J. Biochem. 112, 756-761 (1992)

Preferential Activation of Phospholipase A2 by Low Concentrations of Phosphatidic Acid with Long-Chain Fatty Acids in Rabbit Platelets1 Takashi Sato,2 Tsutomu Hashizume, and Tatsuzo Fujii Department of Biochemistry, Kyoto Pharmaceutical University, Yamaahina-ku, Kyoto, Kyoto 607

The role of phosphatidic acid (PA) in the signal transduction system of platelets was studied using 1-stearoyl 2-arachidonoyl PA (PASA). When PASA was added to rabbit platelets, aggregation occurred. BW756C, a dual inhibitor of cyclooxygenase and lipoxygenase, as well as p-bromophenacyl bromide and mepacrine, inhibitors of phospholipase A,, inhibited the aggregation induced by low concentrations of PASA, but not that induced by high concentrations. PASA also stimulated, in a dose-dependent manner, arachidonic acid liberation, lysophosphatidylcholine and diacylglycerol formation, and mobilization of intracellular Ca2+; all of which were dependent on the presence of Ca2+ in the outer medium. The arachidonic acid liberation was inhibited by p-bromophenacyl bromide or mepacrine, while diacylglycerol formation by low concentrations of PASA was inhibited by BW755C. With platelet membrane fractions or with the platelets made permeable to Ca2+ by pretreatment with ionomycin, PASA caused arachidonic acid liberation in the presence of Ca2+. Furthermore, PASA enhanced the activity of phospholipase A2 partially purified from platelet cytosol acting on l-palmitoyl-2-[14C]arachidonoyl-glycerophosphoethanolamine. These results provide evidence that PASA preferentially potentiates the activation of phospholipase A2 in cooperation with Ca2+, suggesting that PA acts as a positive feedback regulator to potentiate the activation of phospholipase A2 and contributes to the amplification of platelet activation.

Phosphatidic acid (PA) is accumulated as an intermediate of increased inositol phospholipid turnover (1) or generated from membrane phospholipids by the hydrolytic action of activated phospholipase D (2) in certain cells as a response to stimulation. It was originally pointed out that, since PA has an ionophoretic activity for Ca2+, it plays a role in the stimulus-response coupling of the cells (3-6). In fact, ionophoretic action of an exogenous PA has been shown to bring about some physiological responses: contractions of isolated smooth muscle cells (7), release of dopamine from synaptosomes (8) and increase in intracellular cGMP in cultured neuroblastoma cells (9). Recent studies have shown, however, that PA potentiates activation of inositol phospholipid-specific phospholipase C (10-12), Ca2+ mobilization (13, 14), decrease in cAMP (15, 16), superoxide generation (17), and DNA synthesis (10,18-20) in various types of cells independently of its Ca2+ ionophoretic activity, suggesting that it has a role as a second messenger. In platelets, PA was previously shown to be unable to cause aggregation, suggesting that it does not possess such a potentiating effect (21). Recent in vitro studies by Jackowski and Rock (11), however, showed that PA can activate phosphatidylinositol 4,5-bisphosphate-specific phospholipase C, suggesting that this phospholipid may have a role as an initiator in the cellular signal transduction 1

This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan. 1 To whom correspondence should be addressed. Abbreviations: BW755C, 3-amino-l-[/n-(trifluoromethyl)phenyl]2-pyrazoline; PA, phosphatidic acid; PASA) 1-stearoyl 2-arachidonoyl phosphatidic acid.

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system. Similar effects were also reported by Kroll et al. (12). If this hypothesis is true, PA must be generated by hydrolytic action of phospholipase D prior to activation of phospholipase C. However, recent evidence by Huang et al. (22), who found that phospholipase D is activated by elevated cytosolic Ca2+ and most of the PA (about 90%) generated in thrombin-stimulated platelets resulte from the phospholipase C-diacylglycerol kinase pathway, suggests that the phospholipase D-mediated PA formation does not contribute to the early phase of the signal transduction in platelets. In accordance with this view we recently reported that a synthetic didecanoyl PA having medium-length acyl chains activates phospholipase C in cooperation with Ca2+ in rabbit platelets, and suggested that PA contributes to amplification rather than initiation of the platelet activation (23). In the present work, to prove this hypothesis we further investigated a positive feedback role for PA to activate phospholipase A2 in rabbit platelets using exogenous 1-stearoyl 2-arachidonoyl PA (PASA), one of the molecular species of PA produced physiologically under stimulation. MATERIALS AND METHODS Materials—PASA, mepacrine, and leupeptin were purchased from Sigma Chemical (St. Louis, MO). PASA was purified by TLC developed with CHCl3/CH3OH/acetic acid/H2O (30 : 15 : 4 : 2, by volume). 1-Stearoyllysophosphatidic acid was prepared by hydrolyzing 1-stearoyl lysophosphatidylcholine with phospholipase D from Streptomyces chromofuscus (Boehringer Mannheim, Germany). J. Biochem.

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Received for publication, May 6, 1992

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Role for Phosphatidic Acid in Platelet Activation

Vol. 112, No. 6, 1992

reactions were terminated by addition of CHC13/CH3OH/ cone. HC1 (200 : 200 : 1, by volume), then the lipids were extracted and separated by TLC. The radioactivity in each fraction corresponding to arachidonic acid and diacylglycerol derived from [SH]arachidonic acid-labeled platelets and lysophosphatidylcholine from [3H]glycerol-labeled platelets was determined as described previously (28). Preparation of Particulate Fraction—Suspension of the [SH]arachidonic acid-labeled platelets ( l x l O 9 cells/ml) was diluted to fivefold with hypotonic solution (1 mM KHCO,, 1 mM EGTA, 50 ^M leupeptin, 50 ft M APMSF) and lysed by four cycles of freeze-thawing in solid COj/ acetone. Unbroken platelets were removed by centrifugation (1,000 X g for 5 min) and the resulting supernatant was spun at 17,000 Xg at 4'C for 30 min. The particulate fraction was washed once, suspended in KC1 solution (145 mM KC1, 5mM MgCl,, 10 mM HEPES, pH7.2) and adjusted to 350 ng of protein/ml as determined by the method of Lowry et al. (29). Partial Purification of Cytosolic Phospholipase A3 and Assay for the Activity—Washed platelets suspended in 10 mM Tris-HCl buffer (pH 7.4) containing 100 mM NaCl, 1 mM EDTA, 100 //M leupeptin, and 100 /xM APMSF at a concentration of 8 X109 cells/ml were sonicated and centrifuged at 1,500 x g for 10 min to remove unlysed cells. The supernatant was further centrifuged at 100,000 Xg for 30 min and the cytosolic phospholipase A2 in the supernatant obtained was partially purified by sequential column chromatographies on heparin-Sepharose, DEAE-Toyopearl 650M, and butyl-Toyopearl 650M, according to the method of Kim et al. (30). This partial purification resulted in about 25-fold increase in specific activity compared to the heparin-non-binding fraction on the heparin-Sepharose column chromatography. Phospholipase A2 activity was assayed by determining [ U C] arachidonic acid liberated froml-palmitoyl-2-[14C]arachidonoyl-glyceropho8phoethanolamine as a substrate, according to the method of Sundaram et al. (31). To assess the effect of PASA on the partially purified enzyme activity, the substrate (10 j*M) and various concentrations of PASA were dried under N2 gas and sonicated in 100 mM Tris-HCl (pH 8.5), and then the reaction was started by adding 100 ^/M CaCl2 and the partially purified enzyme (5.6 /*g protein/ml). After incubation of the mixture at 37"C for 15 min, the reaction was terminated by addition of isopropanol/heptane/0.5 M H2SO4 (40 : 10 : 1, by volume), then [UC]arachidonic acid liberated was extracted and the radioactivity was determined. RESULTS Effect of PASA on Platelet Aggregation—Upon addition of various concentrations of PASA to washed platelet suspension in the presence of 1 mM Ca2+, dose-dependent aggregation occurred as shown in Fig. 1A. Determination of the amount of the PASA taken up by the cells showed that under these conditions, 12.2±2.2nmol/10° cells (n=3) of PASA was incorporated into the platelets at the concentration of 10 ^M, which is the lowest concentration to induce aggregation. Pretreatment of the platelets with BW755C, a dual inhibitor of cyclooxygenase and lipoxygenase, inhibited the aggregation in the low concentration range .(up to 20 /xM) of PASA, but not at high concentrations ( > 4 0 ^ M ) (Fig. IB).

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A dried sample of each lipid was sonicated in phosphatebuffered saline (140.5 mM NaCl, 10 mM phosphate, pH 7.36) for 4 min at room temperature. p-Bromophenacyl bromide and (4-amidinophenyl)methanesulfonyl fluoride hydrochloride (APMSF) were obtained from Wako Pure Chemical Industries (Osaka). Fura 2-pentaacetoxymethyl ester (fura 2-AM) was from Dohjin Chemical (Kumamoto). [3H]Arachidonic acid (76 Ci/mmol), [3H]glycerol (15 Ci/ mmol), and l-palmitoyl-2-[uC]arachidonoyl-glycerophosphoethanolamine (53.0 mCi/mmol) were from New England Nuclear (Boston, MA). Heparin-Sepharose was from Pharmacia Fine Chemicals (Uppsala, Sweden). DEAEToyopearl 650M and butyl-Toyopearl 650M were from Tosoh (Tokyo). Other reagents were obtained from commercial sources. Preparation of Platelets—Fresh rabbit blood anti-coagulated with one-tenth volume of 1% EDTA was centrifuged at 230 x g at room temperature for 10 rnin to obtain platelet-rich plasma. The platelets from the platelet-rich plasma were washed twice as described previously (24). Finally, the platelets were suspended and adjusted to 5 x 10s cells/ml in modified Tyrode-HEPES buffer (137 mM NaCl, 2.7 mM KC1,1 mM MgCl2, 5.6 mM glucose, 2.9 mM NaH2PO4, 3.8 mM HEPES, pH 7.35). Platelet Aggregation—Platelet aggregation was measured at 37*C in the presence of 1 mM CaCl2 with a light transmission aggregometer (NKK Hema Tracer 1; Niko Bioscience, Tokyo). Incorporation of PASA or Its Lyso-Derivative into Platelets—Washed platelets were treated with PASA or its lyso-derivative at 37'C for 2 min and centrifuged as described above. Each lipid remaining in the supernatant was extracted and determined by the method of Bartlett (25). The amount of the lipid incorporated into the platelets was calculated by subtracting the amount remaining in the supernatant from the total amount in the mixture. Cytosolic Free Ca1+ Concentration—Loading of platelets with a fluorescent dye, fura 2, was carried out essentially according to the method of Pollock et al. (26). Briefly, washed platelets were incubated with 2 fiM fura 2-AM at 37*C for 30 min and then washed once as described above. Fura 2-loaded platelets (2 x 108 cells/ml) were treated at 37"C with various concentrations of PASA in the presence of 1 mM CaCl2 or 1 mM ethylene glycol bisOS-aminoethyl ether) N, W-tetraacetic acid (EGTA). Fluorescence of the suspension was continuously monitored with a spectrofluorometer (F-2000; Hitachi, Tokyo) with excitation at 340 and 380 run, and emission at 500 nm. After each experiment, the cells were lysed with 0.1% Triton X-100 to obtain the maximal fluorescence. Thereafter, the minimum fluorescence was measured after addition of 5 mM EGTA and TrU-base (pH of the lysed cells was adjusted to 8.5). Intracellular Ca2+ concentration was then calculated according to the method of Grynkiewicz et al. (27). Assay for Endogenous Phospholipase Activity—The platelet-rich plasma was incubated with [3H]arachidonic acid (2 fiCi/nA) or [3H]glycerol (80 //Ci/ml) at 37'C for 1 or 3 h, respectively, and washed as described above. The platelets, pretreated with or without 50 ^M BW755C (3-amino-l-[77i-(trifluoromethyl)phenyl]-2-pyrazoline, an inhibitor of both cyclooxygenase and lipoxygenase) at 37'C for 2 min in the presence of 1 mM CaCl2, were exposed to various concentrations of PASA for an appropriate time. The

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T. Sato et al. PASA during the reaction period, we examined the effect of l-stearoyl lysoPA on platelet aggregation. Addition of 10 //M lysoPA to platelet suspension brought about only slight aggregation (10% or less of the full aggregation), whereas that of PASA at the same concentration induced marked aggregation (about 30%), in spite of the larger amount of the lysoPA incorporated (16.1 ± 1.9 nmol/10' cells) than that of PASA at 10 ftM. Furthermore, the concentrations of lysoPA higher than 20 /iM caused the cells to undergo lysis.

PASA 0*0 60 80

10 20 30 40 50 Concentration of PASA (uM)

60 Concentration of P A S A (JJM)

Fig. 1. PAsA-indoced platelet aggregation. Washed platelets were treated at 37"C for 2 min with (A) or without (•) 50 //M BW755C, and then exposed to various concentrations of PAS* in the presence of 1 mM CaCl,. The aggregation pattern as a change in light transmission of the suspension (A) and the extent of aggregation expressed as % of maximum light transmission after 5 min stimulation (B) are shown. Each point represents the mean of two separate experiments.

Concentration of PASA QJM)

Fig. 3. PASA-induced arachidonic add liberation (A) and diacylglycerol formation (B). [8H] Arachidonic acid-labeled platelets were pretreated at 37'C for 2 min with (•, O) or without (A) 50 /iM BW755C, and then exposed to various concentrations of PASA in the presence of 1 mM CaCli (•, A) or 1 mM EGTA (O) for an additional 2 min The radioactivities of [*H] arachidonic acid liberated and ['H]diacylglycerol formed were determined as described in •MATERIALS AND METHODS." Each point represents the mean± SD of three experiments. Significant differences, *p