Choline plasmalogen biosynthesis by transmethylation in human platelets

THROMBOSIS RESEARCH 45; 687-693, 1987 0049-3848/87 $3.00 t .OO Printed in the USA. Copyright (c) 1987 Pergamon Journals Ltd. All rights reserved.

CHOLINE PLASMALOGEN BIOSYNTHESIS BY TRANSMETHYLATION PLATELETS

IN HUMAN

D. Siepi*, 6. Goracci+, G.G. Nenci** and G. Porcellati*§ Istituto di Chimica Biologica * and Istituto di Semeiotica Medica **, Facolta di Medicina e Chirurgia, Universita di Perugia, 06100 Perugia, Italy R. Mozzia, P. Gresele**,

(Received 13.6.1986; Accepted in revised form by

Editor

B.

7.11.1986

Vargaftig)

INTRODUCTION, In the last few years* the interest in the metabolism of choline glycerophospholipids (ChoGpl) in platelets has increased since they represent about 40% of total membrane phospholipids (1) and participate to the release of arachidonic tissues they consist mainly of acid after stimulation (21. As in other i ,2-diacyl-a-glycero-3-phosphochocholine (diacyl-GroPCho) which accounts for about *O% of the lipid class. Choline plasmalogens (ChoPlas) and 1-0-alkyl-2-acyl-a-glycero-3are also present in platelet membranes and each of phosphocholine (alkylacyl-GroPChol them represents around 10% of total ChoGpl (1). Alkylacyl-GroPCho is believed to be the metabolic precursor of alkylacetyl-GroPCho is not known but recently some (PAF-acether) (3). The role of choline plasmalogens evidence has been reported on their contribution in arachidonic acid release during the first minute after stimulation of human platelets with thrombin (41. The synthesis of ChoGpl in platelets has been studied using different labeled precursors and it has been demonstrated that they can be synthesized de nave (5-7) or by methylation of ethanolamineglycerophospholipids (EtnGpl) (8-141. For both pathways, the incorporation of the precursors into ChoPlas has never been considered, since these data refer to the total class of ChoGpl or to diacyl-type only (5-141. The pathway for the synthesis of ChoPlaa in mammalian tissues is still largely unknown, since alkylacyl-GroPCho cannot be desaturated to the corresponding alKeny1. derivatives (15)~ as reported far the biosynthesis of ethanolamine plasmalogens (EtnPlas ) (16).

Key Words: platelets,

plasmalogen,

methylation,

base-exchange,arachidonic

acid.

Abbreviations: AdoMet= S-adenosylmethionine; ChoGpl= choline glycerophospholipids; EtnGpl= ethanolamine glycerophospholipids; ChoPl.as= choline plasmalogens; EtnPlas= ethanolamine plasmalogens; GroPCho= glycerophosphocholine; GroPE tn= glycerophosphoethanolamine. § Prof. 6. Porcellati deceased on July 25,1934.

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In nervous tissue and in rabbit myocardial membranes ChoPlas can be synthesized by meth;flation of EtnPlas, S-adenosyl methionine (AdoMet acting as the methyl grouo donor il7-131. The aim of this work was to demonstrate the capability of human platelpts to synthesize ChoPlas by methylation of the corresponding ethanolamine derivatives. ‘The presence of this pathway might produce a pool of ChoGpl particularly enrlchecj in arachidonic acid.

MATERIALS AND METHODS Preparation of human platelet Iysate. Human blood (40-60 ml) was obtained from healthy volunteers who had fasted for 12 hours and abstained from any drugs for at least 2 weeks. The blood was mixed with 3.3% of trisodium citrate (1O:i by ~01.1~ washed platelets were then prepared as previously described (71 and finally resuspended in a convenient volume of 0.32 M sucrose. Cells were lysed by sonication in melting ice with 10 set pulses (total sonication time 40 secl using an MSE apparatus (100 Wl with a microprobe. Protein concentration was determined according to Lowry et al. (191, using bovine serum albumin as standard. Incubation of platelet lysate with labeled AdoMet. Disrupted platelets (0.2-0.4 mg protein) were incubated in a medium (0.08 ml1 containing 62 mM Tris-HCl pH 8.2, 0.5 mM 1,4-dithioerythritol (DTE, Fluka, Switzerland) and 10 @Zi of S-adenosyl-L-(methyl3H1methionine (labeled AdoMett specific radioactivity 64-70 Ci/mmole , The Radiochemical Cenfre, Amersham, U.K.). Labeled AdoMet was used within two days from its arrival and, in any case* immediately after the vial was opened. The incubation was carried out at 37.C for various time intervals and the reaction was stopped by the addition of 1.35 ml of hexane/isopropanol (3:2 by ~011 to each sample and then the lipids were extracted according to Hara and Radin (20). A convenient amount of ChoPlas, prepared as previously reported (171, was added to the mixture before extraction. Incubation of platelet lysate with labeled ethanolamine. Disrupted platelets (0.8-1.0 ms protein) were incubated at 37°C in a medium (0.5 ml1 containing 66 mM Tris-HCl pH- 8 , 8 mM CaC12, 0.8 mM DTE, 2 mM (l3Hlethan-l-ol-2amine chloride (The Radiochemical Centre, Amersham, U.K., diluted to a Spec. Rad. of 5.H6 mCi/mmol with unlabeled ethanolamine). After 15 min, unlabeled AdoMet at various concentrations was added to the samples and then they were further incubated for 15 min. The same volume of distilled water was additioned to control samples. The reaction was stopped with 8 ml of chloroform/methanol 12:l by ~011 and lipids were extracted according to Folch et al. (21). ChoPlas were added to the mixture before the extraction of lipids. Analysis of lipids and determination of radioactivity. Lipid; were separated by two-dimensional TLC with exposure to HCl fumes between the first ar,li the second run, according to Horrocks (221, and then localized with iodine vapours. This procedure allows the separation of lyso-derivatives produced by acidic hydrolysi? of plasmalogens, from the corresponding acid-stable derivatives (diacyland alkylacyl- types). Standard lyso-EtnGpl was added to the lipid extracts .i&t before TLC analysis for monitoring and excluding a possible contamination of lyso-ChoGpl coming from ChoPlas acidic hydrolysis. The cmr@spanding areas were removed from the and transferred into counting vials. The radioactivity was measured, as plate previously reported, using a Tri-Carb 460 C Packard Scintillation Spectrometer (17).

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RESULTS AND DISCUSS$QJJ Human platelet lysate was incubated with highly labeled AdoMet for various time Ch oGp1 3H-methyl groups into acid-stable intervals and the incorporation of (diacyl-GroPCho and alkylacyl-GroPChol and into ChoPlas was measured. The results are shown in Fig. 1.

min

FIG. 1 Incorporation of 3H-methyl groups into acid-stable ChoGpl (0) and into ChoPIas (0). Human platelet lysate was incubated n with labeled AdoMet as indicated in the text. Results are expressed as pmoles of JH-methyl groups incorporated in the lipid class/ mg protein and represent the means +/S.D. of 4 experiments with platelet preparations from different subjects.

A consistent radioactivity was detected into acid-stable ChoGpl, thus confirming previous reports (8-14) on the possibility of synthesizing these phospholipids by the methylation pathway. The extent of labeling increased with respect to time but not linearly during the considered interval (30 mini. Radioactive methyl groups of AdoMet were also incorporated into ChoPlas, apparently at a lower rate than into acid-stable analogs. Their radioactivity increased almost linearly with time for about 20 min. At longer periods of incubation an unexplained larger scattering of data was observed. No radioactivity was detected in the corresponding areas of TLC plates when non incubated samples or samples incubated with boiled platelet lysate (5 min) were analyzed. These data indicate that human platelets can synthesize ChoPlas by methylation of the cor;\esponding ethanolamine derivatives. After 20 min incubation of platelet lysate with labeled AdoMet, about 40% of ChoGpl radioactivity is due to ChoPlas. Considering the relative Xow content of ChoPlas in platelets (11, their specific radioactivity is certainly higher than that of diacyl-GroPCho. Moreover alkylacyl-GroPCho might also become labeled by methylation thus contributing ‘to the radioactivity of acid-stable ChoGpl. Another series of experiments was performed by incubating human platelet lysate with 3H-ethanolaminet with or without exogenous AdoMets at pH 3 in the presence of 8 mM CaCL

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TABLE

The distribution after AdoMe t lJJM1

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i

of the Radioactivity into E thanolamine and Choline Phosphoglycerides the Incubation of Platelet bysate with Labeled Ethanolamine Acid-stable EtnGpl

EtnPl a5

Acid-stable ChoGpl

ChoPl as

0

165 +/-

17

31 +/-

7

30 f/-

5

a7 f/f-

10

10

183 +/-

Ii

38 +/-

4

36 +/-

6

61 +/-

7

100

185 +/-

32

41 +/-

9

40 +/-

4

66

13

4/-

Data are espressed as picomoles of radioactive precursor/mg protein t mean +/- SD.) performed in duplicate. Differences between the and refer to two experiments incorporation of ethanolamine into EtnPlas and ChoPlas are highly significant 1 Student t-test: ~(0.012). Platelet lysate was incubated with ‘H-ethanolaminet with and without exogenously added AdoMet, as described in the tect.

As shown in Tab-i, labeled ethanolamine was incorporated into acid-stable EtnGpI and into EtnPlas, being the radioactivity about J-fold higher in the former compounds. Without exogenous AdoMet, a consistent radioactivity was detected into ChoGpl where the labeling of ChoPlas was almost the double of that found in acid-stable compounds. The addition of AdoMet, at the concentrations indicated in the table, had practically no effect on the distribution of radioactivity among the phospholipids which have been considered. The presence of radioactivity into ChoPlas can be considered as another evidence for the methylation of EtnPlas in human platelets. Preliminary results, of E tnGp1 to obtained with higher concentrations of Ca‘: resulted in a lower conversion ChoGpl possibly due to the inhibition of phospholipid methyltransferasetsl (14). These experiments have also provided the first evidence for the synthesis of Etn In fact, in our experimental conditions, in platelets. Gpl by “base-exchange” ethanolamine cannot be incorporated into EtnGpl by the de nave synthesis, since it has been demonstrated by Call and Rubert (23) that Ca++ ions strongly inhibit ethanolaminephosphotransferase activity. Since the labeling of ChoPlas is higher than that of the corresponding ethanolamine derivatives, it is possible that EtnPlas synthesized by base-exchange are more readily available to methyltransferase(s1 than acid-stable EtnGpl. However, a different degradation rate of the two phospholipid subclasses cannot be excluded. In other tissues endogenous AdoMet saturates methyltransferases (24); this could explain why the addition of AdoMet did not cause an increase of radioactivity into ChoGpl in our experimental conditions. The methylation of EtnGpl to CnoGpl has been demonstrated in other tissues (25,261 and it has been postulated that this pathway is involved in “signal transduction” (271. Several P.uthors have reported the possibility that EtnGpl may be converted into ChoGpl in platelets and have also tried to correlate phospholipid methylation with platelet function (8-131. Although methyltransferases inhibitors have been reported to reduce the synthesis of thromboxane by platelets stimulated with collagen (281, it remains difficult to visualize a general mechanism linking phospholipid N-methylation to arachidonate release (29). EtnPlas are particularly rich of arachidonic acid (11 and,

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in spite -,f *‘his, they apparently do not seem to be directly involved in-the release of this f>tt: acid after platelet stimulation (30,311. Their conversion to ChoPlas by methyiation could provide a pool of ChoGpl containilng almost exclusively arachidonate, thus representing a reservoir of prostaglandin precursors. EtnPlas methylation might serve to replenish the pool of ChoPlas 1291. In addition, it has been reported that an alkenylacetyl analogue compound of aggregation , although less potently than can induce platelet hosphatidylcholine PAF-acether (32); this compound could be formed by a deacylation-reacylation pathway, as reported for PAF-acether (331.

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of phospholipid

to L&t.

methylation?

acid

platelet Biochim.

B.B. of from