Physiologisches Institut der Universität München, D-80336 Munich, Germany
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ABSTRACT |
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After the rapid extracorporal reduction of plasma low-density lipoprotein (LDL) by LDL apheresis, the percentages of arachidonic acid (AA)-containing species of phosphatidylcholine (PC) were lowered in the plasma of patients with hypercholesterolemia. The same PC species with AA were also decreased in the patient's platelets. Thus the supply of phospholipid-bound AA from LDL to the platelets was probably diminished after the apheresis. We therefore analyzed the concentration dependence of the transfer of phospholipid-bound AA from LDL to the platelets under in vitro conditions. The amount of [14C]AA-PC transferred to platelets strongly increased upon elevation of LDL from 0.1 to 1 mg protein/ml, with a less marked elevation being noted at higher LDL concentrations. After stimulation with thrombin (0.5 U/ml), 7.1% ([14C]AA-PC) and 10.6% ([14C]AA-phosphatidylethanolamine) of the 14C transferred from LDL to the platelets were recovered in the eicosanoids [14C]thromboxane B2 (TxB2) plus 12-[14C]hydroxyeicosatetraenoic acid. Experimental increases and reductions of the [14C]AA-PC import were associated with comparable modifications in the [14C]TxB2 production of the platelets. Accordingly, the import of phospholipid-bound [14C]AA is a necessary prerequisite for the formation of 14C-labeled eicosanoids. In summary, the transfer of phospholipids from LDL to the platelets markedly varies within the physiological range of lipoprotein concentrations. LDL contributes to platelet eicosanoid formation by supplying platelets with phospholipid-bound AA.
[3H]arachidonic acid; 125I-labeled apoprotein B; phosphatidylinositol; sphingomyelin; phospholipase A2
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INTRODUCTION |
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THE HIGHLY POLYUNSATURATED arachidonic acid (AA) is well known as a precursor for a great variety of different paracrine and autocrine factors such as prostaglandins and leukotrienes. For the generation of these bioactive AA metabolites, the polyunsaturated fatty acid has to be liberated first from membrane phospholipids by phospholipase A2 (PLA2; see Refs. 10 and 21). This initial step in eicosanoid production is activated by various extracellular mediators. As free AA is the direct substrate for cyclooxygenases, lipoxygenases, and other metabolizing enzymes, the hydrolysis step represents a means whereby extracellular mediators regulate the amount of free AA according to the demand of a particular physiological situation. This has been studied in particular detail in platelets (25). Furthermore, other products generated by PLA2-mediated degradation of membrane phospholipids such as, for example, lysophosphatidylcholine and lysophosphatidic acid, may fulfill proper functions in cellular signaling by interacting with specific cellular receptors (22, 26).
To cover their demand for AA and other highly polyunsaturated fatty acids (as, for example, eicosapentaenoic and docosahexaenoic acid), cells are in part dependent on a supply of these fatty acids from extracellular donors. In principle, cells can acquire the free polyunsaturated fatty acids from extracellular donors such as albumin by specific uptake mechanisms (27). However, within the plasma compartment, the overwhelming majority of AA is esterified to phospholipids, cholesterol, and triglycerides, with only small amounts being present as free fatty acid (18, 29).
We recently observed that, in patients with hypercholesterolemia, the extracorporal removal of plasma low-density lipoprotein (LDL) by therapeutical LDL apheresis lowered the amount of platelet phospholipid-bound AA (5). This was associated with a decrease in urinary 2,3-dinor thromboxane B2 (TxB2), an indicator of platelet thromboxane A2 (TxA2) production. In the present study, we analyzed the effect of LDL apheresis on the molecular species composition of plasma phosphatidylcholine (PC), the quantitatively predominant phospholipid of plasma lipoproteins (28). Interestingly, the procedure elicited reductions in the percentages of PC species containing AA both in the plasma compartment and in the platelets (see RESULTS). These findings lead us to hypothesize that the decrease in plasma LDL elicited by the apheresis could diminish the transfer of PC-bound AA from LDL to the platelets. Thereby, the percentages of AA-containing species of platelet phospholipids might be lowered.
Previous results indeed indicate that phospholipids are rapidly transferred from LDL to the platelets (3, 12). It is unknown whether the phospholipid transfer varies within the physiological and pathophysiological range of LDL concentrations. In principle, the phospholipid-bound AA transferred from LDL to the platelets could be further metabolized by the platelets to eicosanoid messengers. We therefore tested whether LDL-derived AA-containing phospholipids are utilized by platelets for the production of signaling molecules such as TxA2.
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MATERIALS AND METHODS |
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Materials.
1-Palmitoyl-2-[14C]arachidonoyl-sn-glycero-3-phosphorylcholine
(14C 20:4 PC),
1-acyl-2-[14C]arachidonoyl-sn-glycero-3-phosphorylethanolamine
[14C 20:4
phosphatidylethanolamine (PE)], their
[14C]linoleic acid
analogs,
N-[methyl-14C]sphingomyelin
([14C]SM), and
[3H]AA were obtained
from Amersham (Braunschweig, Germany) or from NEN-Du Pont (Homburg).
125I-labeled apoprotein (apo)
B-LDL [8 × 107
counts · min1
(cpm) · 100 µg LDL
protein
1 · ml
1;prepared
by the Bolton-Hunter method using LDL isolated from healthy
donors] was a kind gift of Dr. E. Koller (Vienna, Austria). Iloprost was generously donated by Schering (Berlin, Germany). TxB2, 12-hydroxyeicosatetraenoic
acid (HETE), pancreatic elastase (type IV), phospholipase C from
Chlostridium welchii and
1-antitrypsin were from Sigma
(Deisenhofen, Germany).
Preparation of platelets. Fresh venous blood obtained from either patients with hypercholesterolemia or healthy individuals was anticoagulated with acid-citrate-dextrose (15 mM citric acid, 90 mM trisodium citrate, 16 mM Na2HPO4, and 160 mM glucose, pH 5.0; 1 part of anticoagulant to 6 parts of blood). The mixture was centrifuged for 10 min at 180 g to obtain platelet-rich plasma. Iloprost (100 nM) and apyrase (50 mg/l) were added, and the platelet-rich plasma was centrifuged at 1,000 g for another 10 min. The pellet was resuspended in Tyrode solution without Ca2+ ("Tyrode buffer": 138 mM NaCl, 2.7 mM KCl, 12 mM NaHCO3, 0.4 mM NaH2PO4, 2 mM MgCl2, 5 mM glucose, 100 nM iloprost, and 50 mg/l apyrase; pH 6.2) and washed two times with the same solution. After the last centrifugation, the platelets were resuspended in a modified Tyrode buffer containing Ca2+ ("Tyrode-Ca2+": 138 mM NaCl, 2.7 mM KCl, 12 mM NaHCO3, 0.4 mM NaH2PO4, 2 mM MgCl2, 5 mM glucose, and 5 mM HEPES; pH 7.35). When stimulated with thrombin (0.5 U/ml), the platelets thus isolated showed the expected functional response, e.g., shape change followed by aggregation.
Molecular species analysis of plasma and platelet PC. The experiments evaluated whether the rapid reduction of LDL in vivo affected the fatty acid composition of diacyl-PC in plasma lipoproteins and platelets. Therefore, the molecular species of diacyl-PC were separated by HPLC in six patients with familial heterozygous hypercholesterolemia before and after LDL apheresis (5). The patients [4 men and 2 women, mean age 53 ± 4 yr (mean value ± SE)] had already been treated by LDL apheresis for 5 mo-6.5 yr. All individuals suffered from angiographically verified coronary heart disease. In three of the six patients, LDL apheresis was performed by the heparin-induced extracorporal LDL precipitation system every fortnight (Plasmat-Secura; Braun, Melsungen, Germany), whereas in another three patients the elevated plasma LDL levels were lowered by immunoabsorption every week. The duration of the LDL apheresis was 2-3 h. Details of the procedures have been described elsewhere (11).
Lipids were extracted (4) from plasma and washed platelets (isolated as detailed above), and phospholipids were separated on TLC plates using the solvent CHCl3-CH3OH-NH3-H2O (90:54:5.5:5.5). After visualization with diphenylhexatriene spray, the spot corresponding to PC was scraped from the plate and eluted from the silica. Subsequently, the phospholipids were dispersed by sonication in 2 ml of 50 mM tris(hydroxymethyl)aminomethane, 30 mM boric acid, 5 mM CaCl2, and 8 units of phospholipase C from C. welchii, pH 7.4. After addition of 4 ml of diethyl ether and a 12- to 16-h incubation under argon, one-dimensional TLC (diethyl ether-hexane, 3:2) was employed 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. Ice-cold H2O (3 ml) and hexane (2 ml) were added. Thereafter, 2 ml of 1 M NaCl were given to the hexane phase, the upper phase was sucked off, and 2 ml of H2O were added. The hexane phase was recovered, and the H2O phase was reextracted two times with hexane. The hexane phases were combined. To separate the different diradylglycerol subclasses (diacyl, alkenylacyl, and alkylacyl), the samples were applied to HPTLC plates (Merck, Darmstadt, Germany) and developed in hexane-diethyl ether (7:3). The spots corresponding to diacylglycerol were scraped off, and the silica was extracted with diethyl ether. 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 × 2.1 mm; Hewlett Packard, Böblingen, Germany) coupled to an HPLC pump (Gilson; obtained from Abimed, Langenfeld, Germany). For detection of the absorption of the peaks at 254 nm, an ultraviolet 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% boron trifluoride in methanol. In addition, derivatization of single molecular species (either obtained from Sigma or synthesized) was used to confirm the identity of the peaks.
Incorporation of 14C-labeled lipoproteins into LDL. 14C-labeled phospholipids were incorporated into LDL particles according to a previously published protocol (12). Briefly, [14C]PC, [14C]PE, and [14C]SM were incorporated into egg PC vesicles and, subsequently, incubated with fresh plasma from healthy donors for 24 h at 37°C under argon. [14C]LDL was isolated by ultracentrifugation at 4°C (17). The specific activities thus obtained were 0.6-9.1 × 104 cpm/nmol of respective phospholipid. Ninety-five percent ([14C]PC), 92% ([14C]PE), and 98% ([14C]SM) of the total lipoprotein-associated 14C were present in the phospholipid fraction originally labeled in the lipid vesicles. Before the start of incubations with platelets, the lipoproteins were extensively dialyzed at 4°C under argon against the Tyrode-Ca2+ buffer. 14C-labeled lipoproteins migrated at the same height as the native lipoproteins as determined by agarose gel electrophoresis.
Washed platelets obtained from healthy donors were suspended with either [14C]LDL or 125I-apo B labeled LDL in Tyrode-Ca2+ buffer at 37°C. After incubation with the labeled LDL, platelets were separated from the lipoproteins by centrifugation and washed one time, and the platelet-associated radioactivity was determined in lipid extracts of platelets (4). To estimate platelet import of 14C-labeled phospholipids, a 40-fold excess of unlabeled LDL was added for 30-40 min after incubation of platelets with labeled LDL. Subsequently, platelets were separated from the lipoproteins by centrifugation and washed one time, and the platelet-associated radioactivity was assessed. In some cases, phospholipids were additionally separated by TLC using the solvent CHCl3-CH3OH-CH3COOH-H2O (90:40:12:2; vol/vol/vol/vol).Determination of [14C]AA
metabolites.
The newly incorporated
[14C]AA-labeled
phospholipids could serve as precursors for the formation of platelet
eicosanoids. To separate and quantify
[14C]AA and its
metabolites, the procedure described in Ref. 31 was employed. After the
incubation of platelets (from healthy donors) with lipoproteins
enriched with either
[14C]PE or
[14C]PC and subsequent
stimulation with thrombin, three volumes of diethyl ether-methanol-0.2
M citric acid (30:4:1; vol/vol/vol) that had been precooled to
20°C were added to the suspensions. The mixture was vortexed
and centrifuged, and the organic layer was evaporated and redissolved
in ethyl acetate. The samples and standards were applied to TLC plates
(20 × 20 cm; Merck) that had been pretreated for 60 min at
80°C. Separation was performed by one-dimensional TLC using the
organic phase of ethyl acetate-isooctane-acetic acid-water
(110:50:20:100; vol/vol/vol/vol) as solvent. In this system,
TxB2, HETE, and AA migrated at
retardation factor (Rf) values
of 0.19, 0.64, and 0.75, respectively. The silica gel from the spots
corresponding to these Rf values
was scraped off, and radioactivities were determined in a liquid
scintillation counter.
Analysis of 3H-labeled eicosanoids. To determine the effect of LDL on platelet eicosanoid formation, the platelets were first labeled with [3H]AA, incubated without or with unlabeled LDL, and subsequently challenged with thrombin. Finally, the 3H-labeled eicosanoids were separated and quantified. Washed platelets from healthy donors were incubated for 90 min at 37°C in 1.5 ml of the Tyrode buffer containing, in addition, 0.3 µCi of [3H]AA [dissolved in ethanol; 1% (vol/vol)]. Thereafter, 0.15 ml of Tyrode buffer containing 30 mg bovine serum albumin were added, and the suspension was incubated for a further 5 min at 37°C. Platelets were centrifuged and washed one time with the Tyrode-Ca2+ buffer. After the incubation in the presence of LDL and thrombin, three volumes of diethyl ether-methanol-0.2 M citric acid (30:4:1; vol/vol/vol) were added to the suspensions, and the amounts of [3H]AA and 3H-labeled eicosanoids were determined by one-dimensional TLC as described above for the respective 14C substances.
Separation of 3H-labeled phospholipids. To analyze whether LDL alters the substrate specificity of platelet PLA2, platelets were labeled with [3H]AA, incubated with LDL, and activated with thrombin as described above. Subsequently, the amount of 3H in major platelet phospholipids was determined. Washed platelets from healthy donors were incubated for 90 min at 37°C in 1.5 ml of the Tyrode buffer with 0.3 µCi of [3H]AA [dissolved in ethanol; 1% (vol/vol)]. Tyrode buffer (0.15 ml) containing 30 mg bovine serum albumin was added, and the suspension was incubated for a further 5 min at 37°C. Platelets were centrifuged and washed one time with the Tyrode-Ca2+ buffer. After incubation with thrombin in the Tyrode-Ca2+ buffer, platelet lipids were extracted. The extract was applied to Silica G60 plates (Merck) previously sprayed with K+ oxalate [1% in CH3OH-H2O (2:3)] and activated for 30 min at 80°C (6). The plates were developed in the first dimension with the solvent CHCl3-CH3OH-NH3-H2O (45:37:6:4) and were dried, and in the second dimension, the platelets were treated with CHCl3-CH3OH-CH3COCH3-CH3COOH-H2O (40:15:15:12:8). The spots corresponding to the different phospholipids were scraped off, and the amount of 3H present in the phospholipid fractions was determined.
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RESULTS |
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The effect of LDL apheresis on the molecular species composition of plasma PC was analyzed in six patients with hypercholesterolemia. Directly after the procedure, total and LDL cholesterol contents of the plasma were reduced by 58 and 66%, respectively. The plasma concentrations of apo B were lowered by 58% (see legend to Fig. 1). Two days after the end of the procedure, all three plasma parameters were again increased. Immediately after apheresis, the percentages of the AA containing species 16:0/20:4- and 18:0/20:4-PC were lowered by 17 and 22% (Fig. 1A), respectively. Two days later, the percentages of these species reincreased toward the preapheresis values. The percentages of the PC species 16:0/18:1 + 18:0/18:2 and of 16:0/22:6 were augmented directly after the procedure (by 8 and 22%, respectively). In Fig. 1B, the effect of the procedure on the species composition of platelet PC is shown. After apheresis, the percentages of two species with AA were diminished by 20% (16:0/20:4-PC) and 24% (18:0/20:4-PC). The percentage of the species 16:0/22:6 in platelet PC was increased after the procedure (by 65%).
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To evaluate whether variations of the extracellular LDL contents would affect the magnitude of transfer of [14C]AA-PC to the platelets, different concentrations of LDL (containing [14C]AA-PC) were incubated with washed platelets from healthy donors. Upon augmenting the LDL concentration from 0.025 to 1 mg/ml, the amount of platelet-associated 14C steeply increased (Fig. 2A). Between 1 and 2 mg/ml of LDL, platelet-associated 14C increased to a lesser extent (by 1.2-fold). When platelets were incubated with LDL enriched in [14C]AA-PE and [14C]SM, platelet-associated 14C again showed a rather marked elevation upon augmenting the lipoprotein concentration from 0.1 to 1 mg/ml. After incubation with 2 mg/ml of LDL containing either [14C]AA-PE or [14C]SM, the amount of platelet-associated 14C was 1.3-fold higher compared with the values obtained at 1 mg/ml (Fig. 2A).
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Under the experimental conditions employed, part of the platelet-associated 14C is due to 14C present in lipoproteins bound to the surface of the platelets (12). The greater part of the bound lipoproteins can be removed by an excess of unlabeled lipoproteins (see below). Accordingly, the platelets were treated with unlabeled LDL after incubation with [14C]phospholipid-LDL. The concentration dependence thus obtained for [14C]AA-PC, [14C]AA-PE, and [14C]SM indicated a steep increase between 0.1 and 1 mg/ml of LDL, whereas the elevation was less pronounced between 1 and 1.5 mg/ml (Fig. 2B). When PC and PE were labeled with [14C]linoleic acid at sn2 (instead of [14C]AA), rather similar concentration dependencies were obtained (data not shown).
Platelets (2 × 108) were incubated for 5 min with LDL labeled in its apoprotein component (125I-apo B-LDL) under the same conditions as with LDL labeled in [14C]phospholipids. After incubation of the platelets with 0.3, 0.5, and 1 mg of 125I-apo B-LDL/ml, platelet-associated 125I was determined to amount to 3.9 ± 1.4, 3.3 ± 0.3, and 3.6 ± 1.2 × 105 cpm/platelets. After adding a 40-fold excess of unlabeled LDL for 30 min, the platelet-associated 125I was 1.0 ± 0.4 (0.3 mg/ml), 0.8 ± 0.3 (0.5 mg/ml), and 1.1 ± 0.2 × 105 cpm/platelets (1 mg LDL protein/ml; mean values ± SD on platelets from 4 different donors).
We then analyzed whether the phospholipid-bound [14C]AA transferred to the platelets was a substrate for the platelet enzymatic machinery mediating the formation of eicosanoids. Platelets were incubated for 5 min with LDL enriched in either [14C]AA-PC or [14C]AA-PE and thereafter were activated for 2 min with thrombin (0.5 U/ml). Of the amount of [14C]PC imported into the platelets, 3.4 and 3.7% were found in [14C]HETE and [14C]TxB2, whereas 1.9% was present in free [14C]AA (Fig. 3A). In platelets that had been incubated with [14C]PE-LDL, 5.1, 5.5, and 3.2% of the [14C]PE taken up was found in [14C]HETE, [14C]TxB2, and [14C]AA, respectively (Fig. 3B). In platelets incubated for 5 min with [14C]LDL and subsequently without thrombin, <0.6% of the imported [14C]PC and [14C]PE was recovered in the 14C-labeled eicosanoids. The possibility was considered that secretory PLA2 liberated from the thrombin-stimulated platelets might release [14C]AA from the donor lipoproteins, which, in turn, might be a substrate for 14C-labeled eicosanoid production by the activated platelets. The supernatant from platelets (2 × 108) that had been activated for 2 min with thrombin was isolated and incubated for another 2 min with LDL enriched with [14C]AA-PC. The amount of free [14C]AA determined was not altered by the presence of the supernatant (not shown).
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Next, we evaluated whether increasing the lipoprotein concentration or prolongation of the incubation time would affect platelet [14C]TxB2 production from the newly incorporated [14C]AA-PC (Table 1). The LDL concentration was augmented from 0.3 to 1 mg/ml, thus covering the physiological range of LDL in human plasma. At 1 mg/ml, the amount of [14C]TxB2 formed in thrombin-activated platelets was 3.9-fold higher compared with 0.3 mg/ml LDL. Concomitantly, the import of [14C]AA-PC was increased by 3.1-fold (Table 1). After 30 min of incubation with [14C]PC-LDL (1 mg/ml), the import was enhanced by 1.6-fold compared with the 5-min time interval. At the same time point, the generation of [14C]TxB2 was augmented by 1.7-fold compared with the 5-min time interval. Preincubation of the platelets with the protease elastase decreased the import of [14C]PC by 46%. Under the same conditions, the formation of [14C]TxB2 was lowered by 51% (Table 1).
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The formation of eicosanoids from AA could be altered by the presence of the lipoproteins per se (1, 30) or by pretreatment with elastase. We therefore determined the 3H-labeled eicosanoid formation of [3H]AA-enriched platelets in the presence of unlabeled LDL and elastase. Neither the two different concentrations of LDL nor pretreatment of the platelets with elastase substantially altered the formation of [3H]HETE, [3H]TxB2, or [3H]AA (Table 2). In the absence of thrombin, the amounts of 3H-labeled eicosanoids and [3H]AA generated were also unaffected by the different experimental conditions shown in Table 2 (data not shown).
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The data presented in Fig. 3 indicate that the percentage of 14C-labeled eicosanoids generated from the newly incorporated [14C]PE are somewhat higher than the percentages observed in [14C]PC-enriched platelets. Accordingly, we tested whether the presence of LDL modified the phospholipid hydrolysis pattern responsible for the release of [3H]AA from thrombin-activated platelets. After preincubation of platelets with [3H]AA, three phospholipid classes were predominantly labeled [PC, PE, and phosphatidylinositol (PI)]. Subsequent to activation of the platelets with thrombin, 6.3 ± 1.3 and 2.2 ± 3.1% of [3H]AA were lost from platelet [3H]PC and [3H]PE, respectively. The amount of [3H]AA-PI was reduced by 14.5 ± 5.5%. Because [3H]PC contained most of the label, the greater part of the total decrease in 3H occurred in [3H]PC. When LDL was added during the stimulation period of platelets with thrombin, the percent reduction in [3H]PC, [3H]PE, and [3H]PI amounted to 6.4 ± 2.1, 3.1 ± 2.5, and 10.1 ± 3.2% (mean values ± SD on platelets from 4 different donors), respectively. Thus the presence of LDL did not substantially modify the characteristics of phospholipid hydrolysis mediating release of [3H]AA.
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DISCUSSION |
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We observed in the present study that the extracorporal removal of the greater part of the plasma LDL particles induced a fall in PC species with AA in the plasma compartment of patients with familial hypercholesterolemia (Fig. 1). Similar decreases in PC species containing AA were found after the apheresis in the platelets. Platelet phospholipids are particularly enriched with AA. This high amount of platelet phospholipid-bound AA is required for the production of bioactive eicosanoids such as TxA2, 12-HETE, and others that are generated from PLA2-mediated hydrolysis of membrane phospholipids and subsequent metabolism. Previous data indicate that the reduction in platelet phospholipid-bound AA after apheresis is associated with a tendency toward a lowered platelet TxA2 production (5). Together, these results could be interpreted to indicate that the LDL particles support the eicosanoid production of the platelets by delivering their precursor AA. Previous results on growth factor-activated fibroblasts indeed show that the LDL-associated AA is utilized for cellular eicosanoid production (16). The uptake of the AA occurred through the endocytosis of LDL particles mediated by the classical LDL receptor.
In human platelets, a similar mechanism is rather unlikely to happen. Platelets are not equipped with the classical LDL receptor and do not exhibit relevant endocytosis of LDL (20). Accordingly, other mechanisms are expected to mediate the uptake of AA into the platelets. In principle, LDL might deliver AA to platelets either in its free form or esterified to phospholipids and/or cholesterol. The amount of free AA in the plasma compartment is very low (18, 29). The concomitant reductions in plasma and platelet PC-bound AA observed after LDL apheresis (Fig. 1) could indicate that, under in vivo conditions, the eicosanoid precursor is delivered to the platelets in part by means of phospholipid transfer.
To evaluate this hypothesis, we analyzed whether phospholipid-bound [14C]AA transferred from LDL to platelets was metabolized to 14C-labeled eicosanoids. Platelets were incubated with LDL enriched with [14C]AA-PC and [14C]AA-PE, and subsequently platelet PLA2 was activated by thrombin. Platelets were analyzed for the presence of the cyclooxygenase and lipoxygenase products [14C]TxB2 and [14C]HETE, respectively. The results indicated that both products were indeed generated after incubation of platelets with labeled lipoproteins and subsequent stimulation with thrombin (Fig. 3 and Table 1). When expressed as percentage of the 14C-labeled phospholipid incorporated, somewhat more 14C-labeled eicosanoids were produced after incubation of platelets with [14C]AA-PE and LDL compared with platelets that had been incubated with [14C]AA-PC and LDL (Fig. 3). The presence of LDL per se could not account for these differences, as unlabeled LDL barely modified the thrombin-induced release of [3H]AA from platelet [3H]PC, [3H]PE, and [3H]PI (see RESULTS). Platelet secretory PLA2 has a higher affinity for PE over PC (14, 21). However, according to recent data, this enzyme does not contribute substantially to the release of AA from membrane phospholipids of thrombin-stimulated platelets, the phospholipid hydrolysis being predominantly mediated by cytosolic PLA2 (2, 24). Thus other mechanisms may be responsible for the somewhat higher hydrolysis of [14C]AA-PE. For example, the newly incorporated 14C-labeled phospholipids may be differentially distributed over the two leaflets of platelet plasma membranes, which, in turn, might affect the accessibility of platelet PLA2 to these substrates.
Experimental conditions that decreased (elastase) or increased platelet phospholipid import (higher LDL concentrations, longer incubation intervals) reduced or enhanced [14C]TxB2 generation, respectively (Table 1). These results thus confirmed that the 14C-labeled eicosanoids were predominantly formed from the newly incorporated 14C-labeled phospholipids. In particular, it is unlikely that 14C-labeled eicosanoids were generated from lipoprotein-associated free [14C]AA subsequently transferred to platelets, as the 14C label was nearly exclusively present in the phospholipid fractions of lipoproteins (see METHODS). In addition, control experiments verified that elastase and the different concentrations of lipoproteins did not grossly modify the formation of the 3H-labeled eicosanoids from phospholipid-bound [3H]AA (Table 2).
Platelets were incubated with different concentrations of 14C-labeled phospholipid-LDL, and the quantities of platelet-associated 14C were determined. In some samples, an excess of unlabeled LDL was added subsequently (Fig. 2). Experiments using 125I-LDL indicated that this procedure effectively removed extracellularly bound particles (see RESULTS). The incorporation of 14C-labeled phospholipids markedly increased within the physiological range of LDL concentrations in human plasma (between 0.3 and 1 mg LDL protein/ml). In particular, the enhancement of [14C]AA-PC transfer within this concentration range was accompanied by an elevated [14C]TxB2 formation (Table 1). Thus the concentration of plasma LDL might partially determine the TxA2 formation of the platelets under in vivo conditions by delivering the eicosanoid precursor AA.
High plasma LDL contents were previously shown to be associated with increased percentages of AA in platelet phospholipids (7, 23). This was accompanied by an augmented production of the platelet eicosanoids HETE and TxB2 (13). In patients with barely measurable LDL levels, in contrast, the percentage of platelet phospholipid-bound AA was found to be reduced (8). After rapid reduction of plasma LDL levels by LDL apheresis, the amount of AA esterified to platelet phospholipids was decreased (Ref. 5 and this study). Similar data were obtained after apheresis in red blood cells (11). Experimental hypercholesterolemia in cholesterol-fed rabbits induced an increase in the AA contents of the plasma compartment and in the platelets (19). Concomitantly, the production of platelet TxB2 was enhanced (15, 19). Accordingly, considerable experimental evidence from studies on patients with hypercholesterolemia and on cholesterol-fed rabbits indicates that the pathological elevation of plasma LDL induces an increase in platelet AA contents. The results of the present study suggest that the augmented platelet AA concentrations in patients with hypercholesterolemia could be in part a consequence of an increased uptake of phospholipid-bound AA originating from the LDL particles. The well-documented antithrombotic effects of LDL-lowering interventions (such as drug therapies or LDL apheresis) may thus be partially explained by a reduced uptake of lipoprotein-derived AA by the platelets. This may contribute to lower the production of prothrombotic eicosanoids such as TxB2.
In conclusion, our data demonstrate that phospholipid transfer from LDL supplies platelets with AA, which, in turn, can be further metabolized to bioactive eicosanoids. Thus, by delivering phospholipid-bound AA, lipoproteins support the eicosanoid production of platelets.
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ACKNOWLEDGEMENTS |
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We thank Robert Bräutigam who participated in the initial phase of the experiments.
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FOOTNOTES |
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This study was supported by a grant from the Deutsche Forschungsgemeinschaft to B. Engelmann (En-178/4-2).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests: B. Engelmann, Physiologisches Institut der Universität München, Pettenkoferstr. 12, D-80336 Munich, Germany.
Received 2 April 1998; accepted in final form 23 July 1998.
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