J. Biochem. 101, 53-61 (1987)

Selective Release of A2 and Lysophosphatidylserine

- Specific from Rat Platelets1

Kazuhiko HORIGOME, Makio HAYAKAWA, Keizo INOUE ,2 and Shoshichi NOJIMA3

Department of Health Chemistry, Faculty of Pharmaceutical Sciences , The University of Tokyo, Bunkyo-ku, Tokyo 113

Received for publication, July 11, 1986

Rat released phospholipase A2 and lysophospholipase upon activation with thrombin or ADP. The release of was energy-dependent and was not in parallel with that of a known lysosomal marker , N-acetyl-f-o glucosaminidase. The phospholipases are derived from other granules (dense granules or a-granules) rather than lysosomal granules of the cells. All of the activities of both phospholipases in the cell free fraction obtained from the activated reaction mixture was recovered in the supernatant after centrifugation at 105,000x g. The degree of of by the phospholipase A2 followed the order: phosphatidylethanolamine (PE) > phosphatidylserine (PS) > phosphatidylcholine (PC). Phospholipase A2 shows a broad pH optimum (> pH 7.0) and absolutely requires Ca 2+. Lysophospholipase was specific to lysophos phatidylserine (lysoPS), and neither lysophosphatidylethanolamine (lysoPE) nor (IysoPC) was hydrolyzed appreciably. Both 1-acyl and 2-acyl-lysophosphatidylserine were equally hydrolyzed. Lysophospholipase activity shows similar pH optimum to phospholipase A2. The lysophospholipase activity was lost easily at 60°C. The activity was reduced by the presence of EDTA, though low but distinct activity was observed even in the presence of EDTA. Addition of Cat} to the mixtures restores the full activity.

Normal serum and plasma from man and several acyl position of a number of sub animals contain phospholipid deacylating activity strates, was attributed to post heparin lipoprotein (1-8). While , acting on the 1- (1-3), serum and plasma also contain phos-

1 This work was supported in part by Grant-in-Aid for Scientific Research (No . 59122004) from the Ministry of Education, Science and Culture of Japan. 2 To whom all correspondence should be addressed . 3 Present address: Faculty of Pharmaceutical Sciences, Teikyo University, Sagamiko-machi, Tsukui-gun, Kana gawa 199-01, Japan. Abbreviations: lysoPS, lysophosphatidylserine; lysoPE, lysophosphatidylethanolamine; lysoPC, lysophosphatidyl choline; PS, phosphatidylserine; PE, phosphatidylethanolamine: PC, phosphatidylcholine; EDTA, ethylene diaminetetraacetic acid; PAF (platelet activating factor), 1-O-alkyl-2-acetyl-sn-3-glycerophosphocholine.

Vol. 101. No. 1, 1987 53 54 K. HORIGOME, M. HAYAKAWA, K. INOUE, and S. NOJIMA pholipase A2 activity (4-8). Though the origin labeled phospholipids by purified snake and function of phospholipase A2 have not been phospholipase A2 (Naja naja) revealed that over established, it has been thought that phospholipase 98 % of the radioactivity in 1-acyl-2-[I-14C]linoleoyl

A2 always circulates in the blood and participates glycerophospholipids was in position 2 and 95 in the turnover of plasma phospholipids, in immu of the radioactivity in 1-[1-14C]palmitoyl-2-acyl nologically mediated membrane damage, or in the glycerophospholipids was in position 1. The spe degradation of bacterial envelope phospholipids cific activity of labeled phospholipids were adjusted during killing of (6, 7). Smith and Silver to 2,000 dpm/nmol by dilution with non-labeled (9) previously reported the release of lysosomal egg yolk PC, PE prepared from egg yolk PC by acidic phospholipase A, from human platelet upon transphosphatidylation, or bovine brain PS. I

stimulation. The activity could only be detected [14C]Palmitoyl-lysophospholipids were prepared by in the presence of detergent. Vadas and Hay (10) treatment of 1-[14C]palmitoyl-2-acylphospholipids reported the release of phospholipase A2 from with phospholipase A2 from (habu, spontaneously aggregated sheep platelets. Release Trimeresurus flavoviridis). 2-[14C]Linoleoyl lysoPS of phospholipase A2 from rabbit peritoneal neu was prepared from 1-acyl-2-[14C]linoleoyl PS by trophils (11) and mouse peritoneal macrophages treatment with Rhizopus delemer lipase, as already (12) stimulated by zymosan were also reported. described (16).

In this paper, we found that rat platelets Platelet Activation-Bloods were taken by

released phospholipase A2 upon stimulation. We cardiac puncture from ether-anaesthetized male

also found that lysophospholipase specific to Wistar rats and mixed with 3.8 % (w/v) sodium

lysoPS together with phospholipase A2, both of citrate in plastic syringes in a ratio of 1 part to 9

which were of a-granule origin, were released when parts of blood. The blood was centrifuged for 10 platelets were stimulated. min at 270x g to prepare platelet-rich plasma.

5-Hydroxy[side-chain-2-14C]tryptamine creatinin

MATERIALS AND METHODS sulfate (serotonin, 0.1 yCi/ml) was added to label the content of the dense granules, and the incu

Chemicals and -[1-14C]Palmitic acid, bation was performed for 30 min at 22?C. After [1-14C]linoleic acid, and 5-hydroxy [side-chain-2 the addition of 1/5 (v/v) of 65 mM citric acid and 14C]tryptamine creatinin sulfate were purchased 85 mM sodium citrate in 2Y. (w/v) glucose (ACD

from the Radiochemical Center, Amersham, U.K. solution), platelets were separated from plasma by Purified phospholipase A2 of Naja naja, thrombin, centrifugation at 1,500 x g for 10 min. Cells were ADP, and p-nitrophenyl-N-acetyl-fl-n-glucosami resuspended in 5 parts HEPES-buffered medium

nide were purchased from Sigma Chemical Com (137 mM NaCl, 2.7 mM KCI, 10 mM HEPES, pany, St. Louis, Mo., U.S.A. A23187 was obtained 0.1 % (w/v) glucose) and 1 part ACD solution, and from Calbiochem-Behring, U.S.A., and NADH washed once more. Washed platelets were finally

from Oriental Yeast Co., Tokyo. Rhyzopus de resuspended in HEPES-buffered medium. lemer lipase (pure grade, 6,000 units/mg) was from The procedure for measuring the release of

Seikagaku Kogyo, Tokyo. All other reagents phospholipases was essentially the same as de used were of analytical grade. scribed previously for the release of platelet gran

Preparation of Substrates--I -[1-14C]Palmitoyl ular proteins (17). Aliquots of the suspension of

2-acyl-glycerophosphocholine and1-acyl-2-[114C] platelets (1 •~ 109 cells/ml) in HEPES-buffered me linoleoyl-glycerophosphocholine were prepared by dium containing 2 mM CaCl2 were incubated with the method of Lands (13) with a slight modifica the indicated concentration of stimuli (thrombin,

tion (14). Both PS and PE were prepared from ADP, A23187, or platelet activating factor (PAF))

the above described labeled PC by transphospha at 37?C. The incubation was terminated after tidylation catalyzed by according cooling in an ice bath, and cells were removed by to the methods of Confurius and Zwaal (15) with centrifugation at 1,200 •~ g for 2 min. The super a slight modification. All labeled substrate thus natant was removed, and aliquots were taken for prepared showed a single spot on silica gel thin measurement of the enzyme activity and [14C] layer plates (Merck, 5724). Hydrolysis of the - serotonin.

J. Biochem. PHOSPHOLIPASE RELEASE FROM PLATELETS 55

Assay of Released Enzymes-Lactate dehydro

genase and N-acetyl-ƒÀ-D-glucosaminidase were measured by the method of Lowry et al. (18) and

that of Day et al. (19) respectively. The results of

all assays were expressed as a percentage of the

total activity measured in the presence of Triton

X-100 at a final concentration of 0.17 % (w/v).

Phospholipase activity was measured using

phospholipids, of which either the sn-1 or sn-2

position was selectively labeled as substrate. The reaction mixture contained 100 mM Tris-HCI (pH

7.4), 4 mM CaCl2, and 401eM sonicated sub

strates. After incubation with the enzyme, the Fig. 1. Release of phospholipase activity from platelets reaction was terminated by adding methanol, and upon stimulation with thrombin. Platelets (1 •~ 109 all lipids were extracted by the methods of Bligh cells/ml) were incubated in the presence of varying con and Dyer (20) and analyzed on thin-layer chro centrations of thrombin at 37?C for 5 min. Aliquots of matography plates as described previously (14). the supernatant obtained by centrifugation of the The radioactive spots corresponding to , reaction mixture were examined for enzyme release. lysophospholipid, and phospholipid were scraped Phospholipase A2 (0), [14C]serotonin (0), N-acetyl-ƒÀ

off from the plate into vials and counted in a glucosaminidase (U), and lactate dehydrogenase (0) Packard Liquid Scintillation Counter Spectrom release were measured as described in " MATERIALS

eter. Phospholipase A2 assay were mainly per AND METHODS." Points are means of duplicate

formed using 1-acyl-2-[14C]linoleoyl PS as substrate determinations.

in a total volume of 0.25 ml. Other conditions

were the same as described above. As phospho unit/ml. A maximum of about 80% of total

lipase Al activity was poor in rat platelets, phos cellular phospholipase (activity detected after soni

pholipase A2 activity was measured in some exper cation) was released. The release of phospholipase

iments by the methods already described (14). The activity almost paralleled the release of serotonin.

reaction was stopped by adding 1.25 ml of Dole's N-Acetyl-(l-D-glucosaminidase, a lysosomal en reagent (21) and the product, free fatty acid, was zyme, was poorly released under the present con

specifically extracted. Lysophospholipase activity ditions. No significant discharge of the cytoplas

was measured by extracting released fatty acids mic enzyme lactate dehydrogenase was observed,

using 2-[14C]linoleoyl lysoPS as a substrate. The either. It is noteworthy here that no phospho

reaction mixture contained 100 mM Tris-HCl (pH lipase activity was detected on intact platelets,

7.4) and Cat} (0.4 mM). Enzyme activities were irrespective of stimulation (data not shown). When

expressed as initial velocity or as percentage of platelets were activated with thrombin, phospho the total phospholipase activity detected after lipase activity associated with cells decreased. The

sonication for two periods of 30 s, with 30 s be activity lost from activated cells was quantitatively

tween each period, using a Branson Sonifier (ap recovered from the culture medium (data not

proximately 80 W). shown). Almost all of the phospholipase activity in the medium was recovered from the supernatant

obtained by centrifugation of the culture medium RESULTS at 105,000 •~ g for 60 min, suggesting that the en Release of Phospholipase and Serotonin from zymes released is not membrane-bound. Rat Platelets upon Stimulation with Thrombin-Rat As is to be expected in any process involving platelets released phospholipase as well as sero an active process like exocytosis (22, 23), secretion tonin when incubated with thrombin (Fig. 1). The of phospholipase as well as that of [14C]serotonin release of phospholipase was concentration de from rat platelets was blocked by metabolic in pendent, being linear up to 0.5 unit/ml thrombin hibitors. Essentially no release was observed in and continuing up to a maximum release at 1 the simultaneous presence of antimycin A (1 ƒÊM)

Vol. 101, No. 1, 1987 56 K. HORIGOME, M. HAYAKAWA, K. 1NOUE, and S. NOJIMA and 2-deoxy-glucose (5 mM) (data not shown) of the position of labeled fatty acid, under the Irreversible aggregation of platelets was observes same experimental conditions where the hydrolysis under the conditions where significant release of proceeded extensively. These findings suggest that phospholipase was induced. both acyl chains were available for the phospho Comparison of Phospholipase Release with Re. lipase(s). LysoPS produced by phospholipase A5 lease of Lysosomal Enzyme in Response to Variouw may be successively hydrolyzed by lysophospho Stimuli"-phospholipase activity was released not only with thrombin but also with A23187 or ADI TABLE IL Positional specificity of the released phos (Fig. 1 and Table I). The release of phospho. pholipase. The incubation mixture contained 100 mM lipase almost paralleled that of serotonin, whereas Tris-HCI (pH 7.4), 4 mM CaC12i 40 nmol of each it was not parallel to lysosomal enzyme release, substrate, and the released enzyme (50,ag protein for ADP induced both serotonin and phospholipase PC, 30 µg for PE and PS) in a total volume of 1.0 ml. release, but not release of N-acetyl-jl-D-glucosamin After incubation for 60 min at 37°C, radioactive prod idase. ucts were extracted and separated as described in " MATERIALS AND METHODS Properties of the Released Phospholipase-The ." Radioactivity in medium of platelets stimulated by thrombin causes fatty acid fraction or lysophospholipid fraction was ex the release of linoleic acid from the 2-position o: pressed as percentage of total radioactivity recovered. All data are corrected for non-enzymatic release of total both PC and PE (Table I1). When phospholipid. label, which was less than 2% in all experiments. containing labeled fatty acid at the 2-position were used as substrates, all of the radioactivity releases was, after thin-layer chromatography, recovered it the fatty acid fraction and none in lyso product, suggesting the enzyme to be of A2 specificity. The specificity was further confirmed using phospho lipids which were labeled in the 1-position with [1-14C]palmitate. After enzymic hydrolysis, the radioactivity increased only in the lyso phospho lipidd fraction, indicating the removal of the non labeled fatty acid in position 2. When PS was used as a substrate, radioactivity was exclusively recovered from the fatty acid fraction, irrespective

TABLE I. Release of phospholipase, serotonin, and lysosomal enzyme in response to various stimuli . Washed platelets (1.0 •~ 109 cells/ml) were stimulated with various stimuli for 5 min at 37?C. Then the extracellular medium was obtained by centrifugation. The amount of phospholipase, [14C]serotonin and N-acetyl-/3-n-glucosaminidase secreted into the medium were determined and are expressed as percentages of the total cellular content . Each value represents the meanl•}S.D. of three experiments.

J. Biochem . PHOSPHOLIPASE RELEASE FROM PLATELETS 57

Fig. 2. Effect of a nonionic detergent, Triton X-100, on the activity of phospholipases released from platelets. Platelets (1 x 108 cells/ml) were incubated with thrombin (2.5 units/ml) for 5 min at 37°C, and whole reaction mixtures were then centrifuged to separate supernatants as described in " MATERIALS AND METHODS." A: The supernatants (4 jig as protein) were incubated with 10 nmol of 1-[14C]palmitoyl lysoPS (0) or 2-[14C]linoleoyl lysoPS (0) for 15 min at 37?C in the presence of various concentrations of Triton X-100. Production of labeled fatty acid was then measured. B: The supernatant (3 ppgas protein) was incubated with 20 nmol of 1-[14C]palmi toyl-2-acyl PS in the presence of various concentrations of Triton X-100. Production of labeled fatty acid (0) and 1-[14C]palmitoyl lysoPS (0) was then measured. C: The supernatants were incubated with 20 nmol of 1-acyl-2-[1iC]linoleoyl PS for 15 min at 37°C in the presence of various concentrations of Triton X-100. Production of labeled fatty acid (0) and 2-[14C]linoleoyl lysoPS (0) was then measured

lipase. The presence of lysophospholipase activity TABLE III. Substrate specificity of lysophospholipase in the medium of activated platelets was confirmed released from activated rat platelets. The incubation by the findings that the culture medium could mixture contained 10 nmol of labeled substrate, re hydrolyze both 1-acyl and 2-acyl lysoPS producing leased enzyme (5 pg protein), 0.4 mM CaCl2, and 10 mM Tris-HCI (pH 7.0) in a total volume of 0.25 ml. radioactive fatty acids in the absence of detergents After incubation for 25 min at 37°C, formation of (Fig. 2A). radioactive free fatty acid was measured as described in Lysophospholipase activity was suppressed by " MATERIALS AND METHODS ." Triton X-100, whereas phospholipase A2 activity was stimulated by the detergent. Production of radioactive 1-acyl lysoPS increased with increase of Triton X-100 when 1-[14C]palmitoyl-2-acyl PS was incubated with the released enzyme (Fig. 2B). At 0.03 %, Triton X-100 gave the highest activity, and the activity decreased at higher concentrations of the detergent. Production of radioactive fatty acid could also be observed under the present conditions. The fatty acid may be released from released enzyme, production of labeled 2-acyl the 1-position of lysoPS by lysophospholipase, lysoPS could not be detected, irrespective of the since no phospholipase A, activity could be de concentration of the detergent, again indicating tected in the preparations (Table II and Fig. 2C). that no appreciable phospholipase A, activity was It is noteworthy here that fatty acid production released (Fig. 2C). was severely suppressed by the presence of 0.03 Substrate specificity of lysophospholipase re Triton X-100. The suppression is compatible with leased from activated platelets was next examined the inactivation of lysophospholipase activities by (Table III). Only lysoPS could be appreciably the detergent under the same conditions. When hydrolyzed, indicating that this lysophospholipase 1-acyl-2-[l-14C]linoleoyl PS was incubated with the is highly specific to lysoPS. The presence of such

Vol. 101, No. 1, 1987 58 K . HORIGOME, M. HAYAKAWA, K. INOUE, and S. NOJIMA

a lysoPS-specific lysophospholipase can explain the poor accumulation of lysophospholipid observed when PS was used as a substrate (Table II). The pH dependence of the phospholipase A2 activity and lysophospholipase activity is shown in Fig. 3, B and D. Both activities could be observed at pH above 5.0 and remained constant over a broad pH range. Phospholipase A2 activity was absolutely dependent on Ca 2+, since no appreciable activity was observed in the presence of EDTA (Fig. 3C), whereas lysophospholipase activity was detected even in the presence of EDTA. Lyso phospholipase activity was heat labile (Fig. 4B),

Fig. 3. requirement of released lysophospho lipase (A) and phospholipase A2 (C), and pH dependence of released lysophospholipase (B) and phospholipase A2 (D). A and C: The enzyme , obtained by the same procedure as described in the legend to Fig. 2, was incubated with 1-[1dC]palmitoyl lysoPS (.) , 2-[14C]li noleoyl lysoPS (0), or 1-acyl-2-[14C]linoleoyl PS (U) suspended in 100 mM Tris-HCI (pH 7.4) containing EDTA or various concentrations of CaC12 for 15 min at 37?C. B: 1-[14C]Palmitoyl lysoPS (•) or 2-[14C]lino leoyl lysoP Fig. 4. Heat inactivation of phospholipase A2 (A) and C in the lysophospholipase (B) activity released from platelets . presence of 0.4 mM CaCI2. The buffers used were 100 The released enzyme (approximately 30 Fig protein) was mM acetate in the range from pH 3 to 6, 100 mM Tris incubated in 0.25 ml of 100 mM sodium acetate buffer , malate from pH 6 to 7, 100 mM Tris-HC1 from pH 7 pH 4.0 (0); 100 mM Tris-HCl, pH 7.0 (0); 0.1 M to 9, and 100 mM glycine-NaOH from pH 9 to 10. glycine-NaOH buffer, pH 9.0 (0) at 60°C. After the D: 1-Acyl-2-[14C]linoleoyl PS (0) was incubated with the indicated time intervals, aliquots were withdrawn to same enzyme preparation as in A and C for 15 min at determine the residual phospholipase A2 and lysophos 37?C in the presence of 4 mM CaC12. The buffers used pholipase activities under the standard conditions de were exactly the same as those described in B. scribed in " MATERIALS AND METHODS ."

TABLE IV. Release of phospholipase A2 and lysophospholipase specific for lysophosphatidylserine in resp onse to th rombin. Platelets (1.0x 109 cells/ml) were incubated in the presence of 2 .5 units/ml of thrombin for 5 min at 37°C. Th en the extracellular medium was obtained by centrifugation . The amount of phospholipase A2 and lysophos pholipase secreted into the medium were determined and are expressed as percentage of the total cellular activity . Totalcellularactiviti es for 1-[14C]palmitoyl-2-lysoPS, l-[14C]palmitoyl-2-lysoPE , and 1-[14C]palmitoyl-2-lysoPC were 7.1, 0.98, 0.046 nmol.min 109 cells platelet equivalent, respectively .

I N .D., not determined.

J. Biochem . PHOSPHOLIPASE RELEASE FROM PLATELETS 59 whereas phospholipase A2 activity was stable under parallel to that of N-acetyl-,B-D-glucosaminidase, a neutral and acidic conditions (Fig. 4A). Phospho lysosomal marker enzyme, suggesting that phos lipase A2 activity was inactivated sharply when pholipase may not come from lysosomes. The incubated at 60?C in a pH 10 solution. phospholipases might be contained in a-granules, More than 50% of the total cell phospho since the dense granules contain mostly low-molec lipase A2 and lysophospholipase activities were ular-weight substances such as serotonin, ADP, released into the medium when platelets were and Ca2+. The idea was supported by the find activated with thrombin and the enzyme activity ing that both phospholipases show high affinity to in the medium was assayed using PS and lysoPS heparin as reported separately (24). Some of the as substrates, respectively (Table IV). It should secretory proteins, including platelet factor 4, which be stressed here that cell homogenate could hy show affinity to heparin, were previously demon drolyze not only lysoPS but also lysophospha strated to be contained in a-granules (25). tidylethanolamine (lysoPE) and lysophosphatidyl Similar release of phospholipase A2 was ob choline (lysoPC). Specific activities of lysophos served with rabbit neutrophils stimulated by f pholipase(s) in the cell homogenates for lysoPS, Met-Leu-Phe and cytochalasin B (26). It is inter lysoPE, and lysoPC were 7.1, 0.98, and 0.046 esting to note here that the total activity in neu nmol/min.109 cells equivalent, respectively. The trophil was greatly enhanced when stimulated. activity hydrolyzing lysoPE and lysoPC could be The authors presumed that the activity may be detected only poorly in the incubation medium of masked by an inhibitor or other means so as not activated platelets, suggesting that there may be to be revealed by conventional sonication. There another lysophospholipase in cells, which can hy is a possibility that platelet phospholipase is also drolyze lysoPE and lysoPC, and was not released regulated by some endogenous inhibitor(s). In upon stimulation. fact, human platelets contain endogenous inhibitor to phospholipase A2 in the particulate fraction

DISCUSSION (27). In rat platelets, however, total cellular phos pholipase activity was not appreciably affected by Phospholipase A2 and lysophospholipase activities stimulation with thrombin. Instead, the localiza were released from rat platelets during activation tion of enzymes was affected, changing from inside by thrombin, ADP, or A23187. The release of the granules to the medium. the enzyme is concentration dependent; the ED50 Lagarde et al. reported that the bulk of phos (0.5 unit/ml) for release of phospholipase A2 ac pholipase AZ of the human platelet was associated tivity is similar to that of serotonin. The lack of with intracellular membrane (28). In our prelim appearance of the cytoplasmic enzyme lactate de inary experiments, more than 50% of the phos hydrogenase in the incubation medium demon pholipase A2 of rat platelets was distributed to the strates that the cell remains intact. The source cytosolic fraction when cells were broken by soni of the enzymes would appear to be in some gran cation, suggesting that part of the phospholipase ules in the platelets, based on the following experi A2 is located in granules in platelets as a soluble mental observations: i) No phospholipase activity form. was detected on platelets without treatment of The characteristics of the released phospho sonication, irrespective of stimulation. The resid lipase A2 are similar to those previously reported ual activity associated with cells (the activity for the phospholipases of rabbit neutrophils (11, detected only after sonication) decreased upon 26), mouse peritoneal macrophages (12), and peri stimulation and the decreased amount was quan toneal exudates (29). The released enzyme is titatively recovered from the medium. ii) The active over a broad range of pH, with an optimum release was energy dependent. iii) The activity in the neutral to alkaline range. The activity of released to medium was almost completely recov the released phospholipase A2 was fully inhibited ered from the supernatants after centrifugation at by 1 mM EDTA, and maximal activation is ob 105,000 X g. served at 4 mM CaC12. Substrate specificity was Platelets contain three kinds of secretory rather broad, since PE, PS, and PC were equally hydrolyzed. granules. The release of phospholipase was not

Vol. 101, No. 1, 1987 60 K. HORIGOME, M. HAYAKAWA, K. INOUE, and S. NOJIMA

Lysophospholipase was also released from 6. Beckerdite-Quagliata, S., Simberkoff, M., & Els platelets upon stimulation. This enzyme might bach, P. (1975) Infec. Inunun. 11, 758-766 also be contained in a-granules, since it similarly 7. Harris, L.K., Weiss, J., Mooney, C., Quagliata, S.B., shows affinity to heparin. The enzyme showed & Elsbach, P. (1980) J. Lipid Res. 21, 617-624 different heat-stability and Ca2+ requirement, indi 8. Etienne, J., Gruber, A., & Polonovski, J. (1980) cating that the activity is on a different protein Biochim. Biophys. Acta 619, 693-698 from phospholipase A2. The enzyme was highly 9. Smith, J.B. & Silver, M.J. (1973) Biochem. J. 131, specific to lysoPS. LysoPC as well as lysoPE was 615-618 hydrolyzed by total platelet homogenates. The 10. Vadas, P. & Hay, J.B. (1980) Life Sci. 26,1721-1729 activity to lysoPE was entirely membrane-bound 11. Traynor, J.R. & Authi, K.S. (1981) Biochim. Bio (unpublished data) and could not be released upon phys. Acta 665, 571-577 stimulation. Existence of at least two different 12. Wightman, P.D., Dahlgren, M.E., Davies, P., & ysophospholipases in platelets was suggested. Bonney, R.J. (1981) Biochem. J. 200,441-444 are widely distributed in 13. Lands, W.E.M. & Merckl, 1. (1963) J. Biol. Chem. procaryotic and eukaryotic cells and are hypo 238,898-904 thesized to have an appreciable role in the metab 14. Arai, H., Inoue, K., Natori-Tamori, Y., Banno, Y., olism of phospholipids by degrading potentially Nozawa, Y., & Nojima, S. (1985) J. Biochem. 97, .ytotoxic lysophospholipids generated by the action 1525-1532 of phospholipase A2. Lysophospholipids are also 15. Confurius, P. & Zwaal, R.F.A. (1977) Biochim. generated in inflammatory foci and may have a Biophvs. Acta 488, 36-42 range of concentration-dependent cytotoxic and 16. Nishijima, M., Akamatsu, Y., & Nojima, S. (1974) noneytotoxic effects on diverse cells. Particularly, J. Biol. Chem. 249, 5658-5667 :he secretion of histamine from mast cells exposed 17. Witte, L.D., Kaplan, K.L., Nossel, H.L., Lages, o antigen, anti-IgE antibody (30), and concana B.A., Weiss, H.J., & Goodman, D.S. (1978) Circ. valin A (31, 32) is strongly and specifically poten Res. 42, 402-409 iated by lysoPS. 18. Lowry, O.H., Roberts, N.R., & Kapphahn, J.I. Recently it has been reported that lysoPS by (1957) J. Biol. Chem. 224, 1047-1064 tself acts as a potent histamine releaser in vivo in 19. Day, H.J., Holmsen, H., & Hovig, T. (1969) Scand. rats and mice (33). No information has been J. Haematol. Suppl. 7, 3-35 available, however, for the possible process of 20. Bligh, E.G. & Dyer, W.J. (1959) Can. J. Biochem. Physiol. 37, 911-917 ysoPS formation. Both platelets and mast cells could be found on the inflammatory sites, and 21. Dole, V.P. & Meinertz, H, (1960) J. Biol. Chem. 235,2595-2599 platelets may provide lysoPS by secretion of phos 22. Palade, G. (1975) Science 189, 347-358 pholipase A2 and stimulus-dependent translocation of PS on platelet membrane (34). Lysophospho 23. Holmsen, H., Robkin, L., & Day, H.J. (1979) Biochem. J. 182, 413-419 ipase specific to lysoPS may function as a scav enger of lysoPS, completing the 24. Horigome, K., Hayakawa, M., Inoue, K., & Nojima, S. J. Biochem. in press process. 25. Kaplan, K.L., Broekman, M.J., Chernoff, A., Lesznik, G.R., & Drilling, M. (1979) Blood 53, REFERENCES 604-618 1. Vogel, W.C. & Bierman, E.L. (1967) J. Lipid Res. 26. Lanni, C. & Becker, E.L. (1983) Am. Assoc. Pathol. 8, 46-53 133,90-94 2. Chajek, T. & Eisenberg, S. (1978) J. Clin. Invest. 61, 27. Ballou, L.R. & Cheung, W.Y. (1983) Proc. Natl. 1654-1665 Acad. Sci. U.S. 80, 5203-5207 3. Groot, P.H.E. & van Tol, A. (1978) Biochem. Bio 28. Lagarde, M., Menashi, S., & Crawford, N. (1981) phys. Acta 530, 188-196 FEBS Lett. 124, 23-26 4. Paysant, M., Bitran, M., Etienne, J., & Polonovski, 29. Vadas, P. (1982) Life Sci.30, 155-162 J. (1969) Bull. Soc. Chi. Biol. 51, 863-873 30. Gomperts, B.D., Cockcroft, S., Bennet, J.P., & 5. Etienne, J., Paysant, M., Grunber, A., & Polonovski, Fertrell, C.M.S. (1980) J. Physiol. (Paris) 76, 383 J. (1969) Bull. Soc. Chi. Biol. 51, 709-716 393

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Vol. 101, No. 1, 1987