© 1995 American Heart Association, Inc.
Articles |
Uptake of Radiolabeled and Colloidal Gold–Labeled Chyle Chylomicrons and Chylomicron Remnants by Rat Platelets In Vitro
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.
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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
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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.
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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.
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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 1
). 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 2). Negative staining
showed that the CM (Fig 1A) and CMR (Fig 1B) 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 1C) or CMRs (Fig 1D). Exposure to pH 4 and pH 9
environments did not dissociate the conjugate.
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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 2). 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 3), 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 4).
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and [14C]cholesterol (
) 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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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 1
. 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 2), 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 6). Table 3
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 4). 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 5,
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 6);
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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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 5), 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 6). Some CM-Au particles were also observed
in the cytoplasm of platelets (Fig 7). 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 7). 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 8A and Table 7), and
some were found in cytoplasmic vesicles that were not stained with
ruthenium red (Fig 8B and Table 7). After 30 minutes, some CM-Au
particles or free gold particles were found in the vacuoles that showed
acid phosphatase activity (Fig 9 and Table 7). 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 10A). A few platelets had taken up CMR-Au particles,
which were found entirely in the open canalicular system (Fig 10B). As
shown in Table 7, 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 8). 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 7).
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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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 3 and 4). 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 3 and 4). 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 1, 5, and 6). 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 2). There was no net hydrolysis
of [14C]CE or [3H]20:4-TG (Tables 1 and 2).
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 9). 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 8B). 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 9). Most of the
CM-Au's were, however, in the open canalicular system, only 11% being
in the cytoplasm (Table 8). 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.
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Acknowledgments |
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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.
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