© 1995 American Heart Association, Inc.
Articles |
Measurement of Platelet-Activating Factor in a Canine Model of Coronary Thrombosis and in Endarterectomy Samples From Patients With Advanced Coronary Artery Disease
From the Division of Cardiology (H.W.M., C.A.H., J.M.M., K.C., J.T.W.), University of Texas Health Science Center, Houston; the Center for Experimental Therapeutics (S.J.G.), Baylor College of Medicine, Houston, Tex; and the Department of Biomathematics (D.A.J.), M.D. Anderson Cancer Center, Houston, Tex.
Correspondence to Dr Howard W. Mueller, Division of Cardiology, University of Texas Health Science Center, 6431 Fannin St, Houston, TX 77030.
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Abstract Platelet-activating factor (PAF, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) is a potent phospholipid mediator of numerous inflammatory and thrombotic responses. The purpose of this study was to determine if PAF synthesis is elevated in damaged coronary arteries after a sustained period of cyclic flow variation (CFV), a phenomenon caused by alternating periods of thrombosis and reperfusion at sites of endothelial injury. Cyclic flow was established and maintained in the left anterior descending coronary arteries (LADs) of 10 dogs. After 8 hours of CFV, the section of damaged LAD containing the thrombus and control sections of the circumflex artery, carotid artery, and saphenous vein was excised, and the total lipids were extracted. The PAF was then purified by silica column chromatography and high-performance liquid chromatography and assayed by both a rabbit platelet bioassay and a PAF radioimmunoassay. With the platelet bioassay, PAF levels of 8.9±4.0 (range, 4.8 to 15.5) pg/mg wet wt were found in the damaged LADs from the 10 dogs. This PAF bioactivity was completely inhibited by a PAF receptor antagonist. When the radioimmunoassay was used, slightly higher PAF levels of 16.3±12.9 (range, 4.5 to 41.8) pg/mg wet wt were observed in the LADs. Overall, these PAF levels were 3- to 64-fold higher than in the control vessels when either assay method was used. Although increases in PAF were observed in the damaged LADs, measurements of PAF in blood samples taken from the LAD and the aorta (control) failed to demonstrate any site-specific increase of PAF in the blood. In related experiments, PAF was also measured in 23 endarterectomy samples taken from the coronary arteries of 16 patients with severe atherosclerosis. The PAF levels in these samples were highly variable (2.9±2.2 [range, 0.3 to 8.5] pg/mg wet wt) and showed no correlation with tissue mass, suggesting that PAF is affected by factors other than the simple presence of atherosclerotic tissue in the vessel. These findings provide direct evidence that PAF is synthesized locally at the site of endothelial injury during thrombosis and that PAF accumulates in the atherosclerotic plaque of some patients with advanced coronary artery disease.
Key Words: platelet-activating factor • coronary thrombosis • cyclic flow variations • unstable angina • atherosclerosis
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Introduction |
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Platelet activating factor (PAF) is a potent phospholipid mediator having the structure 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine.1 2 3 4 Although originally discovered in the supernatant from antigen-challenged IgE-sensitized basophils,5 6 7 subsequent reports have shown that PAF is also produced by other cells in the blood, including neutrophils,8 9 platelets,10 11 macrophages,12 and monocytes.13 The biological effects of PAF are highly varied and include a number of pathological responses in the cardiovascular system. For example, when injected intravenously, PAF causes systemic hypotension,14 15 pulmonary hypertension,14 increased vascular permeability,16 17 thrombocytopenia,14 15 18 19 neutropenia,14 18 19 20 bradycardia,14 capillary clumping of platelets and neutrophils,20 21 and bronchoconstriction.15 Additional in vitro studies have demonstrated that PAF stimulates aggregation and degranulation of both platelets and neutrophils,19 20 22 23 neutrophil chemotaxis and superoxide production,23 and smooth muscle cell contraction.24 Finally, by use of a perfused heart model, PAF has been shown to cause cardiac arrhythmias,25 26 decreased coronary artery blood flow,25 26 27 28 and diminished contractility.25 26 28
In light of this myriad of biological responses, a great deal of scientific interest has focused on the potential involvement of PAF as a mediator in coronary thrombosis and cardiovascular disease. With this role in mind, the present study was undertaken to determine if PAF synthesis is elevated during coronary thrombosis in a canine model of cyclic flow variation (CFV). In this model, blood flow fluctuates in the left anterior descending coronary artery (LAD) because of alternating periods of thrombosis and reperfusion at a site of endothelial damage in the vessel.29 This cyclic flow phenomenon is thought to cause unstable angina in patients with advanced coronary artery disease.30 In previous studies, the involvement of PAF in this CFV model has been suggested through the use of PAF receptor antagonists, which can inhibit CFV, or by infusing PAF into coronary arteries and stimulating cyclic flow.31 32 33 Although these studies provide indirect evidence that PAF mediates coronary thrombosis, efforts to demonstrate a direct relation between PAF synthesis and vascular thrombosis have been hindered by difficulties in quantitatively recovering PAF from in vivo models. These difficulties arise because of the tiny amounts of PAF that are synthesized and also because of its rapid degradation by PAF acetylhydrolase, an enzyme present in serum and numerous cell types.34 35 Despite these potential problems, we have developed a method by which PAF can be extracted and purified from whole blood or tissue with 60% to 75% recovery. By use of this protocol, data have been generated demonstrating that PAF synthesis is elevated at sites of coronary thrombosis after damage to the vascular endothelium. In addition, we have used this methodology to show that PAF accumulates in diseased human coronary arteries.
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Surgical Preparation and Instrumentation for Cyclic Flow Studies
All animal procedures were performed in accordance with institutional guidelines for the proper treatment and handling of laboratory animals. Ten mongrel male dogs weighing
30 kg were
instrumented for cyclic flow experiments essentially as described by
Apprill et al33 using the model originally developed by
Folts and colleagues.29 36 Briefly, the dogs were
anesthetized with 30 mg/kg sodium pentobarbital administered
intravenously and ventilated with room air on a Harvard
ventilator. Body temperature was maintained with a heating pad placed
under the abdomen. To monitor phasic blood pressure and to take aortic
blood samples, a catheter was inserted through the left carotid artery
down to the aortic arch. A second catheter was placed in the left
jugular vein for administration of fluids. A left thoracotomy was then
performed, and the heart was suspended in a pericardial sling. A 15-mm
segment of the LAD was cleared by blunt dissection, and a Doppler
flow probe was placed around the proximal portion of the artery.
Control measurements of blood flow were taken, and the vascular
endothelium was then injured with a pair of
polyethylene-coated vascular forceps. To sample blood from the damaged
LAD, a third catheter was inserted through a diagonal branch of the LAD
up to the juncture with the main artery and immediately distal to the
area of endothelial injury. Finally, a polycarbonate
cylindrical constrictor, which reduced phasic blood flow by 40% to
50%, was placed around the damaged region of the artery, and cyclic
flow was initiated. After 8 hours of continuous cyclic flow, 20-mm
segments of the left saphenous vein and right carotid artery were tied
off, excised, and immediately frozen in liquid nitrogen. The heart was
then removed at the nadir of LAD blood flow, and sections of the
circumflex artery and the damaged LAD containing the thrombus were also
excised and quickly frozen. The total time required for isolation of
all four vessel samples was <5 minutes. To measure the effects of
cyclic flow on blood PAF levels, blood samples (4.5 mL) were drawn at
defined times from the aorta, or from the distal LAD at the nadir of
blood flow, into 0.1 vol of 10 mmol/L EDTA, immediately added to 15 mL
methanol/chloroform (2:1 [vol/vol]), and shaken vigorously. The blood
and tissue samples were stored at -20°C and -80°C, respectively,
before processing. To monitor blood cell concentrations over 8 hours of
CFV, aortic blood samples (5 mL) were drawn at defined intervals into
Vacutainer tubes containing EDTA, and complete blood cell counts were
performed by the in-house clinical pathology laboratory.
Human Endarterectomy Samples
Endarterectomy tissue samples were obtained
during bypass surgery from the LAD, right coronary artery,
obtuse marginal artery, diagonal artery, or ramus artery of 16 patients
with severe coronary artery disease. As a control, samples of
healthy internal mammary artery (10 mm each) were also obtained from
five additional patients. The samples were quickly frozen in liquid
nitrogen and stored at -80°C until PAF isolation.
Preparation of Glassware
While performing mass measurements of PAF, we have found that
standard washing procedures are not effective in removing PAF from the
surfaces of laboratory glassware. This residual contamination
ultimately leads to high background values in assays of PAF. To
minimize this problem, all reusable glassware was brushed out by hand
in hot soapy water and then washed in a mechanical glass washer at a
water temperature of 85°C. The glassware was then treated with 2
mol/L KOH in methanol for 2 hours, rinsed with deionized water, and
washed a second time in the automated glass washer. In later control
experiments, we found that this second washing step could be eliminated
without any increase in background PAF levels. All glassware, both
reusable and disposable, was then silanized with a 10% solution of
SurfaSil in hexane, rinsed once with hexane, and air-dried.
Extraction and Purification of PAF From Tissue and Blood
Samples
Vessel samples obtained from the cyclic flow experiments and
human endarterectomy samples were
homogenized in 19 mL methanol/chloroform/H2O
(2:1:0.8 [vol/vol/vol]) by using three 30-second bursts from a
Polytron homogenizer. A biphasic mixture was then
formed by adding 5 mL each of chloroform and aqueous NaCl (1 mol/L),
and the total lipids were extracted into chloroform three
times.37 Blood samples (4.5 mL) were extracted in the same
manner without homogenization. It is important to
note that the EDTA used during the blood sampling eliminated a
rust-colored precipitate that otherwise carried over into the choroform
phase, and the use of 1 mol/L NaCl during the extraction facilitated
the formation of a well-defined protein wafer at the solvent
interface.
The chloroform extracts (30 mL) were evaporated to dryness and
redissolved in 5 mL chloroform/methanol (4:1 [vol/vol]). The
concentrated extracts, together with a 5-mL rinse, were then applied to
small silica columns (particle size, 32 to 63 µm; pore size, 60 Å;
bed volume, 1.6 mL) preequilibrated in chloroform. The columns were
washed with 50 mL chloroform/methanol/glacial acetic acid (1:1:0.04
[vol/vol/vol]), and the PAF was subsequently eluted with 40 mL
chloroform/methanol/H3PO4 (1:1:0.04
[vol/vol/vol]). Recovery of PAF from these columns, which was
typically 98% to 100%, relied heavily on the use of silica with the
characteristics cited above. To wash the H3PO4
from the eluates, the samples were evaporated to near dryness and
redissolved in 15 mL chloroform/methanol (4:1 [vol/vol]). Nine
milliliters of methanol and 12 mL H2O were then added to
form a biphasic mixture, and the upper phase was adjusted to pH 6 with
concentrated NH4OH. After the mixture was shaken vigorously
and the two phases were allowed to reform, the chloroform phase was
removed, and the upper phase was extracted two more times with
chloroform. The chloroform extracts were combined and concentrated in
preparation for high-performance liquid
chromatography (HPLC).
HPLC purification of the PAF samples was initially performed by a modified method of Hanahan and Kumar,38 using a Waters HPLC instrument and an Econosil silica column (10 µm, 10 mmx250 mm; Alltech). The samples were injected in 100 µL chloroform/methanol (1:1 [vol/vol]), followed by a 100-µL rinse, and eluted isocratically with a solvent system of acetonitrile/methanol/H3PO4 (130:5:1.5 [vol/vol/vol]) at a flow rate of 5 mL/min. Compounds eluting from the column were detected by UV absorbance at 206 nm. The retention time of PAF was determined by injecting a phospholipid standard mixture containing [3H]PAF, and fractions eluting in the PAF "window" were combined and evaporated. The samples were then washed as described above to eliminate the H3PO4, concentrated and evaporated to dryness, and finally solubilized in 0.3 to 0.5 mL Hanks' buffer9 (pH 7.4) containing 1 mg/mL bovine serum albumin (BSA). To facilitate the solubilization, the samples were gently shaken in a sonicating water bath for 30 to 60 seconds and vortexed. After solubilization, the samples were diluted twofold with Hanks' buffer (pH 7.4) without BSA for a final BSA concentration of 0.5 mg/mL. Although this HPLC method yielded very good recovery and separation of PAF from other cellular phosphoglycerides, the polar solvent system drastically reduced column life and necessitated long equilibration times to achieve a stable elution pattern. Therefore, in later experiments, a modified method of Blank and Snyder39 was adopted. In this method, the samples were purified on an Ultrasphere-Si silica column (5 µm, 10 mmx250 mm; Beckman) using a two-component solvent system. Solvent A contained isopropanol/hexane (1:1 [vol/vol]), and solvent B consisted of isopropanol/hexane/H2O (23:23:4 [vol/vol/vol]). The samples were dissolved in solvent A before injection, and the PAF was eluted at a flow rate of 5 mL/min with a linear gradient of 50% B to 100% B over the first 29 minutes of the separation. Fractions containing PAF were pooled, concentrated, and solubilized as described above. Since the eluant did not contain any H3PO4, this HPLC method eliminated the need for a post-HPLC washing step.
To monitor recovery of PAF during the purification protocol, homogenates of control canine arteries were prepared as described above and combined with 0.5 µCi of [3H]PAF, and the radiolabeled PAF was purified in parallel with the other samples. Recovery of this [3H]PAF after purification and solubilization in Hanks' buffer was consistently 60% to 75%. To ensure that low background levels of PAF were maintained throughout the protocol, duplicate blank workups were also carried through the entire purification procedure.
Measurement of PAF
The PAF activity in the purified samples was quantified using a
rabbit platelet bioassay of [3H]serotonin
release. This bioassay was performed as described by Pinckard et
al40 with the following modifications: (1) A gel
filtration buffer41 (pH 6.5) containing (mmol/L) NaCl 129,
sodium citrate 10.9, sodium bicarbonate 8.9, glucose 5.6, Trizma base
10, KCl 2.8, KH2PO4 0.8, and EDTA 2, along with
2.5 mg/mL gelatin, was substituted for the Tyrode's buffer used to
wash the platelets. (2) The washed platelets were stored in gel
filtration buffer (pH 6.5) without EDTA before the assay. (3) The assay
incubation was performed in a HEPES-buffered medium42 (pH
7.5) containing (mmol/L) NaCl 137, KCl 2.7, HEPES 10, glucose 5.6, and
CaCl2 1.3, along with 2.5 mg/mL gelatin. (4) The PAF
samples were added in 100 µL Hanks' buffer containing 0.5 mg/mL BSA
for a final BSA concentration of 0.1 mg/mL. In some experiments, the
ability of a PAF receptor antagonist (Ro 24-4736) to block
the activity in the purified samples was also examined. In this case,
the [3H]serotonin-labeled platelets were
preincubated for 5 minutes in HEPES-buffered medium containing either
antagonist (2.5 µmol/L) or dimethyl sulfoxide (vehicle
control; final concentration, 0.1%) before the addition of stimulus.
As another means of quantification, the purified extracts were also
assayed for PAF by using a radioimmunoassay (RIA) kit from Amersham.
Because of the high cost of this assay, the RIA was only used on tissue
extracts from the first seven dogs and
endarterectomy samples from the first six patients.
During the RIA, the PAF standard included with the assay kit was
diluted in Hanks' buffer (pH 7.4) containing 0.5 mg/mL BSA in order to
mimic the conditions of the purified samples.
Gas Chromatography/Negative Ion Mass Spectrometry
Analysis
To further confirm the identity of the isolated product, the
purified PAF from some samples was converted to the
sn-3-pentafluorobenzoate derivative and analyzed by
gas chromatography/negative ion mass spectrometry
(GC/MS). During this analysis, 1 ng of
1-O-(7,7,8,8-tetradeuterohexadecyl)-2-acetyl-sn-glycero-3-phosphocholine
was added to the sample total lipid extracts as an internal standard
before initiating the purification protocol. To derivatize the PAF, the
samples were digested with phospholipase C43 for 1 hour,
and the reaction was stopped by total lipid extraction.37
The resultant 1-radyl-2-acetylglycerol was then dried under
N2 and redissolved in 200 µL dry
toluene/pyridine/pentafluorobenzoyl chloride (20:1:2 [vol/vol/vol]).
After shaking gently for 2 hours at room temperature, the samples were
dried under N2, and 1.5 mL each of H2O
and hexane was added. After extracting three times into hexane, the
pentafluorobenzoates were purified by thin-layer
chromatography on silica gel G by using a solvent
system of hexane/diethyl ether (3:2 [vol/vol]). The band migrating
with authentic
1-radyl-2-acetyl-sn-glycero-3-pentafluorobenzoate was
scraped from the plate, and the derivatized samples were extracted from
the gel37 and concentrated in hexane.
GC/MS analysis was performed on a Hewlett-Packard 5890 gas chromatograph coupled to a VG Quattro triple quadrupole mass spectrometer used as a single quadrupole instrument for these analyses. The samples were injected with a falling needle injector (Allen Scientific Glass) onto a DB-1 fused silica column (film thickness, 0.25 µm; internal diameter, 30 mx0.32 mm). Helium at a pressure of 10 psi was used as the carrier gas. The oven temperature program was from 220°C to 320°C at 10°C/min starting with the injection, and the injector and interface temperatures were both 275°C. Ionization was by electron capture using methane as the moderator gas, and M- · ions of interest were detected by selected ion monitoring.44 45
Materials
All solvents and reagents were of the highest quality available.
PAF (containing predominantly
1-O-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine)
and phospholipid standards were purchased from Avanti Polar Lipids. The
GC/MS standard,
1-O-hexadecyl-2-acetyl-sn-glycero-3-pentafluorobenzoate,
was synthesized from C-16 PAF as described in the section above.
1-O-Hexadecyl-2-[3H]acetyl-sn-glycero-3-phosphocholine
(10 Ci/mmol) and [3H]serotonin (25.4 Ci/mmol)
were obtained from New England Nuclear; PAF RIA kits, from Amersham;
1-O-(7,7,8,8-tetradeuterohexadecyl)-2-acetyl-sn-glycero-3-phosphocholine,
from Cascade Biochem; BSA, gelatin (type B from bovine skin), and
phospholipase C (Bacillus cereus), from Sigma; silica gel,
from ICN Biomedicals; silica gel G thin-layer
chromatography plates, from Analtech;
pentafluorobenzoyl chloride and pyridine, from Aldrich Chemical Co;
and SurfaSil silanizing reagent, from Pierce. The PAF receptor
antagonist, Ro 24-4736, which was dissolved in dimethyl
sulfoxide before use, was a gift from Dr James Christenson at
Hoffman-LaRoche.
Statistical Analyses
Statistical significance of measured parameters was
evaluated by paired Student's t test or by two-way ANOVA
using SPSS for Windows, version 6.1.
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In Vivo PAF Synthesis After a Sustained Period of CFV
To determine if PAF synthesis is elevated at sites of endothelial damage and thrombosis, 10 dogs were subjected to 8 hours of cyclic flow, and the PAF in the damaged LAD and control vessel samples was subsequently purified and measured. As illustrated in Fig 1
, PAF levels in the damaged LADs
were significantly higher than in the control vessels of all 10 dogs.
By use of the rabbit platelet bioassay, the damaged LADs were found
to contain an average of 8.9±4.0 pg/mg wet wt PAF. These PAF levels
were 3.2-fold (range, 1.4- to 7.7-fold), 6.8-fold (range, 2.1- to
13.0-fold), and 8.2-fold (range, 1.5- to 22.3-fold) greater than PAF
levels in the circumflex artery, carotid artery, and saphenous vein
control samples, respectively. By use of a PAF RIA on the first seven
dogs, a similar trend was observed, although the absolute amounts of
PAF in some of the samples were somewhat higher. With the RIA, the mean
PAF levels in the damaged LADs were 16.3±12.9 pg/mg wet wt. Compared
with the control vessels, these PAF levels were 8.7-fold (range, 2.1-
to 28.4-fold), 26.8-fold (range, 2.67- to 115.5-fold), and 64.0-fold
(range, 3.8- to 410.9-fold) greater than the circumflex artery, carotid
artery, and saphenous vein samples, respectively.
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In five of the cyclic flow dogs, PAF measurements were also performed
on blood samples taken from the LAD downstream from the forming
thrombus and from the aorta (control) to determine if elevated PAF
levels could be detected in the circulation. To optimize recovery of
PAF during these measurements, the extractions were performed on whole
blood rather than plasma or serum, since PAF released into the blood
could conceivably bind to other cells and be lost during separation of
the cells from the fluid phase. If PAF were released into the blood
from a forming coronary thrombus, then a site-specific increase
of PAF in the LAD might be predicted. However, as shown in Fig 2, no significant differences in PAF levels were found
between the LAD and aortic samples, suggesting that PAF accumulation is
limited to the immediate area of thrombosis in the cyclic flow
model.
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Inhibition of the Purified PAF by a Receptor
Antagonist
During the platelet bioassay, the release of
[3H]serotonin from radiolabeled platelets
is mediated through a receptor on the surface of the platelet
membrane. To confirm that the product isolated from the canine
vessel samples was acting through this PAF receptor, labeled
platelets were preincubated with a PAF receptor
antagonist and subsequently challenged with the purified
PAF extracts. As shown in Fig 3, the release of
[3H]serotonin from platelets challenged
with known concentrations of authentic PAF or with the vessel extracts
from three dogs was completely inhibited when the platelets were
preincubated with the PAF receptor antagonist.
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Measurement of Blood Platelet and Neutrophil Levels Over Eight
Hours of CFV
Because of the prolonged period of cyclic flow used in the canine
studies, a separate control experiment was performed to determine if
the blood platelets or neutrophils were being depleted during 8
hours of CFV. As shown in the Table, platelet
concentrations in four dogs remained relatively stable over the 8-hour
period when compared with presurgical control measurements, although
small fluctuations were observed. A two-way ANOVA indicated that these
variations in platelet count were not statistically significant
(P=.33). In contrast to the minimal effects on platelet
levels, the neutrophils, rather than being depleted, exhibited
significant (P<.001) increases of 1.9- to 5.6-fold over
presurgical control values after 6 or 8 hours of cyclic flow. No
significant difference was found between the earlier time points and
the presurgical measurements.
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Measurement of PAF in Endarterectomy Samples
From Patients With Severe Coronary Artery Disease
To determine if PAF accumulates in human atherosclerotic tissue,
23 endarterectomy samples were obtained from 16
patients with severe arterial disease, and the PAF in each
sample was extracted, purified, and assayed. As shown in Fig 4, a wide range of PAF levels (0.3 to 8.5 pg/mg wet wt)
was observed in these tissue samples. Like the canine CFV samples, the
PAF bioactivity in the purified endarterectomy
extracts was completely blocked by PAF receptor antagonists
during the platelet bioassay (data not shown). As a control, PAF
measurements were also performed on sections of internal mammary artery
obtained from five other patients during coronary bypass
surgery. These control samples contained an average of 1.7±0.8 pg/mg
wet wt of PAF (range, 0.8 to 2.7). When the two groups were compared,
16 of the 23 endarterectomy samples had PAF levels
greater than the control mean, 15 of these samples were at least
1.5-fold greater, and 8 samples were at least 2-fold greater than the
control samples. In addition, by comparison with a one-tailed 95%
confidence limit of 2.99 pg/mg wet wt calculated from the internal
mammary artery control samples, 8 of 23
endarterectomy samples exceeded this limit compared
with 0 of 5 control samples. Despite these descriptive differences,
statistical significance could not be demonstrated because of the large
sample variation (P=.125). Interestingly, PAF content varied
not only from patient to patient but also among multiple vessel samples
taken from the same patient. This observation, together with the
finding that PAF levels did not correlate with tissue mass, suggests
that PAF content is affected by factors other than the simple presence
of atherosclerotic tissue in the vessel.
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Identification of PAF by GC/MS
To further confirm the identity of the isolated
product, PAF was purified from four new
endarterectomy samples and converted to the
sn-3-pentafluorobenzoate derivative. The samples were then
analyzed by GC/MS by using selected ion monitoring. In this
analytical technique, the identification of a PAF molecular species is
based on both the retention time of the sample peak and the mass of the
molecular ion contained in that peak. As shown in the top panel of Fig 5, while monitoring a mass-to-charge ratio (m/z) of 552,
a peak of high intensity was observed eluting at 7.54 minutes. Based on
a comparison with authentic standard, this mass and retention time are
consistent with the presence of
1-O-hexadecyl-2-acetyl-sn-glycero-3-pentafluorobenzoate,
the derivative formed from C-16 PAF. The bottom panel in Fig 5
illustrates the elution profile obtained from 1 ng deuterated C-16 PAF
internal standard (m/z, 556) added before purification of the sample.
By comparing the relative areas of the sample and internal standard
peaks, the approximate mass of C-16 PAF in this sample was determined
to be 3.3 ng, or 39.8 pg/mg tissue wet wt. Similar analysis of
the other three samples yielded PAF values of 5.8, 1.0, and 7.7 pg/mg
wet wt (data not shown), further demonstrating the high degree of
variability in PAF content among human
endarterectomy samples previously observed when the
rabbit platelet bioassay was used (Fig 4
). Other results not shown
here suggest that a similar amount of
1-hexadecanoyl-2-acetyl-sn-glycero-3-phosphocholine (C-16
acyl-PAF) was also present in the samples. However, the presence of
other PAF molecular species (C-18) appears to be quantitatively less
significant. Because of the limited amounts of material from the canine
cyclic flow tissue extracts, GC/MS analysis was not performed
on these samples.
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The present study provides direct evidence that PAF accumulates at sites of vascular endothelial damage in a canine model of cyclic flow and also that PAF is present, albeit with a higher degree of variability, in atherosclerotic plaque from patients with severe coronary artery disease. The PAF in the tissue and blood samples was purified by using a carefully designed protocol that allowed us to circumvent several of the major difficulties encountered during PAF isolation. For example, residual PAF contamination of laboratory glassware, which produces high background assay values, was eliminated by an exhaustive base treatment and washing procedure. A second potential obstacle is the presence of PAF acetylhydrolase, which readily degrades PAF in blood and tissue samples.34 35 Since our samples were either immediately frozen in liquid N2 or combined with methanol/chloroform, the influence of this enzyme was minimized, as evidenced by the stability of [3H]PAF in the blood and tissue extracts. A third difficulty is that the newly formed PAF constitutes a tiny fraction of the tissue total lipid extract, thus necessitating a means of removing the milligram levels of extraneous lipid. In the present study, this problem was largely overcome by an initial chromatography step, which used carefully chosen conditions to optimize purification and recovery of PAF.
The identification of PAF in our samples was based on several criteria, including copurification with authentic PAF standard, inhibition of bioactivity by a PAF receptor antagonist, and recognition of PAF by a specific antibody during the RIA, and in the endarterectomy samples, by the additional technique of GC/MS. This latter method is advantageous because it can distinguish between authentic PAF and 1-acyl-2-acetyl-sn-glycero-3-phosphocholine, a structural analogue of PAF containing a fatty acyl group in the sn-1 position. In vitro experiments have demonstrated that synthesis of the 1-acyl compound often accompanies PAF production in a number of different cell types, including platelets and neutrophils.9 10 46 47 48 49 Although the 1-acyl analogue of PAF can elicit the same biological responses as its ether-linked counterpart, its potency is 500- to 1200-fold lower than PAF when compared in a platelet bioassay.50 51 Therefore, even though the 1-acyl analogue has a much lower bioactivity than authentic PAF and despite a 1600-fold greater selectivity of the PAF RIA for 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (Amersham PAF RIA documentation), the presence of the 1-acyl PAF analogue in the canine vessel extracts cannot be excluded at this time since they were not analyzed by GC/MS.
During coronary thrombosis, histological evidence has shown that several cell types are involved in the forming thrombus, including platelets and neutrophils.52 Since both of these cell types, as well as other cells in the blood, are capable of synthesizing PAF,8 9 10 11 12 13 a question is raised with regard to the cellular origin of PAF. From experiments with dogs, two lines of evidence suggest that the neutrophil is a predominant source. First, in a previous study32 designed to examine the effects of a PAF receptor antagonist on coronary cyclic flow, CFV was established and maintained in 10 dogs for an 8-hour period. PAF antagonist was administered at the 30-minute and 8-hour time points, and effects on the CFV were monitored. After the first bolus of drug at 30 minutes, cyclic flow was abolished in only 2 of 10 dogs. However, after the second bolus of drug at 8 hours, cyclic flow was eliminated in 6 of the remaining 8 dogs. This marked increase in drug effectiveness at 8 hours correlated with neutrophil recruitment into the area of endothelial damage. Therefore, this earlier study suggests that PAF synthesis in the canine cyclic flow model may be dependent on neutrophil infiltration into the area of endothelial damage, which in turn requires a sustained period of CFV. Experimental findings presented in the present study showing that PAF is elevated at the site of endothelial damage after 8 hours of cyclic flow support this concept. A second line of evidence implicating the neutrophil is derived from unpublished work in our laboratory, in which PAF synthesis in canine neutrophils and platelets was compared directly. In the presence of [3H]acetate, neutrophils produced 1800- to 6400-fold more radiolabeled product than platelets from the same animal on a per cell basis. Furthermore, structural analysis showed that the platelet product contained 75% to 85% of the less active 1-acyl-2-[3H]acetyl-sn-glycero-3-phosphocholine, whereas the product from the neutrophils contained only 10% to 20% of the 1-acyl analogue (data not shown). Therefore, although extrapolation of in vitro data to an in vivo setting is difficult because of other factors, such as differences in cell size and the relative density of each cell type in the forming thrombus, these data suggest that neutrophils may contribute a disproportionate amount of PAF.
Although PAF synthesis was elevated at the site of
endothelial damage and thrombosis in the canine cyclic
flow model, increases in PAF were not observed in blood samples taken
downstream from the forming thrombus. These findings suggest that the
synthesis and bioactions of nascent PAF may be limited to the immediate
area of endothelial injury during cyclic flow and that
PAF is not released systemically. However, in a previous study using
isolated buffer-perfused rabbit hearts, release of PAF into the
perfusate was reported after a 40-minute period of
ischemia.53 Interestingly, in a clinical study by
Montrucchio et al,54 blood PAF measurements were performed
on 11 patients who were admitted to the hospital with myocardial
infarction within 6 hours of chest pain onset and did not receive
thrombolytic therapy. Of these 11 patients, none had
detectable levels of PAF in their peripheral blood over a
24-hour sampling period. However, in 14 other patients who received
streptokinase, 10 exhibited transient increases of PAF in the blood,
suggesting that the therapy itself can induce PAF release. Since the
dogs in the present study had undergone major surgery during the
experiment, the possibility exists that PAF levels in the blood were
artificially elevated by thrombotic and inflammatory responses to the
surgical procedure and that any increases in blood-borne PAF after 8
hours of cyclic flow were masked by these high background levels. To
examine this possibility, a separate control experiment was performed
on three dogs to compare PAF in aortic blood before surgery and after 8
hours of CFV. After 8 hours of cyclic flow, two of the dogs had blood
PAF levels 1.4- and 1.8-fold higher than presurgical levels, whereas
the blood PAF concentration in the third dog did not change
significantly (data not shown). These results, together with the
increased numbers of blood neutrophils observed over 8 hours of cyclic
flow (Table), suggest that surgery may lead to a general inflammatory
response and increased levels of PAF in the blood of some dogs. At
first glance, this surgery-induced enhancement of PAF in the blood was
a concern, since the increased PAF synthesis observed at the site of
endothelial damage in the LADs (Fig 1) could
conceivably be an artifact of the inflammatory response. However, if
enhanced PAF levels in the blood were responsible for elevations of PAF
in the tissue samples, then a more generalized distribution of PAF
among all the vessel samples would be predicted rather than the
site-specific increases found in the damaged LADs. Furthermore, from
the experiment described above, the dog that showed no change in blood
PAF levels after surgery still exhibited augmented PAF synthesis in the
damaged LAD (2.5- to 4.1-fold over control vessel samples), thus
suggesting that site-specific elevations of PAF in the damaged LADs
were not an artifact of the postsurgical inflammatory response.
Through the use of metabolic inhibitors or
receptor antagonists or by direct measurement, a number of
biological compounds have been implicated in the regulation of CFV,
unstable angina, and vascular thrombosis. These mediators include
thromboxane A2,30 36 52 55 56 57 58
serotonin,30 59 60 61 62 ADP,63
thrombin,64 and 
2-adrenergic
agonists.61 Since CFV and thrombosis are likely the net
effect of these and other mediators working in concert, an unresolved
issue is the role of PAF relative to these other agonists. Previous in
vitro experiments have shown that PAF can activate rabbit and human
platelets in the presence of cyclooxygenase
inhibitors or ADP scavengers.11 65
Furthermore, in canine experiments in which cyclic flow was established
and subsequently blocked by administration of a thromboxane
synthetase inhibitor (UK38485), serotonin
receptor antagonist (ketanserin), or
2-adrenergic antagonist (yohimbine),
infusion of PAF was able to restore cyclic flow.33 Taken
together, these observations suggest that PAF may act independently of
these other mediators to regulate coronary thrombosis.
Alternatively, in another study using an open-chest pig model to
investigate the effects of PAF on coronary
hemodynamics, infusion of PAF into the coronary
vasculature caused significant reductions (
92%) in blood flow. These
reductions were attenuated 25% to 50% by pretreatment with an
antagonist to leukotrienes C4 and
D4 and were almost completely blocked by
indomethacin pretreatment.66 Therefore,
these results indicate that at least some of the vascular effects of
PAF may be mediated through secondary arachidonic acid
metabolites in this model. Although further work will be needed to
resolve the biochemical mechanisms by which PAF exerts its effects,
data presented here provide direct evidence that this potent
mediator is synthesized during coronary thrombosis in the
canine cyclic flow model. This evidence, together with the finding that
PAF accumulates in human atherosclerotic tissue, provides a firm basis
for further investigation into the involvement of PAF in the regulation
of coronary thrombosis, cyclic flow, and vascular disease.
|
Acknowledgments |
|---|
This study was supported by a grant from the American Heart Association, Texas Affiliate, Inc, and by grants HL-17669 and HL-50179 from the National Heart, Lung, and Blood Institute. The authors thank Drs H. Vernon Anderson and Michael S. Sweeney for their help in obtaining human endarterectomy samples and Judy Ober and Dr Salman Akhtar for their help in some of the cyclic flow experiments.
Received January 4, 1995; accepted March 9, 1995.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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 D. C. Tsoukatos, M. Arborati, T. Liapikos, K. L. Clay, R. C. Murphy, M. J. Chapman, and E. Ninio Copper-Catalyzed Oxidation Mediates PAF Formation in Human LDL Subspecies : Protective Role of PAF:Acetylhydrolase in Dense LDL Arterioscler Thromb Vasc Biol, December 1, 1997; 17(12): 3505 - 3512. [Abstract] [Full Text]
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