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
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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
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Materials & Methods
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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).

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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Table 1.   Effect of PAF receptor antagonists on disaggregation of rabbit platelets aggregated by PAF

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.

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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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. open circle  Extracellular [3H]acetate; bullet , intracellular [3H]acetate; square , extracellular [3H]acetyl-PAF; black-square, [3H]acetyl-PAF bound to cells; triangle , extracellular [3H]alkyl-PAF; black-triangle, [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. bullet , Intact rabbit platelets; black-square, membrane fraction; open circle , 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. bullet , Intact platelets; black-square, 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. bullet , Deacetylation activity of intact rabbit platelets; black-square, deacetylation activity of the membrane fraction; open circle , deacetylation activity of the cytosol fraction; square , 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
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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
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Abstract
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Materials & Methods
Results
Discussion
References

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AJP Cell Physiol 274(1):C47-C57
0363-6143/98 $5.00 Copyright © 1998 the American Physiological Society




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