Continuous binding of the PAF molecule to its receptor is
necessary for the long-term aggregation of platelets
Masahiro
Suzuki,
Junko
Sugatani,
Mitsuhiro
Ino,
Masahiko
Shimura,
Masaki
Akiyama,
Ryuta
Yamazaki,
Yasuo
Suzuki, and
Masao
Miwa
Departments of Pharmaco-Biochemistry and Biochemistry, School of
Pharmaceutical Science, University of Shizuoka, Shizuoka 422, Japan
 |
ABSTRACT |
Human and rabbit platelets fully aggregated by
platelet-activating factor (PAF) underwent slow disaggregation but were
rapidly disaggregated by the PAF receptor antagonists WEB-2086,
Y-24180, SM-12502, and CV-3988. Whereas the
1-alkyl-2-[3H]acetyl-sn-glycero-3-phosphocholine
([3H]acetyl-PAF)
specifically bound to platelet receptors underwent slow and spontaneous
dissociation, it dissociated promptly from its receptor when WEB-2086
was added, in parallel with platelet disaggregation and disappearance
of P-selectin on the cell surface. Extracellular
[3H]acetyl-PAF was
rapidly deacetylated by normal rabbit platelets; some of the
[3H]acetyl-PAF was
bound to the cells and a very small amount of [3H]acetate was
detected in the cells. In contrast, when
1-[3H]alkyl-2-acetyl-sn-glycero-3-phosphocholine
was added to the platelets, the radioactivity was rapidly incorporated
into the 1-alkyl-2-acyl-sn-glycero-3-phosphocholine
fraction. These results indicate that
1) continuous binding of PAF to its
receptor is necessary for prolonged platelet aggregation, which may be
mediated through an unknown signaling system for a long-term cell
response rather than a transient signaling system, and
2) most of the
[3H]acetyl-PAF bound
to platelets is metabolized extracellularly by ecto-type PAF
acetylhydrolase, with the lyso-PAF generated being incorporated rapidly
into the cells and converted to
1-alkyl-2-acyl-sn-glycero-3-phosphocholine.
rabbit; receptor; antagonist; polymorphonuclear leukocyte; platelet-activating factor
 |
INTRODUCTION |
PLATELET-ACTIVATING FACTOR (PAF,
1-alkyl-2-acetyl-sn-glycero-3-phosphocholine)
is a lipid mediator with a structure similar to phospholipids, which
are the main components of the membrane lipid bilayer (6, 10). PAF has
potent biological effects, one of which is to stimulate platelets
through its specific receptor (12, 24). The binding of PAF to platelet
receptors triggers intracellular responses such as activation of
phospholipases C, D, and A2 and
many kinases (e.g., protein kinase C, mitogen-activated protein kinase,
G protein receptor kinase, and protein tyrosine kinase) and increases
in cytosolic Ca2+ concentration,
which have been considered to occur transiently in association with
platelet aggregation and serotonin secretion (12, 25). On the other
hand, it has been reported that PAF target cells such as platelets and
polymorphonuclear leukocytes (PMNs) rapidly incorporate exogenous PAF
and convert it to
1-alkyl-2-acyl-sn-glycero-3-phosphocholine via
1-alkyl-2-lyso-sn-glycero-3-phosphocholine
(lyso-PAF). The mechanism responsible involves specific binding of PAF
to its receptor or nonspecific binding to the membrane, its
internalization across the plasma membrane, and its deacetylation by
cytosolic PAF acetylhydrolase (PAF-AH), the resulting lyso-PAF being
converted to alkylacylglycerophosphocholine by transacylase (7, 14, 16,
23). However, it remains to be clarified how PAF molecules pass through
the lipid bilayer of the plasma membrane and become internalized into
PAF target cells, although it has been reported that cell stimulation
enhances transbilayer movement (flipping) (3). Surprisingly, in the
present study, we have demonstrated that platelets fully aggregated by
PAF disaggregate in parallel with the dissociation of PAF from its
receptor. That is, continuous binding of PAF to its receptor is
necessary for prolonged platelet aggregation. The aim of the present
experiments was to clarify in more detail the binding behavior of PAF
to its receptor, which drives the platelet signaling system that causes
shape change and aggregation of platelets.
 |
MATERIALS AND METHODS |
Materials.
1-Hexadecyl-2-acetyl-sn-glycero-3-phosphocholine
(PAF) and
1-hexadecyl-2-N-methylcarbamyl-sn-glycero-3-phosphocholine
(C-PAF) were products of Bachem Feinchemikalien (Bubendorf,
Switzerland) and Calbiochem-Novabiochem (Tokyo, Japan),
respectively.
1-[3H]hexadecyl-2-acetyl-sn-glycero-3-phosphocholine
(1.11 TBq/mmol, [3H]alkyl-PAF),
1-hexadecyl-2-[3H]acetyl-sn-glycero-3-phosphocholine
(370 GBq/mmol,
[3H]acetyl-PAF), and
EN3HANCE spray were purchased from
New England Nuclear Japan (Tokyo). [U-14C]acetic acid
sodium salt (2.1 GBq/mmol) was purchased from American Radiolabeled
Chemicals (St. Louis, MO). WEB-2086
{3-[4-(2-chlorphenyl)-9-methyl-6H-thieno[3, 2-f ][1, 2, 4]triazolo[4,3-a][1,4]diazepin-2-yl]-1-(4-morpholinyl)-1-propanone; Boehringer Ingelheim}, Y-24180
{4-(2-chlorophenyl)-2-[2-(4-
isobutylphenyl)ethyl]-6,9-dimethyl-6H-thieno[3,2-f ][1,2,4]triazolo[4,3-a][1,4]diazepine;
Yoshitomi Pharmaceuticals}, SM-12502
[(+)-cis-3,5-dimethyl-2-(3-pyridyl)thiazolidin-4-one hydrochloride; Sumitomo Pharmaceuticals], and CV-3988
[(rac-3-N-n-octadecylcarbamoyloxy)-2-methoxypropyl 2-thiazolioethyl phosphate; Takeda Chemical Industries] were
dissolved in saline containing 0.1% bovine serum albumin (BSA). Mouse
anti-human P-selectin monoclonal antibody and peroxidase-conjugated
affinity-purified F(ab')2
fragment goat anti-mouse immunoglobulin (Ig) G plus IgM antibody were
obtained from Cambridge Research Biochemicals and Jackson Immuno
Research Laboratories, respectively. Ficoll-Paque and Sepharose 2B were
from Pharmacia. Cycloheximide and actinomycin D were purchased from
Sigma Chemical (St. Louis, MO) and P-L Biochemicals (Milwaukee, WI),
respectively. Chloramphenicol and proteinase K (1,000 U/mg) were
products of Wako Chemical (Tokyo, Japan). Solvents were all of reagent
grade.
Buffers.
Buffers used were as follows: 1)
basic Tyrode solution (consisting of 8.0 g/l NaCl, 0.195 g/l KCl, 0.215 g/l
MgCl2 · 6H2O, 1.02 g/l NaHCO3, and 1.0 g/l
glucose, pH 7.2); 2) Tyrode-gelatin solution without Ca2+ and with
EDTA (same as buffer 1 but containing
2.5 g/l gelatin and 0.373 g/l
Na2EDTA, pH 7.2);
3) Tyrode-gelatin solution with Ca2+ (same as
buffer 1 but containing 0.143 g/l
CaCl2 · 2H2O,
pH 7.2); and 4) 0.05 M
tris(hydroxymethyl)aminomethane (Tris) · HCl-buffered saline with EDTA (consisting of 6.058 g/l Tris, 8.766 g/l NaCl, 0.373 g/l Na2EDTA, and 1.0 g/l glucose,
pH 7.2).
Preparation of rabbit and human platelets and PMNs.
Plastic containers or siliconized glassware was used for all platelet
preparation and stimulation procedures. Washed rabbit platelets were
obtained by a modification of the procedure described by Pinckard et
al. (24). Each 45-ml portion of rabbit or healthy human donor blood was
withdrawn into 5 ml of 41 mM citric acid-85 mM trisodium citrate-2%
glucose solution and centrifuged at 170 g for 10 min. The supernatant
platelet-rich plasma was underlayered with 10 ml of Ficoll-Paque and
then centrifuged at 750 g for 20 min.
The platelet layer was mixed gently with 40 ml of 0.05 M Tris · HCl-buffered saline containing EDTA (pH 7.2)
underlayered with 0.2 ml of Ficoll-Paque and centrifuged at 750 g for 10 min. The platelet layer was
washed three more times as described above. The platelet layer was
again suspended in 40 ml of 0.05 M
Tris · HCl-buffered saline containing EDTA (pH 7.2)
and centrifuged at 750 g for 10 min.
The pellet was resuspended in Tyrode-gelatin buffer (pH 7.2) at a
concentration of 1.25 × 109
cells/ml. Although significant activity of serum PAF-AH remained in the
platelet suspension prepared by the original method, in this study,
PAF-AH activity was not detectable in the washing solution after five
washes. Rabbit and human PMNs were prepared as described previously
(15).
For determination of P-selectin in intact human platelets, gel-filtered
platelets were employed to avoid the influence of possible plasma
inhibitors. Platelet-rich plasma (6 ml) was applied to a Sepharose 2B
column (1.5 × 23 cm), which was equilibrated with Tyrode buffer
(pH 7.2). Fractions containing platelets appeared in the void volume of
the column and were pooled so as to contain 2 × 108 cells/ml.
Platelet aggregation assay.
Platelets (1 × 108 cells,
400 µl) were stimulated in Tyrode-gelatin buffer (pH 7.2) containing
1 mM Ca2+ and 6 mM acetate.
Aggregation activity was measured as the change in light transmission
using an aggregometer (Nikko Hematracer, PAT-2A). The PAF antagonist
WEB-2086 in 0.1% BSA-saline was added to the system 1 min before
addition of the PAF solution.
Immunoassay for P-selectin.
The amount of P-selectin expressed on the platelet cell surface was
determined by enzyme-linked immunosorbent assay. Forty microliters of
gel-filtered human platelets (5 × 105 cells) in Tyrode-gelatin
buffer (pH 7.2) containing 1 mM
Ca2+ and 0.1% BSA were incubated
with 10 µl of 2.5 × 10
6 M PAF plus 2.5 × 10
5 M ADP for 3 min at
25°C in 96-well immunosorbent assay plates (Linbo/Titertek, Flow
Laboratories, McLean, VA). Next, 10 µl of 6 × 10
4 M WEB-2086 in saline or
saline as a control were added to the platelets. After 2 min of
incubation, the reaction was stopped by adding 60 µl of 2% formalin,
and the plates were centrifuged for 3 min at 170 g to allow the platelets to adhere to
the wells. The wells were then washed three times in Tyrode-gelatin
buffer (pH 7.2) containing 1% BSA and incubated with 100 µl of the
same buffer for 3 h. The wells were incubated with 50 µl of
1,000-fold-diluted mouse anti-human P-selectin monoclonal antibody for
1 h at 25°C. After wells were washed three times with the same
buffer, they were incubated with 50 µl of 1,000-fold-diluted
peroxidase-conjugated goat anti-mouse IgG plus IgM antibody for 1 h at
25°C and then washed five times with phosphate-buffered saline.
Finally, the wells were incubated with ortho-phenylenediamine in
citrate-phosphate buffer (pH 5.0) containing 0.01%
H2O2
for 15 min. The optical density of the wells at 492 nm was determined
in a Corona microplate reader (MTP-32, Corona Electric).
Measurement of PAF-AH activity by the trichloroacetic acid
precipitation method.
Intact rabbit platelets (1 × 108 cells, 390 µl) in
Tyrode-gelatin buffer (pH 7.2) containing 1 mM
Ca2+, 6 mM acetate, and 0.1% BSA
either with or without 6.2 × 10
7 M WEB-2086 [or in
Tyrode-gelatin buffer (pH 7.2) containing 1 mM EDTA and 6 mM acetate
either with or without 0.1% BSA], were incubated with 10 µl of
4 × 10
8 M
[3H]acetyl-PAF in
0.1% BSA-saline solution at 37°C for the desired periods. The
amount of [3H]acetate
released was determined essentially as reported previously (19). The
reaction was stopped by addition of 20 µl of 7% BSA-saline solution,
and the mixture was left to stand for 10 min at 0°C. After 80 µl
of 60% trichloroacetic acid (TCA) were added, the reaction mixture was
centrifuged for 5 min at 750 g to
separate the denatured protein. Four hundred microliters of the
supernatant were mixed with 4 ml of scintillation cocktail (Aquasol-2,
DuPont, Boston, MA), and the radioactivity was determined in a liquid scintillation counter (LSC-3100, Aloka, Tokyo, Japan).
Quantitation of intracellular and extracellular
[3H]acetyl-PAF and released
[3H]acetate.
After incubation with 1 × 10
9 M
[3H]acetyl-PAF as
described above, the platelets were cooled on ice,
underlayered with 20 µl of Ficoll-Paque, and then centrifuged at
10,000 g for 1 min at 4°C. The
radioactivity of the supernatant was considered to represent the amount
of extracellular
[3H]acetyl-PAF and
[3H]acetate. The
amount of extracellular
[3H]acetate was
determined by measuring the radioactivity of 400 µl of the
supernatant of a TCA-treated platelet mixture from the first
(Ficoll-Paque) spin that had been mixed with 20 µl of 7% BSA-saline
solution and 80 µl of 60% TCA solution and centrifuged at 750 g for 10 min at 4°C. The cell
pellet from this treatment was resuspended in Tyrode-gelatin buffer (pH
7.2) containing 0.1% BSA and 6 mM acetate and centrifuged at 750 g for 10 min. The pellet was again
resuspended in 500 µl of 0.01 M Tris · HCl buffer (pH 7.0) containing 5 mM MgCl2, 6 mM acetate, and 2 mM EDTA. The radioactivity of the cell suspension was
considered to represent the amount of intracellular
[3H]acetyl-PAF and
[3H]acetate. The
amount of intracellular
[3H]acetate was
determined by measuring the radioactivity of the supernatant, obtained
after a freeze thawed cell suspension had been mixed with TCA and
centrifuged as described above. The amount of unreacted extracellular
[3H]acetyl-PAF and
intracellular
[3H]acetyl-PAF was
calculated by subtracting the amount of
[3H]acetate released.
The amount of
[3H]acetyl-PAF bound
specifically to the platelet receptor was calculated by subtracting the
total 3H radioactivity in the
platelets in the presence of 1 × 10
7 M nonradioactive PAF
from that in the absence of 1 × 10
7 M nonradioactive PAF.
Determination of
[3H]alkyl-PAF and its
radioactive metabolites bound to rabbit platelets.
Platelets (2.5 × 108 cells)
were incubated with 1 × 10
9 M
[3H]alkyl-PAF in
either the presence or absence of 6.2 × 10
7 M WEB-2086 in 1.0 ml of
Tyrode-gelatin buffer (pH 7.2) containing 1 mM
Ca2+, 6 mM acetate, and 0.1% BSA
at 37°C for the desired periods, and then 0.4 ml of the cell
suspension was layered on a 40-µl mixture of dibutyl phthalate and
dinonyl phthalate (5:3, vol/vol) in a microcentrifuge tube and promptly
centrifuged at 10,000 g (TOMY MRX-150,
Tokyo, Japan) for 1 min. After the cell pellet was washed with 0.05 M
Tris · HCl-buffered saline containing 1 mM EDTA and
0.1% BSA (pH 7.2), the cell pellet was dissolved in 100 µl of 1%
Triton X-100 and then the radioactivity of the supernatant plus the
washing fluid and the radioactivity of the cell pellet were measured as
extracellular and intracellular
[3H]alkyl-PAF,
respectively.
Measurement of intracellular
[14C]acetate released from
intact rabbit platelets.
Platelets (1 × 109 cells, 1 ml) in Tyrode-gelatin buffer (pH 7.2) were incubated with 36.8 mM
[U-14C]sodium acetate
(2.1 GBq/ml) dissolved in saline for 20 min at 37°C. The platelets
were then washed three times with 0.05 M
Tris · HCl-buffered saline containing 1 mM acetate
(pH 7.2) and once with 0.05 M Tris · HCl-buffered
saline without acetate (pH 7.2), followed by resuspension in
Tyrode-gelatin buffer (pH 7.2) or Tyrode-gelatin buffer (pH 7.2)
containing 6 mM acetate. The
[14C]acetate-loaded
platelets were incubated with or without 1 × 10
9 M PAF for 20 min, and
then the amount of
[14C]acetate released
from the cells was determined by measuring the radioactivity of the
supernatant after centrifugation at 10,000 g for 2 min.
Isolation of rabbit platelet membrane and cytosol fractions.
The platelet cytosolic fraction and membrane fraction were prepared
essentially according to the procedure of Kramer et al. (14). Platelets
were washed 5 times and then suspended in 0.05 M
Tris · HCl buffer (pH 7.2) containing 2 mM EDTA and
were lysed by freeze thawing 3 times and sonicating 10 times for 15 s
at 4°C in a Bioruptor sonicator (UCD-200TM, COSMO-BIO, Tokyo,
Japan). The cytosol and membrane fractions were separated by
centrifugation at 105,000 g for 1 h at
4°C, and the resulting supernatant fraction represented the
cytosol. The pellet from this centrifugation was resuspended in 0.05 M
Tris · HCl buffer (pH 7.2) containing 2 mM EDTA and
centrifuged again at 105,000 g for 1 h
at 4°C. The resulting pellet was resuspended in 0.05 M
Tris · HCl-buffered saline containing 1 mM EDTA (pH
7.2) and considered to be the membrane fraction.
Treatment of intact rabbit platelets and plasma with proteinase K.
Rabbit platelets (2.5 × 108
cells, 1 ml) in Tyrode-gelatin buffer (pH 7.2) containing 2 mM EDTA and
rabbit plasma were incubated with proteinase K at 37°C. After
incubation for 90 min, the cell suspension was layered on 200 µl of
50% Ficoll-Paque-saline and 15 µl of Ficoll-Paque in a
microcentrifuge tube at 0°C and then promptly centrifuged at 10,000 g for 1 min at 4°C. The
supernatant was transferred carefully, and then the platelets (lower
phase) were washed three times with 900 µl of Tyrode-gelatin buffer
(pH 7.2) containing 2 mM EDTA and 0.2% BSA and two times with 900 µl
of Tyrode-gelatin buffer (pH 7.2) containing EDTA without BSA under the
same conditions as those described in Quantitation of intracellular and extracellular [3H]acetyl-PAF and
released [3H]acetate. The platelet
pellet was resuspended in 1 ml of 8 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic
acid (HEPES)-buffered saline containing 2 mM EDTA, 6 mM acetate, and 5 mM glucose (pH 7.2). After 800 µl of the suspension of proteinase
K-treated platelets were freeze thawed 3 times, the mixture was
sonicated 10 times for 15 s at 4°C. The sonicate was then
centrifuged at 105,000 g for 60 min at
4°C. The resulting supernatant was designated the platelet cytosol
fraction, and the corresponding pellet, representing the platelet
membrane fraction, was resuspended in 8 mM HEPES-buffered saline
containing 2 mM EDTA, 6 mM acetate, and 5 mM glucose (pH 7.2).
Identification of
[3H]acetyl-PAF and its
radioactive metabolites bound to rabbit platelets.
Platelets (2.5 × 108 cells,
1 ml) were incubated with 1 × 10
9 M
[3H]acetyl-PAF in
Tyrode-gelatin buffer (pH 7.2) containing 1 mM Ca2+, 6 mM acetate, and 0.1% BSA
either with or without 6 × 10
7 M WEB-2086. At the
indicated times, the reaction mixture was underlayered with 40 µl of
Ficoll-Paque and centrifuged at 10,000 g for 1 min. The supernatant was
removed carefully, and then the pellet was resuspended in 1 ml of 0.05 M Tris · HCl buffer (pH 7.2). The lipids in the cells
were extracted by the method of Bligh and Dyer (1). Eight hundred
microliters of supernatant were transferred to another tube, and
CHCl3 and
CH3OH were added to bring the
mixture ratio to 1:2:0.8
(CHCl3/CH3OH/H2O).
After centrifugation at 750 g for 10 min, the supernatant was mixed with 1 ml of
CHCl3 and 1 ml of
H2O. The chloroform phase was
removed, and another 2 ml of CHCl3
were added to the aqueous phase. After vigorous mixing, the chloroform
phase was removed and combined with the first fraction. The lipids in
the chloroform extract were separated on a silica gel G plate using a
solvent system of
CHCl3/CH3OH/H2O
(65:35:7, vol/vol/vol). Authentic sphingomyelin, PAF, and
lysophosphatidylcholine were applied to both sides of the samples on
the plate. After plates were exposed to
EN3HANCE, the radioactivity was
determined with an ultrahigh-sensitivity television camera system
(ARGUS-100) (18). The positions of sphingomyelin, PAF, and
lysophosphatidylcholine were detected using Dittmer's reagent (8).
 |
RESULTS |
Disaggregation of PAF-aggregated platelets aggregation and its
enhancement by WEB-2086.
Rabbit platelets aggregated by 1 × 10
9 M PAF and secreted
serotonin showed initial dissociation after they had been fully
aggregated for ~5 min and became almost completely disaggregated
after 2 h of incubation with PAF (Fig.
1A).
These dissociated platelets showed a fully restored aggregatory
response to PAF (Fig. 1B). Although
their specific desensitization to further challenge with PAF was noted
immediately after exposure to PAF, as reported previously (11),
the aggregatory response to PAF was restored time dependently (Fig. 1B).

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Fig. 1.
Restoration of platelet-activating factor (PAF)-inducible aggregation
activity in PAF-treated and -desensitized platelets. Washed rabbit
platelets (1 × 108 cells,
400 µl) in Tyrode-gelatin buffer (pH 7.2) containing 1 mM
Ca2+ were incubated with 1 × 10 9 M PAF
(A) or 1 × 10 10 M PAF
(B) at 37°C. After incubation
for periods of 30, 60, 90, and 120 min, 1 × 10 9 M PAF
(A) or 1 × 10 10 M PAF
(B) was added to the platelets.
Change in light transmission was monitored by an aggregometer (Nikko
Hematracer, PAT-2A).
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|
Furthermore, the PAF receptor antagonist WEB-2086 was found to cause
rapid disaggregation of rabbit platelets that had been aggregated by 1 × 10
9 M PAF (Fig.
2A). The
disaggregation effect occurred regardless of the time of addition of 6 × 10
7 M WEB-2086, but
the disaggregation rate was dependent on the concentration of WEB-2086
to some extent. WEB-2086 also caused disaggregation of human platelets
in platelet-rich plasma after aggregation by 5 × 10
7 M PAF (Fig.
2B). We further investigated the
effect of other PAF receptor antagonists on disaggregation of
PAF-aggregated rabbit platelets. The relative potencies of these
antagonist against platelet disaggregation are given in Table
1. The antagonists inhibited the platelet
aggregation induced by PAF at a concentration of 1 × 10
9 M with the following
rank order of potency: Y-24180 > WEB-2086 > SM-12502 > CV-3988.
This order reflects the inhibitory effect of the agents on platelet
aggregation induced by PAF. On the other hand, these PAF receptor
antagonists did not inhibit disaggregation of platelets aggregated by
ADP and thrombin (data not shown). These observations demonstrate that
the binding of PAF to rabbit and human platelets is reversible and that
the disaggregation induced by PAF receptor antagonists is specific to
platelets aggregated by PAF.

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Fig. 2.
Disaggregation of PAF-aggregated platelets by WEB-2086.
A: washed rabbit platelets (1 × 108 cells, 400 µl) in
Tyrode-gelatin buffer (pH 7.2) containing 1 mM
Ca2+ and 0.1% bovine serum
albumin (BSA) were stimulated with 1 × 10 9 M PAF. WEB-2086 (6 × 10 7 M) was added to
the aggregated platelets at times indicated by arrows.
B: human platelet-rich plasma (1 × 108 cells, 400 µl) was
incubated with 1 × 10 6 M PAF followed by
addition of WEB-2086 (1 × 10 4 M) at times indicated
by arrows.
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|
Next, to investigate whether PAF bound to platelets is displaced by
WEB-2086, rabbit platelets were incubated with or without 5 × 10
9 M PAF for 5, 10, and 20 min, and then 6 × 10
7
M [3H]WEB-2086 was
added to the platelet suspension, followed by incubation for an
additional 1 min. The amounts of
[3H]WEB-2086 bound to
platelets (1.1 ± 0.1, 1.1 ± 0.2, and 1.3 ± 0.1 pmol/108 cells incubated with PAF
for 5, 10 and 20 min, respectively) increased time dependently and were
close to those in the control platelets (1.3 ± 0.1 pmol/108 cells). In addition,
WEB-2086 dissociated
[3H]acetyl-PAF bound
to rabbit platelets in a dose-dependent manner; 6 × 10
7 M WEB-2086 had the
maximal effect on platelets aggregated by 1 × 10
9 M PAF. Furthermore, to
investigate the possibility that new PAF receptors might be synthesized
during PAF stimulation, we examined the effect of RNA-protein synthesis
inhibitors on the aggregatory response of platelets to PAF. Rabbit
platelets were incubated separately with 1 × 10
5 M actinomycin D, 1 × 10
4 M
cycloheximide, and 1 × 10
4 M chloramphenicol at
25°C for 60 min and then stimulated with 1 × 10
9 M PAF. Thirty and sixty
minutes after exposure to 1 × 10
9 M PAF, the aggregatory
responses of actinomycin D-, cycloheximide-, and
chloramphenicol-treated platelets to further challenge with 1 × 10
9 M PAF were almost the
same as those of control platelets (data not shown).
Disappearance of P-selectin on platelet cell surface during
disaggregation by WEB-2086.
Because intracellular Ca2+ change
is involved in platelet activation, the effect of WEB-2086 on
PAF-induced intracellular Ca2+
change during the process of disaggregation was examined using fura
2-loaded platelets. In platelets aggregated by PAF, a transient increase in intracellular Ca2+
caused by PAF was found, but there was no significant change in
platelets disaggregated by WEB-2086 (data not shown). On the other
hand, P-selectin (platelet activation-dependent granular external
membrane protein), which has been reported to be expressed on the cell
surface of activated platelets (5, 17), was confirmed to be expressed
in our experiments using PAF (Fig. 3). In
platelets aggregated by 1 × 10
9 M PAF, the expression
of P-selectin was increased, and the addition of 6 × 10
7 M WEB-2086 to the
platelets reduced the amount of P-selectin expressed on the cell
surface (Fig. 3).

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Fig. 3.
Effect of WEB-2086 on PAF-induced P-selectin expression on surface of
human platelets. Gel-filtered human platelets (5 × 105 cells, 50 µl) in
Tyrode-gelatin buffer (pH 7.2) containing 1 mM
Ca2+ and 0.1% BSA were
preincubated with or without 5 × 10 7 M PAF plus 5 × 10 6 M ADP for 3 min at
37°C, and then WEB-2086 (1 × 10 4 M) in saline (or saline
alone as a control) was added to the platelets. After 2 min of
incubation, platelets were fixed with 60 µl of 2% formalin and
amounts of P-selectin expressed on the platelet surface were measured
as described in MATERIALS AND METHODS.
Values are expressed as means ± SD for 4 experiments.
OD492, optical density at 492 nm.
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Dissociation of
[3H]acetyl-PAF from aggregated
rabbit platelets.
To clarify the relationship between disaggregation by WEB-2086 and
dissociation of PAF from platelets, we investigated the effect of
WEB-2086 on the binding of PAF to rabbit platelets. The amount of
[3H]acetyl-PAF bound
to platelet receptors, calculated by subtracting the radioactivity of
the platelets in the presence of 1 × 10
7 M nonradioactive PAF
from that in the absence of 1 × 10
7 M nonradioactive PAF,
reached a maximum at 5 min and then decreased gradually (Fig.
4). WEB-2086 promptly lowered the amount of
[3H]acetyl-PAF bound
to platelet receptors. The dissociation effect occurred regardless of
the time of addition of WEB-2086, consistent with the disaggregation
effect described above. Interestingly, addition of WEB-2086 to
[3H]acetyl-PAF-aggregated
platelets increased the amount of extracellular [3H]acetate in the
assay medium, in parallel with the dissociation of PAF from the
platelets (Fig. 4).

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Fig. 4.
Effect of WEB-2086 on deacetylation of
[3H]acetyl-PAF in
intact rabbit platelets. Rabbit platelets (1.0 × 108 cells) in Tyrode-gelatin
buffer (pH 7.2) containing 1 mM
Ca2+, 6 mM acetate, and 0.1% BSA
were incubated with 1 × 10 9 M
[3H]acetyl-PAF. As
indicated by arrows, WEB-2086 (6 × 10 7 M, open symbols) was
added to the aggregated platelets 5 (A), 10 (B), and 20 (C) min after addition of PAF.
Extracellular and intracellular
[3H]acetate was
measured by the TCA precipitation method, and amount of
[3H]acetyl-PAF bound
specifically to platelets was calculated as described in
MATERIALS AND METHODS. Results are
expressed as means ± SD for 3 or 4 experiments. Solid symbols
represent measurements without WEB-2086.
|
|
Hydrolysis of exogenous PAF by ecto-type PAF-AH in rabbit platelets.
Rabbit platelets aggregated by C-PAF, which is resistant to PAF-AH
(22), were also found to disaggregate spontaneously (Fig. 5). C-PAF at 5 × 10
10 M had almost
the same aggregatory activity as 2 × 10
10 M PAF, but the rate of
disaggregation of platelets aggregated by C-PAF was rather lower than
that of platelets aggregated by PAF. The disaggregation of rabbit
platelets aggregated by 5 × 10
10 M C-PAF was enhanced
by WEB-2086, similarly to that induced by 2 × 10
10 M PAF. Thus, to
elucidate whether most of the exogenous PAF is deacetylated by
ecto-type PAF-AH, the metabolic fate of
[3H]acetyl-PAF and
[3H]alkyl-PAF was
investigated in the absence or presence of WEB-2086. The cells were
washed five times until PAF-AH activity in the washing buffer was
undetectable to avoid the influence of extracellular (plasma) PAF-AH.
In the absence of WEB-2086, the amount of incorporated [3H]acetyl-PAF and its
metabolites reached a maximum (~40% of the initial amount) at 3 min
and then decreased gradually, whereas the amount of incorporated
[3H]alkyl-PAF and its
metabolites increased with time (Fig. 6,
A and
C). Extracellular
[3H]acetate in the
medium also increased with time, but a trace amount of intracellular
[3H]acetate was
detectable during 30 min of incubation. On the other hand, when rabbit
platelets were incubated with 1 × 10
9 M
1-hexadecyl-2-[3H]arachidonoyl-sn-glycero-3-phosphocholine,
no hydrolysis of
[3H]arachidonic acid
was detected (data not shown). This ruled out the possibility that
phospholipase A2 participated in
the deacetylation of PAF. To further examine the possibility that
radioactive acetate accumulated in the cells had leaked into the
medium,
[14C]acetate-loaded
platelets were incubated in Tyrode-gelatin buffer (pH 7.2) or
Tyrode-gelatin buffer (pH 7.2) containing 6 mM acetate with or without
1 × 10
9 M PAF. The
amount of intracellular
[14C]acetate released
into the medium from both untreated cells and cells treated with PAF
was <5% of the
[14C]acetate
incorporated intracellularly.

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Fig. 5.
Disaggregation of platelets aggregated by PAF and
1-hexadecyl-2-N-methylcarbamyl-sn-glycero-3-phosphocholine
(C-PAF). Washed rabbit platelets (1 × 108 cells, 400 µl) in
Tyrode-gelatin buffer (pH 7.2) containing 1 mM
Ca2+ were incubated with 2 × 10 10 M PAF
(A) or 5 × 10 10 M C-PAF
(B).
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|

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Fig. 6.
Metabolic fate of radioactive
[3H]acetyl-PAF
(A and
B) and
[3H]alkyl-PAF
(C and
D) in rabbit platelets in the
absence (A and
C) and presence
(B and
D) of WEB-2086.
A and
C: rabbit platelets (2.5 × 108 cells) were incubated with 1 × 10 9 M
[3H]acetyl-PAF
(A) or
[3H]alkyl-PAF
(C) in 1.0 ml of Tyrode-gelatin
buffer (pH 7.2) containing 1 mM
Ca2+, 6 mM acetate, and 0.1% BSA
at 37°C. B and
D: after preincubation with 6 × 10 7 M WEB-2086 for 5 min,
rabbit platelets were incubated with 1 × 10 9 M
[3H]acetyl-PAF
(B) or
[3H]alkyl-PAF
(D) in 1.0 ml of Tyrode-gelatin
buffer containing 1 mM Ca2+, 6 mM
acetate, and 0.1% BSA (pH 7.2) at 37°C. At indicated times, cells
were separated from supernatant by centrifugation of the cell
suspension underlayered by a mixture of dibutyl phthalate and dinonyl
phthalate (5:3, vol/vol), and then radioactivity of supernatant and
cells was determined as described in MATERIALS AND
METHODS. Amount of extracellular and intracellular
[3H]acetate released
from [3H]acetyl-PAF
was measured by the TCA precipitation method, as described in
MATERIALS AND METHODS. Extracellular
[3H]acetate; ,
intracellular
[3H]acetate; ,
extracellular
[3H]acetyl-PAF; ,
[3H]acetyl-PAF bound
to cells; , extracellular
[3H]alkyl-PAF; ,
[3H]alkyl-PAF and its
metabolites bound to cells. Results are expressed as means ± SD for
3 or 4 experiments.
|
|
On the other hand, when platelets were incubated with
10
9 M
[3H]acetyl-PAF in the
presence of WEB-2086, the amount of incorporated [3H]acetyl-PAF and its
metabolites decreased to one-third of the amount of intracellular
3H-labeled radioactive lipids in
the absence of WEB-2086 (Fig. 6, A and
B), whereas the amount of
incorporated
[3H]alkyl-PAF and its
metabolites decreased slightly (Fig. 6,
C and
D). In contrast, the amount of
[3H]acetate released
from [3H]acetyl-PAF
into the medium was decreased slightly by WEB-2086. In addition, the
amount of incorporated
[3H]alkyl-PAF was
equal to the total amount of
[3H]acetyl-PAF
incorporated plus the amount of
[3H]acetate released
in the absence and presence of WEB-2086 (Fig. 6,
A-D).
To investigate the possibility that the cytosol PAF-AH released from
intact platelets hydrolyzed extracellular PAF, the deacetylation activity of intact platelets and the membrane and cytosol fractions was
compared by measuring released
[3H]acetate using 1 × 10
9 M
[3H]acetyl-PAF. The
assay was conducted in the presence of 0.1% BSA to avoid any influence
of PAF binding to the membrane (9). The deacetylation activity of the
cytosol fraction was less than one-tenth of the activity of intact
platelets and about one-fifth of the activity of the membrane fraction.
Figure 7 shows plots of PAF deacetylation
activity by intact platelets and the membrane and cytosol fractions
against substrate concentration. The double reciprocal plots revealed a
Michaelis-Menten constant
(Km) value of
1.3 × 10
5 M for
cytosolic PAF-AH and one of 1.1 × 10
6 M for membrane-bound
PAF-AH; thus the PAF-AH of the membrane fraction differed considerably
from that of the cytosol fraction. Because a high concentration of PAF
destroys cells, the apparent Km for intact
platelets could not be obtained. However, the deacetylation activities
of intact platelets at 10
9
M to 10
8 M were similar to
those of the membrane fraction. These results ruled out the possibility
that cytosolic PAF-AH participated in the inactivation of exogenous PAF
by washed rabbit platelets.

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Fig. 7.
Effect of substrate concentration on PAF deacetylation activity in
intact rabbit platelets and their membrane and cytosol fractions.
Rabbit platelets (2.5 × 108
cells) and their subcellular fractions were incubated with various
concentrations of
[3H]acetyl-PAF in 0.4 ml of 0.05 M Tris · HCl-buffered saline containing 1 mM EDTA, 6 mM acetate, and 0.1% BSA (pH 7.2). Amount of
[3H]acetate released
from [3H]acetyl-PAF
was measured as described in MATERIALS AND
METHODS. , Intact rabbit platelets; , membrane
fraction; , cytosol fraction. Results are expressed as means ± SD for 3 or 4 experiments.
|
|
Sensitivity of PAF-AH in intact platelets and rabbit plasma to
proteinase K and diethyl pyrocarbonate.
To clarify whether the ecto-type PAF-AH on the surface of intact
platelets participates in the deacetylation of exogenous PAF, and
furthermore to rule out the possibility that contaminating plasma
PAF-AH might hydrolyze the exogenous PAF, we investigated the effect of
proteinase K and diethyl pyrocarbonate (histidine residue modifier) on
PAF deacetylation activity in intact rabbit platelets and plasma. When
rabbit platelets were incubated with proteinase K, the deacetylation
activity, determined by measuring the amount of released
[3H]acetate, decreased
dose dependently (Fig.
8A).
Whereas treatment with proteinase K decreased the deacetylation
activity in rabbit plasma, the extent of inhibition was rather lower
than that in intact platelets (Fig.
8A). We further investigated the
influence of diethyl pyrocarbonate (0, 0.05, 0.1, 0.2, 0.5, and 1.0 mM) on the deacetylation activity of intact platelets and the membrane and
cytosol fractions and plasma. Diethyl pyrocarbonate suppressed the
deacetylation activity of intact platelets and the membrane and cytosol
fractions dose dependently in comparison with the deacetylation
activity of vehicle-treated platelets, but it suppressed the activity
of plasma PAF-AH slightly (Fig. 8B).
At each concentration of diethyl pyrocarbonate, the extent of
suppression of the deacetylation activity of the membrane fraction was
close to that of intact platelets and higher than that of the cytosol
fraction. These observations indicate that
1) the sensitivities of PAF-AH in
intact platelets toward proteinase K and diethyl pyrocarbonate are
different from those of rabbit plasma and
2) contaminating plasma PAF- AH is
unlikely to participate in the deacetylation of exogenous PAF.

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Fig. 8.
Inactivation by proteinase K and diethyl pyrocarbonate of PAF
acetylhydrolase in intact rabbit platelets, their membrane and cytosol
fractions, and plasma. A: rabbit
platelets (2.5 × 108 cells,
0.4 ml) in 8 mM HEPES-buffered saline containing 2 mM EDTA and 5 mM
glucose (pH 7.2) and rabbit plasma preincubated with various
concentrations of proteinase K or vehicle for 90 min at 37°C were
incubated for 10 min with 1 × 10 9 M
[3H]acetyl-PAF and 2 × 10 5 M
[3H]acetyl-PAF,
respectively. , Intact platelets; , plasma.
B: rabbit platelets (2.5 × 108 cells) and their subcellular
fractions were preincubated with various concentrations of diethyl
pyrocarbonate in 0.4 ml of 0.05 M Tris · HCl-buffered
saline containing 1 mM EDTA (pH 7.2) at 37°C for 10 min and then
incubated with
[3H]acetyl-PAF as
described in A. Amount of
[3H]acetate released
from [3H]acetyl-PAF
was determined by the TCA precipitation method. Relative activity was
calculated on the basis of the activity in the cells or subcellular
fractions after incubation with the vehicle. , Deacetylation
activity of intact rabbit platelets; , deacetylation activity of the
membrane fraction; , deacetylation activity of the cytosol fraction;
, deacetylation activity of rabbit plasma. Results are expressed as
means ± SD for 3 or 4 experiments.
|
|
Identification of metabolites of radioactive PAF bound to rabbit
platelets.
To determine the metabolites of
[3H]alkyl-PAF and
[3H]acetyl-PAF, rabbit
platelets were incubated with radioactive PAF for the desired periods
and then extracted by addition of chloroform and methanol. The
radioactivity associated with the organic phase was separated by
thin-layer chromatography and detected by an ultrahigh-sensitivity
television camera. The radioactive metabolites of
[3H]alkyl-PAF were
alkylacylglycerophosphocholine and lyso-alkylglycerophosphocholine, as
reported previously (7, 14, 16, 23) (data not shown). In contrast, no
radioactive metabolite of
[3H]acetyl-PAF was
detected in the cells (Fig. 9). Even after
60 min of incubation,
[3H]acetyl-PAF was
detectable, indicating that the PAF bound specifically to the receptor
([3H]acetyl-PAF
incorporated in the absence of WEB-2086 minus that in the presence of
WEB-2086) and the portion of PAF bound nonspecifically to the platelets
([3H]acetyl-PAF
incorporated in the presence of WEB-2086) was essentially unmetabolized.

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Fig. 9.
Identification of radioactive PAF bound to rabbit platelets. Rabbit
platelets were incubated with 1 × 10 9 M
[3H]acetyl-PAF in
Tyrode-gelatin buffer (pH 7.2) containing 1 mM
Ca2+, 6 mM acetate, and 0.1% BSA.
At indicated times, radioactivity of
[3H]acetyl-PAF and its
metabolites in the cells was determined with an ultrahigh-sensitivity
television camera system (ARGUS-100) as described in
MATERIALS AND METHODS. PC,
1-alkyl-2-acyl-sn-glycero-3-phosphocholine;
SM, sphingomyelin. Results from 1 of 5 experiments are shown.
|
|
Metabolism of exogenous
[3H]acetyl-PAF in rabbit PMNs
and human platelets and PMNs.
To clarify whether the deacetylation of most of the exogenous PAF by
ecto-type PAF-AH was specific for rabbit platelets, we investigated the
metabolism of
[3H]acetyl-PAF in
rabbit and human PMNs and human platelets. In rabbit PMNs and human
platelets, extracellular
[3H]acetate was
released into the medium, but a trace amount of intracellular
[3H]acetate was
detected (Fig. 10,
A and
B). In human PMNs, accumulation of
intracellular
[3H]acetate was
observed, but most of the
[3H]acetate was
detected extracellularly (Fig. 10C).
These results indicate that PAF-AH is likely to function on the outer
cell surface as an ecto-type enzyme in rabbit and human PMNs and human
platelets.

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Fig. 10.
PAF deacetylation activity in rabbit polymorphonuclear leukocytes
(PMNs; A), human platelets
(B), and human PMNs
(C). Rabbit PMNs (1.25 × 106 cells), human
platelets (4 × 108 cells),
and human PMNs (2.5 × 106
cells) were incubated with 1 × 10 9 M
[3H]acetyl-PAF under
the same conditions as those described in Fig. 5. Extracellular (open
symbols) and intracellular (solid symbols)
[3H]acetate levels
were measured by the TCA precipitation method as described in
MATERIALS AND METHODS. Results are
expressed as means ± SD for 3 or 4 experiments.
|
|
 |
DISCUSSION |
PAF at very low concentrations plays important roles in diverse
pathophysiological reactions such as cell activation (2, 31). It has
been recognized that platelet aggregation occurs following activation
of an intracellular signaling system that includes cell surface
expression of P-selectin, a cell adhesion molecule needed for mediating
the interaction of platelets (5, 17). The present study demonstrated
that 1) platelet activation by PAF
is associated with the expression of P-selectin on the cell surface and
2) continuous binding of PAF to its
receptor maintains P-selectin expression on the platelet surface with
prolonged platelet aggregation (Figs. 1-3). PAF receptor
antagonists are well known to inhibit PAF-induced actions in a
competitive manner when added to target cells before PAF in vitro (26).
In this study, when PAF receptor antagonists (WEB-2086, Y-24180,
SM-12502, and CV-3988) were added to platelets already aggregated by
PAF, they were able to enhance the disaggregation of the platelets
(Fig. 2 and Table 1). The potency of PAF receptor antagonists in
disaggregating platelets aggregated by PAF paralleled their ability to
inhibit platelet aggregation induced by PAF, i.e., their binding
affinity with the PAF receptor (Table 1). Verapamil and
8-diethylaminooctyl-3,4,5-trimethoxy-benzoate (TMB-8) have also been
reported to disaggregate PAF-aggregated platelets, reducing the
phosphorylation of 40- and 20-kDa proteins, an event
related to reduction of the intracellular
Ca2+ level (13). Because WEB-2086
is distinct from verapamil and TMB-8, it did not significantly affect
the level of intracellular Ca2+
and the extent of protein phosphorylation but did disrupt the binding
of [3H]PAF to the
platelets and displaced it, resulting in disaggregation (Figs. 2 and
4). In addition, this ruled out the possibility that a cryptic PAF
receptor might move to the cell surface and/or that new PAF
receptors might be synthesized during the 2 h of platelet stimulation,
since 1) there was no significant
difference in the specific binding site for PAF between control
platelets and PAF-treated platelets (11) and
2) even in rabbit platelets treated
with actinomycin D, cycloheximide, and chloramphenicol (4), the aggregatory response to PAF was restored time dependently. Therefore, a
possible explanation for the disaggregation of platelets aggregated by
PAF is that WEB-2086 disrupts the binding of PAF to its receptor, reducing the expression of the adhesion molecule P-selectin on the
platelet cell surface. Thus PAF receptor antagonists may represent a
novel therapeutic means of not only preventing platelet aggregation by
PAF but also disaggregating platelets that have been aggregated by PAF.
When PAF is released from its cell of origin into blood, it is rapidly
metabolized to an inactive form by PAF-AH (half time = 7.3 min) (27).
In blood, PAF-AH exists not only in plasma/serum but also in
circulatory cells such as PMNs, macrophages, and platelets (28).
Exogenous
[3H]alkyl-PAF bound to
platelets and PMNs is promptly converted to
alkylacylglycerophosphocholine (7, 14, 16, 23). Because a very small
amount of intermediate lyso-PAF is accumulated at an earlier stage,
cytosolic PAF-AH is considered to participate in the deacetylation of
PAF as the rate-limiting step (7, 14, 16, 23). Although this process
requires the transbilayer movement of exogenous PAF, the mechanism is
unclear (30). In this study on the behavior of PAF bound to rabbit
platelets, platelets fully aggregated by PAF were found to be
disaggregated in parallel with the decrease in the amount of PAF on its
receptors. The completely disaggregated platelets were thereafter
reaggregated to a similar extent by the same dose of PAF (Fig. 1). This
observation indicates that the binding of PAF to its receptor is
necessary for maintenance of platelet aggregation, although it remains
to be clarified whether this mechanism is universal for platelet
aggregation by other agonists such as ADP and thrombin. It has been
reported that, in previous experiments using twice-washed platelets,
PAF-AH activity in the cytosol fraction was higher than that in the
membrane fraction, suggesting the participation of cytosol PAF-AH in
the metabolism of exogenous PAF. However, the second washing buffer
contained a detectable amount of PAF-AH activity, which may have
originated from plasma. Accordingly, in the present study, platelets
washed an additional three times were used because PAF-AH activity was not detected in the fifth washing buffer. Ecto-type PAF-AH on intact
platelets was strongly suppressed by proteinase K and diethyl pyrocarbonate, compared with PAF-AH in plasma (Fig. 8). These results
exclude the possibility that the contaminating plasma PAF-AH was
operating under our experimental condition. Thus, in the absence of
plasma PAF-AH, we investigated the possibility that membrane-bound
PAF-AH might participate in the deacetylation of exogenous PAF. The
intracellular and extracellular behavior of
[3H]acetyl-PAF and its
radioactive products in the absence and presence of WEB-2086 indicated
that PAF was able to bind to platelets using three different
components, A,
B, and
C (Fig. 6). Component
A was the specific receptor of PAF and was blocked by
the PAF antagonist WEB-2086 (26). The binding of PAF to
components B and
C was nonspecific.
Component B was associated with rapid
deacetylation of PAF. PAF that bound to component
C was metabolized slowly. These binding modes are
identical to those proposed by Homma et al. (11). In rabbit platelets,
PAF bound nonspecifically to membranes was deacetylated about four
times faster than PAF bound specifically to the receptor. This has also
been observed in myelogenous cells (21). On the other hand, the rate of
deacetylation of [3H]acetyl-PAF in
intact platelets and the membrane fraction was faster than that in the
cytosol fraction, in spite of apparent suppression of the deacetylation
activity caused by PAF binding to the membrane (Fig. 7). Moreover, the
relationship between substrate concentrations at
10
9-10
8
M and deacetylation activity in intact platelets was similar to that in
the membrane fraction, although PAF at a concentration higher than
10
7 M destroys cells and
thus the deacetylation activity cannot be determined (Fig. 7). These
findings strongly indicate that membrane-bound PAF-AH, but not
intracellular PAF-AH, participates in the inactivation of exogenous
PAF. In fact, platelets aggregated by PAF were disaggregated faster
than those aggregated by C-PAF (Fig. 5), whereas WEB-2086 enhanced the
disaggregation of platelets aggregated by both agents to a similar
extent. Furthermore, the finding that the amount of intracellular
[3H]acetate was rather
small or undetectable in cells incubated with or without PAF for any
period indicates that exogenous PAF is deacetylated on the outer
surface of intact platelets. No radioactive metabolite (except
[3H]acetyl-PAF) was
detected in the platelets even after 60 min of incubation with
[3H]acetyl-PAF (Fig.
9). Figure 10 shows that most of the
[3H]acetate released
from [3H]acetyl-PAF
after incubation with rabbit and human PMNs and human platelets was
present in the medium. These results indicate that ecto-type PAF-AH,
not only in rabbit platelets but also in human platelets and rabbit and
human PMNs, may participate in the rapid inactivation of exogenous PAF.
We and others have reported previously that the action of PAF in blood
appears to be controlled by serum PAF-AH and that an abnormality in the
level of blood PAF due to deficiency or low activity of serum PAF-AH
might cause more severe symptoms (19, 29). Thirty-eight subjects with
PAF-AH deficiency (3.8%) were found among 1,000 healthy Japanese
adults. However, although the probability of occurrence of PAF-AH
deficiency is significantly higher in certain groups such as severely
asthmatic children (11.4%) and individuals with hornet-sting anamnesis
(13%) (I. Fujii, T. Shimizu, H. Mochizuki, K. Tokuyama, A. Morikawa,
J. Sugatani, and M. Miwa, unpublished observations),
subjects with the enzyme deficiency do not always have severe symptoms
such as thrombosis. We have previously elucidated the occurrence of a
factor(s) releasing newly synthesized PAF from human PMNs in serum
(PAF-releasing factor) (20). If released PAF plays a pathophysiological
role, a PAF-inactivation system in blood other than serum PAF-AH is necessary. The present results indicate that ecto-type PAF-AH is
present on both rabbit and human platelets and PMNs. PAF-AH activity in
platelets and PMNs from subjects with PAF-AH deficiency was similar to
that in platelets and PMNs from normal subjects (19). Therefore,
ecto-type PAF-AH in target cells such as platelets and PMNs may
function to control the level of PAF in blood of individuals with serum
PAF-AH deficiency.
 |
ACKNOWLEDGEMENTS |
We would like to thank Dr. Donald J. Hanahan for reading our manuscript
and for useful discussions. We gratefully acknowledge the assistance of
Yoshihiro Okamoto, Hiroko Nagabuchi, Yoshihiko Wakazono, Ikuko Fujii,
Hitosi Yoshida, and Takahiro Iwai. We are also grateful to Dr. Takashi
Suzuki for helpful discussions.
 |
FOOTNOTES |
This work was supported by a grant-in-aid for Scientific Research from
the Ministry of Education, Science, and Culture of Japan.
Address for reprint requests: M. Miwa, Dept. of Pharmaco-Biochemistry,
School of Pharmaceutical Science, Univ. of Shizuoka, Yada 52-1,
Shizuoka 422, Japan.
Received 8 May 1997; accepted in final form 11 September 1997.
 |
REFERENCES |
1.
Bligh, E. G.,
and
W. J. Dyer.
A rapid method of total lipid extraction and purification.
Can. J. Biochem. Physiol.
37:
911-918,
1959.
2.
Braquet, P.,
L. Touqui,
T. Y. Shen,
and
B. B. Vargaftig.
Perspectives in platelet-activating factor research.
Pharmacol. Rev.
39:
97-145,
1987[Medline].
3.
Bratton, D. L.,
E. Dreyer,
J. M. Kailey,
V. A. Fadok,
K. L. Clay,
and
P. M. Henson.
The mechanism of internalization of platelet-activating factor in activated human neutrophils: enhanced transbilayer movement across the plasma membrane.
J. Immunol.
148:
514-523,
1992[Abstract/Free Full Text].
4.
Bruce, I. J.,
and
R. Kerry.
The effect of chloramphenicol and cycloheximide on platelet aggregation and protein synthesis.
Biochem. Pharmacol.
36:
1769-1773,
1987[Medline].
5.
Celi, A.,
G. Pellegrini,
R. Lorenzet,
A. D. Blasi,
N. Ready,
B. C. Furie,
and
B. Furie.
P-selectin induces the expression of tissue factor on monocytes.
Proc. Natl. Acad. Sci. USA
91:
8767-8771,
1994[Abstract].
6.
Chao, W.,
and
M. S. Olson.
Platelet-activating factor: receptors and signal transduction.
Biochem. J.
292:
617-629,
1993[Medline].
7.
Chilton, F. H.,
J. T. O'Flaherty,
J. M. Ellis,
C. L. Swendsen,
and
R. L. Wykle.
Metabolic fate of platelet-activating factor in neutrophils.
J. Biol. Chem.
258:
6357-6361,
1983[Abstract/Free Full Text].
8.
Dittmer, J. C.,
and
R. L. Lester.
A simple, specific spray for the detection of phospholipids on thin-layer chromatograms.
J. Lipid Res.
5:
126-127,
1964[Free Full Text].
9.
Grigoriadis, G.,
and
A. G. Stewart.
Albumin inhibits platelet-activating factor (PAF)-induced responses in platelets and macrophages: implications for the biologically active form of PAF.
Br. J. Pharmacol.
107:
73-77,
1992[Abstract].
10.
Hanahan, D. J.
Platelet-activating factor: a biologically active phosphoglyceride.
Annu. Rev. Biochem.
55:
483-509,
1986[Medline].
11.
Homma, H.,
A. Tokumura,
and
D. J. Hanahan.
Binding and internalization of platelet-activating factor 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine in washed rabbit platelets.
J. Biol. Chem.
262:
10582-10587,
1987[Abstract/Free Full Text].
12.
Izumi, T.,
and
T. Shimizu.
Platelet-activating factor receptor: gene expression and signal transduction.
Biochim. Biophys. Acta
1259:
317-333,
1995[Medline].
13.
Khan, S. N.,
P. A. Lane,
and
A. D. Smith.
Disaggregation of PAF-acether-aggregated platelets by verapamil and TMB-8 with reversal of phosphorylation of 40K and 20K proteins.
Eur. J. Pharmacol.
107:
189-198,
1985[Medline].
14.
Kramer, R. M.,
G. M. Patton,
C. R. Pritzker,
and
D. Deykin.
Metabolism of platelet-activating factor in human platelets: transacylase-mediated synthesis of 1-O-alkyl-2-arachidonoyl-sn-glycero-3-phosphocholine.
J. Biol. Chem.
259:
13316-13320,
1984[Abstract/Free Full Text].
15.
Lotner, G. Z.,
J. M. Lynch,
S. J. Betz,
and
P. M. Henson.
Human neutrophil-derived platelet activating factor.
J. Immunol.
124:
676-684,
1980[Free Full Text].
16.
Malone, B.,
T.-C. Lee,
and
F. Snyder.
Inactivation of platelet activating factor by rabbit platelets: lyso-platelet activating factor as a key intermediate with phosphatidylcholine as the source of arachidonic acid in its conversion to a tetraenoic acylated product.
J. Biol. Chem.
260:
1531-1534,
1985[Abstract].
17.
McEver, R. P.,
K. L. Moore,
and
R. D. Cummings.
Leukocyte trafficking mediated by selectin-carbohydrate interactions.
J. Biol. Chem.
270:
11025-11028,
1995[Abstract/Free Full Text].
18.
Miwa, M.,
M. Matsumoto,
M. Tezuka,
S. Okada,
S. Ohsuka,
and
H. Fujiwake.
Quantitative fluorographic detection of 3H and 14C on two-dimensional thin-layer chromatographic sheets by an ultra-high-sensitivity TV camera system.
Anal. Biochem.
152:
391-395,
1986[Medline].
19.
Miwa, M.,
T. Miyake,
T. Yamanaka,
J. Sugatani,
Y. Suzuki,
S. Sakata,
Y. Araki,
and
M. Matsumoto.
Characterization of serum platelet-activating factor (PAF) acetylhydrolase: correlation between deficiency of serum PAF acetylhydrolase and respiratory symptoms in asthmatic children.
J. Clin. Invest.
82:
1983-1991,
1988[Medline].
20.
Miwa, M.,
J. Sugatani,
T. Ikemura,
Y. Okamoto,
M. Ino,
K. Saito,
Y. Suzuki,
and
M. Matsumoto.
Release of newly synthesized platelet-activating factor (PAF) from human polymorphonuclear leukocytes under in vivo conditions: contribution of PAF-releasing factor in serum.
J. Immunol.
148:
872-880,
1992[Abstract/Free Full Text].
21.
O'Flaherty, J. T.,
M. C. Chabot,
J. Redman, Jr.,
D. Jacobson,
and
R. L. Wykle.
Receptor-independent metabolism of platelet-activating factor by myelogenous cells.
FEBS Lett.
250:
341-344,
1989[Medline].
22.
O'Flaherty, J. T.,
J. R. Redman, Jr.,
J. D. Schmitt,
J. M. Ellis,
J. R. Surles,
M. H. Marx,
C. Piantadosi,
and
R. L. Wykle.
1-O-Alkyl-2-N-methylcarbamyl-glycerophosphocholine: a biologically potent, non-metabolizable analog of platelet-activating factor.
Biochem. Biophys. Res. Commun.
147:
18-24,
1987[Medline].
23.
O'Flaherty, J. T.,
J. R. Suries,
J. Redman,
D. Jacobson,
C. Piantadosi,
and
R. L. Wykle.
Binding and metabolism of platelet-activating factor by human neutrophils.
J. Clin. Invest.
78:
381-388,
1986[Medline].
24.
Pinckard, R. N.,
R. S. Farr,
and
D. J. Hanahan.
Physicochemical and functional identity of rabbit platelet-activating factor (PAF) released in vivo during IgE anaphylaxis with PAF released in vitro from IgE sensitized basophils.
J. Immunol.
123:
1847-1857,
1979[Medline].
25.
Prescott, S. M.,
G. A. Zimmerman,
and
T. M. McIntyre.
Platelet-activating factor.
J. Biol. Chem.
265:
17381-17384,
1990[Free Full Text].
26.
Shen, T. Y.,
S.-B. Hwang,
T. W. Doebber,
and
J. C. Robbins.
The chemical and biological properties of PAF agonists, antagonists, and biosynthetic inhibitors.
In: Platelet-Activating Factor and Related Lipid Mediators, edited by F. Snyder. New York: Plenum, 1987, p. 153-190.
27.
Stafforini, D. M.,
T. M. McIntyre,
M. E. Carter,
and
S. M. Prescott.
Human plasma platelet-activating factor acetylhydrolase: association with lipoprotein particles and role in the degradation of platelet-activating factor.
J. Biol. Chem.
262:
4215-4222,
1987[Abstract/Free Full Text].
28.
Stafforini, D. M.,
S. M. Prescott,
G. A. Zimmerman,
and
T. M. McIntyre.
Platelet-activating factor acetylhydrolase activity in human tissues and blood cells.
Lipids
26:
979-985,
1991[Medline].
29.
Stafforini, D. M.,
K. Satoh,
D. L. Atkinson,
L. W. Tjoelker,
C. Eberhardt,
H. Yoshida,
T. Imaizumi,
S. Takamatsu,
G. A. Zimmerman,
T. M. McIntyre,
P. W. Gray,
and
S. M. Prescott.
Platelet-activating factor acetylhydrolase deficiency: a missense mutation near the active site of an anti-inflammatory phospholipase.
J. Clin. Invest.
97:
2784-2791,
1996[Abstract/Free Full Text].
30.
Tokumura, A.,
T. Tsutsumi,
J. Yoshida,
and
H. Tsukatani.
Translocation of exogenous platelet-actvating factor and its lyso-compound through plasma membranes is a rate-limiting step for their metabolic conversions into alkylacylglycerophosphocholines in rabbit platelets and guinea-pig leukocytes.
Biochim. Biophys. Acta
1044:
91-100,
1990[Medline].
31.
Zimmerman, G. A.,
T. M. McIntyre,
S. M. Prescott,
and
D. M. Stafforini.
Platelet-activating factor: antagonists, terminators, molecular mimics, and microbial opportunism.
J. Intern. Med.
239:
463-466,
1996[Medline].
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