic donors (total plasma cholesterol levels of 6.85 and 7.03 mmol/l plasma, respectively). Molecular species of three major arachidonic acid containing phospholipids were ana-

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Table 3 Percentages of molecular species of platelet diacyl PC and diacyl PE in six patients with hypercholesterolemia as determined before and after LDL apheresis Diacyl PC

18:2/20:4 16:0/20:5 16:0/22:6 18:1/20:4 16:0/20:4 18:0/22:6 16:0/18:2 16:0/22:4 18:0/20:4 18:1/18:1 16:0/18:1+18:0/18:2 18:0/22:4 20:0/20:4 18:0/18:1 18:0/16:0

Diacyl PE

Pre

Post

48 h Post

Pre

Post

48 h Post

— 0.34 9 0.10 1.009 0.36 2.1090.40 9.349 1.02 — 13.19 0.72 — 6.489 0.69 2.559 0.48 40.69 2.89 0.839 0.26 0.329 0.08 13.89 0.85 8.679 0.61

— 0.579 0.06 1.739 0.32*a,c 1.679 0.40 7.109 0.83*a,c — 13.49 0.58 — 5.089 0.86 3.049 0.57 41.79 3.18 1.179 0.24 0.459 0.18 12.39 0.61 10.39 1.43

— 0.20 90.09 0.77 9 0.19 1.51 9 0.28 9.08 9 1.12 — 13.1 90.40 — 5.86 90.82 2.46 9 0.42 42.8 92.69 0.76 90.16 0.30 90.03 14.2 90.76 8.87 90.99

0.49 90.16 0.31 90.12 1.75 90.34 4.16 90.55 13.0 90.76 4.24 9 0.86 8.11 91.01 2.59 91.04 40.0 92.85 5.09 9 1.36 13.1 9 2.11 0.55 9 0.08 — 4.11 90.56 —

0.28 90.11 0.37 90.06 1.96 90.48 4.82 90.78 14.7 9 0.60 3.31 9 0.96 7.44 9 1.16 2.62 90.74 37.2 9 3.52 4.07 9 0.80 14.7 92.06 0.97 90.25 — 4.16 91.02 —

0.439 0.23 0.27 90.14 1.73 90.35 4.679 0.66 13.9 9 0.91 4.53 9 0.80 8.44 9 0.92 3.869 1.28 38.3 9 3.41 4.51 9 1.21 12.1 9 2.01 0.60 9 0.11 — 3.48 90.32 —

Mean9S.E.M. * PB0.05; a vs. pre; c vs. 48 h post.

lyzed before and 1 h after heparin application. Table 7 indicates that heparin administration barely affected the sum of species with arachidonic acid of diacyl PC, diacyl PE and alkenylacyl PE. In order to evaluate whether LDL apheresis affected the synthesis of platelet TXA2, the production of TXA2 was estimated by measuring the content of the stable TXA2 metabolite 2,3-dinor-TXB2 in urine samples collected by the patients over a 24 h interval immediately before apheresis, over the 24 h period directly after apheresis as well as over a time interval between 24 h and 48 h after the procedure. Urinary content of 2,3-dinor-TXB2 was lowered in four of the six patients at 24 h post while it remained unchanged in the remaining two patients (Fig. 3). Overall, the amount of urinary 2,3-dinor-TXB2 tended to be reduced after apheresis (Fig. 3). The percentage alteration in urinary 2,3-dinorTXB2 neither correlated with the reduction in total and LDL cholesterol induced by apheresis (not shown) nor with the individual amount of heparin administered to the single patients (r =0.01, N.S.). However, the percentage change in urinary 2,3-dinor-TXB2 at 24 h post as compared to pre was strongly correlated (r=0.89, PB 0.05) to the percentage change in the amount of total platelet phospholipid bound arachidonic acid (Table 6).

4. Discussion The effect of rapid extracorporal removal of LDL particles by LDL apheresis on platelet phospholipid structure was investigated in six patients with het-

erozygous familial hypercholesterolemia. All patients had been subjected to apheresis for prolonged time intervals and were currently treated with the HMGCoA reductase inhibitor simvastatin. The moderately elevated preapheresis values for total and LDL cholesterol in plasma were reduced by about two thirds following the procedure and still lowered two days after apheresis (Section 3). Plasma C:P ratio and the percentages of plasma SM were decreased after apheresis, while the PC:SM ratio and plasma LPC were elevated (Tables 1 and 2). As discussed earlier [15] these alterations can be mostly attributed to the changes in lipoprotein pattern elicited by apheresis, i.e. the relatively higher proportion of HDL as compared to LDL at post. HDL particles indeed exhibit higher ratios of PC:SM and increased percentages of LPC in comparison to LDL [21]. Furthermore, the C:P ratio of HDL is lower than the one of LDL. No alterations of the cholesterol content of the platelets were noticed after apheresis (Table 1). These data could indicate that cholesterol molecules are not rapidly exchanged between plasma lipoproteins and platelets under in vivo conditions. In line with this observation it has been suggested that the cholesterol content of platelets is mainly determined by the rate of cholesterol synthesis in megakaryocytes, the precursors of platelets [22]. Inhibition of cholesterol synthesis by lovastatin has indeed been shown to be able to reduce platelet cholesterol levels [7]. In contrast to the rise in plasma PC:SM induced by apheresis in plasma and red blood cell membranes (this study and [15]), the PC:SM ratio of platelets was reduced after the procedure (Table 2). It is unknown at present whether this effect is

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104

Table 4 Percentages of molecular species of platelet diacyl PS and diacyl PI in six patients with hypercholesterolemia as determined before and after LDL apheresis Diacyl PS

16:0/20:4 16:0/18:2 18:0/20:4 16:0/18:1+18:0/18:2 18:0/18:1

Diacyl PI

Pre

Post

48 h Post

Pre

Post

48 h Post

4.099 1.76 5.429 2.57 37.69 3.87 11.9 9 0.99 40.0 9 6.02

6.639 1.15 3.849 1.28 25.79 4.27* 16.39 0.93* 46.39 3.83

5.86 91.17 2.33 91.16 27.1 93.55* 15.7 91.12* 44.4 95.78

18.3 91.94 — 53.4 9 2.67 27.4 9 4.88 —

13.0 9 2.93* — 46.3 94.13 38.5 95.76 —

11.9 9 2.57* — 51.7 92.56 37.1 9 2.52 —

Means 9S.E.M. * PB0.05 (vs. pre).

a consequence of an altered metabolism of platelet diacyl PC or related to changes in phospholipid exchange between lipoproteins and platelets. Substantial amounts of diacyl PC are rapidly transferred from LDL to platelets under in vitro conditions [6]. The transfer of diacyl PC from LDL to platelets saturates at or slightly above the upper physiological range of plasma LDL concentration (between 1.5 and 2 mg/ml LDL protein (unpublished observation)). We thus favour the view that—as a consequence of the reduction in plasma LDL—fewer PC molecules are transferred from LDL to platelets, thereby inducing a fall in platelet diacyl PC at post. Interestingly, lovastatin treatment has recently been reported to reduce platelet PC:SM [7]. Accordingly, both rapid and prolonged reductions of plasma LDL are associated with lowering of platelet PC:SM. In further experiments it was analyzed whether the rapid removal of LDL particles by LDL apheresis affected the molecular species composition of platelet glycerophospholipids. The procedure elicited significant decreases in the percentages of the species 16:0/20:4 of diacyl PC, diacyl PI and alkenylacyl PE while the level of 18:0/20:4 was reduced in diacyl PS (Tables 3–5). Concomitantly the percentages of species with oleic acid and linoleic acid or other fatty acids were elevated after apheresis. Esterified arachidonic acid was significantly lowered by apheresis in diacyl PC, diacyl PI and alkenylacyl PE (Figs. 1 and 2). Interestingly, the strongest reduction was observed in diacyl PI. It has previously been shown that the platelets of patients with hypercholesterolemia contain increased amounts of PI associated arachidonic acid [8,9]. In addition, experimental hypercholesterolemia was accompanied by a rise in the same parameter [23]. Apheresis elicited reductions of arachidonic acid levels in diacyl PC and alkenylacyl PE but not in diacyl PE and even increased the percentage of arachidonic acid in alkenylacyl PC. The modifications in the former three phospholipids resembled those previously observed following LDL apheresis in red blood cells and plasma lipoproteins [18]. In three of the six patients in

this study, the molecular species composition of red blood cell alkenylacyl PC was analyzed before and after LDL apheresis. The sum of species with arachidonic acid in the plasmalogen subgroup of red blood cells tended to be increased following apheresis (from 44.8 9 3.79 (pre) to 53.4 9 2.57 (post) and 52.1 9 2.91% (48 h post), mean9 S.E.M.). Taken together the pattern of changes induced by the procedure in platelet arachidonic acid (esterified to PC and PE) was thus strikingly similar to the one observed in red blood cells. These findings are in agreement with the view that the effects of apheresis on phospholipid species of platelets (and red blood cells) are related to modifications in exchange of lipids (e.g. phospholipids or free fatty acids) between lipoproteins and the blood cells. Due to the reduction of plasma LDL the transfer of arachidonic acid (either in the esterified or in the free form) from lipoproteins to the blood cells is obviously decreased. This could indicate that LDL particles are responsible for the delivery of arachidonic acid to cells under in vivo conditions in accordance with previous conclusions [24]. It is rather unlikely that the fall in plasma VLDL after apheresis [11,18] contributed to the decrease in platelet arachidonic acid. In parallel experiments it was indeed observed that the levels of VLDL cholesterol and VLDL apo B reached preapheresis values 6 h after the end of the procedure (unpublished observation). In contrast, the decrease in the percentages of arachidonic acid of diacyl PC, diacyl PS and alkenylacyl PE was still evident two days after apheresis (Figs. 1 and 2). For the following reasons it is unlikely that heparin which was present during 2/3 of the time of apheresis contributed to the observed effects: (i) heparin administration per se did not affect phospholipid bound arachidonic acid in diacyl PC and alkenylacyl PE (Table 7) while apheresis clearly reduced the same parameters (Figs. 1 and 2); (ii) the amount of heparin administered to the different patients was not related to the fall in TXA2 observed after apheresis (Section 3); and (iii) previous data indicate that administration of unfrac-

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105

Table 5 Percentages of molecular species of platelet alkenylacyl PC and alkenylacyl PE in six patients with FH as determined before and after LDL apheresis Alkenylacyl PC

16:0/22:6 18:1/20:4 16:0/20:4 16:0/18:2 16:0/20:3 16:0/22:4 18:0/20:4 18:1/18:1 16:0/18:1+18:0/18:2 18:0/22:4 20:0/20:4 18:0/18:1 Means9 S.E.M. * PB0.05; a vs. pre;

b

Alkenylacyl PE

Pre

Post

48 h Post

Pre

Post

48 h Post

2.81 9 0.59 — 41.19 6.86 3.58 9 1.37 — — 16.9 91.68 16.1 93.06 24.0 95.16 — — 3.54 9 1.23

3.4690.34 — 48.29 4.49 3.399 0.93 — — 14.49 1.87 15.79 3.61 19.49 1.57 — — 5.1491.54

2.91 90.40 — 49.8 9 4.63 2.59 90.87 — — 16.7 9 1.63 17.1 9 1.97 18.6 9 2.14 — — 3.36 91.13

1.36 90.22 5.25 90.94 35.5 9 3.81 3.26 9 1.07 4.50 9 1.07 2.11 90.76 32.5 9 4.58 0.76 9 0.19 5.92 90.94 2.03 9 0.22 1.10 9 0.44 1.22 90.41

1.91 90.21 5.22 90.83 26.6 92.32*a 4.63 9 0.36 8.11 9 1.18*a 2.54 9 0.59 29.4 93.92 1.42 90.28 5.38 9 0.84 2.12 90.37 0.83 90.21 0.84 90.18

1.16 90.29 8.1491.23 28.6 92.91*a 4.57 9 1.16 5.4390.84 4.08 91.08 23.6 9 3.09 1.08 9 0.21 8.98 91.67*a,b 2.4390.57 0.69 90.22 0.53 90.21

vs. post.

tionated heparin (as used in the present study) increases urinary 2,3-dinor TXB2 under in vivo conditions [25] while the same parameter was found to be rather decreased after apheresis in this study (see below). It might be argued that changes in platelet activation could account for the modifications of platelet phospholipids induced by apheresis. The reduction of plasma LDL might be expected to mitigate the proposed agonist-like effects of LDL on platelets. This, in turn, is supposed to decrease phospholipase A2 activity. However, under these conditions, phospholipid bound arachidonic acid is unlikely to be reduced. On the other hand activation of platelets during the procedure of LDL apheresis itself cannot be excluded. Enhanced activation of platelets is associated with increased proTable 6 Changes in the total amount of platelet phospholipid bound arachidonic acid as induced by LDL apheresis in six patients with hypercholesterolemia Molecular species of platelet phospholipids with 20:4 (nmol/2× 108 platelets) Patient

Pre

Post

1 2 3 4 5 6 Mean9 S.D.

94.7 68.8 97.4 73.6 62.3 77.9 79.19 14.1

67.2 56.8 93.7 69.7 44.4 66.3 66.49 16.3*

The values represent the sum of arachidonic acid containing molecular species of diacyl subgroups of PC, PE, PS and PI as well as of the alkenylacyl subgroups of PC and PE. The contribution of the alkylacyl subgroups of PC and PE were neglected. * PB0.05 (vs. pre).

duction of TXA2. However, TXA2 production tended to be reduced following apheresis (Fig. 3). Accordingly, modifications in the activation status of platelets are unlikely to contribute significantly to the effects of apheresis on platelet phospholipids. The synthesis of TXA2, an important autocrine and paracrine substance eliciting platelet activation and vasoconstriction, requires liberation of arachidonic acid from platelet phospholipids by phospholipase A2 and subsequent metabolism catalyzed by cyclooxygenase. Stimulation of platelet phospholipase A2 cleaves arachidonic acid from diacyl PC and diacyl PI [1], probably also from phospholipids containing an ethanolamine head group [20,26,27]. The percentages of arachidonic acid in diacyl PC, diacyl PI and alkenylacyl PE are reduced after apheresis (Figs. 1 and 2), in agreement with previous findings observed in red blood cells [18]. Accordingly, it was evaluated whether LDL apheresis affected production of platelet TXA2. The levels of Table 7 Effect of heparin administration on the sum of percentages of arachidonic acid in platelet phospholipid subgroups of two hypercholesterolemic donors (A and B) Sum of percentages of species with arachidonic acid

Diacyl PC Diacyl PE Alkenylacyl-PE

Before heparin

After heparin

A

B

A

B

25.2 63.3 64.7

23.3 65.8 63.0

25.4 64.8 63.7

22.8 65.5 65.0

50 I.U. (unfractionated) heparin were injected as a bolus intravenously. Venous blood was taken for analysis before and 1 h after the injection.

106

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urinary 2,3-dinor TXB2 (an estimate of platelet TXA2 synthesis) tended to be reduced after apheresis (Fig. 3). The magnitude of change of phospholipid bound arachidonic acid following apheresis was positively related to the alterations in urinary 2,3-dinor TXB2 in the different donors (see Section 3). The varying efficacy of apheresis to lower platelet arachidonic acid may be responsible for the differential effects of apheresis on platelet TXA2 production in different donors observed in this as well as in previous investigations [28,29]. The results thus suggest that the decreased availability of the eicosanoid precursor arachidonic acid in distinct platelet phospholipids may contribute to the reduction in TXA2 production observed in some of the patients.

Acknowledgements The excellent technical assistance of Susanne Zieseniss, Ulrike Reinhardt and Monica Laliberte is gratefully acknowledged. This study was supported by grants from the Wilhelm Sander-Stiftung and the Deutsche Forschungsgemeinschaft (En 178/4-1) to BE.

References [1] Nozawa Y, Nakashima S, Nagata K. Phospholipid-mediated signaling in receptor activation of human platelets. Biochim Biophys Acta 1991;1082:219. [2] Surya II, Akkerman JWN. The influence of lipoproteins on blood platelets. Am Heart J 1993;125:272. [3] Spector AA, Hoak JC, Warner ED. Utilization of long-chain free fatty acids by human platelets. J Clin Inv 1970;49:1489. [4] Surya II, Gorter G, Akkerman JWN. Arachidonate transfer between platelets and lipoproteins. Thromb Haemost 1992;6:719. [5] Bereziat G, Chambaz J, Trugnan G, Pepin D, Polonovski J. Turnover of phospholipid linoleic and arachidonic acid in human platelets from plasma lecithins. J Lipid Res 1978;19:495. [6] Engelmann B, Ko¨gl C, Kulschar R, Schaipp B. Transfer of phosphatidylcholine, phosphatidylethanolamine and sphingomyelin from low and high density lipoproteins to human platelets. Biochem J 1996;315:781. [7] Hochgraf E, Levy Y, Aviram M, Brook JG, Cogan U. Lovastatin decreases plasma and platelet cholesterol levels and normalizes elevated platelet fluidity and aggregation in hypercholesterolemic patients. Metabolism 1994;43:11. [8] Mosconi C, Colli S, Tremoli E, Galli C. Phosphatidylinositol (PI) and PI-associated arachidonate are elevated in platelet total membranes from type II hypercholesterolemic subjects. Atherosclerosis 1988;72:129. [9] Chetty N, Naran NH. Platelet hyperreactivity in hyperlipidemia with specific reference to platelet lipids and fatty acid composition. Clin Chim Acta 1992;213:1. [10] Davi G, Averna M, Catalano I et al. Increased thromboxane biosynthesis in type IIa hypercholesterolemia. Circulation 1992;85:1792. [11] Richter WO, Jacob BG, Ritter MM, Su¨hler K, Vierneisel K, Schwandt P. Three-year treatment of familial heterozygous hypercholesterolemia by extracorporeal low-density lipoprotein immunoadsorption with polyclonal apolipoprotein B antibodies. Metabolism 1993;7:888.

[12] Lorenz RL, Uedelhoven WM, Fischer S, Ruetzel A, Weber PC. A critical evaluation of urinary immunoreactive thromboxane: feasibility of its determination as a potential vascular risk indicator. Biochim Biophys Acta 1989;993:259. [13] Bligh EG, Dyer WJ. A rapid method of total lipid extraction and purification. Can J Biochem Physiol 1959;37:912. [14] Engelmann B, Streich S, Scho¨nthier UM, Richter WO, Duhm J. Changes of membrane phospholipid composition of human erythrocytes in hyperlipidemias. I. Increased phosphatidylcholine and reduced sphingomyelin in patients with elevated levels of triacylglycerol-rich lipoproteins. Biochim Biophys Acta 1992;1165:32. [15] Kulschar R, Engelmann B, Bra¨utigam C, Duhm J, Thiery J, Richter WO. Fast transmission of alterations in plasma phosphatidylcholine/sphingomyelin ratio and lyso phosphatidylcholine levels into changes of red blood cell membrane phospholipid composition after low density lipoprotein apheresis. Eur J Clin Invest 1995;25:258. [16] Bra¨utigam C, Engelmann B, Reiss D et al. Plasmalogen phospholipids in plasma lipoproteins of normolipidemic donors and patients with hypercholesterolemia treated by LDL apheresis. Atherosclerosis 1996;119:77. [17] Engelmann B, Duhm J, Scho¨nthier UM, Streich S, Op den Kamp JAF, Roelofsen B. Molecular species of membrane phospholipids containing arachidonic acid and linoleic acid contribute to the interindividual variability of red blood cell Na + −Li + countertransport: in vivo and in vitro evidence. J Membrane Biol 1993;133:99. [18] Engelmann B, Bra¨utigam C, Kulschar R et al. Reversible reduction of phospholipid bound arachidonic acid after low density lipoprotein apheresis. Evidence for rapid incorporation of plasmalogen phosphatidylethanolamine into the red blood cell membrane. Biochim Biophys Acta 1994;1196:154. [19] Arvidson GAE. Structural and metabolic heterogeneity of rat liver glycerophosphatides. Eur J Biochem 1968;4:478. [20] Takamura H, Narita H, Park HJ, Tanaka K-I, Matsuura T, Kito M. Differential hydrolysis of phospholipid molecular species during activation of human platelets with thrombin and collagen. J Biol Chem 1987;262:2262. [21] Skipski VP. In: GJ Nelson (ed). Blood Lipids and Lipoproteins: Quantification, Composition and Metabolism. New York: Wiley, 1975;471 – 583. [22] Schick B, Schick PK. The effect of hypercholesterolemia on guinea pig platelets, erythrocytes and megakaryocytes. Biochim Biophys Acta 1985;833:291. [23] Dalal KB, Ebbe S, Mazoyer E, Carpenter D, Yee T. Biochemical and functional abnormalities in hypercholesterolemic rabbit platelets. Lipids 1990;25:86. [24] Habenicht AJR, Salbach P, Go¨rig M et al. The LDL receptor pathway delivers arachidonic acid for eicosanoid formation in cells stimulated by platelet-derived growth factor. Nature 1990;345:634. [25] Landolfi R, De Candia E, Rocca B et al. Effects of unfractionated and low molecular weight heparin on platelet thromboxane biosynthesis ‘in vivo’. Thromb Haemost 1994;72:942. [26] Purdon AD, Patelunas D, Smith JB. Evidence for the release of arachidonic acid through the selective action of phospholipase A2 in thrombin-stimulated human platelets. Biochim Biophys Acta 1987;920:205. [27] Colard O, Breton M, Pepin D, Chevy F, Bereziat G, Polonovski J. Arachidonate cannot be released directly from diacyl-sn-glycero-3-phosphocholine in thrombin-stimulated platelets. Biochem J 1989;259:333. [28] Brook G, Winterstein G, Aviram M. Platelet function and lipoprotein levels after plasma-exchange in patients with familial hypercholesterolemia. Clin Sci 1983;64:637. [29] Bro¨ijersen A, Eriksson M, Larsson PT, Beck O, Berglund L, Angelin B. Effects of selective LDL-apheresis and pravastatin therapy on platelet function in familial hypercholesterolemia. Eur J Clin Invest 1994;24:488.