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1 Biomedical Engineering and 2 Pulmonary Medicine, Vanderbilt University, Nashville, Tennessee 37235; and 3 Department of Biology, Boston University, Boston, Massachusetts 02118
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ABSTRACT |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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We previously reported that platelets release a soluble factor that decreases the solute permeability of cultured bovine aortic endothelial monolayers. This factor was characterized as heat stable, trypsin sensitive, and not serotonin, adenosine, ADP, or ATP [F. R. Haselton and J. S. Alexander. Am. J. Physiol. 263 (Lung Cell Mol. Physiol. 7): L670-L678, 1992]. We now report its identity as lysophosphatidic acid (LPA). Endothelial permeability decreases rapidly, reversibly, and repeatedly when exposed to platelet supernatants. Continuous exposure produces a sustained decrease in permeability. Methanol extracts of platelet supernatants also decrease endothelial permeability. Treatment of methanol extracts of platelet supernatants with phospholipase B or alkaline phosphatase, which modify the structure of LPA, abolishes the permeability-decreasing activity. However, activity is unaffected by treatment with phospholipase A2. This pattern of enzyme inactivation is consistent with the structure of LPA. Furthermore, synthetic 1-oleoyl-LPA rapidly and significantly decreases endothelial permeability in a concentration-dependent manner. Platelet activation does not appear to be required to produce activity in supernatants from platelet isolations, since P-selectin expression is not increased and thromboxane B2 is <14 pg/6,000 platelets. Our data show that platelets release a methanol-extractable compound with an enzyme degradation profile consistent with LPA, which decreases the permeability of endothelial monolayers in vitro. In vivo, LPA derived from platelets may be an important mediator of the transport barrier formed by the vascular endothelium.
endothelial barrier
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INTRODUCTION |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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ENDOTHELIAL CELLS form a barrier to the exchange of solutes across the vascular wall. This barrier can be pharmacologically regulated by a variety of vasoactive hormones and autacoids through receptor-mediated second messenger-controlled pathways (7, 9, 10, 33). We recently reported that human platelets release a factor that significantly decreases the solute permeability of cultured bovine endothelial monolayers in a rapid and reversible manner (7). Several other reports have also demonstrated that platelets release similar factors that decrease endothelial permeability in vitro (20, 26).
All three reports agree that platelets release one or more agents that decrease endothelial permeability; however, there is disagreement regarding the identity of the agent. In the original report of Shepard et al. (26), they found that neither serotonin, prostacyclin, or thromboxane A2 (TxA2) mediated the permeability-decreasing effects of platelets. In a subsequent report, Paty et al. (20) reported that the factor responsible for the decreased permeability produced by platelets was adenosine. We, however, have shown that this platelet effect was not attenuated by treatments that remove or block adenosine, ATP ADP, serotonin, or norepinephrine. Instead, we previously reported our data to be consistent with a heat-insensitive, trypsin-labile protein with a molecular mass >100 kDa (7).
Most studies on the permeability-decreasing effects of platelets have
focused on components of the dense and 
-granules released by
activated platelets. However, platelets also release several important
vasoactive mediators not found in granules derived from membrane
phospholipids. These products include platelet-activating factor,
phosphatidic acid, phosphatidylcholine, the hydroxyheptadecatrienoates, and hydroxyeicosatetraenoates. These compounds play important roles
in platelet activity, neutrophil chemotaxis, and smooth muscle
contraction and might also help to regulate vascular permeability (16,
22).
Recently, several published reports have shown that some of the effects of plasma, serum, and other platelet-conditioned products, which had been previously attributed to high-molecular-weight proteins, e.g., albumin, are actually mediated by lysophosphatidic acid (LPA), a bioactive lipid mediator released by platelets and bound to albumin (17, 23, 27). Here we demonstrate that platelet-derived LPA produces a rapid, significant, and reversible decrease in endothelial solute permeability in vitro, which suggests that platelet-derived LPA may be important for the maintenance and regulation of the in vivo endothelial barrier as well.
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MATERIALS AND METHODS |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Materials
1-Oleoyl-LPA was purchased from Sigma, and unless otherwise noted, all other biological and tissue culture supplies were purchased from Sigma Chemical (St. Louis, MO).Isolation of Human Platelets and Platelet-Conditioned Medium
Human platelets were isolated from whole blood collected by venipuncture using 3.2% sodium citrate (10% by vol) as an anticoagulant with 5 µM D-phenylalanyl-prolyl-arginine chloromethyl ketone (PPACK, Chemica Alta, Edmonton, Canada) added to inhibit thrombin-mediated platelet activation. Blood was centrifuged at 300 g for 5 min, and the platelet-rich plasma was removed. The platelet concentration was determined using a Coulter counter. Leupeptin (1 µg/ml) was added to the platelet-rich plasma, and the plasma was incubated for 2 h at room temperature. Platelets were then removed by centrifugation at 2,500 g for 10 min, and the supernatant was decanted and frozen at
20°C.
Preparation of Lipid Extracts From Platelet-Conditioned Supernatants
Lipid extracts were prepared from human platelet supernatants by freeze-drying 5-ml aliquots of platelet-conditioned supernatants (Hetosicc lyophilizer, Hitechnology, Scandinavia) and extracting the residual lyophilized material in 100% methanol with agitation for 10 min at 25°C. This methanol extract was separated from the insoluble protein material by centrifugation (300 g, 5 min), and the insoluble material was extracted four additional times. The pooled methanol extracts (~50 ml) were evaporated to 500 µl by heating the extracts to 35°C under a stream of compressed air. Samples were stored at20°C. The residual methanol-insoluble material was dried
under a stream of compressed air until dry and stored at
20°C. Platelet methanol extracts were diluted to yield a
final dilution equivalent to the original platelet-rich plasma isolated
for each fraction (6,000 platelets/µl).
Enzymatic Degradation of Phospholipids
To demonstrate that the agent responsible for the permeability-decreasing effects of platelet supernatants was LPA, 50-µl aliquots of methanol extracts from platelet supernatants were incubated with agitation for 18 h at 37°C with 10 U of phospholipase A2, (PLA2, Sigma P-0790) in 1 ml of PLA2 incubation buffer [100 mM tris(hydroxymethyl)aminomethane (Tris) · HCl, pH 8.0, 2 mM CaCl2], or with 1 U phospholipase B (PLB, Sigma P-8914) in 1 ml of PLB incubation buffer, (100 mM Tris · HCl, pH 8.0) for 18 h at 37°C or with 10 U of alkaline phosphatase (Sigma) for 18 h at 37°C in alkaline phosphatase buffer (50 mM MgCl2, 150 mM NaCl, 150 mM Tris · HCl, pH 8.5). These samples were then diluted equivalent to 6,000 platelets/µl in the original platelet preparation for each sample and assayed for their effects on endothelial permeability as described below (see Cell-column chromatographic measurement of endothelial monloayer permeability). Because PLA2 cleaves the acyl chain in the two position of the phospholipid, it will not degrade LPA; conversely, PLB will remove acyl groups in both the two and three positions and degrades lysophosphatidic acid.Endothelial Cell Culture Methods
Well-established techniques were used to isolate and culture bovine aortic endothelial cells and bovine fetal aortic endothelium. Briefly, cultures of adult and fetal aortic endothelial cells were obtained from thoracic aorta by means of 0.1% collagenase treatment. Monolayer cultures were established in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and were subcultured using 0.1% trypsin-EDTA. Endothelial cell identity was verified by indirect immunofluorescent assay for the presence of factor VIII-related antigen and uptake of DiI-labeled low-density lipoprotein. Cells were used during the vigorous proliferative phase of their in vitro lifespan. The fetal bovine cell lines (AG-7680 and AG-7681) are available from the Cell Repository at the Coriell Institute for Medical Research (Camden, NJ).Microcarrier Culture Methods
Cells were cultured on microcarrier beads as previously described (10). Cells were seeded on Cytodex-3 microcarrier beads at a density of 2 × 104 cells/cm2. Cell attachment was achieved by intermittent stirring overnight. Microcarrier cultures were maintained at 60 revolutions/min and fed three times a week. Cultures were used for these assays between 7 and 30 days postseeding.Cell-Column Chromatographic Measurement of Endothelial Monolayer Permeability
We used a previously reported cell-column permeability assay (1, 7, 9, 10) to measure changes in endothelial permeability and the time course of these changes. Cell columns consisted of a 2.0-cm height by 0.65-cm diameter water-jacketed column of bovine aortic endothelial cells or bovine fetal aortic endothelium monolayers cultured on porous microcarrier beads. Columns were perfused continuously at 1 ml/min with Hanks' balanced salt solution (HBSS)-0.5 % bovine serum albumin (BSA) and 15 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) (pH 7.35) at 37°C. Permeability under different conditions was measured by indicator dilution analysis of the elution of a 50-µl bolus of tracers injected over the column. The tracer mixture contained an impermeant tracer (or flow tracer) blue dextran (10 mg/ml) with a molecular mass of 2,000 kDa, and permeant tracers, cyanocobalamin (B12, mol wt 1,355) or sodium fluorescein (NAFl, mol wt 342). A Gilson 203 fraction collector was used to collect eluant into 96-well plates, and the absorbance of each well read at 620, 540, and 492 nm on a plate reader (Titertek MCC 340, Flow labs). These absorbance values were used to reconstruct the concentration time curves of the elution of these tracers at the outlet of cell columns. Impermeant and permeant tracers have different elution patterns, and with suitable analysis, they can be used to estimate the permeability of the cell barrier to the permeant tracers cyanocobalamin and sodium fluorescein (10). The elution profiles are different because the permeant tracers depend on the properties of the mobile phase of the column plus the paracellular junctional permeability properties of the endothelial monolayer and the diffusive motion of the tracer within the porous microcarrier beads. On the other hand, the elution of blue dextran depends only on the flow-phase properties of the column. All permeabilities are reported as × 10
5 cm/s.
Platelet Activation Assays
Measurement of TxB2 in platelet samples by mass spectrometry. Levels of TxB2 in samples of platelet supernatants were measured by mass spectrometry as described by Knapp et al. (15). Briefly, known amounts of tetradeuterated internal standards were added to 100-µl aliquots of platelet-conditioned supernatants. These samples were derivatized to the corresponding methoxime, trimethylsilyl ether-pentafluorobenzyl esters, and quantified with negative ion-chemical ionization gas chromatography-mass spectrometry in the selected ion-monitoring mode, monitoring at mass-to-charge ratio (m/z) 614 for the endogenous compounds and m/z 618 for the tetradeuterated internal standards.
Measurement of platelet activation using fluorescence-activated cell sorting. Fluorescence-activated cell sorting (FACS) was used to measure platelet activation as determined by the percentage of platelets exhibiting P-selectin above the activation threshold of 600 P-selectin copies/µm2 of platelet surface. Platelets samples were collected during the platelet incubation period at baseline, after 60 min of incubation and after 120 min of incubation. A platelet sample was treated with 1 µM PMA as a positive control. Platelet samples were then fixed by adding phosphate-buffered paraformaldehyde to achieve a final concentration of 1%. Samples containing 2 × 106 platelets (in ~10 µl of buffer) were adjusted to 100 µl total volume with antibody buffer (10 mM HEPES, 143 mM NaCl, 5 mM KCl, 0.5 mM NaHPO4 and 1 mM MgCl2) and 20 µg of anti-CD62-phycoerythrin conjugate (Becton Dickinson Immunocytochemistry Systems, San Jose, CA). These samples were incubated in the dark for 30 min, washed twice in HBSS with 1% paraformaldehyde, and stored in the dark until used, usually no longer than 1 h. A total of 20,000 platelets from each sample were analyzed using a Becton Dickinson FACScan flow cytometer. The FACScan was formatted for two-color analysis with the light scatter and fluorescence channels set at logarithmic gain using 1,024 channels and a four-decade scale. The FACScan uses two 15-mW air-cooled lasers producing single-line excitations of 488 and 540 nm. Light scatter and fluorescence channels were standardized daily using QC3 841 bead standards, a dual-color reference standard of known size and fluorescence. Amplifier settings were adjusted for optimal measurement sensitivity and reproducibility. Statistics. Cell-column permeability results were examined for statistical significance by comparing the baseline permeability measurements with cell-column permeability values obtained following treatments. Repeated-measures analysis of variance was used to test for a significant difference (P < 0.05) among groups. Significant differences between groups was determined using Tukey's modified t-test.| |
RESULTS |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Cell columns still responded to platelet-released material after 10 cycles of platelet material treatment alternated with baseline
treatments (Fig. 1). Exposure of cultured
endothelial cell monolayers to platelet-released material produced the
expected rapid decrease in cell-column permeability. This effect was
completely reversible, and the effect could be reproduced many times by
reexposing monolayers to platelet-released material alternated with
baseline perfusate. The average baseline value was 11.6 ± 0.87 (means ± SD; × 105 cm/s), and the average
of the platelet-derived treatment state was 8.28 ± 0.98.
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The effect of continuous exposure to platelet-derived material was
studied by continuously perfusing cell columns with platelet material.
As shown in Fig. 2, cell columns
continuously exposed to platelet-derived material showed a rapid
decrease in endothelial permeability, which remained below the initial
baseline value for 
2 h tested (Fig. 2). The average over all
treatment time point was 0.66 ± 0.11 (means ± SD; because in
some experiments permeability was measured at more frequent intervals
n varied with
n = 4 except
n = 3 at 70, 100, 145, n = 2 at 130).
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Elution profiles are altered by treatment with platelet supernatant
methanol extracts (Fig. 3). Elution
profiles from the same cell column under the conditions of baseline
perfusate and after addition of a methanol extract of platelet
supernatant medium for 5 min are shown in Fig. 3. The shapes of the
curves for the large-molecular-weight tracer blue dextran (open
triangles) are similar under both conditions, whereas there are
dramatic changes in the shape of the small molecular permeant tracers,
cyanocobalamin (open squares) and sodium fluorescein (filled
triangles). In Fig. 3B, both
cyanocobalamin and sodium fluorescein have higher peak values, narrower
elution profiles, and less tracer in the tail of their curves. After
treatment, the cell layer excludes more of the permeant tracer from the
microcarrier bead interior. This column had a total estimated cell
surface area of 130 cm2, and
computer analysis of the relative shapes of the impermeant and permeant
tracers found a permeability decrease of nearly 67%. In this
experiment, when platelet supernatant methanol extracts were added to
the perfusate, permeability decreased from baseline values of 17.1 and
24.2 × 105 cm/s for
cyanocobalamin and sodium fluorescein, respectively (Fig.
3A), to 5.22 and 7.55 × 105 cm/s, respectively
(Fig. 3B). Methanol vehicle
treatments had no effect on permeability (data not shown).
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To determine whether a methanol-soluble factor present in platelet supernatants decreased endothelial permeability, we compared the permeability effects of untreated platelet supernatants with methanol extracts of platelet supernatants. Platelet supernatants diluted to give a dilution equivalent to 6,000 platelets/µl significantly decreased cell-column permeability compared with untreated controls within 10 min (n = 25; Fig. 4). Methanol extracts of platelet supernatants also reduced endothelial permeability to a similar degree of that produced by unextracted platelet supernatant (n = 19). The material that remained insoluble in methanol, consisting mostly of albumin and immunoglobulin, decreased endothelial permeability slightly but not significantly (n = 7).
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To show that LPA is responsible for the decrease in in vitro endothelial permeability, we treated methanol extracts of platelet supernatants with PLA2, PLB, and alkaline phosphatase (Fig. 5). Treatment of methanol extracts with 10 U of PLA2 did not block the response produced by LPA (n = 7); however, treatment of methanol extracts with 1 U of PLB (n = 10) or 10 U of alkaline phosphatase (n = 5) abolished the permeability-decreasing effect observed with methanol-extracted material (n = 10).
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To determine whether LPA itself modulated endothelial permeability, we
investigated the direct effects of synthetic 1-oleoyl-LPA on
cell-column permeability. Ten minutes of LPA treatment decreased endothelial permeability in a concentration-dependent manner (Fig. 6; n = 4 at 106 M,
n = 3 at
107 M, and
n = 2 at
108 M).
n values differ because the order of
LPA concentration treatments was randomized, and complete concentration
protocols were not completed within each of the four columns tested.
Endothelial permeability was significantly decreased within 10 min by
106 M LPA but not by lower
concentrations of LPA.
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We compared the time course of continuous platelet-derived material
with LPA by treating cell columns with
106 M LPA for 2 h. A pattern similar to that produced by platelets (Fig.
2) was also observed for continuous LPA treatment (Fig. 7; n = 3, last two time points n = 2 and 1, respectively). The average over all treatment time points was 0.63 ± 0.06 (means ± SD) of baseline.
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We characterized platelet activation by both the release of TxB2 and the surface expression of P-selectin. We found no remarkable changes in the surface expression of P-selectin produced by our platelet preparation procedure, whereas phorbol 12-myristate 13-acetate (1 µM) produced significant increases in platelet P-selectin expression. The percentage of P-selectin-positive platelets in the platelet isolate was 0.03% without incubation, 0.46% after 60 min of incubation, 1.79% after 120 min of incubation, and 91.5% after 120 min of incubation and activation with phorbol 12-myristate 13-acetate.
The concentration of TxB2 in stored platelet material ranged from 1.5 to 66.6 pg/6,000 platelets with an average concentration of 14.4 ± 5.14 pg/6,000 platelets (means ± SE; n = 14). Most isolates measured had relatively low levels of TxB2, but three had higher levels (~50), indicating that the platelets may have been partially activated during these particular isolations. However, a thrombin-activated platelet sample had a much higher concentration of TxB2 (265 pg/6,000 platelets). These stored samples had permeability-decreasing effects similar to other platelet supernatants. The permeability decrease observed with these isolates was similar to that of Fig. 4 and our previous report (0.69 ± 0.05; n = 14 of baseline). Furthermore, the measured TxB2 concentration of each sample was not correlated with the permeability-decreasing effect observed in cell-column experiments with the isolate. Therefore, our data suggest that the platelets used in these studies were not activated by our isolation procedures and indicate that activation is not required to produce the observed platelet effect of decreased endothelial monolayer permeability.
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DISCUSSION |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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There are several lines of evidence indicating that LPA is the factor responsible for the permeability-decreasing activity of platelet-conditioned supernatants. First, we observe that the methanol extracts of platelet-conditioned supernatants produce a rapid and reversible decrease in endothelial permeability, which was qualitatively identical to that produced by untreated platelet supernatant (Fig. 4). Whereas the permeability effects of methanol extracts are not significantly different from supernatants, some activity is still associated with the methanol-insoluble fraction (Fig. 4, right bar). This residual activity could be due to the presence of other permeability-decreasing factors released by platelets or possibly due to the incomplete extraction of LPA. Bjerve et al. (4) showed that lipid extractions with methanol typically fail to completely extract lysophospholipids, like LPA, unless butanol-based extraction procedures are used. Additional experiments using acidified butanol as a solvent showed that no activity remains after extraction with butanol (n = 4, data not shown).
Second, the activity of platelet methanol extracts, like that of LPA,
is sensitive to treatment with PLB, but not
PLA2 (23, 27). The fact that
alkaline phosphatase also abolished activity of this factor indicates
that the structure of the active factor in platelet methanol extracts
has a phosphomonoester that is apparently critical to the permeability
activity of the platelet factor. The sensitivity of the factor to these
enzymes strongly suggests that the agent present in platelet methanol
extracts is a lysolipid with a three-position phosphomonoester, which
identifies this factor as a LPA. These experiments do not however
identify which lipid groups are esterified to the phosphoglycerate
backbone, or, as has been observed in some studies, if different lipids produce different activities (28, 30). However, 1-oleolyl LPA produces
a permeability decrease similar to platelet supernatants and methanol
extracts (see Figs. 4, 6, and 7), suggesting that esterified oleic
acid-containing lysolipids exhibit these effects. Alone, pure
1-oleoyl-LPA produces a rapid (<5 min), significant, dose-dependent,
and reversible decrease in endothelial permeability qualitatively
identical to methanol extracts of platelet supernates and platelet
supernates. The effect of LPA is apparently dose dependent between
108 and
106 M but is significant
only at 106 M.
The biological effects of LPA are mediated through a specific 38- to 40-kDa cell surface receptor that specifically binds LPA (30, 31), and cells that are sensitive to LPA express this receptor. LPA receptors have been described in neuroblastoma, brain, and lung cells but not in human neutrophils (13). Binding of LPA to its receptor mobilizes several second messenger pathways, including phospholipases C (21) and D (29, 32), and importantly LPA leads to the activation of the rho protein signal transduction pathway (23). Activation of the rho system in cultured cells produces membrane ruffling, actin stress fiber formation, and formation of focal adhesions (3, 12, 14, 18, 24). It has been previously reported that endothelial permeability changes are associated with structural changes in the actin cytoskeleton (9, 25) and that agents that promote actin polymerization enhance the junctional barrier (2). Similarly, drugs that disassemble the actin cytoskeleton reduce this barrier (2, 10). In epithelia, rho protein has been shown to regulate tight junction and perijunctional actin organization (19). It seems highly likely that the enhancement of barrier observed with both platelet-conditioned supernatants and LPA also involve signal transduction through the rho system.
Although the time course of the rho pathway has not been well characterized, we found that the response of endothelial permeability to platelet-conditioned supernatants is rapid in onset and can be rapidly reversed by removal of platelet supernatant, and this on/off effect can be reproduced at least 10 times in a single cell column (Fig. 1). We also observed a sustained decrease with continuous application of platelet-derived material (Fig. 2). This suggests that the permeability factor in platelet-conditioned supernatant is an as yet unappreciated short-term and/or long-term modulator of endothelial solute permeability.
To date, platelets have been reported to synthesize LPA only when they
are stimulated, for example by thrombin or phorbol ester (5, 6), and it
is estimated that the concentration of LPA in serum is near
106 M (5). However, it is
not known whether platelets constitutively release LPA in plasma
without stimulation. We detected LPA activity in methanol extracts of
both normal platelet extracts and platelet-poor plasma (data not
shown), both prepared in the presence of PPACK thrombin inhibitors.
Furthermore, platelets did not release
TxB2 or increase the surface
expression of P-selectin during the supernatant-conditioning procedures. This suggests that platelets may secrete LPA without activation.
Our conclusion that LPA is responsible for the permeability effects of
platelets conflicts with that reported by Paty et al. (20), who
suggested adenosine was responsible for this effect. We have previously
reported that adenosine acts as a barrier-enhancing agent (8, 9) and
have also considered adenosine as a possible platelet-derived mediator
in this model. Several lines of evidence suggest that adenosine is not
responsible for the barrier-enhancing effects of platelets. Adenosine
(104 M) significantly
decreased endothelial permeability, and this effect was blocked by
8-phenyltheophylline (105
M), an adenosine A2 antagonist.
However, 8-phenyltheophylline failed to prevent the barrier-enhancing
response of platelet-conditioned medium (7). Similarly, treatment of
platelet releasates with adenosine deaminase also failed to abolish its
effects on permeability (7). In addition the permeability factor
identified in this report is methanol soluble, which is one
characteristic of phospholipid mediators. This also argues that at
least under the conditions used here, adenosine is not the permeability
factor because adenosine is methanol insoluble. The differences in the
results reported by Paty et al. (20) and those reported here could
however be due to the differences in the concentration of platelets
used in these studies. We have previously reported that at
106 M, adenosine fails to
reduce endothelial permeability. Paty et al. (20) used
platelet-conditioned medium at a dilution equivalent to 7 × 104 platelets/µl (>10 times
the concentration used here), thus it is possible that sufficient
platelet-derived adenosine was released in this previous study to
account for their permeability results. Holmsen et al. (11) showed that
6,000 platelets/µl, equivalent to that used in our study, could only
release adenosine to achieve a concentration of
106 M. Another critical
difference between these studies was the method used to prepare
platelets and the steps taken to prevent platelet activation. We used
PPACK (105 M) to inhibit
thrombin-mediated platelet activation, which was not used in the study
by Paty et al. (20). Therefore, the platelets in that study could have
been activated through this pathway.
Our conclusion of effects of LPA is of course dependent on the purity of the 1-oleoyl-LPA reagent purchased from Sigma. Preliminary mass spectroscopy of this reagent (data not shown) suggests that the one lot tested contains a mixture of C18:0 and C16:0 LPA. After purification, modification, and derivatization of an authentic C18:1 LPA from Sigma, there were two predominant peaks in the total GC trace at the appropriate retention time for the bis-pentafluorobenzoyl (PFB) derivatives (data not shown). The majority of the total ion current is present as the molecular anion (m/z 746), corresponding to C18:0LPA. Preliminary assignments for the other observed fragments include: 1) fluorine ejection (m/z 728), 2) CO2 ejection (m/z 702), 3) loss of PFB group (m/z 534), and 4) loss of the alkyl chain (m/z 464) (fragment also seen in the complete spectrum of a peak corresponding to C16:0LPA). The lower mass fragments at m/z 167, 196, and 211 correspond to different fragments of the PFB group that retained charge after heterolytic cleavage. Reconstructed ion current profiles support these assignments. The relative abundance is approximately 1:4.
As noted by others, our previous observation that the factor was trypsin sensitive and >100 kDa in size (7) can be attributed to the albumin-binding characteristics of LPA (5, 23, 27). Albumin binding of LPA is important for its biological activity, and trypsin degradation of albumin apparently interferes with this bioactivity.
Biologically active LPA is present in platelet-conditioned supernatants, and LPA decreases the permeability of endothelial monolayers in vitro at concentrations similar to these values reported in serum. It is highly likely that platelet-derived LPA is present in plasma under normal and perhaps during certain pathological conditions. From these in vitro studies, it appears that LPA may be an important autacoid regulating vascular permeability in vivo.
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ACKNOWLEDGEMENTS |
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Outstanding technical assistance was provided by Pat Price, Angela Koeper, and Charlene Finney. Norman Purvis of Cytometry Associates (Brentwood, TN) performed the flow cytometry.
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FOOTNOTES |
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This study was sponsored in part by National Institutes of Health Grants HL-40554, EY-10086, HL-19153, HL-55198, and HL-47615.
Address for reprint requests: R. Haselton, Box 1510B, Vanderbilt Univ., Nashville, TN 37235 (E-mail; haselton{at}vuse.vanderbilt.edu).
Received 8 October 1996; accepted in final form 15 August 1997.
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REFERENCES |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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