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Arteriosclerosis, Thrombosis, and Vascular Biology. 1995;15:972-981

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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1995;15:972-981.)
© 1995 American Heart Association, Inc.

Articles

Uptake of Radiolabeled and Colloidal Gold–Labeled Chyle Chylomicrons and Chylomicron Remnants by Rat Platelets In Vitro

Ning Xu; Li Zhou; Rolf Odselius; Åke Nilsson

From the Department of Cell Biology 1 (N.X., L.Z., Å.N.), Electron Microscopy Unit (R.O.), and Department of Medicine (Å.N.), University Hospital of Lund, Sweden.

Correspondence to Åke Nilsson, MD, PhD, Department of Internal Medicine, University Hospital of Lund, S-221 85 Lund, Sweden.


*    Abstract
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 *Abstract
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Abstract This study examined the uptake of chyle chylomicrons (CMs) and chylomicron remnants (CMRs) by rat platelets in vitro. CMs and CMRs were doubly labeled with [3H]arachidonate ([3H]-20:4) and [14C]cholesterol and were incubated with platelets for up to 4 hours. A significant uptake (binding and/or internalization) of CMs by the platelets occurred, as indicated by the parallel increase of [3H]20:4 and [14C]cholesterol in platelets with incubation time. Addition of unlabeled CMs, VLDLs, LDLs, and HDLs decreased the uptake of labeled CMs. The competition experiments suggested that there is both a saturable binding and a nonspecific uptake of CMs. During incubation with CMs, the proportion of [3H]20:4 in phospholipids decreased and that in 1,2-x-diacylglycerol increased. The data indicated that a phospholipase C–mediated degradation of phosphatidylcholine and phosphatidylethanolamine occurred, whereas [3H]20:4 in triglyceride and 14C in cholesteryl ester did not change. Electron microscopic studies after incubation with colloidal gold–labeled CMs (CM-Au's) demonstrated an accumulation of CM-Au particles in the open canalicular system of the platelets. Some CM-Au particles were localized in cytoplasmic vacuoles that were not stained by ruthenium red. Some CM-Au's or free gold particles were in vacuoles that showed acid phosphatase activity, indicating that some true endocytosis of CM occurred. The uptake of [3H]-20:4– and [14C]cholesterol-labeled CMRs was low compared with the uptake of CMs. After incubation with colloidal gold–labeled CMRs (CMR-Au's), only a few platelets contained CMR-Au in their open canalicular systems, and no CMR-Au particles were seen in the cytoplasm or in acid phosphatase–positive vacuoles. Rat platelets can thus interact with CMs by a process that leads to a sequestration in the open canalicular system and endocytosis and a net degradation of CM phospholipids. The conversion of CMs to CMRs counteracts this interaction.


Key Words: lipoproteins • metabolism • endocytosis • electron microscopy • cytochemistry


*    Introduction
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 *Introduction
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 down arrowDiscussion
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Platelets can take up or bind the different lipoprotein classes, ie, LDLs, HDLs, and VLDLs, although they do not express the apo BE receptor.1 2 3 4 Binding of LDLs and HDLs was found to be independent of the state of platelet activation and was saturable.4 Electron microscopic examination of the interaction of colloidal gold–labeled HDLs with platelets indicates that HDLs become closely associated with the surface open canalicular system and endocytic vesicles.3 The interaction of platelets with the lipoproteins may influence platelet activity, with LDLs enhancing and apo E–containing HDLs attenuating agonist-stimulated platelet activation.5 6 Chylomicrons (CMs) isolated from normolipidemic individuals after a fatty meal or from type V hyperlipoproteinemia patients inhibit platelet activation.7 8 There are, however, only a few studies on the interaction between platelets and CMs or chylomicron remnants (CMRs),7 8 although a role for the postprandial lipoproteins has been postulated in the development of cardiovascular diseases.9 10 11 Whether any significant uptake of CMRs by platelets occurs in vivo by means of an apo E–mediated binding to LDL receptor–related protein (LRP), which has a key function in the rapid clearance of CMRs by the liver,12 13 or through some other mechanism is not known. If such an uptake occurs it may be a source of arachidonic acid (AA) (20:4, n-6) for the platelets, since AA accumulates in CMR acylglycerols because of the partial resistance of this fatty acid to lipoprotein lipase.14 Recent studies have demonstrated that platelets not only bind and sequester [3H]cholesteryl ester–labeled liposomes in the open canalicular system, but also that liposomes are transferred to acid phosphatase– and esterase-containing vacuoles and degraded.15 This indicates that a true endocytosis of liposomes occurs in platelets.

The present experiments were designed to study in vitro whether rat platelets take up and degrade chyle CMs and CMRs. In a biochemical study, [3H]arachidonic acid ([3H]20:4)– and [14C]cholesterol– doubly labeled CMs ([3H,14C]CMs) and CMRs ([3H,14C]CMRs) were used. In a morphological study we examined by electron microscopy the uptake of colloidal gold–labeled CMs and CMRs by platelets. Cytochemical staining procedures using ruthenium red as a marker for the surface coat of plasma membranes16 and ß-glycerophosphate as a substrate to detect acid phosphatase activity were used to see whether engulfed CMs and CMRs were transferred to lysosomal structures in platelets.


*    Methods
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 up arrowIntroduction
 *Methods
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Materials
[3H]20:4 (100.0 Ci/mmol, [5,6,8,9,11,12,14,15-3H(N)]-20:4, NET-298) and [14C]cholesterol (51.4 mCi/mmol, [4-14C]-cholesterol, NEC-018) were obtained from Du Pont NEN Research Products. Intralipid (20% [wt/vol]) was obtained from Kabi Vitrum AB. Tetrachloroauric acid (HAuCl4), potassium phosphotungstate, agar 100 resin, lead citrate, uranyl acetate, and ruthenium red were purchased from Link Nordiska. Thin-layer chromatography (TLC) plates (silica gel DG) were from Riedel-deHaën. Enzymatic kits for triglycerides (TGs) and total cholesterol determination (Triglycerides GPO-PAP and Cholesterol-PAP) were from Boehringer Mannheim GmbH. Human albumin (fraction V) and other reagents were obtained from Sigma Chemical Co. Male Sprague-Dawley rats (Møllegaard Ltd) weighing 250 to 350 g were housed in mesh stainless steel cages at a constant room temperature (22°C) with a 12-hour light-dark cycle and were provided standard rodent chow (ALTROMIN NR. 324, Altromin Spezialfutter-werke, GmbH) and water ad libitum for 5 days before initiation of the experiments. All the animal work was conducted in compliance with the recommendations of the Guide for the Care and Use of Laboratory Animals and approved by the Lund University Medical Faculty Animal Care Committee.

Preparation of Chyle CMs and CMRs
The mesenteric lymph ducts of rats (weighing 250 to 280 g) were cannulated and a gastric fistula was inserted in each.17 Twenty-four hours after surgery, 2 mL Intralipid was infused through the gastric fistula for 1 hour. The chyle was collected for 6 hours on ice in the presence of Na2EDTA (2 mmol/L). CMs were floated by ultracentrifugation at 25 000 rpm for 2 hours at 4°C using a Beckman SW 40 Ti swing-out rotor after the chyle was adjusted to d=1.063 kg/L and layered under EDTA/saline (d=1.006 kg/L: 188 mmol/L NaCl, 1 mmol/L Na2EDTA). The particles with Svedberg's flotation rate of more than 400 were harvested from the top layer of the tubes and then resuspended in EDTA/saline and filtered through a 0.45-µm Millipore filter. CMRs were prepared by injecting 2 mL of the CMs (approximately 10 mg TG/mL) into eviscerated rats (weighing 250 to 300 g) through the right jugular vein.18 The blood was collected by aortic puncture after the CMs were allowed to circulate for 30 minutes. Acid-citrate-dextrose solution (ACD: 2.2 g trisodium citrate, 0.8 g citric acid, and 2.45 g dextrose in 100 mL distilled water; blood:ACD, 6:1 [vol/vol]) was used as the anticoagulant. After the blood cells were removed by centrifugation at 3000g for 30 minutes, the plasma was adjusted to d=1.063 kg/L, layered under EDTA/saline, and then ultracentrifuged at 32 000 rpm for 18 hours at 4°C using a Beckman SW 40 Ti swing-out rotor. The CM and CMR suspensions were adjusted to 10 mg TG/mL CMs and 0.5 mg TG/mL CMRs respectively, stored at 4°C under nitrogen, and used within 2 weeks. For preparing [3H]20:4– and [14C]cholesterol-doubly labeled CMs and CMRs or [3H]20:4–labeled CMs ([3H]20:4-CM), 50 µCi [3H]20:4 and 50 µCi [14C]cholesterol or 50 µCi [3H]20:4, respectively, were dried under nitrogen and mixed with 2 mL Intralipid. The procedures for the preparation of labeled CMs and CMRs were the same as those described above for unlabeled CMs and CMRs.

Platelet Isolation
Rat blood was drawn into plastic syringes containing ACD solution (blood:ACD, 6:1 [vol/vol]). Platelet-rich plasma was separated and pooled after centrifugation at 200g for 15 minutes at room temperature and was then centrifuged at 150g for 15 minutes to remove residual erythrocytes and leukocytes. Platelet pellets were washed twice with Tyrode's buffer A ([mmol/L] NaH2PO4 0.02, NaCl 136, KCl 2.68, NaHCO3 11.9, and glucose 5.4, pH 6.5) and then resuspended in Tyrode's buffer B (Tyrode's buffer A containing 2.0 mmol/L CaCl2 and 1.0 mmol/L MgCl2, pH 7.35).

Incubation of Rat Platelets With [3H,14C]CMs, [3H,14C]CMRs, or [3H]20:4-CMs
For determining the uptake and degradation of radiolabeled CMs or CMRs by platelets, 0.9 mL of platelet suspension (2.7x109 cells) and 0.1 mL of [3H,14C]CMs (about 1 mg TG) or 0.1 mL of [3H,14C]CMRs (about 50 µg TG), respectively, were incubated for 5, 30, 60, 120, and 240 minutes at 37°C under gentle shaking. After incubation, the platelet pellets and the medium were separated by centrifugation at 3000g for 15 minutes. The platelet pellets were washed twice with cold Tyrode's buffer A. To determine the effects of unlabeled CMs, VLDLs, LDLs, HDLs, and human albumin on the uptake of labeled CMs, 0.5 mL of platelet suspension (1.2x109 cells) was incubated with 20 µL of labeled CMs (about 200 µg TG) and different amounts of unlabeled CMs, VLDLs, LDLs, HDLs, or human albumin. At the end of incubation, the platelet pellets were recovered by centrifugation at 3000g for 15 minutes and washed once with cold Tyrode's buffer A. Aliquots were taken for determination of total radioactivity by use of a Packard 460 CD liquid scintillation system. In another series of experiments, 0.9 mL of platelet suspension (about 2.7x109 cells) was incubated with 0.1 mL of [3H]20:4-CMs (about 1 mg TG) for 5, 10, 30, and 60 minutes. After incubation the platelet pellets were separated and washed twice with cold Tyrode's buffer A. The total lipids in the platelets and in the medium were extracted for determination of the distribution of [3H]20:4 in neutral lipids and phospholipid subclasses in platelets and in medium by TLC.

Preparation of Colloidal Gold
Colloidal gold was prepared by the reduction of HAuCl4 with trisodium citrate.19 In brief, 100 mL 0.01% freshly prepared HAuCl4 solution was heated to boiling and 3.2 mL 1% sodium citrate was poured rapidly into the boiling solution. After 5 minutes of boiling, the solution obtains a brilliant red color, indicating the formation of gold particles. The solution (pH 5.5) contained about 1012 particles per milliliter, with a particle diameter of 19±1.8 nm (mean±SEM). Colloidal gold was viewed on a Formvar-coated grid, without staining, in a JEOL JEM-100 CX electron microscope at 60 kV.

Conjugation of Colloidal Gold to CMs and CMRs
To determine the optimal concentration of CMs or CMRs in relation to the amount of stabilized gold particles, a series of CM and CMR solutions with increasing concentration of lipoproteins were prepared and mixed with 0.5 mL of colloidal gold solution. Five minutes later, 0.1 mL 10% NaCl was added and rapidly mixed. The best concentration of CMs and CMRs for conjugation with colloidal gold was 10% more than the minimum amount of CMs or CMRs that prevented the flocculation of gold particles by NaCl. Conjugates were made by the method of Handley et al20 with slight modifications. In brief, 5 mL colloidal gold solution (pH 5.5) was rapidly mixed with 0.5 mL freshly dialyzed and diluted CMs (2.5 mg TG/mL) or CMRs (64 µg TG/mL) in 50 mmol/L EDTA (pH 5.8). After 20 minutes, the conjugates thus formed were separated by centrifugation at 5000 rpm for 30 minutes at 4°C. The top layer and precipitates were discharged, and the conjugates (supernatants) were collected and then concentrated by a concentrator (Centricon-30, Amicon Inc), stored at 4°C, and used within 48 hours. The conjugates were routinely examined by negative-staining electron microscopy before use. The stability of the CMs and CMRs conjugated to colloidal gold (CM-Au and CMR-Au, respectively) were determined by subjecting the conjugates to pH extremes of 4.0 (adjusted with 100 mmol/L acetic acid) or 9.0 (adjusted with 200 mmol/L Na2CO3) for 24 hours at 4°C and then viewing them by electron microscopy with negative staining.

Negative Staining of CMs, CMRs, CM-Au's, and CMR-Au's
Following the method of Forte and Nordhausen,21 a 5-µL sample of diluted CMs (1.5 to 2 mg TG/mL) or CMRs (10 to 50 µg TG/mL) was applied to the freshly glow-discharged Formvar-coated grids for 15 to 20 seconds. Excess sample was then removed by blotting paper. A droplet of 2% potassium phosphotungstate with 0.2% sucrose (pH 7.3) was immediately added. After 10 minutes, excess stain was removed with blotting paper, and the grid was air dried and viewed in the electron microscope at 60 kV.

Incubation of Platelets With CM-Au's or CMR-Au's
Platelets were incubated in Tyrode's buffer B in the presence of an excess of CM-Au's or CMR-Au's for 1 hour at 4°C. Unbound CM-Au or CMR-Au conjugates were removed by washing with Tyrode's buffer A and resuspended in Tyrode's buffer B. Some samples were subsequently fixed at 4°C with 2.5% glutaraldehyde. Other samples were incubated at 37°C after the incubation at 4°C. After 10, 30, 60, and 90 minutes, samples were obtained and fixed with 2.5% glutaraldehyde in sodium cacodylate buffer (100 mmol/L, pH 7.3) for 30 minutes. Part of the samples were postfixed in 1% osmium tetroxide at 4°C for 1 hour and were then dehydrated in graded ethanol solutions, impregnated in propylene oxide, and embedded in agar 100 resin. Thin (60-nm) sections were stained with lead citrate and uranyl acetate and observed in the electron microscope.

Cytochemistry
Some prefixed samples were stained with ruthenium red following the method of Luft.16 In brief, the samples were postfixed with 1% osmium tetroxide in cacodylate buffer (100 mmol/L, pH 7.3) containing 0.1% ruthenium red for 3 hours at room temperature and were dehydrated and embedded as described above. Thin sections (about 70 nm) without electron staining were used for electron microscopic observations. Acid phosphatase activity was detected following the method of Robinson and Karnovsky22 with the modifications described by Menard et al.23 In brief, the prefixed samples were washed three times in cacodylate buffer and twice in acetate buffer (100 mmol/L, pH 5.0). Cytochemical reactions were carried out in freshly prepared acetate buffer (100 mmol/L, pH 5.0, filtered through a 0.22-µm Millipore filter) with 2 mmol/L ß-glycerophosphate, 2 mmol/L CeCl3, 5% sucrose, and 0.0001% Triton X-100 at 37°C with constant gentle shaking for 90 minutes. The medium was replaced once with freshly prepared medium during incubation. As controls, platelets were incubated in medium without substrate. After incubation, the platelets were washed twice in acetate buffer and then twice in cacodylate buffer containing 5% sucrose. The cells were refixed in 2.5% glutaraldehyde-cacodylate buffer for 1 hour at room temperature and washed overnight at 4°C in the same buffer. Postfixation was made with 1% osmium tetroxide–cacodylate buffer for 1 hour, and the samples were dehydrated and embedded as described above. The thin sections (about 70 nm), either stained with uranyl acetate and lead citrate or unstained, were examined by electron microscopy.

Separation of VLDL, LDL, and HDL
VLDLs (d<1.006 kg/L), LDLs (d=1.006 to 1.063 kg/L), and HDLs (d=1.063 to 1.210 kg/L) were isolated by sequential flotation.24 Stock solution (d=1.35 kg/L, 1354 g/L KBr and 153 g/L NaCl) was used for density adjustment. In brief, 24 mL plasma obtained from fasting rats was layered under EDTA/saline (d=1.006 kg/L: 188 mmol/L NaCl, 1 mmol/L Na2EDTA) and centrifuged at 37 000 rpm for 18 hours at 10°C in a Beckman L5-65 ultracentrifuge with an SW 40 Ti swing-out rotor. The top layer containing VLDLs was collected. LDLs were obtained after adjustment of the infranatant to d=1.063 kg/L by addition of stock solution and centrifugation at 32 000 rpm for 24 hours at 10°C. Preparation of HDLs was carried out after removal of the LDL fraction and adjustment of the density to 1.21 kg/L by addition of stock solution and centrifugation at 38 000 rpm for 48 hours at 10°C. All lipoprotein fractions were extensively dialyzed against saline (150 mmol/L NaCl, 1 mmol/L Na2EDTA, pH 8.6) and stored at 4°C before using.

Separation of Lipid Subclasses
To determine degradation of [3H,14C]CM, [3H]20:4-CM, and [3H,14C]CMR lipids by platelets, total lipids of platelets and medium were extracted according to the method of Bligh and Dyer25 using chloroform:methanol (1:1, vol/vol) containing 0.005% butylated hydroxytoluene. Cholesteryl ester (CE), TG, free fatty acid (FFA), 1,2-x- and 1,3-x-diacylglycerol (1,2-x-DG and 1,3-x-DG), monoacylglycerol (MG), and phospholipids (PLs) were separated by TLC plates that were developed in light petroleum/diethyl ether/acetic acid (80:20:1, vol/vol/vol). Phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, lysophosphatidylcholine, sphingomyelin, phosphatidic acid, and cardiolipin were separated by TLC plates that were developed in chloroform/methanol/acetic acid/water (100:80:12:1.2, vol/vol/vol/vol). The lipid spots were visualized by iodine vapor and scraped into scintillation counting vials. One milliliter of methanol:water (1:1, vol/vol) and 9 mL of toluene:Instagel (1:1, vol/vol) were added and radioactivity was determined in a Packard 460 CD liquid scintillation system, with computerized external standard used for quench correction.

Chemical Analysis
The concentrations of TG and total cholesterol in lipoprotein fractions were determined by the respective enzymatic assay methods using Boehringer test-combination kits according to the manufacturer's protocols. The protein content of lipoproteins was determined by use of the procedure of Lowry et al.26

Statistical Analysis
Values are reported as mean±SEM. One-way ANOVA followed by unpaired Student's t test was used for statistical analysis. A probability value of less than .05 in a two-tailed test was considered significant. All experiments were performed at least in triplicate.


*    Results
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 *Results
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Characterization of CMs, CMRs, CM-Au's, and CMR-Au's
The TG concentrations of prepared CMs and CMRs were, respectively, 7.90 to 11.09 mmol/L and 0.34 to 0.56 mmol/L, and the concentrations of total cholesterol were 1.16 to 1.55 mmol/L and 0.91 to 1.40 mmol/L, indicating that 90% to 96% of the CM TG was hydrolyzed during formation of the CMRs. The TG concentration of CMs was about 20-fold higher than that in CMRs, whereas the amounts of CM and CMR cholesterol added were similar during incubation with platelets. The number of lipoprotein particles were thus similar in the incubations with CMs and CMRs. In [3H]20:4- and [14C]cholesterol-radiolabeled CMs, 72% of [3H]20:4 was in TG, 23% in PL, and the remaining part in CE, DG, MG, and FFA. Seventy-five percent of the [14C]cholesterol was in CE and 23% in free cholesterol (Table 1Down). In the CMRs, 81% of the [3H]20:4 was in TG, 7.3% in 1,2-x-DG, and only 5% in PL. Ninety percent of the [14C]cholesterol was in CE and 9% in free cholesterol (Table 2Down). Negative staining showed that the CM (Fig 1ADown) and CMR (Fig 1BDown) particles appear spherical. CMs have a particle diameter range of 40 to 180 nm, and the range for CMRs is 18 to 80 nm. CM-Au's or CMR-Au's remain as individual particles, with the gold particles bound to the surface of the CMs (Fig 1CDown) or CMRs (Fig 1DDown). Exposure to pH 4 and pH 9 environments did not dissociate the conjugate.


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  		Table 1. Percent Distribution of Radioactivity in Incubations With Chylomicrons


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  		Table 2. Percent Distribution of Radioactivity in Incubations With Chylomicron Remnants



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  		Figure 1. Photograph shows electron microscopic observations of chylomicrons, chylomicron remnants, and their conjugates with colloidal gold. Rat chylomicron particles (A, original magnification x75 000), chylomicron remnant particles (B, original magnification x66 000), colloidal gold–labeled chylomicron particles (C, original magnification x62 500), and colloidal gold–labeled chylomicron remnant particles (D, original magnification x70 950) that were contrasted by negative staining appear spherical.

Uptake of Labeled CMs and CMRs by Rat Platelets In Vitro
When washed rat platelets were incubated with doubly labeled [3H,14C]CMs or [3H,14C]CMRs for up to 240 minutes, the percentage of [3H]20:4 and [14C]cholesterol in platelets increased with time (Fig 2Down). In the CMs there was a parallel uptake of [3H]20:4 and [14C]cholesterol by the platelets. At all time intervals the percent uptake of both [3H]20:4 and [14C]cholesterol during incubations with CMs was much higher than during incubation with CMRs. Increasing the mass of unlabeled CMs significantly decreased the percent uptake of [3H,14C]CMs (Fig 3Down), indicating that the CMs were taken up by a saturable process. The specificity of the interaction between CMs and platelets was studied by adding increasing amounts of other lipoprotein classes. The uptake of labeled CMs by the platelets was inhibited by VLDL, LDL, and HDL, whereas human serum albumin did not affect the uptake. Addition of VLDL and LDL (100 µg/mL) reduced the uptake of CMs by 50%, whereas HDL (about 100 µg/mL) reduced the uptake by 65% (Fig 4Down).



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  		Figure 2. Graph shows time course for the uptake of [3H]- and [14C]-labeled chylomicrons and [3H]- and [14C]-labeled chylomicron remnants by rat platelets in vitro. The figure shows the percentage uptake of [3H]arachidonic acid ({bullet}) and [14C]cholesterol ({circ}) accumulated into platelets, which were incubated with chylomicrons () or chylomicron remnants (----) for up to 240 minutes. A 0.9-mL aliquot of platelet suspension (2.7x109 cells) was incubated with 0.1 mL [3H]- and [14C]-labeled chylomicrons (about 1 mg triglyceride) or 0.1 mL of [3H]- and [14C]-labeled chylomicron remnants (about 50 µg triglyceride) at 37°C. Data are presented as the percentage uptake of added chylomicrons or chylomicron remnants per 109 cells. Values are mean±SEM (n=6).



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  		Figure 3. Graph shows effect of unlabeled chylomicrons on the uptake of labeled chylomicrons in platelets. The figure shows the effect of unlabeled chylomicrons on the uptake of [3H]arachidonate-labeled chylomicrons ([3H]20:4-CMs) by washed platelets. A 0.5-mL aliquot of platelet suspension (1.2x109 cells) was incubated with 20 µL [3H]20:4-CMs (200 µg triglyceride) with addition of different amounts of unlabeled chylomicrons for 120 minutes. Data are presented as the percentage of uptake compared with controls (100%) containing only 20 µL [3H]20:4-CMs. Values are mean±SEM (n=4).



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  		Figure 4. Graph shows effects of VLDLs, LDLs, and HDLs on platelet uptake of [3H]arachidonate-labeled chylomicrons ([3H]20:4-CMs). A 0.5-mL aliquot of washed platelets (1.2x109 cells) was incubated with 20 µL [3H]20:4-CMs (200 µg triglyceride) with addition of different amounts of VLDLs, LDLs, and HDLs at 37°C for 120 minutes. Data are presented as the percentage of uptake compared with controls (100%) containing only 20 µL [3H]20:4-CMs. Values are mean±SEM (n=4).

Degradation of 3H- and 14C-Labeled CM and CMR Lipids During Incubation With Platelets
The percent distribution of [3H]20:4 and [14C]cholesterol in added CMs and in platelets and medium after 120 minutes of incubation is shown in Table 1Up. In both platelets and medium more of the [3H]20:4 was in 1,2-x-DG and 1,3-x-DG and in MG and less in PL than in the added CMs. The percentage of [3H]20:4 in platelets was higher in TG and FFA and lower in CE and PL than in the medium. The percentage of [3H]20:4 in TG of medium plus platelets did, however, not differ from that of the added CMs. The data thus indicated that there was a net hydrolysis of PL, with formation of partial acylglycerols during incubation, whereas a degradation of [3H]20:4-TG could not be demonstrated. More of the [14C]cholesterol in platelets was in unesterified cholesterol and less in CE compared with the medium. However, the percentage of [14C]cholesterol in CE in medium exceeded that in the added CMs, indicating that a transfer of unesterified [14C]cholesterol from the CMs to the platelets had occurred, and the total [14C]CE radioactivity did not decrease during the incubation. In the [3H,14C]CMRs, more of the [3H]20:4 was in 1,2-x-DG and less in PL in the added lipoproteins than in the incubation of the platelets with [3H,14C]CMs. After incubation of the platelets with [3H,14C]CMRs (Table 2Up), the percentages of [3H]20:4 in FFA, 1,2-x-DG, MG, and PL were higher and the percentage in TG was lower in platelets than in the medium. There was, however, little or no change in the distribution of total [3H]20:4 (platelets plus medium) during incubation, indicating that some transfer of [3H]20:4-PL and [3H]20:4-DG from CMRs to platelets, rather than any significant net degradation of PL or TG, occurred. When the distributions of [3H]20:4 in the medium and in the added CMRs were compared, more [3H]20:4 was seen to be in 1,3-x-DG and less in 1,2-x-DG in the medium than in CMRs, probably because of an isomerization of 1,2-x-DG during the incubation. More of the [14C]cholesterol was in unesterified cholesterol and less was in CE in the platelets than in the medium. The ratio of [14C]CE to unesterified [14C]cholesterol was higher in medium than in platelets. However, the distribution of [14C]cholesterol between free cholesterol and CE in cells plus medium remained unchanged. There was thus no evidence for any net hydrolysis of [14C]CE during the incubations. In another series of experiments we determined the degradation of [3H]20:4-CMs in platelets that were incubated for 5, 10, 30, and 60 minutes (Tables 3 through 6DownDownDownDown). Table 3Down shows the percentage distribution of [3H]20:4 in platelets that were incubated with [3H]20:4-CMs for different times; the [3H]20:4 contents in CE and DG were increased with time, and there was a slight decrease of [3H]20:4 in TG. There was no significant change in neutral lipids in the medium at different time intervals, except that the [3H]20:4 in PL was decreased with incubation time (Table 4Down). The [3H]20:4 in platelet PL subclasses and that remaining in the medium PL subclasses was different after different incubation times. As shown in Table 5Down, there were increases of [3H]20:4 in platelet phosphatidylethanolamine, phosphatidic acid, and cardiolipin and a decrease of [3H]20:4 in phosphatidylinositol and phosphatidylserine. The [3H]20:4 contents in medium PL subclasses were also changed after incubation (Table 6Down); there was an obvious decrease of [3H]20:4 in phosphatidylethanolamine and there were some increases in phosphatidylserine, lysophosphatidylcholine, sphyngomyelin, phosphatidic acid, and cardiolipin. The possibility that metabolites of [3H]20:4, eg eicosanoids, may migrate as phosphatidic acid and cardiolipin was not excluded. The 3H in these PL fractions may thus have been overestimated.


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  		Table 3. Percent Distribution of [3H]Arachidonic Acid in Platelet Lipid Classes


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  		Table 4. Percent Distribution of [3H]Arachidonic Acid of the Medium Between Lipid Classes


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  		Table 5. Percent Distribution of [3H]Arachidonic Acid Between Platelet Phospholipid Subclasses


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  		Table 6. Percent Distribution of Phospholipid [3H]Arachidonic Acid of the Medium Between Phospholipid Subclasses

Incubation of Platelets With CM-Au's or CMR-Au's
After incubation of platelets with CM-Au's at 4°C for 1 hour, the morphology of platelets was similar to that of control platelets. Round and homogeneous CM particles of varying sizes, labeled by colloidal gold, were found to adhere to the surface of some of the platelets, but minimal internalization of CM-Au's was observed. When the platelets were incubated at 37°C for 10 to 60 minutes, more CM-Au particles were bound to platelets (Fig 5Down), and many CM-Au particles were observed in the open canalicular system of the platelets. This accumulation was accompanied by a slight tendency towards irregularity in cell shape and dilation of the open canalicular system (Fig 6Down). Some CM-Au particles were also observed in the cytoplasm of platelets (Fig 7Down). In 14.5% to 22.6% of the total number of platelets, the open canalicular system was filled with CM-Au particles after 30 minutes (Table 7Down). In electron microscopic cytochemistry, both the membranes of whole platelets and the open canalicular system were stained as layers of electron-dense deposits after treatment with ruthenium red. The ruthenium red–stained surface coat appeared granular, and the open canalicular system was stained in a way similar to that of the external membranes. Many CM-Au particles were found in the open canalicular system (Fig 8ADown and Table 7Down), and some were found in cytoplasmic vesicles that were not stained with ruthenium red (Fig 8BDown and Table 7Down). After 30 minutes, some CM-Au particles or free gold particles were found in the vacuoles that showed acid phosphatase activity (Fig 9Down and Table 7Down). After platelets had been incubated with CMR-Au's at 4°C and then for another 10 minutes at 37°C, CMR-Au particles were only occasionally found in platelets. After 30 minutes, some platelets were very active and appeared irregular in shape with long pseudopodia. Some CMR-Au particles were bound to the surface of the cell and pseudopodia (Fig 10ADown). A few platelets had taken up CMR-Au particles, which were found entirely in the open canalicular system (Fig 10BDown). As shown in Table 7Down, 28% to 35% of examined platelet profiles contained CM-Au particles after incubation with CM-Au's. Of the total amount of CM-Au particles in platelets, 71% were in the open canalicular system, 11% in the cytoplasm, and 18% on the platelet surface (Table 8Down). Only 8% to 9% of the examined profiles contained CMR-Au particles after incubation with CMR-Au's. No profiles contained CMR-Au particles or free gold particles in the cytoplasm or in acid phosphatase–positive vacuoles (Table 7Down).



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  		Figure 5. Photograph shows electron microscopic observations of colloidal gold–labeled chylomicron (CM-Au) particles bound on platelet surface. Platelets were incubated with CM-Au particles for 30 minutes at 4°C and warmed to 37°C for 10 minutes (A, original magnification x31 540) and 30 minutes (B, original magnification x29 000). Some CM-Au particles were found bound on the platelet surface (indicated by arrows).



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  		Figure 6. Photograph shows electron microscopic observation of colloidal gold–labeled chylomicron (CM-Au) particles in the open canalicular system (OCS). Platelets were incubated with CM-Au particles at 37°C for 30 minutes. The shapes of platelets are slightly irregular and the CM-Au particles are seen in dilated cisternae of the OCS (shown by arrows). Some of them appear to be bound on the surface of the OCS (shown by the arrow head). (Original magnification x34 000).



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  		Figure 7. Photograph shows electron microscopic observation of colloidal gold–labeled chylomicron (CM-Au) particles in platelet cytoplasm. The platelet shown, which was incubated with CM-Au particles at 37°C for 30 minutes, contains two CM-Au particles in the cytoplasmic vacuoles (shown by arrows). (Original magnification x85 000).


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  		Table 7. Morphometric Distribution of Platelets Containing Colloidal Gold–Labeled Chylomicron or Colloidal Gold–Labeled Chylomicron Remnant Conjugates



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  		Figure 8. Photograph shows electron microscopic observations of ruthenium red staining of platelets. Platelets were incubated with colloidal gold–labeled chylomicron particles at 37°C for 30 minutes. The plasma membrane and the membrane of the open canalicular system were positively stained. Some colloidal gold–labeled chylomicron particles were seen in the open canalicular system (shown by arrow [A]; original magnification x28 000), and some were seen in a cytoplasmic vesicle (shown by arrowhead [B]; original magnification x39 000).



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  		Figure 9. Photograph shows electron microscopic observations of acid phosphatase cytochemistry staining of platelets. Platelets were incubated with colloidal gold–labeled chylomicron particles at 37°C for 30 minutes (A [original magnification x58 500] and B [original magnification x85 000]) and 90 minutes (C [original magnification x108 900]). A colloidal gold–labeled chylomicron–containing vesicle with acid phosphatase activity appears in the platelet (A). Free gold particle–containing vacuoles with acid phosphatase activity are found in platelets (B and C).



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  		Figure 10. Photograph shows electron microscopic observations of platelets incubated with colloidal gold–labeled chylomicron remnant (CMR-Au) particles at 37°C for 30 minutes. The shape of platelets appears irregular, and some CMR-Au particles are associated with platelet surfaces (A [original magnification x14 500]). Few platelets were found to take up CMR-Au particles, and those were only in the open canalicular system (B [original magnification x14 500]).


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  		Table 8. Morphometric Distribution of Colloidal Gold–Labeled Chylomicron Particles in Platelets


*    Discussion
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
 *Discussion
down arrowReferences
 
The present study shows that rat platelets take up (bind and/or internalize) CMs more efficiently than CMRs in vitro. The conversion of chyle CMs to CMRs thus counteracts rather than favors the interaction with platelets. This rapid hepatic uptake of CMRs is probably mediated by the LRP,27 a multifunctional receptor that binds apo E, CMRs, ß-VLDL, protease-antiprotease complexes, and lipoprotein lipase,12 28 29 30 31 and by the LDL receptor, which recognizes apo E with high affinity.32 33 Earlier studies have shown that platelets do express high-affinity binding sites for LDL, but these appear to be different from the apo BE receptor.2 34 LRP has so far not been demonstrated in platelets. The low uptake of CMRs and the lack of a net degradation of CMR [14C]CE and [3H]20:4-TG during the incubation observed in this study support the idea that no significant apo BE– or LRP-mediated endocytosis of CMRs occurs in platelets. Furthermore, in vivo experiments in normal rats with radioactive CMs have failed to show any significant uptake of CMRs by platelets (N.X., MD, and Å.N., MD, PhD, unpublished data, 1994).

Unlabeled CMs, VLDLs, LDLs, and HDLs competed with labeled CMs for uptake by platelets (Figs 3Up and 4Up). At a concentration of 100 µg protein of either VLDL, LDL, or HDL, 40% to 50% inhibition was seen. Only a slight increase in inhibition was seen at higher concentrations (Figs 3Up and 4Up). Koller et al4 demonstrated a rapid binding of HDL3s to platelets and found the process to be independent of divalent cations and insensitive to temperature. VLDLs also bind to the platelet membranes, the binding being nonspecific and nonsaturable.4 Furthermore, platelets may take up lipid emulsions and phosphatidylcholine liposomes15 35 as well as a wide variety of foreign particulate material, such as latex spherules,36 37 viruses, and bacteria,38 39 40 that may be either sequestered in the open canalicular system or endocytosed and subjected to lysosomal degradation. In our experiments, Intralipid inhibited the uptake of CMs, but high concentrations were required (data not shown). Our data thus indicate that the uptake of CMs occurs both by saturable binding sites, which may be common for several lipoprotein classes, and by nonspecific mechanisms for particulate uptake.

During incubation of CMs labeled with [3H]20:4, there was a decrease in the 3H of total PLs, phosphatidylcholine, and phosphatidylethanolamine and an increase in the percentage of [3H]20:4 in DG (Tables 1Up, 5Up, and 6Up). CM PLs may thus be exposed to platelet phospholipase C,41 which has been found both in the platelet cytosol and plasma membranes.42 43 In the CMRs the percentages of PL and DG radioactivity in platelets were higher than in the medium, but it was not possible to conclude whether a hydrolysis and a reesterification of [3H]20:4 or only a transfer of radioactive PL and DG from CMRs to platelets occurred (Table 2Up). There was no net hydrolysis of [14C]CE or [3H]20:4-TG (Tables 1Up and 2Up).

The origin of the platelet AA pools necessary for eicosanoid formation44 45 46 is not known. Because platelets are unable to synthesize AA from the precursor, linoleic acid (18:2, n-6),47 the incorporation of AA into platelet PLs must be either an integral part of platelet formation in bone marrow cells, ie, megakaryocytes, which contain high levels of AA and actively incorporate exogenous AA into PLs in vitro,48 49 or the result of transfer from plasma lipoproteins and/or FFA in blood circulation. Our study shows that in vitro platelets may acquire AA from CM PLs. Moreover, 1,2-DxG, a compound that may activate protein kinase C, is formed. The interesting possibility that this process may influence the activity state of platelets after a high-fat meal needs further investigation. The possibility remains, however, that it is an in vitro phenomenon that does not occur in vivo because of the rapid hydrolysis of CM TG and the rapid transfer of CM PLs to HDLs during CM metabolism.

The morphological study demonstrated that CMs were bound to platelets and sequestered in the open canalicular system, but some were internalized (Figs 7 through 9UpUpUp). The use of the electron-dense tracer ruthenium red, which can detect the membrane of the open canalicular system,16 shows that some CM particles appeared in the cytoplasm without being surrounded by a ruthenium red–positive membrane (Fig 8BUp). Some CM-Au particles and free colloidal gold particles were found in connection to structures that exhibited a positive acid phosphatase reaction, suggesting that there was a transfer of CM material to lysosomal structures (Fig 9Up). Most of the CM-Au's were, however, in the open canalicular system, only 11% being in the cytoplasm (Table 8Up). When platelets were incubated with liposomes containing labeled CE,15 both a sequestration in the open canalicular system and a significant lysosomal degradation of CE occurred. The absence of the net degradation of TG and CE and the small proportion of CM-Au's found in acid phosphate–positive structures in this study indicate that the lysosomal degradation of CMs is a slow process. In the incubations with CMRs, only a few platelets contained CMR-Au particles in the open canalicular system, and no evidence for internalization of particles was seen. The visual evidence thus supported the biochemical observations and demonstrated that platelets interact with CMRs less effectively than with CMs. No evidence for an endocytic catabolism of CMRs mediated by lipoprotein receptors or other ligands was thus observed.

In summary, the study thus shows that platelets are able to interact with chyle CMs by a mechanism that leads to a significant sequestration of CMs in the open canalicular system, to some internalization of CMs, and to a phospholipase C–mediated degradation of CM PLs. Although the rapid conversion of CMs to CMRs by lipoprotein lipase may normally counteract this interaction, older studies showed that lipid particles appeared in platelets after infusion of Intralipid or after a high-fat meal,35 50 suggesting that some uptake of TG-rich lipoproteins and artificial lipid emulsion particles may actually occur also in vivo.


*    Acknowledgments
 
This study was supported by grants from the Swedish Medical Research Council (Nr-3969), the Medical Faculty, University of Lund, The Albert Påhlsson Foundation, the Nutrition Foundation of the Swedish Margarine Industry, the Crafoord Foundation, and the Anna and Sven-Erik Lundgren Foundation.

Received November 30, 1994; accepted March 29, 1995.


*    References
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
 up arrowDiscussion
 *References
 

  1. Brook JG, Aviram M. Platelet lipoprotein interactions. Semin Thromb Hemost. 1988;14:258-265. [Medline] [Order article via Infotrieve]
  2. Aviram M, Brook JG, Lees AM, Lees RS. Low-density lipoprotein binding to human platelet: role of charge and specific amino acids. Biochem Biophys Res Commun. 1981;99:308-318. [Medline] [Order article via Infotrieve]
  3. Martin-Nizard F, Richard B, Torpier G, Nouvelot A, Fruchart JC, Duthilleul P, Delbart C. Analysis of phospholipid transfer during HDL binding to platelets using a fluorescent analog of phosphatidylcholine. Thromb Res. 1987;46:811-825. [Medline] [Order article via Infotrieve]
  4. Koller E, Koller F, Doleschel W. Specific binding sites on human platelets for plasma lipoprotein. Z Physiol Chem. 1982;363:395-405.
  5. Bruckdorfer KR. The effects of plasma lipoproteins on platelet responsiveness and on platelet and vascular prostanoid synthesis. Prostaglandins Leukot Essent Fatty Acids. 1989;38:247-254. [Medline] [Order article via Infotrieve]
  6. Desai K, Bruckdorfer KR, Hutton RA, Owen JS. Binding of apo E rich high density lipoprotein particles by saturable sites on human blood platelets inhibits agonist-induced platelet aggregation. J Lipid Res. 1989;30:813-840.
  7. Aviram M, Furman B, Brook JG. Chylomicrons from patients with type V hyperlipoproteinemia inhibit platelet function. Atherosclerosis. 1985;56:157-167. [Medline] [Order article via Infotrieve]
  8. Nordøy A, Lagard M, Renaud S. Platelets during alimentary hyperlipidemia induced by cream and cod liver oil. Eur J Clin Invest. 1984;14:339-345. [Medline] [Order article via Infotrieve]
  9. Lippel K, Tyroler H, Eder H, Gotto A, Vahouny G. Relationship of hypertriglyceridemia to atherosclerosis. Atherosclerosis. 1981;1:406-417.
  10. Zilversmit DB. Atherogenesis: a postprandial phenomenon. Circulation. 1979;60:473-485. [Abstract/Free Full Text]
  11. Simpson HS, Williamson CM, Olivecrona T, Pringle S, Maclean J, Lovimer AR, Bonnefous F, Bogaievsky Y, Packard CJ, Shepherd J. Postprandial lipemia, fenofibrate and coronary artery disease. Atherosclerosis. 1990;85:193-202. [Medline] [Order article via Infotrieve]
  12. Beisiegel U, Weber W, Ihrke G, Herz J, Stanley KK. The LDL-receptor-related protein, LRP, is an apolipoprotein E-binding protein. Nature. 1989;341:162-164. [Medline] [Order article via Infotrieve]
  13. Beisiegel U, Weber W, Bengtsson-Olivecrona G. Lipoprotein lipase enhances the binding of chylomicrons to low density lipoprotein receptor-related protein. Proc Natl Acad Sci U S A. 1991;88:8342-8346. [Abstract/Free Full Text]
  14. Nilsson Å, Landin B, Schotz MC. Hydrolysis of chylomicron arachidonate and linoleate ester bonds by lipoprotein lipase and hepatic lipase. J Lipid Res. 1987;28:510-517. [Abstract]
  15. Male R, Vannier WE, Baldeschwieler JD. Phagocytosis of liposomes by human platelets. Proc Natl Acad Sci U S A. 1992;89:9191-9195. [Abstract/Free Full Text]
  16. Luft JH. Ruthenium red and violet, II: fine structural localization in animal tissues. Anat Res. 1971;171:369-416. [Medline] [Order article via Infotrieve]
  17. Warshaw AL. A simplified method of cannulating the intestinal lymphatic of the rat. Gut. 1972;13:66-67. [Free Full Text]
  18. Sherrill BC, Dietschy JM. Characterization of the sinusoidal transport process responsible for uptake of chylomicrons by the liver. J Biol Chem. 1978;253:1859-1867. [Free Full Text]
  19. Frens G. Controlled nucleation for the regulation of the particle size in monodisperse gold suspensions. Nat Phys Sci. 1973;241:20-22.
  20. Handley DA, Arbeeny CM, Witte LD, Chien S. Colloidal gold-low density lipoprotein conjugates as membrane receptor probes. Proc Natl Acad Sci U S A. 1981;78:368-371. [Abstract/Free Full Text]
  21. Forte TM, Nordhausen RW. Electron microscopy of negatively stained lipoprotein methods in enzymology. Methods Enzymol. 1986;128:442-457. [Medline] [Order article via Infotrieve]
  22. Robinson JM, Karnovsky MJ. Ultrastructural localization of several phosphatases with cerium. J Histochem Cytochem. 1983;31:1197-1208. [Abstract]
  23. Menard M, Meyers KM, Prieur DF. Demonstration of secondary lysosomes in bovine megakaryocytes and platelets using acid phosphatase cytochemistry with cerium as a trapping agent. Thromb Haemost. 1990;63:127-132. [Medline] [Order article via Infotrieve]
  24. Havel RJ, Eder HS, Bragdon JH. The distribution and chemical composition of ultracentrifugally separated lipoprotein in human serum. J Clin Invest. 1955;34:1345-1353.
  25. Bligh EG, Dyer WJ. A rapid method for total lipid extraction and purification. Can J Biochem Physiol. 1959;37:911-918.
  26. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951;193:265-275. [Free Full Text]
  27. Herz J, Hamann U, Rogne S, Myklebost O, Gausepohl H, Stanley K. Surface location and high affinity for a 500-kd liver membrane protein closely related to the LDL-receptor suggest a physiological role as lipoprotein receptor. EMBO J. 1988;7:4119-4127. [Medline] [Order article via Infotrieve]
  28. Brown MS, Herz J, Kowal RC, Goodstein JL. The low-density lipoprotein receptor-related protein: double agent or decoy? Curr Opin Lipidol. 1991;2:65-72.
  29. Kowal RC, Herz J, Goldstein JL, Esser V, Brown MS. Low density lipoprotein receptor-related protein mediates uptake of cholesteryl esters derived from apoprotein E-enriched lipoproteins. Proc Natl Acad Sci U S A. 1989;86:5810-5814. [Abstract/Free Full Text]
  30. Kowal RC, Herz J, Weisgraber KH, Mahley RW, Brown MS, Goldstein JL. Opposing effects of apolipoproteins E and C on lipoprotein binding to low density lipoprotein receptor-related protein. J Biol Chem. 1990;265:10771-10779. [Abstract/Free Full Text]
  31. Nykjær A, Bengtsson-Olivecrona G, Lookene A, Moestrup SK, Petersen CM, Weber W, Beisiegel U, Gliemann J. The {alpha}2-macroglobulin receptor/low density lipoprotein receptor-related protein binds lipoprotein lipase and ß-migrating very low density lipoprotein associated with the lipase. J Biol Chem. 1993;268:15048-15055. [Abstract/Free Full Text]
  32. Windler EET, Greeve J, Daerr WH, Greten H. Binding of rat chylomicrons and their remnants to the hepatic low-density-lipoprotein receptor and its role in remnant removal. Biochem J. 1988;252:553-561. [Medline] [Order article via Infotrieve]
  33. Windler E, Chao Y-S, Havel RJ. Regulation of the hepatic uptake of triglyceride-rich lipoproteins in rat. J Biol Chem. 1980;255:8303-8307. [Free Full Text]
  34. Aviram M, Lees AM, Lees RS. Low density lipoproteins in human platelets. Clin Res. 1980;28:384A. Abstract.
  35. Hovig T, Grøttum KA. Lipid infusions in man: ultrastructural studies on blood platelet uptake of fat particles. Thromb Diathes Haemorrh. 1973;29:450-460. [Medline] [Order article via Infotrieve]
  36. White JG. Uptake of latex particles by blood platelets. Am J Pathol. 1972;69:439-458. [Medline] [Order article via Infotrieve]
  37. Lewis JC, Maldonado JE, Mann KG. Phagocytosis in human platelets: localization of acid phosphatase-positive phagosomes following latex uptake. Blood. 1976;47:833-840. [Abstract/Free Full Text]
  38. Fajardo LF, Tallent C. Malarial parasites within human platelets. JAMA. 1974;229:1205-1207.
  39. Clawson CC. Platelet interaction with bacteria, III: ultrastructure. Am J Pathol. 1973;70:449-464. [Medline] [Order article via Infotrieve]
  40. Kemona H, Wysocka J, Mantur M, Prokopowicz J. Phagocytic activity of platelets in patients with pneumonia. Acta Haematol Pol. 1989;20:201-205. [Medline] [Order article via Infotrieve]
  41. Randell E, Mulye H, Mookerjea S, Nagpurkar A. Evidence for phosphatidylcholine hydrolysis by phospholipase C in rat platelets. Biochim Biophys Acta. 1992;1124:273-278. [Medline] [Order article via Infotrieve]
  42. Baldassare JJ, Henderson PA, Fisher JG. Isolation and characterization of one soluble and two membrane-associated forms of phophoinositide-specific phospholipase C from human platelets. Biochemistry. 1989;28:6010-6016. [Medline] [Order article via Infotrieve]
  43. Banno Y, Yu A, Nakashima T, Homma Y, Takenawa T, Nozawa Y. Purification and characterization of a cytosolic phosphoinositide-phospholipase C ({gamma}2-type) from human platelets. Biochem Biophys Res Commun. 1990;167:396-401. [Medline] [Order article via Infotrieve]
  44. Surya II, Gorter G, Mommersteeg M, Akkerman JWN. Enhancement of platelet functions by low density lipoproteins. Biochim Biophys Acta. 1992;1165:19-26. [Medline] [Order article via Infotrieve]
  45. Bills TK, Smith JB, Silver MJ. Metabolism of [14C]arachidonic acid by human platelets. Biochim Biophys Acta. 1976;424:303-314. [Medline] [Order article via Infotrieve]
  46. Takamura H, Narita H, Park HJ, Tanaka K, Mutsuura T, Kito M. Differential hydrolysis of phospholipid molecular species during activation of human platelet with thrombin and collagen. J Biol Chem. 1987;262:2262-2269. [Abstract/Free Full Text]
  47. Needleman SW, Spector AA, Hoak JC. Enrichment of human platelet phospholipids with linoleic acid diminishes thromboxane release. Prostaglandins. 1982;24:607-622. [Medline] [Order article via Infotrieve]
  48. Schick BP, Schick PK, Chase PR. Lipid composition of guinea pig platelets and megakaryocytes: the megakaryocytes as a probable source of platelet lipids. Biochim Biophys Acta. 1981;663:239-248. [Medline] [Order article via Infotrieve]
  49. Schick PK, Schick BP, Foster K, Block A. Arachidonate synthesis and uptake in isolated guinea-pig megakaryocytes and platelets. Biochim Biophys Acta. 1984;795:341-347. [Medline] [Order article via Infotrieve]
  50. Nordøy A. Plasma and platelet lipids in man. Haemostasis. 1973;74:103-117.



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