Proc. Nati. Acad. Sci. USAVol. 82, pp. 371-375, January 1985Biochemistry

Inhibition of human platelet phospholipase A2 activity byunsaturated fatty acids

(arachidonic acid metabolism/endogenous inhibitor)

LESLIE R. BALLOU AND WAI YIU CHEUNGDepartments of Biochemistry, St. Jude Children's Research Hospital and the University of Tennessee Center for the Health Sciences, Memphis, TN 38101

Communicated by Martin Gibbs, September 27, 1984

ABSTRACT Phospholipase A2 (PLA2; phosphatide 2-acylhydrolase, EC 3.1.1.4) activity from human platelets in-creases significantly when the enzyme is separated from anendogenous inhibitor(s). The inhibitor, associated mainly witha particulate fraction, has now been identified as a mixture ofunsaturated fatty acids. Treatment of the inhibitor with tryp-sin, RNase, DNase, or heat did not diminish its inhibitory ac-tivity, which was extractable by organic solvents. Incubationof PLA2 with phospholipids or various neutral lipids, includ-ing saturated fatty acids, had little or no effect on enzymaticactivity. In contrast, unsaturated fatty acids such as palmito-leic acid (16:1), oleic acid (18:1), linoleic acid (18:2), linolenicacid (18:3), arachidonic acid (20:4), all of which were detectedin the particulate fraction, or longer chained unsaturated fattyacids inhibited PLA2 activity by 50% at -5 x 10-7 M. Thelevel of unsaturated fatty acids in the inhibitor fraction wasequivalent to i0-4 M, apparently sufficient to effectively in-hibit PLA2 activity. Methylation of unsaturated fatty acidscaused a complete loss of inhibitory activity, and subsequentdemethylation restored the activity, suggesting that a free car-boxyl group was necessary. Inhibition ofPLA2 by unsaturatedfatty acids appeared to be noncompetitive. PLA2 absolutelyrequired Ca2+ for activity; the inhibition by unsaturated fattyacids was not reversed by Ca2+. The finding that unsaturatedfatty acids are potent inhibitors of PLA2 would explain its gen-erally low activity in human platelet extracts and its markedincrease of activity during the course of enzyme purification.

The mechanism by which human platelet phospholipase A2(PLA2; phosphatide 2-acylhydrolase, EC 3.1.1.4) activity isregulated is not fully understood. Platelet activation and sub-sequent release of arachidonate from membrane phospholip-id is a Ca2'-dependent process, as are the activities of phos-pholipase C (1-3) and PLA2 (4-12), enzymes that generatearachidonate, which serves as the substrate for the synthesisof prostaglandins and thromboxanes (13, 14). Some investi-gators suggest that activation of phospholipase C results inthe conversion of phosphatidylinositol (PtdIns) to a diglycer-ide, which is metabolized to phosphatidic acid by a diglycer-ide kinase (4, 7, 8). Others argue that the diglyceride generat-ed by phospholipase C from PtdIns is hydrolyzed to arachi-donic acid, stearic acid, and glycerol by a diglyceride lipase(1-3, 15). On the other hand, activation of PLA2 results inthe direct release of arachidonate primarily from phosphati-dylcholine (PtdCho) (5, 6, 9-12). Although the in vitro activi-ties of phospholipase C and diglyceride lipase have been re-ported to be sufficient to account for the arachidonate re-leased from activated platelets (1, 2), the activity of PLA2 isgenerally found to be low, and the relative importance ofthese pathways with regard to arachidonate release in vivoremains unclear. However, our recent finding (12) that plate-

let PLA2 activity is much higher than previously realizedsuggests that the enzyme activity reported in the literature isprobably underestimated.

In that report, we showed that total PLA2 activity from ahuman platelet particulate fraction increased dramaticallyduring the course of purification and that the increase in en-zyme activity was due to the removal of an endogenous in-hibitor, probably a lipid, that was associated with the partic-ulate fraction. We now present evidence showing that theinhibitor activity is in fact associated with a mixture of unsat-urated fatty acids that appear to inhibit PLA2 activity non-competitively.

MATERIALS AND METHODSMaterials. All lipid standards were obtained from Nu Chek

Prep. TLC plates (silica gel G and H) were purchased fromAnaltech (Newark, DE). 1-Palmitoyl-2-[1-'4C]arachidonyl-sn-glycerol-3-phosphatidylcholine (51.6 mCi/mmol; 1 Ci =37 GBq) was supplied by New England Nuclear. DEAE-cel-lulose (DE-52) was purchased from Whatman; Affi-Gel 501was from Bio-Rad; EGTA, dithiothreitol, and trypsin inhibi-tor were from Sigma; RNase, DNase, and trypsin were fromWorthington; and all organic solvents were from Fisher.PLA2 Purification. Human platelets were obtained from

recently outdated platelet concentrates by centrifugation at200 x g to remove contaminating erythrocytes. The superna-tant fluid was then centrifuged at 4850 x g for 10 min tosediment the platelets. PLA2 was partially purified with aslight modification of the previous procedure (12). ActivePLA2 fractions from the DEAE-cellulose column werepooled and applied to an Affi-Gel 501 column (1 x 2 cm)equilibrated with 50 mM Tris HCl (pH 7.5) containing 1 mMEGTA (buffer A). The column was washed with several col-umn volumes of the same buffer; no PLA2 activity appearedin the wash buffer. The column was eluted with 10 ml of 10mM dithiothreitol in buffer A; PLA2 activity was recoveredin the eluent front in -2 ml. One advantage of this modifica-tion is that the column effectively concentrated the enzymefrom a large volume of eluent. Another advantage, and per-haps more significant, is that the enzyme thus prepared wasstable during storage. The enzyme was aliquoted and storedin a frozen state for several weeks in 20% (vol/vol) glycerolat -80°C without an appreciable loss of activity.

Fractions collected from the DEAE-cellulose column pos-sessing inhibitory activity were pooled and designated as theinhibitor fraction.PLA2 Assay. A standard reaction mixture (100 ,ul) con-

tained 100 mM Tris HCl (pH 7.5), 100 ,uM CaC12, 1 mM di-thiothreitol, 18 AM [14C]arachidonyl PtdCho, PLA2, and,where indicated, the "inhibitor" or various lipids. The sub-strate was first dried under N2 and then dissolved in 10 ,ul of

Abbreviations: PtdIns, phosphatidylinositol; PtdCho, phosphatidyl-choline; PtdSer, phosphatidylserine; PtdEtn, phosphatidylethanola-mine; PLA2, phospholipase A2; Me2SO, dimethyl sulfoxide.

371

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372 Biochemistry: Ballou and Cheung

dimethyl sulfoxide (Me2SO) prior to addition to the reactionmixture. The assay was initiated either by the addition ofPLA2 or labeled PtdCho, as indicated in the legends to thefigures or tables. In either case, PLA2 activity was not signif-icantly different. The incubation was carried out at 30'C for5 min; the amount of substrate hydrolyzed was usually <3%.All of the radioactivity that disappeared from the substratewas accounted for in the [14C]arachidonate released. No oth-er radioactive compounds were detected in the reaction mix-ture using several solvent systems that clearly separated ara-chidonate from diacylglycerol, phosphatidic acid, and lyso-phospholipids. The [14C]arachidonate isolated by TLC wasassayed in a liquid scintillation counter (12).

Lipid Analysis. Lipids were extracted from the inhibitorfraction by the method of Bligh and Dyer (16) and densito-metrically quantitated by TLC. Polar lipids were co-chroma-tographed with known standards on silica gel H plates devel-oped in a polar solvent system of chloroform/methanol/ace-tic acid/water, 50:25:8:3 (vol/vol). The lipids werevisualized by spraying the plates with 3% cupric acetate in20% ortho-phosphoric acid and charring them at 100'C for 10min. The sensitivity of this method is at least 0.1 Ag ofPtdCho. Neutral lipids with standards were chromato-graphed on silica gel G plates developed in a nonpolar sol-vent system of either pentane/diethyl ether/acetic acid,80:20:1 (vol/vol), or ethyl acetate/acetic acid, 99:1 (vol/vol), and visualized as described above. Lipids pnrified byTLC were recovered from the uncharred plate by removingthe silica gel and extracting it with chloroform/methanol, 1:1(vol/vol). The silica gel was removed by centrifugation andthe organic solvent was dried under N2.

Analysis of Fatty Acid Methyl Esters. After TLC, the fattyacid component of the inhibitor fraction was removed fromthe plate and extracted as described above. The purified fat-ty acids were dissolved in 1 ml of anhydrous methanol towhich 1 drop of acetyl chloride was added. The mixture wasincubated at 220C for 12 hr under N2; the methylated samplewas dried under N2 and redissolved in 50-100 Al of carbondisulfide. The derivatized sample was analyzed by GLC us-ing a Chromosorb W (acid washed) column (183 x 1.3 cm)coated with 10% SP-2330 (Supelco, Bellefonte, PA). The ini-tial column temperature was maintained at 160°C for 3 minand was increased at a rate of 5°C/min until 220°C wasreached. The final column temperature was maintained for10 min. A solution with a known composition of fatty acidmethyl ester was analyzed in parallel to serve as standard.

RESULTSNature of the Inhibitor from Human Platelets. We demon-

strated previously the presence of an endogenous inhibitorof human platelet PLA2 (12). To assess the general nature ofthe inhibitor, we subjected samples of the inhibitor fractionto various treatments as shown in Table 1. The inhibitor frac-tion isolated from the DEAE column (12) was not clearlyseparated from the PLA2 and thus had some PLA2 activity,which was abolished by boiling. Boiling, however, did notaffect the inhibitory activity nor did treatment with DNase,RNase, or trypsin. In fact, the trypsinized fraction wasslightly more inhibitory than the untreated one, presumablybecause the trace of PLA2 activity associated with the inhibi-tor fraction was inactivated by trypsin. In the control tubes,RNase, DNase, or trypsin appeared to reduce PLA2 activityslightly. These findings suggest that the inhibitor was not as-sociated with a protein or a nucleic acid-like substance(platelets are known not to contain RNA or DNA). Next, asample of the inhibitor fraction was extracted with chloro-form/methanol (16); the solution was centrifuged to separatethe organic phase from the aqueous phase. At the interfaceof these phases a distinct layer of denatured protein was

Table 1. Nature of the inhibitor from human plateletPLA2 activity,pmol/min per

Tube Addition mg of proteinA PLA2 1610 ± 69B Inhibitor 442 ± 67C Inhibitor + PLA2 658 ± 39D Inhibitor (boiled) 0E Inhibitor (boiled) + PLA2 316 ± 59F Inhibitor (RNase treated) + PLA2 677 ± 41G RNase + PLA2 1452 ± 63H Inhibitor (DNase treated) + PLA2 638 ± 52I DNase + PLA2 1450 ± 58J Inhibitor (trypsin treated) + PLA2 234 ± 20K Trypsin + trypsin inhibitor + PLA2 1509 ± 55L Inhibitor "protein fraction" + PLA2 1731 ± 74M Inhibitor "lipid fraction" + PLA2 322 ± 31

All assays were performed by using the standard reaction mix-ture, except that the concentration of Ca2l was 1 mM. Whenapplicable, 100 ng of RNase, DNase, or trypsin was present; theinhibitor fraction was 20 ul. Tubes D and E were boiled for 30 minand cooled to 30'C prior to addition of PLA2; tubes F, H, and J werepreincubated for 30 min at 220C prior to the addition of PLA2. TubeK received 10 /ig of trypsin inhibitor prior to the addition of PLA2.Tube L contained 5 ug of denatured protein obtained from theaqueous phase of the chloroform/mnethanol extract, whereas tube Mcontained 5 Ag of lipid from the organic phase of the extract. In allcases, the PLA2 (3 ;g) assay was initiated by the addition of labeledPtdCho. The values represent the means ± SEM of triplicate assaysfrom two separate experiments.

formed. A sample of this protein extract was neutralized andassayed for its ability to inhibit PLA2; none was observed. Asample of the organic phase was dried under N2 and assayedfor its ability to inhibit PLA2; significant inhibition was ob-served. These data indicate that the inhibitory activity wasprobably associated with a lipid(s).

Effect of Lipids on PLA2 Activity. A lipid extract of theinhibitor fraction was analyzed by TLC and found to containphospholipids [phosphatidylethanolamine (PtdEtn), PtdIns,phosphatidylserine (PtdSer), PtdCho] and neutral lipids, amajority of which was cholesterol. First, we examined theeffects of individual phospholipids on PJ,,A2 activity (Fig. 1).At low concentrations (10-6 M to 10- M), PtdSer had noeffect, but PtdEtn or Ptdlns increased PLA2 activity 10%.At 102 M, they all inhibited enzymatic activity 30-40%.PtdCho inhibited PLA2 activity 50% at -40 ,uM and 95% at10-2 M; however, an aliquot of the inhibitor fraction that ef-

120'

2_080

tS 60 \

a. 40 PtCho

20

6 5 4 3 2-log phospholipid (M)

FIG. 1. Effect of phospholipids on PLA2 activity, A standardreaction mixture contained 10-6 to 10-2 M PtdIns, PtdSer, PtdEtn,or PtdCho, which was added in 10 p1 of Me2SO. The assay wasinitiated by the addition of 4 pg of PLA2. One-hundred percentPLA2 activity was 1906 pmol/min per mg of protein.

Proc. NatL Acqd. Sci. USA 82 (1985)

Proc. NatL Acad Sci. USA 82 (1985) 373

fectively suppressed PLA2 activity did not contain a detect-able quantity of PtdCho (<0.1 pg). The "inhibition" of PLA2activity by PtdCho at high concentrations was presumablydue to dilution of the labeled substrate in the assay mixture.

Since phospholipids present in the inhibitor fraction couldnot account for the inhibitory activity, we examined the ef-fects of various neutral lipids on PLA2 activity (Table 2).fliglyceride, cholesterol, cholesterol ester, phosphatidicacid, and alcohols of saturated fatty acids had little or noeffect on PLA2 activity. Saturated fatty acids such as palmit-ic (16:0), stearic (18:0), or behenic (22:0) inhibited PLA2 ac-tivity by about 25%, whereas arachidic acid (20:0) inhibitedenzymatic activity about 40%o. More effective, however,were the unsaturated fatty acids such as palmitoleic (16:1),oleic (18:1), linoleic (18:2), linolenic (18:3), arachidonic(20:4), docosatetraenoic (22:4), and docosahexaenoic (22:6);they all abolished the PLA2 activity. Interestingly, alcoholsof unsaturated fatty acids such as docosatetraenol and doco-sallexaenol stimulated PLA2 activity 2- to 3-fold. These re-sults show that of the neutral lipids tested, the unsaturatedfatty acids were the most effective inhibitors and suggestthat they may well be the active components in the inhibitorfraction.

Fatty Acid Coinpos1tion of the Inhibitor Fraction. To exam-ine if unsaturated fatty acids were in fact the endogenousinhibitors, the fatty acids present in the inhibitor fractionwere isolated by TLC, methylated as described earlier, andanalyzed by GC. Fig. 2 shows a representative elution pro-file of the free fatty acid derivatives obtained from the inhibi-tor fraction. Approximately 44% of the sample consisted ofunsaturated fatty acids: 29.9%o oleic acid (18:1), 4.4% linoleicacid (18:2), 3.7% arachidonic acid (20:4)i 3.3% of an uniden-tified long chain fatty acid, 2.6% palmitoleic acid (16:1), and<1% tricosanoic acid (20:3) and linolenic acid (18:3). Satu-rated fatty acids made up -54% of the sample: 28.5% palmit-

Table 2. Effect of various neutral lipids on the activity of humanplatelet PLA2

Addition

NoneCholesteryl pentadecanoateTricosanolDiglycerideBehenyl alcoholPhosphatidic acidCholesterolTricosanoic acid (23:0)Cholesteryl heptadecanoateBehenic acid (22:0)Stearic acid (18:0)Palmitic acid (16:0)Arachidic acid (20:0)Palmitoleic acid (16:1)Oleic acid (18:1)Linoleic acid (18:2)Linolenic acid (18:3)Arachidonic acid (20:4)Docosatetraenoic acid (22:4)Docosahexaenoic acid (22:6)DocosatetraenolDocosahexaenol

PLA2activity, %

100105 ± 0.1100 ± 10.798 ± 3.193 ± 5.591 ± 2.289 ± 0.488 + 4.387 ± 1.280 ± 3.475 ± 4.475 ± 4.961 ± 3.2

0

0

0

0

0

0

0

275 ± 5.5

200 ± 16.5

The reaction mixture contained the standard components and 3 ,gof PLA2, and 1 mM neutral lipid was added in 10 ,ul of Me2SO. Thereaction was initiated by the addition of labeled substrate. Thevalues represent the means + SEM of triplicate assays from twoseparate experiments. The numbers in the parenthesis refer tocarbon number and number of double bonds in the fatty acid.One-hundred percent activity was 1442 pmol/min per mg of protein.

10Time, min

FIG. 2. Fatty acid composition of the inhibitor fraction. The in-hibitor fraction from a DEAE-cellulose column (12) was extractedand prepared for GC. A representative profile of the fatty acid com-position from the inhibitor fraction is shown. The compound with aretention time of 19 min has not been identified.

ic acid (16:0), 22.6% stearic acid (18:0), and 2.6% myristicacid (14:0). The fatty acid composition of the inhibitor frac-tion from three samples averaged 42% + 5%. These datademonstrated that the inhibitor fraction indeed contained theunsaturated fatty acids that effectively suppressed PLA2 ac-tivity.To determine the amount of unsaturated fatty acids in the

inhibitor fraction we applied a membrane fraction preparedfrom 1 x 101l platelets to a DEAE-cellulose column (see ref.12). The inhibitor fraction was pooled from the column, andthe fatty acids were isolated by TLC and analyzed by GC.From this procedure, we obtained -100 pg of fatty acids, 42fug of which were unsaturated fatty acids. Ifwe assumed that1 ml of packed platelets contained 1 x 1011 cells and thatmost of the platelet lipid was associated with the particulatefraction, the value of 42 Mg of unsaturated fatty acid per 1 mlofpacked platelets would be equivalent to a concentration of=1 X lo- M (the average molecular weight of fatty acid wastaken as 300). This level of unsaturated fatty acid would ap-pear to be more than sufficient to suppress most, if not all,PLA2 activity in vitro. This estimation may not accuratelyreflect their cellular concentration; some fatty acids, espe-cially arachidonic acid, may have been released from theparticulate fraction during the course of PLA2 purification,giving an apparently higher value (see Discussion). Never-theless, these findings strongly support the notion that theactive components of the inhibitor fraction consisted ofknown unsaturated fatty acids.To explore further the active components of the inhibitor

fraction, a lipid extract of the inhibitor fraction was methyl-ated. Table 3 shows that methylation not only rendered theinhibitor inactive but also caused it to become slightly stimu-latory (tube C). Saponification of the methylated sample toremove the methyl groups restored a majority of the inhibi-tory activity (tube D), suggesting that a free carboxyl groupwas necessary for inhibitory activity. In another experiment,we isolated the fatty acids from the inhibitor fraction by

Biochemistry: Ballou and Cheung

374 Biochemistry: Ballou and Cheung

Table 3. Loss of inhibitory activity by methylationPLA2 activity,pmol/min per

Tube Addition mg of protein

A None 1838 ± 64B Inhibitor fraction 480 ± 25C Inhibitor fraction (methylated) 2220 + 138D Inhibitor fraction (methylated, then 950 ± 41

saponified)E Fatty acid fraction 698 ± 2F Fatty acid fraction (methylated) 1728 + 53G Oleic acid (18:1) 331 ± 43H Oleic acid (methylated) 2867 ± 81I Stearic acid (18:0) 1489 ± 24J Stearic acid (methylated) 1691 ± 31

Samples of the inhibitor fraction (20 Al, tubes B-D), the fattyacids isolated from the inhibitor fraction (2 AM, tubes E and F),oleic acid (2 AM, tubes G and H), and stearic acid (2 ,M, tubes I andJ) either were left untreated or were methylated. All samples wereevaporated to dryness under N2, dissolved in 10 A.l of Me2SO, andthen assayed for their ability to inhibit PLA2 activity. The fatty acidspresent in the inhibitor fraction were isolated by lipid extraction andTLC. Tube D contained a sample of inhibitor fraction that wasmethylated, evaporated to dryness, saponified in 33% KOH by re-flux at 70'C for 1 hr, and neutralized with 1 M HCl. All assays wereinitiated by the addition of PLA2 (3 pg). Values represent the means± SEM of duplicate assays from two separate experiments.

TLC. This sample, which contained a mixture of saturatedand unsaturated fatty acids (tube E), inhibited PLA2 activitysignificantly. When methylated, the inhibitory activity in themixture was lost (tube F). When oleic acid (18:1) was meth-ylated (tube H) it not only lost its inhibitory activity but alsobecame stimulatory. Stearic acid (18:0) (tube J), which wasonly slightly inhibitory, became less inhibitory followingmethylation.

Effectiveness of Unsaturated Fatty Acids as Inhibitors ofHuman Platelet PLA2. Unsaturated fatty acids present in theinhibitor fraction were examined individually for their abilityto inhibit PLA2 activity. Fig. 3 shows that all unsaturatedfatty acids tested inhibited PLA2 activity, with 50% inhibi-tion ranging from 2 x 10-6 M for oleic acid to 2 x 10-7 M forarachidonic acid. In increasing order of potency, their effec-tiveness was oleic acid (18:1) < docosatetraenoic acid (22:4)< linoleic acid (18:2) < docosahexaenoic acid (22:6) < lino-lenic acid (18:3) < palmitoleic acid (16:1) < arachidonic acid(20:4). Thus, these data clearly indicated that unsaturatedfatty acids were effective inhibitors ofPLA2 activity and thatthe fatty acids present in the inhibitor fraction could accountfor all of its inhibitory activity.Mechanism of PLA, Inhibition by Unsaturated Fatty Acid.

PLA2 activity was measured as a function of substrate con-centration in the absence or presence of 10-6 M linolehicacid (18:3), a concentration that inhibited PLA2 activity sig-nificantly (Fig. 4). Without linolenic acid, PLA2 activity in-creased rapidly as a function of substrate concentration,reaching 50% of maximal activity at 1 AM PtdCho and 100%at 2 ,M. No further increase in PLA2 activity was observedat higher substrate concentrations. In the presence of lino-lenic acid, PLA2 activity was inhibited at all substrate con-centrations tested; moreover, the enzymatic activity neverexceeded 40% of maximum, which was reached at 5 ,MPtdCho. The inhibition of PLA2 by linolenic acid was notdiminished by increasing the substrate concentration, sug-gesting that the inhibition of PLA2 by linolenic acid was non-competitive, probably due to a direct interaction of linolenicacid with the enzyme at an allosteric site(s).PLA2 absolutely requires Ca2+ for activity (5-7, 9-12)

with half-maximal activity at -1 /iM Ca2+ under our assay

80-

_0

60-

20-

-log unsaturated fatty acid (M)

FIG. 3. Effect of unsaturated fatty acids on PLA2 activity. Astandard reaction mixture contained the indicated amounts of fattyacid. The reaction was initiated by the addition of 6 ;kg of PLA2. x,Oleic acid (18:1); A, palmitoleic acid (16:1); a, linoleic acid (18:2); a,linolenic acid (18:3); e, arachidonic acid (20:4); o, docosahexaenoicacid (22:6); a, docosatetraenoic acid (22:4). One-hundred percentPLA2 activity was 2577 pmol/min per mg of protein.

conditions. Inhibition of PLA2 by unsaturated fatty acid wasnot due to chelation of Ca2 , which was present at 0.1 mM inthe reaction mixture, a concentration significantly higherthan that of fatty acids required to inhibit the enzyme. Rais-ing the concentration of Ca2+ from 0.1 to 1.0 mM did notdiminish the inhibitory effect of these fatty acids.

DISCUSSIONThe data presented here clearly demonstrate that the activecomponent of the endogenous inhibitor of human plateletPLA2 (12) is a mixture of unsaturated fatty acids. In fact, allof the unsaturated fatty acids that were detected in the inhib-itor fraction suppressed PLA2 activity significantly at low(10-7 M) concentrations. Other investigators (17-21) haveshown that unsaturated fatty acids inhibited prostaglandinbiosynthesis in various tissues, and Franson et al. (18k re-ported that oleic acid inhibited PLA2 from rabbit polymor-phonuclear leukocyte granules. The present finding that ole-ic acid and a number of other unsaturated fatty acids inhibit-ed the enzyme from human platelet suggests that theinhibition may be characteristic of PLA2 in other tissues.

In resting human platelets, free arachidonate is barely de-tectable (22-24); upon stimulation by thrombin, its level in-

100 j y +60

);50-

4040

1~30-0L

20

10

C0 2 10 20

PtdCho, juM

FIG. 4. PLA2 activity as a function of substrate concentration inthe presence (e) or absence (o) of linolenic acid. Each reaction mix-ture contained the standard components and the indicated concen-trations of labeled PtdCho. Linolenic acid (18:3) was used as an in-hibitor at a concentration of 1 AtM. The reaction was initiated by theaddition of 6 Ag of PLA2. One-hundred percent PLA2 activity was2404 pmol/min per mg of protein.

Proc. NatL Acad ScL USA 82 (1985)

Proc. Natl. Acad. Sci. USA 82 (1985) 375

creased to 0.28 ,gmol per 1011 platelets (23) (equivalent to 280/iM using our assumption that the volume of 1011 platelets is-1 ml). The levels of other free unsaturated fatty acids suchas oleate and linoleate reached 12 and 3 mM in both unstimu-lated and stimulated platelets (24), respectively. Our estima-tion of free unsaturated fatty acid in a particulate fraction ofplatelets was about 0.1 mM. Although this level may havebeen overestimated because of the potential release of thesefatty acids during the isolation of PLA2 from the particulatefraction, the value is lower than that from whole fresh plate-lets. Others (25) have determined the level of free unsaturat-ed fatty acids in the particulate fraction isolated from freshplatelets. Recalculation of their values indicates that the lev-el is also about 0.1 mM (assuming that 101" platelets give 26mg of membrane protein). These findings would explain ourprevious observation that PLA2 activity markedly increasedduring its purification, apparently as a result of the removalof these unsaturated fatty acids from the enzyme (12). Theeffective inhibition ofPLA2 by unsaturated fatty acids wouldalso explain the general observation of low PLA2 activity inplatelet extracts (1, 2). The apparently low level of PLA2activity has been used as an argument that the enzyme doesnot play a potentially significant role in the release of arachi-donate by stimulated platelets. Our finding that the level ofPLA2 activity may be much greater than generally realizedsuggests that its potential role in arachidonate release de-serves re-examination.

In the experiments presented herein, we have preparedPLA2 from outdated platelets. In other experiments, wehave isolated the enzyme from fresh platelets and have ob-served a comparably low level of PLA2 activity in the plate-let extract and a marked increase of enzymatic activity afterhaving removed an inhibitor from the enzyme during thecourse of purification. This suggests that the findings pre-sented in this paper are not a procedural artifact of agedplatelets.

Stimulation of the platelet results in a rapid release of ara-chidonic acid, a process thought to be initiated by the avail-ability of Ca2+ (9, 13), which activates PLA2. In this study,we found that platelet PLA2 was inhibited by unsaturatedfatty acids in the presence of Ca2+. These unsaturated fattyacids appear to be present in unstimulated platelets in suffi-cient levels to effectively inhibit PLA2 activity. Although wedo not know whether they are accessible to the enzyme invivo, our results indicate that they are associated with thesame particulate fraction as the enzyme. This raises an ap-parently perplexing question. If the PLA2 is inhibited by theunsaturated fatty acids in situ, how is the inhibition over-come when the intracellular level of Ca2+ in platelets israised in response to various agonists? In stimulated plate-lets the transient accumulation of arachidonate appears suf-ficient to inhibit PLA2 activity; does this constitute a mecha-nism for feedback control? Indeed, do the findings here rep-resent a laboratory phenomenon or do they have somephysiological relevance? Answers to these questions mayprove to be important in understanding the mechanism regu-

lating PLA2 activity in human platelets and, perhaps, in oth-er types of cells.

We thank Keith Kunkel and staff of the St. Jude blood bank fortheir assistance in the acquisition of human platelets, Dr. CharlesRock for valuable suggestions, Mark Bierner for editorial assist-ance, and Vicki Gray for typing the manuscript. This research wassupported by Grants CA 21765 and GM 28178 from the NationalInstitutes of Health and by ALSAC. L.R.B. is the recipient of Na-tional Research Service Award CA 09346.

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Biochemistry: Ballou and Cheung