1011

The Effect of Inhibitorsof Platelet Aggregation on the Metabolism of Platelet-Activating Factor (PAF) in W a s h e d Rabbit Plateletsl C. O'Neill a,*, A.J. Ammit u, R. Korlh b, S. Fleming a, and X. Wells a aHuman Reproduction Unit, Royal North Shore Hospital of Sydney, St. Leonards, 2065, NSW Australia and blnserm U200, Universite Paris, Sud, F-92140 Clamart, France

The rabbit platelet metabolizes platelet-activating factor (PAF) intracellularly. PAF is deacetylated to produce lysoPAF which, in turn, can be acylated to produce 1-Oalkyl-2-acyl-sn-glycero-3-phosphocholine (alkylacyl GPC). Some PAF receptor antagonists have been shown to inhibit this metabolic conversion. In the present study we examined whether the PAF receptor antagonists SRI 63-441 and WEB 2086 would inhibit the metabolism of PAF by intact rabbit platelets. In addition, we examined whether iloprost, a stable analogue of prostaglandin I2 {PGI2), and a potent inhibitor of platelet activation induced by a range of agonists, would also inhibit PAF metabolism. We found that SRI 63~141 and WEB 2086 caused an almost complete inhibition of the conversion of PAF to alkylacyl GPC. Iloprost caused up to a 50% inhibition of PAF metabolism compared to antagonistfree controls. Iloprost {and PGI2) is thought to inhibit platelet response by elevation of cAMP, while receptor antagonists act by blocking PAF binding to its receptor. Since iloprost caused partial inhibition of PAF metabolism, the results of this study suggest that inhibition of PAF metabolism does not occur solely due to competitive inhibition of PAF binding to its receptor. Lipids 26, 1011-1014 {1991}.

of PAF by rabbit platelets {7,8} and a variety of other cell types, preventing the production of both lysoPAF and alkylacyl GPC. The dose of antagonist required is generally higher than that needed to inhibit platelet activation {8). It has been suggested that the PAF receptor antagonist prevents internalization of PAF by rabbit platelets, and thus exposure of PAF to intracellular metabolic enzymes {7,8}. In the case of human platelets, however, PAF receptor antagonists, such as WEB 2086 or ginkgolides, did not inhibit PAF metabolism, nor did they inhibit serum acetylhydrolase {6,9). The present study, examined {for the first time) the capacity of the PAF receptor antagonists SRI 63-441 and WEB 2086 to inhibit the metabolism of PAF by washed rabbit platelets. Since other agents which activate platelets, such as thrombin, can also promote metabolism of PAF by platelets {10), the ability of the prostaglandin I2 (PGI2) stable analogue, iloprost {ZK 36374}, which is a very potent inhibitor of platelet activation, was also examined.

MATERIALS AND METHODS

Preparation ofplatelets. Blood (7 vol) from a male New Zealand white rabbit was collected into I vol of acidified citrate dextrose (ACD; pH 6.4, 4~ (6). The platelets were Platelet-activating factor (PAF) is generally acknowledg- centrifuged at 100 • g for 15 rain to produce platelet-rich ed to have a short half-life in vivo. This is due to the broad plasma (PRP). They were then washed three times in distribution of the enzyme PAF acetylhydrolase (1), both Tyrodes buffer containing ACD {9:1, v/v; pH 6.4) (6). After extracellularly and intracellularly. Acetyl hydrolysis the last wash, the platelets were resuspended {250 • results in the conversion of PAF to lysoPAF, the latter be- 103/~L) in Tyrodes buffer. Tyrodes buffer contained 137 ing cytotoxic (2). To avoid cell and tissue damag~ rapid mM NaC1, 2.6 mM KC1, 11.9 mM NaHCO3, 1.0 mM processing of lysoPAF is therefore required. LysoPAF in MgC12, 0.41 mM NaH2PO 4, 0.5 mM dextrose, and 5.0 cells is usually reacylated via a transacylase to form mM H E P E S (Sigma Chemical Ca, St. Louis, MO). The 1-O-alkyl-2-acyl-sn-glycero-3-phospho. buffer was supplemented with 0.25% (w/v) bovine serum choline (alkylacyl GPC). The fatty acid utilized in this albumin {BSA, CSL, Melbourne. Australia) and adjusted esterification in most cells is preferentially, but not ex- to pH 7.4. This medium is referred to as Tyrodes-BSA (6}. clusively, arachidonic acid (3,4). Membrane alkylacyl GPC [3I-I]PAF and inhibitors. [3H]PAF (1-O-[hexadecyl-l', may then serve as a precursor for PAF synthesis via 2'-3H(N)]-; 56.7 Ci/mmol) was obtained from N E N phospholipase A2 and acetyltransferase (E.C. 2.3.1.67), Research Products (Wilmington, DE). [3H]PAF in thus creating the PAF cycle (5). However, this pattern of chloroform was placed into siliconized glass test tubes, metabolism may not be universal. Activation of washed the solvent was evaporated under N2, and PAF was human platelets by PAF does not result in this type of resuspended in Tyrodes-BSA, Three inhibitors were used cellular PAF metabolism (6). in the study: i) SRI 63-441 which is cis-(+_}-l-[2-hydroxy Some PAF receptor antagonists inhibit the metabolism [tetrahydro-5- [(octadecylaminocarbonyl)oxy] methyl]furan-2-yl] methoxy-phosphinyloxy]ethyl] quinolinium hydroxide, inner salt (Sandoz Research Institute, East Hanover, N J); ii) WEB 2086 which is 3-[4-(2-chlorophenyl)1Based on a paper presented at the Third International Conference 9-methyl-6H-thieno [3,2-f] [1,2,4]triazolo-[4,3-a] [1,4]on Platelet Activating Factor and Structurally Related Alkyl Ether diazepin- 2-yl]-l-(4-morpholinyl}-l-propanone (Boehringer Lipids, Tokyo, Japan, May 1989. Ingelheim, Ingelheim, Germany); and iii) iloprost which *To whom correspondence should be addressed. Abbreviations: ACD, acidifiedcitrate dextrose; alkylacylGPC, 1-O- is 5-{E)-{1S,5 S,6R,7 R)-7-hydroxy-6-{E}-{3S,4RS}-3-hydroxyalkyl-2-acyl-sn-glycero-3-phosphocholine; BSA, bovine serum 4-methyl-oct-l-en-6-yn-yl-bicyclo-3.3.0-octano-3-ylidenalbumin; HPLC, high-performance liquid chromatography; PAF, pentanoic acid {Schering AG, Berlin, Germany). Iloprost platelet-activating factor, 1-O-alkyl-2-acetyl-sn-glycero-3-phospho- was provided as a 0.1 mg/mL saline solution, and SRI 63choline; PGI 2, prostaglandin I2; PRP, platelet-rich plasma. 441 and WEB 2086 were prepared as 1 mg/mL solutions LIPIDS,Vol. 26, No. 12 (1991)

1012

C. O'NEILL ET AL. in phosphate buffered saline The inhibitor concentrations required were attained by serial dilution with saline. The potency of the three inhibitors was tested in a platelet aggregation assay as previously described (11). P A F metabolism assay. The washed platelet suspension (250 ~L) was placed into a 37~ teflon chamber and stirred using a magnetic stirrer (approximately 500 rpm). [3H]PAF (5/~) i n Tyrodes-BSA was added to give a final concentration of 1.3 nM. Immediately thereafter,45 ~L of saline containing the appropriate concentration of inhibitor was added. For zero time assessment, 125 ~L of the platelet suspension was removed and added immediately to 19 vol of methanol to stop metabolism and to extract the lipids.The remaining platelet suspension was incubated, and the reaction was stopped at various time points by addition to methanol. Lipid extraction. The addition of methanol caused precipitation of proteins which were removed by centrifugation {2,500 • g, 4~ 30 rain).The supernatant {1 vol) was added to H20 and chloroform {0.8:0.95,v/v)to effectphase separation.The chloroform phase was recovered and solvent was removed in a rotary evaporator; the lipids were resuspended in 100 ~L of methanol and chromatographed by high-performance liquid chromatography (HPLC). Chromatography. Ion exchange H P L C was used to separate P A F from its metabolites {adapted from ref.12), utilizingan L K B system with a W h a t m a n PartisilS C X (covalentlybound benzene sulfonateresidues)column (250 • 4.6 mm: i0 ~ M silica;Activon, Sydney, Australia).The mobile phase was CH3CN/CH3OH/H20 {300:150:35,v/v/v) at a flow rate of 1.5 mL/min. Fractions (1.5mL) were collected for 40 rain and the radioactivity in each fraction was quantitated by scintillationcounting. Figure I shows a typicalelutionprofilefor a number of phospholipids, including [ZH]lysoPAE The putative acylated derivative of

lysoPAF (alkylacyl GPC) eluted at approximately 11-12 rain, PAF at 18-21 re_in and lysoPAF at 28-31 min. Over 85% of all counts recovered were associated with these three regions. Counts eluting at the solvent front were generally less than 10%. RESULTS

The firstexperiments were designed to determine the time dependence of P A F metabolism by washed rabbit platelets.These experiments were done in the absence of P A F antagonists {Fig. 2). A significant amount of P A F conversion had already occurred by 15 rain when most of the PAF-derived labelwas found in the lysoPAF fraction. P A F degradation proceeded in an almost linear fashion between 15 and 65 rain, and by 80 rain most of the label migrated close to the phosphatidylcholine fraction.Based on the results of previous studies, it appeared likely that the latter fraction was alkylacyl G P C {13).Only a small amount of lysoPAF was present at this time, confirming that lysoPAF does not accumulate in platelets. Further experiments used 80 rain as the end-point, and the conversion of [3H]PAF to putative alkylacyl G P C was used as a measure of metabolic activity. Figure 3 shows the relativepotency of inhibitionof P A F induced plateletaggregation by the three antagonists used in this study. Both iloprostand W E B 2086 were more potent than SRI 63-441, while iloprostwas significantly more potent than W E B 2086 {iloprost ICs0 13.9 nM; W E B 2086 ICs0 55 nM). Figure 4 shows that the P A F receptor antagonists SRI 63-441 and W E B 2086 almost completely inhibited conversion of [3H]PAF to putative alkylacyl G P C at the highest concentrations tested. The amount of lysoPAF present was not different from the zero time control or inhibitor~freepreparation (resultsnot shown). There was

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FIG. 1. Typical elution of [3H]PAF and [3H]lysoPAF (broken line) and phospholipid standards (solid line) from a Whatman PartisU strong cation~xchange column with aeetonitriielmethanol/water (300:150:35, v/v/v) at a flow rate of 1.5 mLJmin.

LIPIDS,Vol. 26, No~ 12 (1991)

1013 I N H I B I T O R S OF PAF CELLULAR M E T A B O L I S M

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FIG. 4. The effect of PAF inhibitors (iloprost O-O;, W E B 2086 9 9 ; SRI 63-441 e - - e ) on the conversion of [oH]PAF to putative aikylacyl GPC relative to its conversion in the absence of inldbitors, /.e., % conversion with inhibitor/% conversion without inhibitor X 100. Each point is the mean of three replicates, except for W E B 2086 g, which was the mean of four replicates.

the maximum inhibition of metabolism was at a 1,000-fold higher dose than its non-effective concentration. Iloprost caused significant inhibition of metabolism at lower doses than SRI 63-441 and WEB 2086. In contrast to that of the two receptor antagonists, the maximum inhibition caused by iloprost was only approximately 50% of that of antagonist-free controls. WEB 2086 and SRI 63-441 caused a 90% inhibition of metabolism as compared with controls. As with the receptor antagonists, iloprost displayed its maximum inhibitory effect at a 1,000-fold higher dose than its non-effective concentration.

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FIG. 2. Redistribution of label from [3H]PAF by wnshod rabbit platelets as percentage of radioactivity added as a function of time. PAF ( 9 - 9 ), lysoPAF ( 9 - - 9 ), or alkylacyl GPC fraction (O-O). Each point is the mean of three replicates.

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FIG. 3. The effect of inereeslng concentrations of the PAF inldbitors, iloprast (O-O), W E B 2086 ( 9 1 4 9 and SRI 63-441 ( 9 - 9 on the platelct aggregation induced by the ECs0 for PAF (0.023 p~l) after 15 rain incubation with citrated rabbit blood. Each point is the mean of at least three replicates. The platelet aggregation index is l--(platelct count at 15 min/platelct count at 0 min). A n index of 0 signifies no aggregation, and 1 is complete aggregation.

little difference in the relative potencies of response of the two receptor antagonists (which is in contrast to the relative potencies of inhibition of platelet aggregation by these two agents; Fig. 3). For both of these antagonists,

The action of PAF on blood platelets is receptor mediated (6,9,14). This signal is transduced by a GTPase (G protein) (15,16) which appears to activate phospholipase C (17). The consequent generation of inositol polyphosphates and diacylglycerol is well documented, as is the resulting increase in cellular calcium concentration and activation of protein kinase C (for a review, see ref. 18). PAF receptor antagonists compete with PAF for platelet receptors, but do not stimulate GTPase activity upon binding (19), thus inhibiting signal transduction and preventing cell activatio~L By comparison, floprost is a p ~ tent stimulant of adenylate cyclase (20), resulting in increased cellular cAMP concentrations. Treatment of platelets with dibutyryl cAMP suppresses their response to PAF, and also suppresses PAF induced GTPase activity (19). cAMP also inhibits diacylglycerol dependent activation of protein kinase C (21). Thus, by different mechanisms, the PAF receptor antagonists and iloprost prevent PAF induced activation of cells by inhibition of its signal transduction and secondary messengers. LIPIDS, Vol. 26, Na 12 (1991)

1014

C. O'NEILL E T AL. It was suggested t h a t the binding of PAF to its recel~ tor stimulates the internalization of PAF {and other phospholipids}, resulting in its exposure to metabolic enzymes {8,22}. The PAF receptor antagonist, BN 52021, inhibited conversion of PAF to putative alkylacyl GPC in intact platelets, but not with cell lysates {7}. A possible explanation for the inhibition of PAF metabolism by receptor antagonists is that the antagonists compete with PAF for the receptor, thus reducing the amount of PAF t h a t can be internalized. It was shown t h a t structural analogues of PAF, U66985 and lysoPAF had only marginal effects on the metabolism of PAF {10}. However, these agents also compete for the receptor, b u t had either minimal or no inhibitory effect on platelet activation, respectively {10}. Furthermore, desensitization of platelets to PAF, by prior exposure in the absence of calcium, actually enhanced the metabolism of PAF upon stimulation of platelets with thrombin, collagen or A23187 {9}.From such results it appears t h a t the effects of receptor antagonists on PAF metabolism are not simply due to competitive inhibition at the receptor level, b u t are also dependent upon inhibition of platelet activation. If this is the case. it may be expected t h a t other inhibitors of platelet activation would also inhibit PAF metabolisrrL To test this hypothesis we have examined the effect of iloprost on PAF metabolism. Iloprost was the most potent inhibitor of platelet aggregation, with an ICs0 of 13.9 nM {Fig. 3}, and was also the most potent inhibitor of PAF metabolism by platelets {Fig. 4}. All three inhibitors showed a similar pattern of response at low concentrations. For iloprost, however, a maximum response of only about 50% inhibition of metabolism was observed. F u r t h e r increases in the concentration caused no additional effect. At low doses, iloprost stimulates intracellular platelet cAMP levels {23}. At higher concentrations, however, while there was a rapid production of cAMP, the concentration subsequently returned to normal {14}. Such a biphasic response is consistent with our inability to completely inhibit PAF metabolism with iloprost. It was not determined in this s t u d y whether iloprost affected PAF binding to platelets, although it previously has been shown t h a t iloprost increases intracellular cAMP {24} and t h a t dibutyryl cAMP may indirectly reduce PAF binding by platelets {19}. Since iloprost caused partial inhibition of metabolism, the results of this s t u d y suggest t h a t inhibition of PAF metabolism does not occur solely due to competitive inhibition of PAF binding to its receptor. I n situ, there is extensive extracellular metabolism of PAF to lysoPAF by s e r u m acetylhydrolase {1}. The resulting lysoPAF can cross membranes independently of the PAF receptor {8}, albeit at a slower rate t h a n PAF. Therefore, the inhibition of metabolism by PAF antagonists at the cellular level may be of limited significance However, PAF binding molecules in serum (molecular weight 160-180 kilodalton} which protect PAF from metabolism by serum acetylhydrolase and, hence, limit extracellular metabolism, has been reported {25}. If PAF were bound to a molecule protecting it from serum

LIPID& Vol. 26, Na 12 (1991)

acetylhydrolase, the presence of a high concentration of an inhibitor of PAFs action {and hence cellular metabolism} would extend the half-life of PAF in situ considerably. This could facilitate PAFs local accumulation and potentially accentuate its action. ACKNOWLEDGMENT This work was funded by a grant from the Special Programme of Research, Development and Research Training in Human Reproduction, World Health OrganisatiorLWe thank Schering AG, Boehringer Ingelheim and Sandoz Research Institute for the gifts of antagonists, and Mrs. D. Gillespiefor her help in the preparation of the manuscript. REFERENCES 1. Blank, M.L., Lee, T:a, Fitzgerald, V., and Snyder,F. (198DJ. BioL Chem. 256, 175-178. 2. Hoffman, D.R., Hajdu, J., and Snyder, F. {1984} Blood 63, 545-552. 3. Kumar, H., King, R.J., Martin, H.M., and Hanahan, D.J. {1987} Biochim. Biophys. Acta 917, 33-41. 4. Ramesha, C.S., and Pickett, W.C. {1986}J. Biol. Chem. 261, 519-523. 5. Snyder, F. ~1987}in New Horizons in Platelet Activating Factor Research {Winslow, E.M., and Lee, J., eds.} pp. 13-26, J. Wiley and Sons. 6. Korth, R., Nunez, D., Bidault~ J., and Benveniste, J. {1988}Eur. J. Pharmacol. 152, 101-110. 7. Lachach~ H., Plantavid, M., Simon, M.F., Chap, H., Braquet, P., and Douste-Blazy, J. (1985) Biochem. Biophys. Res. Commun. 132, 460-466. 8. Lamant, V., Manco, G., Braquet, P., Chap, H., and Douste-Blazy, L. {1987}Biochem. Pharmacol. 36, 2749-2752. 9. Korth, R., Hirafuji, C., Keraly, D., Delantier, J., and Benveniste, J. {1989}Br. J. Pharmacol. 98, 653-661. 10. Homma, H., Kumar, R., and Hanahan, D.J. {1987}Arch. Biochem. Biophys. 252, 259-268. 11. Collier, M., O'Neill, C., Ammit, A.J., and Saunders, D.M. {1988} Hum. Reprod 3, 993-998. 12. Gross, R.W., and Sobel, B.E. {1980}J. Chromatogr. 197, 79-85. 13. Bussolino, F., Breviaro, F., Aglietta, M., Sanavio, F., Bosia, A., and Dejana, E. (1987} Biochim. Biophys. Acta 927, 43-54. 14. Hwang, S-B., Lee, S-C., Cheah, M.J., and Shen, T.Y. {1983} Biochemistry 22, 4756-4763. 15. Houslay, M.D., Bojanic, D., and Wilson, A. {1986}Biochem. J. 234, 737-740. 16. Hwang, S-B., Lain, M-H., and Pong, S-S. {1986}J. Biol. Chem. 261, 532-537. 17. Exton, J.H. {1990}J. Biol. Chem. 265, 1-4. 18. Braquet, P., Touqui,L., Shen, TY., and Vargaftig, B.B. {1987}Pha~ macoL Rev. 39, 97-145. 19. Homma, H., and Hanahan, D.J. {1988}Arch. Biochem. Biophys. 262, 32-39. 20. Molina, Y., and Lapetina, E.G. {1989)Proa Natl. Aca~ Sci. USA 86, 868-870. 21. Kroll, H.M., Zaroic~ G.B., and Schafer, A.I. {1988}Biochim. Biophys. Acta 970, 61-67. 22. O'Flaherty, J.T., Surles, J.R., Redman, J., Jacobsen, D., Piantadosi, C., and Wylde, R.L. {1986)J. Clin. Invest. 78, 381-388. 23. Ashby, B. {1988}Second Messengers Phosphoproteins 12, 45-57. 24. Gorman, R.R., Bunting, S., and Miller, O.V. {1977}Prostaglandins 13, 377-388. 25. Matsumota M., and Miwa, M. {1985)Adv.Prostaglandin Thromboxane Leukotriene Res. 15, 705-706.

[ReceivedSeptember 15, 1989, and in revised form October 3, 1991; Revision accepted October 11, 1991]