Metabolism of Platelet-activating Factor in Human Platelets

02130-The present study demonstrates that inactivation of exogenous l-O-alkyl-2-acetyl-en-glycero-3-phospho-choline (alkylacetyl-GPC; platelet-activating factor) by human platelets is mediated by the sequential action of two enzymes, 1) a Ca2+-independent acetylhydrolase recovered in the cytosolic fraction of platelets that deacylates alkylacetyl-GPC forming alkyllyso-GPC and 2) a CoA-independent, N-ethylmaleimide-sensi-tive transacylase associated with platelet membranes that incorporates a long-chain fatty acid into alkyllyso-GPC to produce alkylacyl-GPC. Separation of platelet phospholipids and subsequent resolution into individ- ual molecular species by high-performance liquid chromatography revealed that the newly formed alkylacyl- GPC was exclusively alkylarachidonoyl-GPC and that the arachidonoyl group for acylation of alkyllyso-GPC wm provided by phosphatidylcholine. We conclude the previously described platelet arachidonoyl transacylase (Kramer, R. M., and Deykin, D. (1983) J. in the metabolism of platelet-activating factor.

Platelet-activating factor, identified as 1-0-alkyl-2-acetylsn-glycero-3-phosphocholine (l), is synthesized and released by a variety of cells and tissues upon stimulation (2,3). Among the many biological activities described (2,3), alkylacetyl-GPC' has a potent effect on platelets (4-6) and neutrophils (7,8) inducing aggregation and secretion of granular constituents. It was found that both neutrophils (9) and platelets (10,11) rapidly convert added alkylacetyl-GPC to the biologically inactive alkylacyl-GPC.' In human neutrophils the newly incorporated fatty acid at the 2-position consisted of predominantly (>75%) arachidonic acid (12). We have recently described a transacylase in human platelets that is responsible for incorporation of arachidonate into plasmenylethanolamine (13). In this present study we examined the * This study was supported by National Heart, Lung and Blood Institute Grant HL-18586-07 and the Veterans Administration. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The term "acyl" is used throughout the paper to designate longchain acyl groups and is not used in reference to acetyl groups. possible involvement of this enzyme in the inactivation of platelet-activating factor. We found that conversion of alkylacetyl-GPC to alkylacyl-GPC by human platelets involves a transacylase-catalyzed transfer of endogenous esterified arachidonate to alkyllyso-GPC (produced as an intermediate after deacetylation of alkylacetyl-GPC) leading to the formation of alkylarachidonoyl-GPC.
Labeling and Preparation of Platelets-Blood was obtained from healthy volunteers and processed as described previously (16). The platelet-rich plasmas from four different donors were pooled and washed platelets prepared as described (13). For labeling of platelets the platelet-rich plasma was incubated with ["Clarachidonic acid (1 jtCi/lO ml) for 30 min at 37 "C resulting in approximately 10,OOO cpm of esterified ["C]arachidonate/108 platelets. Platelets suspended to 10s/ml in 120 mM NaCl, 2 mM EGTA, 30 mM Tris/HCl, pH 7.5 (buffer B), were frozen in solid C02/acetone, and stored at -70 'C. After thawing, platelets were sonicated for 6 X 15 s at 4 "C using a microprobe sonicator (Heat Systems-Ultrasonics Inc., Plainview, NY) at an output setting of 3. The resulting lysate (containing less than 4% unbroken platelets (13)) was then centrifuged at 100,000 X g for 60 min at 4 "C. The supernatant containing the platelet cytosolic fraction was collected, and the pellet enriched in platelet membranes was resuspended in buffer B by brief sonication in a waterbath type sonicator (RAI Research Corp.). Platelet cytosolic fractions and membranes could be stored at -70 "C for several months without loss of enzymatic activity.
Other Methods-Protein was determined, after adding 1 mg/ml of dodecyl sulfate to samples, according to Bradford (18) using the dye reagent from Bio-Rad. Platelets alkyllyso-GPC never exceeded 5% of the total radioactivity. When [3H]alkylacetyl-GPC was added to platelet cytosol, on the other hand, its hydrolysis was paralleled by appearance of [3H]alkyllyso-GPC and less than 20% of the total radioactivity was recovered as [3H]alkylacyl-GPC after 30 min (Fig.  1B). Degradation of [3H]alkylacetyl-GPC was inhibited >65% after preincubation of platelet lysate for 20 min at 4 "C with 5 mM diisopropyl fluorophosphate. In the presence of 5 mM CaCl, hydrolysis of [3H]alkylacetyl-GPC was decreased by 30%.

Conversion of [3H]Alkykzcetyl-GPC by
When incubated with platelet membranes less than 25% of the added [3H]alkylacetyl-GPC was degraded within 30 min (Fig. IC) (Fig. 2). This reaction was not affected by preincubation of membranes with 5 mM diisopropyl fluorophosphate. To determine whether formation of alkylacetyl-GPC may be catalyzed by platelet transacylase, acylation of alkyllyso-GPC was further characterized in the presence of Caz+, cofactors for fatty acid activation or the sulfhydryl reagent N-ethyl-
Acylation of [3H]alkyllyso-GPC was studied as a function of added platelet membranes. As indicated in Fig. 3 conversion of [3H]alkyllyso-GPC to [3H]alkylacyl-GPC was optimal in the presence of 0.5 mg/ml of membrane protein (equivalent to approximately 1 X lo9 platelets/ml). Formation of alkylacyl-GPC as a function of alkyllyso-GPC concentration is presented in Fig. 4. Acylation of alkyllyso-GPC reached a maximum of 0.8 nmol/min/mg at 0.1 mM of added alkyllyso-GPC and decreased at higher concentrations (20.15 mM) of alkyllyso-GPC. The Lineweaver-Burk plot of the data gave apparent KM and Vmax values of 12 p~ and 0.87 nmol/min/ mg.

Identification of [3HlAlkylucyl-GPC-Platelet transacylase
was previously found to mediate selective transesterification of arachidonate from endogenous platelet phosphatidylcholine to lysoplasmenylethanolamine (13). In order to examine further the possible involvement of this enzyme in the for- mation of alkylacyl-GPC, the nature and origin of the fatty acid(s) incorporated into [3H]alkyllyso-GPC were identified as follows. Platelet membranes were incubated with saturating amounts (see Fig. 4) of radiolabeled hexadecyllyso-GPC to obtain a maximal yield of hexadecylacyl-GPC. After extraction of lipids, the platelet PC fraction containing the newly formed [3H]hexadecylacyl-GPC was isolated and then further analyzed by HPLC, resolving PC into individual molecular species. As previously demonstrated (14), within any phospholipid class, molecular species elute from a reverse- 6. Identification of fatty acid incorporated into hexadecyllyso-GPC. Platelet membranes from 1 X los plateleta prelabeled with ["Clarachidonic acid (containing 1 X lo6 dpm of esterified [14C]arachidonate) were incubated for 30 min at 37 "C without (A) or with ( B ) [3H]hexadecyllyso-GPC (1 X lo6 dpm, 100 mol) in 0.75 ml of buffer B. After extraction of lipids, the PC fraction was isolated and further separated into individual molecular species by HPLC as detailed under "Experimental Procedures." The eluting solvent from the reverse-phase column was collected for peaks detected by ahsorption at 205 nm (see Fig. 5), dried under nitrogen, and counted for 3Hand "C radioactivity. According to Ref. 14, peak 10 and 20 can be identified as palmitoylarachidonoyl-GPC and stearoylarachidonoyl-GPC, respectively. The data shown are representative of three separate experiments.

Transfer of armhidonate from molecular species of PC to hexadecyllyso-GPC
Membranes from 1 X lo9 of ["Clarachidonic acid-labeled platelets (containing 60,000 dpm of PC-esterified ["Clarachidonate) were incubated with or without 100 nmol of hexadecyllyso-GPC in 0.75 ml of buffer B for 30 min at 37 "C. The reactions were stopped with 15 ml of chloroform/methanol (2:l) and hexadecyllyso-GPC added to control membranes. The lipids were extracted and the PC fraction further fractionated into molecular species by two-step HPLC as detailed under "Experimental Procedures." As shown in Fig. 6, palmitoylarachidonoyl-GPC, stearoylarachidonoyl-GPC, and hexadecylarachidonoyl-GPC elute as peak 10, 20, and 16, respectively. The total 14C radioactivity recovered in the various molecular species after phase column according to chain length and degree of unsaturation of the fatty-acyl moieties and can be identified by their retention time. Fig. 5 shows that the generated [3H] hexadecylacyl-GPC eluted as one peak indicating the presence of only one molecular species. The newly formed hexadecylacyl-GPC could also be detected spectrophotometrically (Fig.  5) and its retention time, expected to be longer than for the corresponding 1-palmitoyl analog, suggested that it may be hexadecyllinoleoyl-GPC or hexadecylarachidonoyl-GPC (14). When membranes were preincubated with N-ethylmaleimide formation of hexadecylacyl-GPC as monitored by increased UV absorption and appearance of radioactivity in peak 16 was inhibited >95%.
To determine whether arachidonate was transferred from platelet phospholipids to hexadecyllyso-GPC, membranes from [14C]arachidonic acid-labeled platelets (1 x 10') were incubated with [3H]hexadecyllyso-GPC (100 nmol) for 30 min. Analysis of extracted platelet phosphatides by TLC revealed that although 5 nmol of hexadecylacyl-GPC were produced, the distribution of ["Clarachidonate among the major platelet lipids, including total PC, phosphatidylethanolamine, plasmenylethanolamine, phosphatidylserine, phosphatidylinositol, and neutral lipids remained unchanged and was 61, 12, 3, 9, 7, and 8%, respectively, in membranes incubated with or without hexadecyllyso-GPC. However, resolution of the total PC fraction into individual molecular species by HPLC (Fig. 6) demonstrated that the newly formed [3H]hexadecylacyl-GPC contained "C radioactivity (5% of the total 14C radioactivity in PC) thus identifying the incorporated fatty acid as arachidonate. The gain of "C radioactivity in the newly formed [3H] hexadecyl-['4C]arachidonoyl-GPC was accompanied by a concomitant loss of radiolabel from arachidonate-containing species of PC, particularly palmitoylarachidonoyl-GPC and stearoylarachidonoyl-GPC (Table I).

DISCUSSION
Alkylacetyl-GPC (platelet-activating factor) has multiple biological activities and there is increasing evidence that potent enzymatic mechanisms exist to metabolize alkylacetyl-GPC into a biologically inactive molecule (2). Inactivation of alkylacetyl-GPC was found to occur via deacetylation (19) thus removing the acetyl group that is crucial for expression of its biological activities (20). The enzyme involved, alkylacetyl-GPC/acetylhydrolase, has been detected in the soluble fraction of several tissues with highest activities in kidney and lung (19) and is also present in plasma (19,21) and leukocytes (22). Recent studies, however, indicate that in neutrophils and platelets, alkylacetyl-GPC is converted to a long-chain fatty acyl analog, alkylacyl-GPC (9)(10)(11)(12). We now demonstrate that the transformation of alkylacetyl-GPC by human platelets is mediated by the sequential action of two enzymes, 1) a cytosolic acetylhydrolase that first removes acetate from the 2-position of alkylacetyl-GPC and 2) a membrane-bound transacylase that subsequently acylates the newly formed alkyllyso-GPC with arachidonate to form alkylarachidonoyl-GPC. The enzyme responsible for hydrolysis of alkylacetyl-GPC, acetylhydrolase, exhibits properties similar to the intracellular acetylhydrolase characterized by Blank et al. (19), including lack of stimulation by Ca2+ and inhibition by diisopropyl fluorophosphate. Our results indicate that formation of alkylarachidonoyl-GPC from the intermediate alkyllyso-GPC is catalyzed by platelet arachidonoyl transacylase. Thus, acylation of alkyllyso-GPC by platelet membranes occurs in the absence of added free fatty acids and cofactors (e.g. CoA) and is inhibited by N-ethylmaleimide.
Human platelets contain significant amounts of alkylarachidonoyl-GPC (30). Stimulation of platelets induces release of arachidonic acid from platelet PC (31) with. subsequent conversion to biologically active compounds (32) and also promotes synthesis of platelet-activating factor (26,33). It can thus be speculated that alkylarachidonoyl-GPC may serve as a common precursor for both alkylacetyl-GPC and arachidonic acid metabolites. In support of this hypothesis are recent findings of Swendson et al. (34) demonstrating that in stimulated rabbit neutrophils, 40% of the arachidonic acid released from choline-containing glycerophospholipids originated from alkyl ether-linked species.
We conclude that the enzyme arachidonoyl transacylase plays an important role in the metabolism of platelet activating factor and is responsible for synthesis of alkylarachidonoyl-GPC, a likely precursor of platelet-activating factor.