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Mobilization of arachidonic acid in human platelets kinetics and Ca2+ dependency
370
Biochimica et Biophysics @ Elsevier/North-Holland
BBA
Acta, 488 (1977) 370-380 Biomedical Press
57037
MOBILIZATION
OF ARACHIDONIC
ACID IN HUMAN PLATELETS
KINETICS AND Ca” DEPENDENCY
SUSAN Boston School
RITTENHOUSE-SIMMONS, VA Hospital of Medicine,
(Received
March
FRANCIS
and Departments of Medicine Boston, Mass. 02130 (U.S.A.)
A. RUSSELL
and DANIEL
and Biochemistry,
Boston
DEYKIN University
7th, 1977)
Summary
The release and transfer of [3H]arachidonic acid in human platelets was examined with regard to (1) varied doses of thrombin, (2) varied periods of incubation with thrombin, (3) the presence of 4 mM Ca”, (4) the degree of exposure of platelet phosphatidylethanolamine to the surface and (5) the release of serotonin from platelet granules. The effect of thrombin on the incorporation of [3H]arachidonic acid and [‘4C]glycerol into platelet phosphatides was also studied. [3H]Arachidonic acid was released in pre-labeled platelets exposed to thrombin from phosphatidylcholine and phosphatidylinositol and appeared increasingly esterified in alk-l-enyl-acyl-glycerophosphoethanolamine. Maximum transfer occurred with 5 U/ml of thrombin and 15 min of incubation and was dependent upon the presence of Ca”, whereas serotonin was maximally released with 0.04 U/ml of thrombin. No preferential exposure of plasmalogen (as opposed to diacyl) ethanolamine at the pIatelet surface occurred during incubation with thrombin. The stimulated incorporation of [ 3HJarachidonic acid into alk-I-enyl-acyl-glycerophosphoethanolamine was not accompanied by a stimulation of [ “C]glycerol uptake into this phosphatide. We suggest that, rather than stimulating de novo synthesis, perturbation of the platelet activates a phospholipase AZ activity leading to turnover of arachidonic acid in platelet plasmalogen. Such turnover may help provide substrate for conversion by cyclooxygenase and lipoxydase to biologically active metabolites.
Introduction
The human platelet contains substantial quantities of arachidonic acid bound in acyl linkage to phosphatides [ 1,2]. We have shown [ 3,4] that platelets incorporate radiolabeled arachidonic acid into several of their phosphatides. When
371
subsequently exposed to thrombin, such platelets lose much of their radiolabel from PC * and PI. In a brief preliminary communication [ 41, we noted that the ethanolamine plasmalogen of resting cells contains an insignificant portion of radiolabel. However, when pre-labeled platelets are exposed to thrombin, a marked shift in distribution of arachidonic acid label occurs, producing a greatly elevated content of the labeled fatty acid in alk-l-enyl-GPE and no comparable change in diacyl-GPE or PS. The source of radiolabel is apparently PC and possibly PI. The purpose of the present paper is to describe the kinetic relationship between the release of arachidonic acid and the incorporation of arachidonic acid into alk-1-enyl-acyl-GPE and to report how these changes compare with the course of serotonin release and the exposure of PE to the medium. In addition, we have accumulated evidence to indicate that newly-labeled alk-l-enylacyl-GPE is the result of specific acylation of existing ethanolamine plasmalogen, possibly occurring in conjunction with the activation of a phospholipase A2 which hydrolyzes this phosphatide prior to acylation. Materials Radioisotopes were obtained from New England Nuclear. All solvents employed were Spectrograde or redistilled (diisobutylketone). Highly purified human (Ythrombin (1 nM = 0.11 U/ml) was kindly provided by Dr. J.W. Fenton, II. Lipid standards were supplied by Applied Science Laboratories, Supelco and NuChek. Eicosatetraynoic acid was kindly given us by Dr. W.E. Scott of Hoffman-LaRoche, Inc. Methods
and Results
In the majority of experiments, blood was drawn on acid-citrate-dextrose anticoagulant [5] from normal human donors. The blood was spun in siliconized tubes at 900 rev./min for 15 min in a Sovall GLC-1 centrifuge at room temperature. Platelet-rich plasma was removed and incubated with radioisotopes, as described below, or applied directly to Sepharose 2B columns equilibrated to pH 6.8-7.0 with a buffer system previously described [6,7], from which Ca*’ had been omitted. The number of cells in the excluded volume were counted by a Coulter device. Platelet-rich plasma (6.0ml) was incubated for 15 min with 6.25 PCi 5, 6, 8, 9, 11, 12,14,15 [3H]arachidonic acid (72 Ci/mmol) bound to 10% delipidated bovine serum albumin [ 81, as described previously [ 41. Aliquots of incubation mixture were then sedimented directly by centrifugation at 4500 rev./min for 5 min (Sorvall RC-5, HS-5 rotor). The lipids of the pelleted cells were extracted in the lower phase after with chloroform/methanol (2 : 1, v/v), recovered mixing with 2/10 ~01s. 0.73% NaCl, and resolved on silica-impregnated paper, as described below. More than 99% of the radioactivity was present in the lower * The
following abbreviations are used in the text: diacyl-GPE, diacylglycerophosphoethanolamine: alk-l-enyl-acyl-GPE, alk-1-enyl-acyl-glycerophosphoethanolamine; PC. phosphatidylcholine; PE, phosphatidylethanolamine: PI. phosphatidylinositol: PS. phosphatidylserine; (NO*)3 phS. trinitrobenzene sulfonic acid; (N02)~ ph PE, trinitrophenyl derivative of PE.
372
chloroform phase. The remaining platelet-rich plasma was passed through a Sepharose 2B column. Unincorporated radiolabel, bound to albumin, was well separated from platelets obtained in the excluded volume. These platelets were used in most subsequent studies. Incorporation of radiolabel into filtered platelets was assessed by mixing aliquots with H,O/Aquasol (1 : 10, v/v) and counting in a Packard Scintillation Counter. Results were analyzed using suitable controls for quenching. Lipids were chromatographed at 15°C in two directions, as described by Wuthier [9], on dried Whatman SG 81 paper. The solvent system employed in direction 1 was chloroform/methanol/diisobutyl ketone/acetic acid/H,0 (45 : 15 : 30 : 20 : 4; by vol.) and in direction 2 was chloroform/methanol/diisobutyl ketone/pyridine/H,O (30 : 25 : 25 : 35 : 8; by vol). Prior to chromatography in the second direction, the lipid track was sprayed with 5 mM HgCl, [l] and dried under vacuum in the presence of hot desiccant for 1 h to hydrolyze the plasmalogen 0 J-unsaturated ether linkage, allowing separation of monoacyl-GPE from diacyl-GPE on subsequent chromatography. Following chromatography, the papers were dried, sprayed with aqueous 0.005% Rhodamine 6G, and viewed immediately under ultraviolet light. Lipid spots were cut out and counted in H,O/Aquasol (1 : 10, v/v) or assayed for phosphorus [9]. Platelet lipid samples, chromatographed in two directions and exposed or unexposed to HgC12, were subjected to spraying with a solution of ninhydrin or 0.4% of the aldehyde, ketone, and vinyl ether reagent, dinitrophenylhydrazine in 0.1 M HCl. The phosphorus content of the spots indicated by the staining reagents was analyzed, as was the phosphorus in those relevant regions of the paper which would serve as blanks. Three ninhydrin-positive spots were seen: one corresponding to standard PS, and two in the PE lane, corresponding to monoacyl-GPE and diacyl-GPE. The neutral lipid region of the PE lane was dinitrophenylhydrazine-positive. When water was substituted for the HgCl* spray (a) only two ninhydrin-stained spots were visible: PS and PE; (b) one dinitrophenylhydrazine-reactive spot was found, corresponding to PE. Only when the lipid track had been sprayed with HgClz did radioactivity appear in the monoacyl-GPE region, varying primarily with prior treatment of [ 3H]arachidonic acid-labeled platelets with thrombin. In no case was radioactivity found in the neutral lipid region of the PE lane. Thus the fatty aldehyde derived from alk-lenyl-acyl GPE contained no radiolabel. The ratio of phosphorus for mono-acyl-GPE/diacyl GPE was 0.97 of-0.06 (n = 4), indicating complete breakdown of alk-1-enyl-acyl-GPE by HgCl*, since the plasmalogen content of PE is about 50% [ 11. In addition, a quantitative determination of aldehyde in the neutral lipid region of the PE lane was made by the method of Rapport [lo] for experiments in which HgClz spraying was either included or omitted in the chromatographic procedure. After HgClz spraying, the aldehyde found was equimolar with monoacyl-GPE phosphorus. “H-Labeled monoacyl-GPE extracted from paper chromatograms was subjected to alkaline hydrolysis with 15% KOH in 50% methanol-H20 at 60°C for 1 h. The chloroform-extracted hydrolysate was chromatographed in one direction on silica paper in the chloroform/methanol/diisobutylketone/acetic acid solvent described. More than 95% of 3H was found in the chloroform fraction,
373
and of this, more than 90% migrated as neutral lipid, the remainder being in unhydrolyzed monoacyl-GPE. For studies comparing de novo synthesis of phosphatides with fatty acid uptake, filtered platelets (unlabeled) were added to siliconized tubes (4.6 * lo8 cells) containing delipidated bovine serum albumin (final concn. 0.5%) with or without bound oleic acid (0.6 PM) and Ca” (4 mM). Immediately thereafter, human thrombin (2.5 U/ml) or buffer (pH 7) was added. After 15 s, glycerol-2[‘“Cl (11.8 mCi/mmol, 1.9 mM, final concn.) or albumin-bound [“Hlarachidonic acid, 4 nM (described above) were added and the mixtures were incubated 30 min more at 37°C. EDTA (5 mM), pH 6.5, was added to each tube, and the cells were sedimented at 4500 rev./min for 5 min. The pellets were extracted with chloroform/methanol. Whereas the presence of 0.6 PM oleic acid inhibited the incorporation of [3H]arachidonic acid (4 nM) into phosphatides of resting cells by 20-30%, it had no significant inhibitory effect on arachidonic acid incorporation into alk-l-enyl-acyl-GPE when thrombin was present. In a series of parallel experiments, platelet-rich plasma (2.2 - 10' cells), buffered at pH 6.8, was incubated for 30 min with [‘4C]glycerol (0.2 mM) or [3H]arachidonic acid (16 nM) in the absence of thrombin. Cells were sedimented and their lipids extracted and resolved. In the absence of thrombin, incorporation of [‘4C]glycerol into alk-1-enyl-acyl-GPE was very low, but similar for incubated filtered and plasma-suspended platelets. The amounts of [‘4C]glycerol incorporated into diacyl-GPE for plasma and filtered platelets were also similar to each other. The uptake of [3HJarachidonic acid into alk-lenyl-acyl-GPE of plasma platelets was only 20% of that found in this phosphatide of filtered platelets. In contrast, incorporation of [3H]arachidonic acid into diacyl-GPE was about 70% of that in diacyl-GPE of filtered platelets. The slight depression of 30% in the latter case can be accounted for by isotope dilution, since plasma itself contains (unlabeled) arachidonic acid. Such dilution cannot, however, account for the 80% less incorporation of arachidonic acid into alk-l-enyl-acyl-GPE of plasma platelets. Filtered platelets derived from platelet-rich plasma incubated with [“HIarachidonic acid were treated for 5 min with freshly-prepared acetylsalicylic acid (100 pg/mll’), indomethacin (60 pug/ml-‘) or 5, 8, 11, 14-eicosatetraynoic acid (3 lug/ml-‘). Acetyl-salicylic acid and indomethacin have been shown to be inhibitors of platelet cyclooxygenase [ 111 and eicosatetraynoic acid acts as an inhibitor of cyclooxygenase and lipoxydase [ll]. Both enzymes normally utilize non-esterified arachidonic acid in platelets. Treated and untreated platelets were incubated further with 4 mM Ca2+ and 2.5 U/ml-’ thrombin for 15 min. Quantitation of radiolabel in resolved phosphatides showed no consistent effect of any inhibitors on thrombin-promoted release of arachidonic acid from PC and PI, or transfer to alk-1-enyl-acyl-GPE. Such a finding tends to exclude the possibility that labeled prostaglandin products might be responsible for the label found in alk-l-enyl-acyl-GPE. For kinetic studies, filtered platelets pre-labeled with 13H]arachidonic acid were incubated at 37°C in 0.5% delipidated bovine serum albumin, pH 7.0, in the presence and absence of 4 mM CaCl* and 2.5 U/ml of thrombin, for up to 30 min. At various times after the addition of thrombin to labeled platelets,
374
ice-cold EDTA was added and the cells were sedimented. The release of radiolabel to the supernatant and the distribution of remaining radioactivity among the lipid fractions of the pelleted cells were determined. In other experiments, the response of labeled platelets to O-10 U/ml of thrombin after 15 min of incubation was ascertained as above. Prior to release reaction studies, platelet-rich plasma (7.0 ml) was incubated with 2 FCi [‘4C]5-hydroxytryptamine (creatinine sulfate salt, final concentration, 1 FM) for 15 min at 3’7”C, with gentle shaking. Incubated mixtures were then filtered through Sepharose 2B. [‘4C]Serotonin not associated with platelets was removed by this procedure. Incorporation of serotonin was 60-70%. Platelets pre-labeled with [ i4C] serotonin were incubated with varied concentrations of thrombin for 15 min as above, sedimented, and the extent of release of radiolabel to the medium was assessed. In addition, platelets obtained after Sepharose filtration were incubated with Ca’+ and varied doses of thrombin for 15 min at pH 7. Subsequently, (NO,), phS at pH 7 was added (final concentration, 3 mM) and cells were incubated 15 min further. Cells were pelleted after the addition of EDTA-glycine, pH 6.5. It was possible, using the above-described chromatography system, to separate diacyl-GPE and monoacyl-GPE not only from other phosphatides, but from their (NO,), phS derivatives. The extent of formation of (N0,)3 ph-PE derivatives was determined by phosphorus analysis. When pre-labeled filtered platelets were incubated with thrombin, both the decrease of [ 3H]arachidonic acid in PC, and the incorporation of radiolabel into alk-l-enyl-acyl-GPE were considereably diminished if Ca*’ was omitted from the incubation medium (Fig. 1). The extent of depression was progressive with time. The kinetics of loss from PI appears to be complex, in that measurable reincorporation of arachidonic acid into the PI pool occurred with time when Ca*’ had not been added. Mobilization of labeled arachidonic acid in the presence of Ca2+ was stimulated by increased concentrations of human thrombin. Changes were minimal above 5 U/ml (Fig. 2). [3H] Arachidonic acid in PS varied slightly, increasing by 5% with time in the absence of Ca*’ and decreasing by, at most, 10% when Ca2’ was included. Diacyl-GPE showed a 5% loss of [3H]arachidonic acid for incubations without Ca*+ and a lo-15% loss with Ca*+. In the presence of Ca”, loss of radioactivity from diacyl-GPE after 15 min was only 15% of the gain observed in alk-1-enyl-acyl-GPE over the same period. Values for PS and diacyl-GPE have been omitted from Figs. 1 and 2 for reasons of clarity. The loss of radiolabel from PC and PI could be accounted for by the increased presence of label in alk-1-enyl-acyl-GPE (as arachidonic acid) and in the incubation media as arachidonic acid and cyclooxygenase and lipoxydase metabolites. Such metabolites and fatty acids were resolved by high performance liquid chromatography. Fig. 3. represents the cumulative results of six experiments in which the percentage of radiolabel which was present in platelets as alk-l-enyl-acyl-GPE is correlated with the percentage of total platelet radioactivity which was released to the medium. The data include values for released radioactivity from platelets merely shaken for different periods of time at 37°C and not exposed to thrombin, as well as for cells exposed to thrombin..The dotted line is the extrapolation to the limiting case of zero release, and goes through the origin. This
375
L_
0
I
I
20
30
I
IO
Time (min)
1
I
I
0
5
IO
Thrombin (U/ml)
Fig. 1. Change in content of [ 3Hlarachidonic acid in phosphatides with time following exposure of labeled platelets to thrombin. 3H-Labeled platelets were obtained by incubation of platelet-rich plasma with [3Hlarachidonic acid. Platelets were gel-filtered and incubated with thrombin (2.5 U/ml) with and without 4 mM 0%. shaking at 150 rev./min. No significant change in isotopic distribution was found for platelets incubated without thrombin and CaB. (0). PC - Ca2+; (0). PC + Ca2+: (A), alk-1-enyl-acyl-GPE + Ca*+: (A). alk-l-enyl-acyl-GPE - CaZ+: (0). PI - Ca*+. , Cm), PI + Ca*+. Radioactivity given as thousands of dpm/109 platelets. Fig. 2. Change in content of [3Hlarachidonic acid in phosphatides following exposure of labeled platelets to thrombin in varied doses. Labeled gel-filtered platelets were incubated with thrombin and 4 mM Ca” for 15 min. shaking at 150 rev./min. (0). PC; (A), alk-lenyl-acyl-GPE; (w). PI. Radioactivity as in Fig. 1.
finding suggests that platelets, preincubated with [3H]arachidonic acid but not perturbed, do not transfer radiolabel into alk-l-enyl-acyl-GPE. Mobilization of incorporated arachidonic acid and transfer to alk-1-enyl-acyl-GPE is thus triggered by perturbation of the platelet. We next examined the effect of gel filtration of the 3H-labeled plasma platelets on transfer of label to alk-l-enyl-acylGPE (Table I). Gel filtration of 3H-labeled platelet-rich plasma caused minimal transfer. Gel-filtration prior to incubation with radiolabel was associated with greater radioactivity in alk-1-enyl-acyl-GPE which may reflect the more vigorous shaking of the filtered platelets, rather than activation attributable to gel-filtration per se. A partial activation of the platelets, whether due to the removal of plasma factors or contact with Sepharose beads, cannot be unequivocally ruled out. However, in other experiments (unpublished) we had found that washing of the platelets caused greater activation (as measured by transfer of radiolabel to alk-lenyl-acyl-GPE) than did gel-filtration. Thrombin caused maximal transfer of label to this ethanolamine plasmalogen. In parallel experiments (Fig. 4) we compared three phenomena promoted by thrombin: (1) the labeling of alk-l-enyl-acyl-GPE with [3H]arachidonic acid, (2) the release to the medium of serotonin from the platelets and (3) the by the non-penetrating opening of PE to the platelet surface, as detected
376
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4
8
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16
20
24
28
32
36
0 %
of total lncorporoted
‘H released
to medium
Thrombtn
(U/ml)
Fig. 3. Transfer of [3Hlarachidonic acid to labeled platelets as a function of label released to the incubation medium. Labeled gel filtered platelrts were incubated for different periods of time and different shaking frequencies at 37’ C and pH 7, with and without thrombin. Radioactivity in the platelet-free medium and in platelet alk-1-rnyl-awl-GPE was then quantitated. Fig. 4. Percentage of maximum serotonin release, (NO*)jph-PE formationand transfer of [3H1arachidonic acid to alk-1-enyl-acyl-GPE achieved with varied low doses of thrombin. Gel-filtered platelets. labeled with [14Clserotonin or [ 3Hlarachidonic acid. were incubated with Ca*+ and thrombin for 15 min. Release of 14 C to the medium or transfer of 3H to alk-l-enyl-acyl-GPE were assessed with regard to the maximum effect achievable with thrombin (at 2.5 U/ml). In addition. thrombin-treated filtered platelets were exposed to trinitrobrnzrne sulfonate for 15 min. The amount of reaction product ((N02)3 ph-PE) in the platelet lipid extracts was assayed by phosphorus analysis. (a), alk-l-enyl-acyl-GPE - 3H: (?). serotonin -- 14C: (A). (NO2 )3 ph-PE.
reagent, (NO,), phS [12,13]. All are plotted as the percentage of the maximum response achieved with thrombin (2.5 U/ml) seen at varied low concentrations of thrombin. Maximum release of serotonin was 90%. The surface exposure of PE induced by thrombin was similar to the release of serotonin, which was very sensitive to thrombin, achieving its greatest level at 0.04 U/ml. In contrast, the release of arachidonic acid and its transfer to alk-1-enyl-acyl-GPE did not reach half its maximum extent until 10 times that amount of thrombin had been
TABLE
I
DISTRIBUTION ~____ Phosphatidr
OF [“H]ARACHIDONIC PRP a
ACID GFPb
AMONG
PLATELET GFP ’ --.--
PC PI PS PPE DPE
66 7 11 0.6 12
64 10 12 1.0 11
58 11 12 3.2 12
PHOSPHATIDES _ GFP fhrombin
(%)
__ ____
49 3 14 17 14
a Platelet-rich plasma (PRP) was incubated with [3Hlarachidonic acid at 37°C for 15 min, 50 rev./ min. b PRP incubated as in (a) was filtered through Sepharose to yield labeled gel-filtered platelets (GFP). c Unlabeled GFP were incubated with [ 3H]arachidonic acid for 15 min at 37@C. 150 rcv./min. d Labeled GFP. as in (b) were incubated with thrombin (2.5 U/ml) and 4 mM Ca2+ for 15 min at 150 rev./min. PPE, alk-l-enyl-acyl-GPE; DPE, diacyl-GPE.
_
377
added. The degree of exposure of PE increased only two-fold under our incubation conditions, going from 8.5% to 15% of the total platelet PE. We further examined the labeling of PE with (NO,), phS to determine if there was preferable exposure of one of the species of PE: diacyl-GPE or alk-l-enyl-acyl-GPE, in the course of treatment with thrombin. The phosphorus ratios of monoacylGPE/diacyl-GPE and of (N0,)3 ph-monoacyl-GPE/(NO*), ph-diacyl-GPE for resting and thrombin-activated platelets were compared, and were consistently 0.97. Thus, no disproportionate accessibility of surface alk-l-enyl-acyl-GPE or diacyl-GPE was measurable, with or without thrombin. Since the amount of total PE in the platelet which is in the form of alk-l-enyl-acyl-GPE is about 50% [ 11, accessibility of the two ethanolamines was equivalent. Incubation of filtered platelets with [14C]glycerol led to incorporation of this radiolabel into a number of phosphatides. The majority of the radioactivity was recovered in PC (69%) followed, in decreasing order, by PI (17%), diacylGPE (6.9%), PS (4.0%) and alk-1-enyl-acyl-GPE (2.9%). Thus, the incorporation of 14C into the ethanolamine plasmalogen of control cells was 0.04 of that into PC (whereas the molar ratio of alk-1-enyl-acyl-GPE to PC is 0.35). This was the case whether uptake of radiolabel was studied in filtered platelets or platelet-rich plasma. As shown in Table II, the addition of thrombin to filtered platelets caused a marked depression in incorporation of [ 14C]glycerol into PC, as had been anticipated from previous reports based on experiments with washed platelets [14,15]. The effect of thrombin on the uptake of [‘Hlarachidonic acid and on that of [‘4C]glycerol into several other platelet phosphatides is also shown in Table II. Whereas incorporation of [‘4C]glycerol into alk-lenyl-acyl-GPE was depressed by thrombin, incorporation of [ 3H] arachidonic acid rose, as noted before for platelets pre-incubated with arachidonic acid and then exposed to thrombin. When the ratio of ‘H/14C incorporated into cells in the presence of thrombin is divided by that ratio in the absence of thrombin, a number representing the proportionality of the effect of thrombin on [ 14C]glycerol and [ 3H]arachidonic acid uptake into various phosphatides is obtained. A perfectly proportionate effect would give a ratio of 1. PC and diacyl-GPE approach this ratio most closely. Alk-1-enyl-acyl-GPE has a much greater ratio (7.7) and PS and PI, much less (0.28,0.29). TABLE
II
EFFECT
OF
GLYCEROL
THROMBIN INTO
ON
THE
PHOSPHATIDES
INCORPORATION OF
GEL-FILTERED
OF
[3H]ARACHIDONIC
ACID
AND
t14C1-
PLATELETS
Platelets (2.3 . lo* cells/ml) were incubated for 30 min with the radiolabeled compounds added 15 s after the addition of buffer (pH 7) or thrombin (T); concentrations: Ca*+. 4 mM: (where Present) thrombin. 2.5 U/ml; [ *4C]glycerol. 0.2 mM; r3HJarachidonic acid, 4 nM. PPE, alk-l-enyl-acul-GPE; DPE. diacyl-GPE. Phosphatide
PPE DPE PS PI PC
3H/‘4C
14C/‘4C
3H/3H
-T
+T
+T/-T
+T/-T
+T/-T
31 114 252 63 85
238 89 71 18 99
1.7 0.78 0.28 0.29 1.2
0.45 0.54 1.1 1.2 0.25
3.4 0.42 0.29 0.35 0.29
378
Discussion It appears that thrombin-stimulated acylation of alk-l-enyl-acyl-GPE does not require de novo synthesis of this phosphatide, but probably is dependent upon either the prior formation of alk-l-enyl-GPE resulting from the action of a phospholipase AZ, or a transacylating mechanism that does not involve lysophosphatides. In addition, our data indicate the existence of an acylating activity in human platelets which, in the presence of thrombin, exhibits a specificity for arachidonic acid, as opposed to oleic acid. We conclude that it is unlikely that de novo synthesis is the major determinant of the increment in arachidonic acid incorporation into ethanolamine plasmalogen for the following reasons: [‘4C]glycerol uptake into alk-l-enyl-acyl-GPE, used as a gauge of de novo synthesis, was comparable for plasma platelets and filtered platelets although there was more than three times more uptake of [3H]arachidonic acid into filtered platelet alk-lenyl-acyl-GPE. The data in Fig. 3 and Table i indicate that, in the limiting unperturbed state, platelets incorporate no arachidonic acid into alk-1-enyl-acyl-GPE and that the incubated filtered platelets derived from labeled plasma are slightly activated. Consequently, one might well assume that a small amount of phospholipase A2 was active in incubated filtered platelets and that incorporation of [“Hlarachidonic acid which was observed was dependent on the transient formation of lyso-ethanolamine plaswhile the plasmalogen malogen (i.e., with fatty acid absent at glycerol-sn-2, linkage was still intact at glycerol-sn-1). By extension, one could argue that a similar, magnified, phenomenon occurred when filtered platelets were incubated with thrombin (Table II), during which [3H]arachidonic acid incorporation rose more than three-fold, and [ “C]glycerol incorporation fell by half. The only precedent in the literature for such an activation of which we have knowledge is that cited by Wykle, et al. [16] for rat testis enzyme studies. These investigators demonstrated a tendency for unsaturated fatty acids to build up in ether-linked lipid classes via deacylation-reacylation reactions. Rats on an essential fatty acid-deficient diet preferentially incorporated [“Hlarachidonic acid into the 2-position of alk-1-enyl-acyl-GPE rather than into diacylGPE. It was conjectured that plasmalogens might serve as a reservoir for prostaglandin precursors during essential fatty acid deficiency [ 17,181. The dependency on Ca” of the thrombin-induced decrease in [3H]arachidonic acid in PC and PI is consistent with the observations of Derksen and Cohen [19], who examined the release of arachidonic acid from endogenous substrates in platelet membranes. They found a virtually complete dependency of the phospholipase A2 on added Ca”. The progressively increasing extent to which [3H]arachidonic acid mobilization was subject to added Ca2+ (Fig. 1) might be explained by depletion of the internal Ca” stores [20] which the platelet is known to have. Recent work by Jesse and Cohen [21] has indicated that 72% of the arachidonic acid hydrolyzed from endogenous phosphatides by plasma membrane phospholipase A at pH 9.5 comes from diacyl-GPE, with the remainder coming from PC. No hydrolysis of alk-1-enyl-acyl-GPE was observed under these conditions. In contrast, we have found only a lo-15% loss of [“Hlarachidonic acid from diacyl-GPE at pH 7 (3,4] after the activation of platelets by thrombin, a
379
major loss of this labeled fatty acid from PC, and an implied deacylation of alklenyl-acyl-GPE followed by acylation with labeled fatty acid, Clearly, several platelet fractions should be examined closely at a variety of pH’s with regard to ethanolamine plasmalogen-specific fatty acid hydrolysis. It is possible, for example, that alk-lenyl-acyl-GPE-phospholipase A is not bound to the plasma membrane but is associated with a particulate fraction not examined by Jesse and Cohen. We are presently undertaking such experiments. In addition, we are exploring another suggestion which we made above: that a PC-alk-l-enyl-acylGPE transacylating activity might exist which would not lead to generation of lyso-phosphatides. We have found that in vitro incorporation of arachidonic acid into alk-lenyl-acyl-GPE becomes pronounced during thrombin-induced activation of platelets. It is known [l] that the endogenous level of arachidonic acid in plasmalogen phosphatide of human platelets is high enough to account for essentially all of the sn-2-fatty acid in this phosphatide. Therefore, if the entry of labeled arachidonic acid is contingent on the function of phospholipase A2 acting on alk-1-enyl-acyl-GPE, then unlabeled arachidonic acid must be liberated from alk-1-enyl-acyl-GPE during activation. Our study of the course of release of arachidonic acid from PC and PI showed half maximal release at 2.5 min. However, Hamberg et al. [22] demonstrated that washed human platelets incubated with thrombin formed the prostaglandin metabolites PHD and HETE half-maximally in less than 1 min. Since there is little if any free arachidonic acid within the normal platelet, and virtually all of the arachidonic acid is bound to phosphatides [23], this fatty acid must be hydrolyzed from some phosphatide(s) before being acted upon by cyclooxygenase. Should a liberation of arachidonic acid from alk-1-enyl-acyl-GPE significantly precede that from PC and PI, in keeping with the kinetics reported by Hamberg et al., there would be compelling reason to view alk-l-enyl-acyl-GPE as a precursor phosphatide. Thus it is possible that the turnover of ethanolamine plasmalogen contributes to the pool of free arachidonic acid for utilization by cyclooxygenase and/or lipoxydase and therefore helps provide biologically active metabolites [11,241. We have indicated in Fig. 4 that the binding of thrombin to a few sites on the platelet membrane (120 out of a possible 50000 [25]) is sufficient to promote both maximum release of dense granule components such as serotonin, and the maximum exposure of PE at the platelet surface. To initiate the maximum mobilization of arachidonic acid from PC, PI and alk-1-enyl-acyl-GPE however, 100 times the concentration of thrombin employed to induce the release reaction (0.04 U/ml) is required, as has also been found by Minkes et al. [26] since this work was completed. This finding would tend to support the independence of the latter phenomenon with respect to the generation of prostaglandin intermediates from liberated arachidonic acid. Yet such an implication is valid only if the small quantities of cyclooxygenase metabolites produced during the exposure of platelets to low concentrations of thrombin are insufficient to promote the release reaction. The requirements are at present under investigation.
380
Acknowledgments Supported by an award from the Veterans Administration 5231822. We would also like to thank Dr. Manfred L. Karnovsky Medical School for helpful comments regarding the manuscript.
MRIS No. of Harvard
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Biochimica et Biophysics @ Elsevier/North-Holland
BBA
Acta, 488 (1977) 370-380 Biomedical Press
57037
MOBILIZATION
OF ARACHIDONIC
ACID IN HUMAN PLATELETS
KINETICS AND Ca” DEPENDENCY
SUSAN Boston School
RITTENHOUSE-SIMMONS, VA Hospital of Medicine,
(Received
March
FRANCIS
and Departments of Medicine Boston, Mass. 02130 (U.S.A.)
A. RUSSELL
and DANIEL
and Biochemistry,
Boston
DEYKIN University
7th, 1977)
Summary
The release and transfer of [3H]arachidonic acid in human platelets was examined with regard to (1) varied doses of thrombin, (2) varied periods of incubation with thrombin, (3) the presence of 4 mM Ca”, (4) the degree of exposure of platelet phosphatidylethanolamine to the surface and (5) the release of serotonin from platelet granules. The effect of thrombin on the incorporation of [3H]arachidonic acid and [‘4C]glycerol into platelet phosphatides was also studied. [3H]Arachidonic acid was released in pre-labeled platelets exposed to thrombin from phosphatidylcholine and phosphatidylinositol and appeared increasingly esterified in alk-l-enyl-acyl-glycerophosphoethanolamine. Maximum transfer occurred with 5 U/ml of thrombin and 15 min of incubation and was dependent upon the presence of Ca”, whereas serotonin was maximally released with 0.04 U/ml of thrombin. No preferential exposure of plasmalogen (as opposed to diacyl) ethanolamine at the pIatelet surface occurred during incubation with thrombin. The stimulated incorporation of [ 3HJarachidonic acid into alk-I-enyl-acyl-glycerophosphoethanolamine was not accompanied by a stimulation of [ “C]glycerol uptake into this phosphatide. We suggest that, rather than stimulating de novo synthesis, perturbation of the platelet activates a phospholipase AZ activity leading to turnover of arachidonic acid in platelet plasmalogen. Such turnover may help provide substrate for conversion by cyclooxygenase and lipoxydase to biologically active metabolites.
Introduction
The human platelet contains substantial quantities of arachidonic acid bound in acyl linkage to phosphatides [ 1,2]. We have shown [ 3,4] that platelets incorporate radiolabeled arachidonic acid into several of their phosphatides. When
371
subsequently exposed to thrombin, such platelets lose much of their radiolabel from PC * and PI. In a brief preliminary communication [ 41, we noted that the ethanolamine plasmalogen of resting cells contains an insignificant portion of radiolabel. However, when pre-labeled platelets are exposed to thrombin, a marked shift in distribution of arachidonic acid label occurs, producing a greatly elevated content of the labeled fatty acid in alk-l-enyl-GPE and no comparable change in diacyl-GPE or PS. The source of radiolabel is apparently PC and possibly PI. The purpose of the present paper is to describe the kinetic relationship between the release of arachidonic acid and the incorporation of arachidonic acid into alk-1-enyl-acyl-GPE and to report how these changes compare with the course of serotonin release and the exposure of PE to the medium. In addition, we have accumulated evidence to indicate that newly-labeled alk-l-enylacyl-GPE is the result of specific acylation of existing ethanolamine plasmalogen, possibly occurring in conjunction with the activation of a phospholipase A2 which hydrolyzes this phosphatide prior to acylation. Materials Radioisotopes were obtained from New England Nuclear. All solvents employed were Spectrograde or redistilled (diisobutylketone). Highly purified human (Ythrombin (1 nM = 0.11 U/ml) was kindly provided by Dr. J.W. Fenton, II. Lipid standards were supplied by Applied Science Laboratories, Supelco and NuChek. Eicosatetraynoic acid was kindly given us by Dr. W.E. Scott of Hoffman-LaRoche, Inc. Methods
and Results
In the majority of experiments, blood was drawn on acid-citrate-dextrose anticoagulant [5] from normal human donors. The blood was spun in siliconized tubes at 900 rev./min for 15 min in a Sovall GLC-1 centrifuge at room temperature. Platelet-rich plasma was removed and incubated with radioisotopes, as described below, or applied directly to Sepharose 2B columns equilibrated to pH 6.8-7.0 with a buffer system previously described [6,7], from which Ca*’ had been omitted. The number of cells in the excluded volume were counted by a Coulter device. Platelet-rich plasma (6.0ml) was incubated for 15 min with 6.25 PCi 5, 6, 8, 9, 11, 12,14,15 [3H]arachidonic acid (72 Ci/mmol) bound to 10% delipidated bovine serum albumin [ 81, as described previously [ 41. Aliquots of incubation mixture were then sedimented directly by centrifugation at 4500 rev./min for 5 min (Sorvall RC-5, HS-5 rotor). The lipids of the pelleted cells were extracted in the lower phase after with chloroform/methanol (2 : 1, v/v), recovered mixing with 2/10 ~01s. 0.73% NaCl, and resolved on silica-impregnated paper, as described below. More than 99% of the radioactivity was present in the lower * The
following abbreviations are used in the text: diacyl-GPE, diacylglycerophosphoethanolamine: alk-l-enyl-acyl-GPE, alk-1-enyl-acyl-glycerophosphoethanolamine; PC. phosphatidylcholine; PE, phosphatidylethanolamine: PI. phosphatidylinositol: PS. phosphatidylserine; (NO*)3 phS. trinitrobenzene sulfonic acid; (N02)~ ph PE, trinitrophenyl derivative of PE.
372
chloroform phase. The remaining platelet-rich plasma was passed through a Sepharose 2B column. Unincorporated radiolabel, bound to albumin, was well separated from platelets obtained in the excluded volume. These platelets were used in most subsequent studies. Incorporation of radiolabel into filtered platelets was assessed by mixing aliquots with H,O/Aquasol (1 : 10, v/v) and counting in a Packard Scintillation Counter. Results were analyzed using suitable controls for quenching. Lipids were chromatographed at 15°C in two directions, as described by Wuthier [9], on dried Whatman SG 81 paper. The solvent system employed in direction 1 was chloroform/methanol/diisobutyl ketone/acetic acid/H,0 (45 : 15 : 30 : 20 : 4; by vol.) and in direction 2 was chloroform/methanol/diisobutyl ketone/pyridine/H,O (30 : 25 : 25 : 35 : 8; by vol). Prior to chromatography in the second direction, the lipid track was sprayed with 5 mM HgCl, [l] and dried under vacuum in the presence of hot desiccant for 1 h to hydrolyze the plasmalogen 0 J-unsaturated ether linkage, allowing separation of monoacyl-GPE from diacyl-GPE on subsequent chromatography. Following chromatography, the papers were dried, sprayed with aqueous 0.005% Rhodamine 6G, and viewed immediately under ultraviolet light. Lipid spots were cut out and counted in H,O/Aquasol (1 : 10, v/v) or assayed for phosphorus [9]. Platelet lipid samples, chromatographed in two directions and exposed or unexposed to HgC12, were subjected to spraying with a solution of ninhydrin or 0.4% of the aldehyde, ketone, and vinyl ether reagent, dinitrophenylhydrazine in 0.1 M HCl. The phosphorus content of the spots indicated by the staining reagents was analyzed, as was the phosphorus in those relevant regions of the paper which would serve as blanks. Three ninhydrin-positive spots were seen: one corresponding to standard PS, and two in the PE lane, corresponding to monoacyl-GPE and diacyl-GPE. The neutral lipid region of the PE lane was dinitrophenylhydrazine-positive. When water was substituted for the HgCl* spray (a) only two ninhydrin-stained spots were visible: PS and PE; (b) one dinitrophenylhydrazine-reactive spot was found, corresponding to PE. Only when the lipid track had been sprayed with HgClz did radioactivity appear in the monoacyl-GPE region, varying primarily with prior treatment of [ 3H]arachidonic acid-labeled platelets with thrombin. In no case was radioactivity found in the neutral lipid region of the PE lane. Thus the fatty aldehyde derived from alk-lenyl-acyl GPE contained no radiolabel. The ratio of phosphorus for mono-acyl-GPE/diacyl GPE was 0.97 of-0.06 (n = 4), indicating complete breakdown of alk-1-enyl-acyl-GPE by HgCl*, since the plasmalogen content of PE is about 50% [ 11. In addition, a quantitative determination of aldehyde in the neutral lipid region of the PE lane was made by the method of Rapport [lo] for experiments in which HgClz spraying was either included or omitted in the chromatographic procedure. After HgClz spraying, the aldehyde found was equimolar with monoacyl-GPE phosphorus. “H-Labeled monoacyl-GPE extracted from paper chromatograms was subjected to alkaline hydrolysis with 15% KOH in 50% methanol-H20 at 60°C for 1 h. The chloroform-extracted hydrolysate was chromatographed in one direction on silica paper in the chloroform/methanol/diisobutylketone/acetic acid solvent described. More than 95% of 3H was found in the chloroform fraction,
373
and of this, more than 90% migrated as neutral lipid, the remainder being in unhydrolyzed monoacyl-GPE. For studies comparing de novo synthesis of phosphatides with fatty acid uptake, filtered platelets (unlabeled) were added to siliconized tubes (4.6 * lo8 cells) containing delipidated bovine serum albumin (final concn. 0.5%) with or without bound oleic acid (0.6 PM) and Ca” (4 mM). Immediately thereafter, human thrombin (2.5 U/ml) or buffer (pH 7) was added. After 15 s, glycerol-2[‘“Cl (11.8 mCi/mmol, 1.9 mM, final concn.) or albumin-bound [“Hlarachidonic acid, 4 nM (described above) were added and the mixtures were incubated 30 min more at 37°C. EDTA (5 mM), pH 6.5, was added to each tube, and the cells were sedimented at 4500 rev./min for 5 min. The pellets were extracted with chloroform/methanol. Whereas the presence of 0.6 PM oleic acid inhibited the incorporation of [3H]arachidonic acid (4 nM) into phosphatides of resting cells by 20-30%, it had no significant inhibitory effect on arachidonic acid incorporation into alk-l-enyl-acyl-GPE when thrombin was present. In a series of parallel experiments, platelet-rich plasma (2.2 - 10' cells), buffered at pH 6.8, was incubated for 30 min with [‘4C]glycerol (0.2 mM) or [3H]arachidonic acid (16 nM) in the absence of thrombin. Cells were sedimented and their lipids extracted and resolved. In the absence of thrombin, incorporation of [‘4C]glycerol into alk-1-enyl-acyl-GPE was very low, but similar for incubated filtered and plasma-suspended platelets. The amounts of [‘4C]glycerol incorporated into diacyl-GPE for plasma and filtered platelets were also similar to each other. The uptake of [3HJarachidonic acid into alk-lenyl-acyl-GPE of plasma platelets was only 20% of that found in this phosphatide of filtered platelets. In contrast, incorporation of [3H]arachidonic acid into diacyl-GPE was about 70% of that in diacyl-GPE of filtered platelets. The slight depression of 30% in the latter case can be accounted for by isotope dilution, since plasma itself contains (unlabeled) arachidonic acid. Such dilution cannot, however, account for the 80% less incorporation of arachidonic acid into alk-l-enyl-acyl-GPE of plasma platelets. Filtered platelets derived from platelet-rich plasma incubated with [“HIarachidonic acid were treated for 5 min with freshly-prepared acetylsalicylic acid (100 pg/mll’), indomethacin (60 pug/ml-‘) or 5, 8, 11, 14-eicosatetraynoic acid (3 lug/ml-‘). Acetyl-salicylic acid and indomethacin have been shown to be inhibitors of platelet cyclooxygenase [ 111 and eicosatetraynoic acid acts as an inhibitor of cyclooxygenase and lipoxydase [ll]. Both enzymes normally utilize non-esterified arachidonic acid in platelets. Treated and untreated platelets were incubated further with 4 mM Ca2+ and 2.5 U/ml-’ thrombin for 15 min. Quantitation of radiolabel in resolved phosphatides showed no consistent effect of any inhibitors on thrombin-promoted release of arachidonic acid from PC and PI, or transfer to alk-1-enyl-acyl-GPE. Such a finding tends to exclude the possibility that labeled prostaglandin products might be responsible for the label found in alk-l-enyl-acyl-GPE. For kinetic studies, filtered platelets pre-labeled with 13H]arachidonic acid were incubated at 37°C in 0.5% delipidated bovine serum albumin, pH 7.0, in the presence and absence of 4 mM CaCl* and 2.5 U/ml of thrombin, for up to 30 min. At various times after the addition of thrombin to labeled platelets,
374
ice-cold EDTA was added and the cells were sedimented. The release of radiolabel to the supernatant and the distribution of remaining radioactivity among the lipid fractions of the pelleted cells were determined. In other experiments, the response of labeled platelets to O-10 U/ml of thrombin after 15 min of incubation was ascertained as above. Prior to release reaction studies, platelet-rich plasma (7.0 ml) was incubated with 2 FCi [‘4C]5-hydroxytryptamine (creatinine sulfate salt, final concentration, 1 FM) for 15 min at 3’7”C, with gentle shaking. Incubated mixtures were then filtered through Sepharose 2B. [‘4C]Serotonin not associated with platelets was removed by this procedure. Incorporation of serotonin was 60-70%. Platelets pre-labeled with [ i4C] serotonin were incubated with varied concentrations of thrombin for 15 min as above, sedimented, and the extent of release of radiolabel to the medium was assessed. In addition, platelets obtained after Sepharose filtration were incubated with Ca’+ and varied doses of thrombin for 15 min at pH 7. Subsequently, (NO,), phS at pH 7 was added (final concentration, 3 mM) and cells were incubated 15 min further. Cells were pelleted after the addition of EDTA-glycine, pH 6.5. It was possible, using the above-described chromatography system, to separate diacyl-GPE and monoacyl-GPE not only from other phosphatides, but from their (NO,), phS derivatives. The extent of formation of (N0,)3 ph-PE derivatives was determined by phosphorus analysis. When pre-labeled filtered platelets were incubated with thrombin, both the decrease of [ 3H]arachidonic acid in PC, and the incorporation of radiolabel into alk-l-enyl-acyl-GPE were considereably diminished if Ca*’ was omitted from the incubation medium (Fig. 1). The extent of depression was progressive with time. The kinetics of loss from PI appears to be complex, in that measurable reincorporation of arachidonic acid into the PI pool occurred with time when Ca*’ had not been added. Mobilization of labeled arachidonic acid in the presence of Ca2+ was stimulated by increased concentrations of human thrombin. Changes were minimal above 5 U/ml (Fig. 2). [3H] Arachidonic acid in PS varied slightly, increasing by 5% with time in the absence of Ca*’ and decreasing by, at most, 10% when Ca2’ was included. Diacyl-GPE showed a 5% loss of [3H]arachidonic acid for incubations without Ca*+ and a lo-15% loss with Ca*+. In the presence of Ca”, loss of radioactivity from diacyl-GPE after 15 min was only 15% of the gain observed in alk-1-enyl-acyl-GPE over the same period. Values for PS and diacyl-GPE have been omitted from Figs. 1 and 2 for reasons of clarity. The loss of radiolabel from PC and PI could be accounted for by the increased presence of label in alk-1-enyl-acyl-GPE (as arachidonic acid) and in the incubation media as arachidonic acid and cyclooxygenase and lipoxydase metabolites. Such metabolites and fatty acids were resolved by high performance liquid chromatography. Fig. 3. represents the cumulative results of six experiments in which the percentage of radiolabel which was present in platelets as alk-l-enyl-acyl-GPE is correlated with the percentage of total platelet radioactivity which was released to the medium. The data include values for released radioactivity from platelets merely shaken for different periods of time at 37°C and not exposed to thrombin, as well as for cells exposed to thrombin..The dotted line is the extrapolation to the limiting case of zero release, and goes through the origin. This
375
L_
0
I
I
20
30
I
IO
Time (min)
1
I
I
0
5
IO
Thrombin (U/ml)
Fig. 1. Change in content of [ 3Hlarachidonic acid in phosphatides with time following exposure of labeled platelets to thrombin. 3H-Labeled platelets were obtained by incubation of platelet-rich plasma with [3Hlarachidonic acid. Platelets were gel-filtered and incubated with thrombin (2.5 U/ml) with and without 4 mM 0%. shaking at 150 rev./min. No significant change in isotopic distribution was found for platelets incubated without thrombin and CaB. (0). PC - Ca2+; (0). PC + Ca2+: (A), alk-1-enyl-acyl-GPE + Ca*+: (A). alk-l-enyl-acyl-GPE - CaZ+: (0). PI - Ca*+. , Cm), PI + Ca*+. Radioactivity given as thousands of dpm/109 platelets. Fig. 2. Change in content of [3Hlarachidonic acid in phosphatides following exposure of labeled platelets to thrombin in varied doses. Labeled gel-filtered platelets were incubated with thrombin and 4 mM Ca” for 15 min. shaking at 150 rev./min. (0). PC; (A), alk-lenyl-acyl-GPE; (w). PI. Radioactivity as in Fig. 1.
finding suggests that platelets, preincubated with [3H]arachidonic acid but not perturbed, do not transfer radiolabel into alk-l-enyl-acyl-GPE. Mobilization of incorporated arachidonic acid and transfer to alk-1-enyl-acyl-GPE is thus triggered by perturbation of the platelet. We next examined the effect of gel filtration of the 3H-labeled plasma platelets on transfer of label to alk-l-enyl-acylGPE (Table I). Gel filtration of 3H-labeled platelet-rich plasma caused minimal transfer. Gel-filtration prior to incubation with radiolabel was associated with greater radioactivity in alk-1-enyl-acyl-GPE which may reflect the more vigorous shaking of the filtered platelets, rather than activation attributable to gel-filtration per se. A partial activation of the platelets, whether due to the removal of plasma factors or contact with Sepharose beads, cannot be unequivocally ruled out. However, in other experiments (unpublished) we had found that washing of the platelets caused greater activation (as measured by transfer of radiolabel to alk-lenyl-acyl-GPE) than did gel-filtration. Thrombin caused maximal transfer of label to this ethanolamine plasmalogen. In parallel experiments (Fig. 4) we compared three phenomena promoted by thrombin: (1) the labeling of alk-l-enyl-acyl-GPE with [3H]arachidonic acid, (2) the release to the medium of serotonin from the platelets and (3) the by the non-penetrating opening of PE to the platelet surface, as detected
376
.
6.
4-
. /
2-
,/*
;I/'* . ..
.
. l
,,/',
,
,
,
,
,
,
,
,
4
8
I2
16
20
24
28
32
36
0 %
of total lncorporoted
‘H released
to medium
Thrombtn
(U/ml)
Fig. 3. Transfer of [3Hlarachidonic acid to labeled platelets as a function of label released to the incubation medium. Labeled gel filtered platelrts were incubated for different periods of time and different shaking frequencies at 37’ C and pH 7, with and without thrombin. Radioactivity in the platelet-free medium and in platelet alk-1-rnyl-awl-GPE was then quantitated. Fig. 4. Percentage of maximum serotonin release, (NO*)jph-PE formationand transfer of [3H1arachidonic acid to alk-1-enyl-acyl-GPE achieved with varied low doses of thrombin. Gel-filtered platelets. labeled with [14Clserotonin or [ 3Hlarachidonic acid. were incubated with Ca*+ and thrombin for 15 min. Release of 14 C to the medium or transfer of 3H to alk-l-enyl-acyl-GPE were assessed with regard to the maximum effect achievable with thrombin (at 2.5 U/ml). In addition. thrombin-treated filtered platelets were exposed to trinitrobrnzrne sulfonate for 15 min. The amount of reaction product ((N02)3 ph-PE) in the platelet lipid extracts was assayed by phosphorus analysis. (a), alk-l-enyl-acyl-GPE - 3H: (?). serotonin -- 14C: (A). (NO2 )3 ph-PE.
reagent, (NO,), phS [12,13]. All are plotted as the percentage of the maximum response achieved with thrombin (2.5 U/ml) seen at varied low concentrations of thrombin. Maximum release of serotonin was 90%. The surface exposure of PE induced by thrombin was similar to the release of serotonin, which was very sensitive to thrombin, achieving its greatest level at 0.04 U/ml. In contrast, the release of arachidonic acid and its transfer to alk-1-enyl-acyl-GPE did not reach half its maximum extent until 10 times that amount of thrombin had been
TABLE
I
DISTRIBUTION ~____ Phosphatidr
OF [“H]ARACHIDONIC PRP a
ACID GFPb
AMONG
PLATELET GFP ’ --.--
PC PI PS PPE DPE
66 7 11 0.6 12
64 10 12 1.0 11
58 11 12 3.2 12
PHOSPHATIDES _ GFP fhrombin
(%)
__ ____
49 3 14 17 14
a Platelet-rich plasma (PRP) was incubated with [3Hlarachidonic acid at 37°C for 15 min, 50 rev./ min. b PRP incubated as in (a) was filtered through Sepharose to yield labeled gel-filtered platelets (GFP). c Unlabeled GFP were incubated with [ 3H]arachidonic acid for 15 min at 37@C. 150 rcv./min. d Labeled GFP. as in (b) were incubated with thrombin (2.5 U/ml) and 4 mM Ca2+ for 15 min at 150 rev./min. PPE, alk-l-enyl-acyl-GPE; DPE, diacyl-GPE.
_
377
added. The degree of exposure of PE increased only two-fold under our incubation conditions, going from 8.5% to 15% of the total platelet PE. We further examined the labeling of PE with (NO,), phS to determine if there was preferable exposure of one of the species of PE: diacyl-GPE or alk-l-enyl-acyl-GPE, in the course of treatment with thrombin. The phosphorus ratios of monoacylGPE/diacyl-GPE and of (N0,)3 ph-monoacyl-GPE/(NO*), ph-diacyl-GPE for resting and thrombin-activated platelets were compared, and were consistently 0.97. Thus, no disproportionate accessibility of surface alk-l-enyl-acyl-GPE or diacyl-GPE was measurable, with or without thrombin. Since the amount of total PE in the platelet which is in the form of alk-l-enyl-acyl-GPE is about 50% [ 11, accessibility of the two ethanolamines was equivalent. Incubation of filtered platelets with [14C]glycerol led to incorporation of this radiolabel into a number of phosphatides. The majority of the radioactivity was recovered in PC (69%) followed, in decreasing order, by PI (17%), diacylGPE (6.9%), PS (4.0%) and alk-1-enyl-acyl-GPE (2.9%). Thus, the incorporation of 14C into the ethanolamine plasmalogen of control cells was 0.04 of that into PC (whereas the molar ratio of alk-1-enyl-acyl-GPE to PC is 0.35). This was the case whether uptake of radiolabel was studied in filtered platelets or platelet-rich plasma. As shown in Table II, the addition of thrombin to filtered platelets caused a marked depression in incorporation of [ 14C]glycerol into PC, as had been anticipated from previous reports based on experiments with washed platelets [14,15]. The effect of thrombin on the uptake of [‘Hlarachidonic acid and on that of [‘4C]glycerol into several other platelet phosphatides is also shown in Table II. Whereas incorporation of [‘4C]glycerol into alk-lenyl-acyl-GPE was depressed by thrombin, incorporation of [ 3H] arachidonic acid rose, as noted before for platelets pre-incubated with arachidonic acid and then exposed to thrombin. When the ratio of ‘H/14C incorporated into cells in the presence of thrombin is divided by that ratio in the absence of thrombin, a number representing the proportionality of the effect of thrombin on [ 14C]glycerol and [ 3H]arachidonic acid uptake into various phosphatides is obtained. A perfectly proportionate effect would give a ratio of 1. PC and diacyl-GPE approach this ratio most closely. Alk-1-enyl-acyl-GPE has a much greater ratio (7.7) and PS and PI, much less (0.28,0.29). TABLE
II
EFFECT
OF
GLYCEROL
THROMBIN INTO
ON
THE
PHOSPHATIDES
INCORPORATION OF
GEL-FILTERED
OF
[3H]ARACHIDONIC
ACID
AND
t14C1-
PLATELETS
Platelets (2.3 . lo* cells/ml) were incubated for 30 min with the radiolabeled compounds added 15 s after the addition of buffer (pH 7) or thrombin (T); concentrations: Ca*+. 4 mM: (where Present) thrombin. 2.5 U/ml; [ *4C]glycerol. 0.2 mM; r3HJarachidonic acid, 4 nM. PPE, alk-l-enyl-acul-GPE; DPE. diacyl-GPE. Phosphatide
PPE DPE PS PI PC
3H/‘4C
14C/‘4C
3H/3H
-T
+T
+T/-T
+T/-T
+T/-T
31 114 252 63 85
238 89 71 18 99
1.7 0.78 0.28 0.29 1.2
0.45 0.54 1.1 1.2 0.25
3.4 0.42 0.29 0.35 0.29
378
Discussion It appears that thrombin-stimulated acylation of alk-l-enyl-acyl-GPE does not require de novo synthesis of this phosphatide, but probably is dependent upon either the prior formation of alk-l-enyl-GPE resulting from the action of a phospholipase AZ, or a transacylating mechanism that does not involve lysophosphatides. In addition, our data indicate the existence of an acylating activity in human platelets which, in the presence of thrombin, exhibits a specificity for arachidonic acid, as opposed to oleic acid. We conclude that it is unlikely that de novo synthesis is the major determinant of the increment in arachidonic acid incorporation into ethanolamine plasmalogen for the following reasons: [‘4C]glycerol uptake into alk-l-enyl-acyl-GPE, used as a gauge of de novo synthesis, was comparable for plasma platelets and filtered platelets although there was more than three times more uptake of [3H]arachidonic acid into filtered platelet alk-lenyl-acyl-GPE. The data in Fig. 3 and Table i indicate that, in the limiting unperturbed state, platelets incorporate no arachidonic acid into alk-1-enyl-acyl-GPE and that the incubated filtered platelets derived from labeled plasma are slightly activated. Consequently, one might well assume that a small amount of phospholipase A2 was active in incubated filtered platelets and that incorporation of [“Hlarachidonic acid which was observed was dependent on the transient formation of lyso-ethanolamine plaswhile the plasmalogen malogen (i.e., with fatty acid absent at glycerol-sn-2, linkage was still intact at glycerol-sn-1). By extension, one could argue that a similar, magnified, phenomenon occurred when filtered platelets were incubated with thrombin (Table II), during which [3H]arachidonic acid incorporation rose more than three-fold, and [ “C]glycerol incorporation fell by half. The only precedent in the literature for such an activation of which we have knowledge is that cited by Wykle, et al. [16] for rat testis enzyme studies. These investigators demonstrated a tendency for unsaturated fatty acids to build up in ether-linked lipid classes via deacylation-reacylation reactions. Rats on an essential fatty acid-deficient diet preferentially incorporated [“Hlarachidonic acid into the 2-position of alk-1-enyl-acyl-GPE rather than into diacylGPE. It was conjectured that plasmalogens might serve as a reservoir for prostaglandin precursors during essential fatty acid deficiency [ 17,181. The dependency on Ca” of the thrombin-induced decrease in [3H]arachidonic acid in PC and PI is consistent with the observations of Derksen and Cohen [19], who examined the release of arachidonic acid from endogenous substrates in platelet membranes. They found a virtually complete dependency of the phospholipase A2 on added Ca”. The progressively increasing extent to which [3H]arachidonic acid mobilization was subject to added Ca2+ (Fig. 1) might be explained by depletion of the internal Ca” stores [20] which the platelet is known to have. Recent work by Jesse and Cohen [21] has indicated that 72% of the arachidonic acid hydrolyzed from endogenous phosphatides by plasma membrane phospholipase A at pH 9.5 comes from diacyl-GPE, with the remainder coming from PC. No hydrolysis of alk-1-enyl-acyl-GPE was observed under these conditions. In contrast, we have found only a lo-15% loss of [“Hlarachidonic acid from diacyl-GPE at pH 7 (3,4] after the activation of platelets by thrombin, a
379
major loss of this labeled fatty acid from PC, and an implied deacylation of alklenyl-acyl-GPE followed by acylation with labeled fatty acid, Clearly, several platelet fractions should be examined closely at a variety of pH’s with regard to ethanolamine plasmalogen-specific fatty acid hydrolysis. It is possible, for example, that alk-lenyl-acyl-GPE-phospholipase A is not bound to the plasma membrane but is associated with a particulate fraction not examined by Jesse and Cohen. We are presently undertaking such experiments. In addition, we are exploring another suggestion which we made above: that a PC-alk-l-enyl-acylGPE transacylating activity might exist which would not lead to generation of lyso-phosphatides. We have found that in vitro incorporation of arachidonic acid into alk-lenyl-acyl-GPE becomes pronounced during thrombin-induced activation of platelets. It is known [l] that the endogenous level of arachidonic acid in plasmalogen phosphatide of human platelets is high enough to account for essentially all of the sn-2-fatty acid in this phosphatide. Therefore, if the entry of labeled arachidonic acid is contingent on the function of phospholipase A2 acting on alk-1-enyl-acyl-GPE, then unlabeled arachidonic acid must be liberated from alk-1-enyl-acyl-GPE during activation. Our study of the course of release of arachidonic acid from PC and PI showed half maximal release at 2.5 min. However, Hamberg et al. [22] demonstrated that washed human platelets incubated with thrombin formed the prostaglandin metabolites PHD and HETE half-maximally in less than 1 min. Since there is little if any free arachidonic acid within the normal platelet, and virtually all of the arachidonic acid is bound to phosphatides [23], this fatty acid must be hydrolyzed from some phosphatide(s) before being acted upon by cyclooxygenase. Should a liberation of arachidonic acid from alk-1-enyl-acyl-GPE significantly precede that from PC and PI, in keeping with the kinetics reported by Hamberg et al., there would be compelling reason to view alk-l-enyl-acyl-GPE as a precursor phosphatide. Thus it is possible that the turnover of ethanolamine plasmalogen contributes to the pool of free arachidonic acid for utilization by cyclooxygenase and/or lipoxydase and therefore helps provide biologically active metabolites [11,241. We have indicated in Fig. 4 that the binding of thrombin to a few sites on the platelet membrane (120 out of a possible 50000 [25]) is sufficient to promote both maximum release of dense granule components such as serotonin, and the maximum exposure of PE at the platelet surface. To initiate the maximum mobilization of arachidonic acid from PC, PI and alk-1-enyl-acyl-GPE however, 100 times the concentration of thrombin employed to induce the release reaction (0.04 U/ml) is required, as has also been found by Minkes et al. [26] since this work was completed. This finding would tend to support the independence of the latter phenomenon with respect to the generation of prostaglandin intermediates from liberated arachidonic acid. Yet such an implication is valid only if the small quantities of cyclooxygenase metabolites produced during the exposure of platelets to low concentrations of thrombin are insufficient to promote the release reaction. The requirements are at present under investigation.
380
Acknowledgments Supported by an award from the Veterans Administration 5231822. We would also like to thank Dr. Manfred L. Karnovsky Medical School for helpful comments regarding the manuscript.
MRIS No. of Harvard
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