American Journal of Hematology 1 : 59-70 (1976)
THE EFFECT OF THROMBIN ON THE UPTAKE AND TRANSFORMATION OF ARACHIDONIC ACID BY HUMAN PLATELETS Francis A. Russell, M.S. and Daniel Deykin, M.D. Boston Veterans Administration Hospital, and Departments of Medicine, Boston University and Tufts University Schools of Medicine, Boston, Massachusetts
Washed human platelets take up arachidonic acid from plasma and incorporate the fatty acid into the major classes of complex lipids. Thrombin impairs net incorporation. It activates endogenous phospholipases which liberate arachidonic acid from phospholipids. As a consequence of thrombin induced aggregation platelets release arachidonic acid intermediates formed by the action of platelet fatty acid cyclooxygenase and by platelet fatty acid lipoxygenase. Cyclooxygenase, but not lipoxygenase, is inhibited by aspirin and indomethicin. Analysis of the pathways of arachidonic acid metabolism may furnish new insight into platelet function and into disorders of primary hemostasis. Key words: platelets, arachidonic acid, thrombin, phospholipases, cyclooxygenase, prostaglandins
Prostaglandins are formed and released by platelets during aggregation. ( 1 -3). Recent studies by Hamberg and Samuelsson and their associates (4-8) have indicated that certain prostaglandin intermediates are themselves potent initiators of platelet aggregation. In this study we have extended our previous experiments (9) on the effects of thrombin on the incorporation of longchain free fatty acids into platelet lipids to include arachidonic acid, the direct precursor of platelet prostaglandins. Our data indicate that washed human platelets take up arachidonic acid from plasma and incorporate it into the major classes of complex lipids. Thrombin impairs the net incorporation of arachidonic acid into the major lipid classes. It activates phospholipases which release newly incorporated arachidonic acid from phospholipids, and it causes the time-dependent release into plasma of metabolites of arachidonic acid consistant with the newly described lipoxygenase and fatty acid cyclooxygenase pathways of transformation of arachidonic acid.
Address reprint requests to Daniel Deykin, M.D., Boston VA Hospital, 150 S. Huntington Ave., Jamaica Plain, Mass. 021 30.
59
0 1976 Alan R. Liss, Inc., 150 Fifth Avenue, New York, N.Y. 10011
60
Russell and Deykin
METHODS Preparation of Washed Human Platelets
Suspensions of washed human platelets containing 0.6-1.5 X lo9 plateletsfml were prepared from normal subjects as previously described (9). Lipids
(5,6,8,9, 11, 12, 14, 15 [3H]) arachidonic acid (specific activity 72 Ci/mmole) was obtained from New England Nuclear Corporation, Boston, Mass. Prior to use, the arachidonic acid was dissolved in acetone at a final concentration of 100 ,uCi/ml. 5,8,11,14eicosatetraynoic acid was the gift of Dr. W. E. Scott, Hoffman, La Roche, Inc., Nutley, N. J. Prostaglandin standards were the gift of Drs. J. E. Pike, Upjohn Company, Kalamazoo, Mich. and E. W. Salzman, Beth Israel Hospital, Boston, Mass. Thrombin
Human thrombin was obtained from two sources. Commercial human thrombin was purchased from Ortho Diagnostics, Raritan, New Jersey. Highly purified thrombin (specific activity > 1,500 units/mg) was the gift of Dr. Robert Rosenberg, Beth Israel Hospital, Boston, Mass. In preliminary experiments we observed that at final concentrations of thrombin up to 15 U/ml there was no difference in the effects of either preparation on platelet aggregation or on the metabolism of arachidonic acid. At concentrations in excess of 15 U/ml commercial thrombin, but not the purified thrombin, inhibited aggregation. In experiments in w h c h the concentration of thrombin was 5 U/ml we used either preparation. In dose-response experiments we used the highly purified thrombin. Incubation Techniques
Three types of incubations were conducted. In all experiments in which thrombin was added, massive platelet aggregation occurred within 2 min. In experiments in which thrombin was omitted no aggregation occurred. A. Incorporation of fatty acids into platelets from prelabeled plasma. [3 HI arachidonic acid was complexed t o albumin in defibrinogenated dialyzed plasma, pH 6.8, (prepared as previously described [9]) by introducing the acetone solution (2.5pl/ml of plasma) slowly under the surface of the plasma with constant swirling. The acetone was driven off by incubation for 30 min at 37°C under a stream of nitrogen. Reactions were initiated by the addition of 1.5 ml of the platelet suspension (prewarmed to 37°C for 5 min before addition) to siliconized flasks containing 0.4 ml of the labeled plasma t o which 3.4 pmoles of CaC12 had been added, followed by the addition of either 0.1 ml of buffer or 0.1 ml of thrombin (final concentration 5 U/ml). In certain experiments thrombin was added 15 min after the incubations were begun. Under the conditions of these experiments the final amount of added arachidonic acid was approximately 1.5 X lo-' moles/flask. Incubations were terminated at various time intervals by immersing the flasks in an ice bath for 120 sec and then transferring the chilled contents of the flasks to iced centrifuged tubes. The platelets were then separated from the incubation medium by centrifugation at 2,500 X g for 10 min at 4°C. The platelets were then resuspended in a solution of phosphate buffer: dialyzed (unlabeled) plasma, pH 6.5 (1 :1 vol/vol), and were washed twice to remove unreacted and dissociable fatty acids. The platelets were then suspended finally in 2 ml of the washing solution, and the platelets and the incubation media were each transferred separately to extraction flasks
61
Platelet Arachidonic Acid Metabolism
containing 50 ml of chloroform: methanol (2: 1, vol/vol). Lipids were extracted by a modified Folch procedure (9). The lower (chloroform) phase was washed three times with 0.154 M NaC1.
B. Release of radioactive metabolitesfrom prelabeled platelets. [3 HI arachdonic acid was complexed to albumin as described in (A) except that 5 or 10 p1 of the acetone solution were added /ml of defibrinogenated plasma. Equal volumes of platelet suspensions and labeled plasma were incubated for 15 min at 37°C. The platelets were separated from the plasma by centrifugation. They were washed once with phosphate buffer, pH 6.5, and once again with the buffer-plasma solution as in (A) and resuspended in phosphate buffer, pH 6.8. One ml of the suspension was added to siliconized flasks containing 0.9 ml defibrinogenated plasma, pH 6.8, t o which 3.4 ymoles of CaC12 had been added. Incubations were initiated by the addition of 0.1 ml of buffer or thrombin. In one set of flasks platelets were immediately separated from the medium. The platelets were then washed, and the platelets and the medium were extracted separately. A second set of flasks was incubated with buffer for 15 min at 37°C and then processed, and a third set was incubated with thrombin for 1 5 min and then processed. In some experiments, the time of incubation with thrombin or the concentration of thrombin were varied. C. Release of radioactive metabolites from prelabeled platelets in the presence of inhibitors.Preliminary incubations, washing, and resuspension of the platelets were per-
formed as in (B). Prior to the addition of thrombin or buffer indomethacin (final concentration 150 pg/ml), aspirin (150 pg/ml), or eicosatetraynoic acid (final concentration 5 yg/ml were incubated with the platelets at 37°C for 5 min. Thrombin (final concentration 5 U/ml) was then added and the incubations were continued for an additional 15 min. Lipid Analysis
Phospholipids in the platelet samples were resolved by bi-directional thin-layer chromatography. (System A) Thin layer plates were coated (0.5 mm) with a slurry prepared from 50 gm silica gel H, 0.5 gm (N&),S04 and 110 ml of water. A portion of the chloroform extract of the platelets was evaporated at reduced temperature under vacuum to a volume of 100 p1 and was applied under nitrogen to a corner of the plate. Phospholipids were resolved with chloroform: methanol: acetic acid: water (1 50:90:24: 8.5, vol/vol). When the solvent front had reached a point 1 cm from the top of the plate, the plate was removed and dried at room temperature for 60 min. It was then rotated 90” and rechromatographed in a tank newly equilibrated with the same solvent system. Phospholipids were visualized by iodine vapor. With this system sphmgomyelin, lecithin (PC), phosphatidylinositol (PI), phosphatidylserine ( P S ) , and phosphatidylethanolamine (PE) were well resolved. An additional, as yet unidentified lipid (“X”) distinct from lysophosphatidylethanolamine was present between PS and PE. Platelet neutral lipids from a separate portion of the chloroform extract were resolved by one-dimensional chromatography (System B) using plates coated (0.25 mm) with a slurry made from 30 gm silica gel H and 65 ml of water. The solvent system was petroleum ether: diethyl ether: acetic acid (150:50:2 vol/vol). With this system phospholipids remained at the origin, and diglycerides (DG), free fatty acids (FFA), triglycerides (TG), and cholesteryl esters (CE) were well resolved. Medium lipids were separated by two methods. Routinely, the chloroform extract
62
Russell and D e y k i n
was concentrated by evaporation as described above, and the lipids were resolved by System B. The plates were marked into 4 zones: l(r.f., 0.0-0.09); 2(r.f., 0.10-0.31); 3(r.f., 0.32-0.56);4(r.f., 0.57-1 .O). In those experiments in which prostaglandin intermediates and derivatives were to be further examined, medium samples were handled differently. The chloroform extracts were divided into 2 portions, each of which was concentrated to 100 pl, applied to adjacent lanes of thin-layer plates and resolved with System €3 as above. One lane was scraped into zones 1,2,3, and 4 as described above for determination of total radioactivity in each zone. The duplicate adjacent zones were scraped into flasks and the lipids eluted with successive washes of petroleum ether: diethyl ether (1 : 1 vol/vol). The washes from each zone were pooled and concentrated to 100 pl. Methyl esters were formed by the addition of 5.4 X gm of diazomethane in 1.5 ml of dry ethyl ether. The samples were mixed, allowed to stand for 1 hr and reduced to 100 p1 by evaporation as previously described. The methyl esters were separated by System C, a modification of the method of Hamberg and Samuelsson. Thin layer plates were coated (0.5 mm) with a slurry composed of 60 gm silica gel G and 80 ml of water. Lipids were resolved with the upper phase of a mixture of ethyl acetate: 2,2,4-trimethylpentane: HzO (1OO:lOO: 150 vol/vol). The plates were divided into 16 1-cm bands each of w h c h was scraped and counted. In this system methyl arachidonate traveled with the solvent front. Radioactivity
Radioactivity in all samples was determined in a Packard Instruments Tri-Carb model #3385. Absolute activity was determined with an external standard and a computer derived quench calibration curve. RESULTS
Washed human platelets take up labeled arachidonic acid from plasma and incorporate it into all major lipid classes (Table I). Of the total radioactivity, 88%was incorporated into phospholipids and 12% into neutral lipids. Over half of the total radioactivity was present in lecithin. Only 3.8% remained as free fatty acid, and 1.4%was recovered in the area of the unknown lipid. TABLE 1. Platelet Lipid Distribution of Incorporated Arachidonic Acid ~
~~
Lipid class
Radioactivity* (DPM/ 10’ platelets)
PC PI PS PE “X” DG TG FFA
60,447 t 3,033 15,392 t 765 8,650i 359 9,617k 276 1,518i- 561 2,757 t 415 3,345 t 948 4,136 f 1,034
Total
% 54.8 14.0
7.8 8.7 1.4 2.5 3.0 3.8
110,285 i 4,466
*Mean ? SD, 3 experiments. In each experiment points determined in duplicate; 15 min incubations.
63
Platelet Arachidonic Acid Metabolism
The effect of thrombin on the time course of incorporation of arachidonic acid into total platelet lipids and into individual lipid classes is shown in Fig. 1. When thrombin was added at the onset of incubation, incorporation of arachidonic acid into the major lipid classes was reduced at all time points. In contrast thrombin stimulated incorporation of arachidonic acid into the unknown lipid. When thrombin was added after 15 min of incubation, total radioactivity in the platelets declined reaching a minimum withm 3 min. There was concomitant release of radioactivity from all major lipid classes. In contrast there was a sharp, prompt, and sustained stimulation of incorporation of radioactivity into the unknown lipid.
PI
0
PS
10
20
30
0
10
20
30
MINUTES Fig. 1. Effect of thrombin on [ 3 H ] arachidonic acid incorporation into platelet lipids (mean of triplicate experiments, each in duplicate). Abbreviations: PC = lecithin; PI = phosphatidylinositol; PS = phosphatidylserine; PE = phosphatidylethanolamine.
To examine thrombin-induced release of arachidonic acid and its derivatives from washed prelabeled platelets, platelets were first incubated with arachidonic acid, washed, and reincubated in unlabeled plasma in the presence or absence of thrombin. The effect of increasing the concentration of thrombin on release of radioactivity into the medium is shown in Table 11. In the absence of thrombin platelets released 4% of their radioactivity during 15 min. Increasing concentrations of thrombin caused progressively greater release of radioactivity into the medium, reaching a peak at a final concentration of 10 U/ml. The effect of thrombin on the distribution of radioactivity among the major phospholipid classes, the unknown lipid, and in the medium is shown in Table 111. Thrombin (5 Ujml) caused the release of radioactivity from PC, PI, and PS but enhanced the radio-
64
Russell a n d Deykin TABLE 11. Effect of Thrombin Concentration on Release of Radioactivity from Prelabeled Platelets Thrombin (units/ml)
0 0.5 1.o 2 .o 5 .O 10.0 25 .O
Medium radioactivity *
c%)
4.0 5.6 7.5 13.5 21.9 30.0 21.1
1.1 1.3 2.9 f 4.8 t 6.8 t 6.0 k 2.2 f
t t
*Mean f 1 SD of 4 experiments, each performed in duplicate. Initial total radioactivity: 227,550 2 17,443 DPM/109 platelets. % medium radioactivity: (medium radioactivity/platelet radioactivity + medium radioactivity) X 100. Platelets prelabeled with [3 H] a a c h i d n o i c acid, washed, and reincubated with thrombin for 15 min.
activity in the unknown lipid. Thrombin caused a release of 21.5% of the total radioactivity into the medium. When the medium radioactivity was analyzed with solvent System B, radioactivity was recovered in zones 1 , 2 , and 3. In other experiments (not shown) prostaglandins E l , Ez and Fza when added to plasma, extracted, and resolved with solvent System B were recovered exclusively at the origin. The time course of thrombin-induced release of radioactivity into the medium is shown in Fig. 2. The total release was linear for 1 0 min with little net additional release thereafter. The pattern of release in the different zones was not uniform. Radioactivity first appeared in zone 3 , reaching a peak at lOmin,declining thereafter. During the first 5 min, release into zone 2 was less than in zones 1 and 2, but thereafter the rate accelerated so that by 15 min of incubation radioactivity in zone 2 exceeded that in either zone 1 or 3. T o examine further the nature of the derivatives of arachidonic acid released into each zone by thrombin, two types of experiments were performed. In the first, lipids from each zone were extracted, treated with diazomethane, and the methyl esters of the lipids from each zone were separately chromatographed using solvent System C. The radioactivity from zone 1 was recovered near the origin with System C (r.f., 0.06). However, there was marked variability of recovery of methyl esters from this area. Since the radioactivity from this zone rarely exceeded 4% of the total, it was not investigated further. The radioactivity from zone 2 was recovered primarily in a single band; r.f., 0.72 (Fig.3A). The radioactivity from zone 3 was found in two approximately equal peaks, zone 3-A, r.f., 0.41, and zone 3-B,at the solvent front, r.f., 1.0 (Fig. 3B). In the second set of experiments, platelets were incubated with aspirin or indomethacin, both of which have been shown to inhibit platelet cyclooxygenase (5) and with eicosatetraynoic acid, which inhibits both the cyclooxygenase and lipoxygenase pathways of arachidonic acid metabolism. The data shown in Table IV indicate that none of the in-
Platelet Arachidonic Acid Metabolism
65
TABLE 111. The Effect of Thrombin on Distribution of Platelet and Medium Radioactivity 0 min O", no thrombin
15 min 37"C, no thrombin
PC
85,090 +1,631* (57.7)
81,492 t 2,205 (57.1)
56,127 f 6,631 (40.0)
PI
13,712 -f 7,274 (9.3)
16,589 f 2,822 (11.6)
9,715 f 432 (6.9)
PS
11,258 2 4,229 (7.6)
6,383 f 3,561 (4.5)
7,352 f 5,156 (5.2)
PE
17,156 f 1,241 (1 1.6)
18,876 f 2,780 (13.2)
15,348 f 2,140 (10.9)
"X"
1,732 f 923 (1 .2)
1,630 f 561 (1.2)
7,164 f 2,961 (5.1)
Platelet total
144,343 t 27,138 (97.9)
136,986 f 26,256 (95.9)
110,902f 14,134 (78.5)
Zone 1
252 (0.6) 292 f 38 (0.2) 1,549 f 436 (1.1) 327 f 48 (0.2)
1,095 f 75 (0.8) 366 f 86 (0.3) 3,889 f 145 (2.7) 345 t 51 (0.3) 5,689 f 922 (4.1
5,149 f 3,420 (3.6) 13,122 t 4,365 (9.3) 11,731 f 2,500 (8.3) 487 f 145 (0.3) 30,608 f 10,405 (21.5)
142,675 f 27,238 (100)
141,510 ?r 24,539 (100)
Zone 2 Zone 3 Zone 4 Medium total
878
f
3,112 f 647 (2.1)
Total radioactivity 147,455 t 27,785 (100)
15 min 37"C, 5 U/ml thrombin
*Mean f 1 SD 3 experiments; each point in duplicate. DPM/109 platelets. Number in parenthesis: [3 H] -arachidonic acid, washed and reincubated as indicated. %of total radioactivity. Platelets prelabeled with
hibitors prevented the release by thrombin of radioactivity from prelabeled platelets into the medium. Aspirin and indomethicin did not alter the distribution of radioactivity between zones2 and 3. In contrast, eicosatetraynoic acid suppressed the appearance of radioactivity into zone 2 and potentiated release into zone 3. The pattern of radioactivity in zones 2 and 3 was further resolved with System C. None of the inhibitors altered the pattern of radioactivity in zone 2. Whether enhanced by aspirin or suppressed by eicosatetraynoic acid, zone 2 radioactivity remained primarily in a single peak. In contrast, as shown in Table V, aspirin and eicosatetraynoic acid altered the ratio between zone 3A and zone 3B. In the absence of inhibitors, the radioactivity released by thrombin was equally distributed between compounds 3A and 3B. Both aspirin and eicosatetraynoic acid inhibited the appearance of radioactivity in zone 3A and enhanced appearance of radioactivity in zone 3B. The effect of aspirin on the distribution of radioactivity in zones 3, and 3 is shown in Fig. 3.
Russell and Deykin
35 -
Zone 2 Thrombin
-Zone
1
Zone 3 Thrombin 1 Thrombin 0 Control,
Total
I
0
2
4
6
8
10
12
14
16
M/NU TES Fig. 2. Time course of release of radioactivity into the medium lipids analyzed by System B (see Methods). (Mean of duplicate experiments.)
These experiments indicated, therefore, that eicosatetraynoic acid inhibited appearance of radioactivity in zones 2 and 3A, but enhanced appearance of radioactivity in zone 3B. Aspirin impaired appearance of radioactivity in zone 3A but enhanced appearance of radioactivity in zone 2. From these data we have concluded that radioactivity in zone 2 reflects the action of the lipoxygenase pathway of arachidonic acid metabolism, zone 3A reflects the cyclooxygenase pathway, and zone 3B represents methyl arachidonate (previously shown to have an r.f. of 1.O in System C).
DISCUSSION The data presented in Table I indicate that arachidonic acid is taken up by platelets and incorporated into complex lipids. The distribution of labeled arachidonic acid is similar to that which we have previously described for palmitic acid (10). As shown in Fig. 1 , thrombin impairs the net uptake of arachidonic acid, it stimulates the prompt liberation of newly incorporated arachidonic acid from the major phospholipid classes; and it causes the appearance of radioactivity in a lipid we have not previously encountered. Free fatty acids are incorporated into platelet lipids in a series of steps (1 1, 12). They are first taken up by the plasma membrane and then transferred to internal sites within the platelet where they may be either oxidized or incorporated into lipids. We have previously shown (9) that aggregation of platelets by thrombin does not impair the initial binding of free fatty acids by platelets, a rapid, nonenergy dependent process. We found that thrombin impaired the subsequent incorporation of the saturated fatty acids, palmitic
67
Platelet Arachidonic Acid Metabolism
9
9
10
\ $
8
8 6 4
2 0
SOLVENT M/GRAT/ON fcm/ Fig. 3. Effect of aspirin on distribution of lipids in zone 2 and zone 3 radioactivity analyzed by System C (see Methods). (Mean of duplicate determination.)
and stearic, into phospholipids but that it enhanced the uptake of oleic acid, particularly into lecithin. We have postulated that thrombin impairs de novo assembly of platelet complex lipids but that the enhanced incorporation of oleic acid reflects acylation of newly exposed monoacyl receptors. In our present studies we have observed that thrombin causes the net release of arachidonic acid from complex lipids. These findings indicate that for arachidonic acid, in contrast to palmitic, stearic, oleic, and linoleic acids, phospholipase activity plays a major role in determining the net effect of thrombin on fatty acid incorporation into platelet lipids. Thrombin causes the appearance of radioactivity in a lipid we have not previously characterized. The chromatographic migration of the lipid is clearly distinct from lysophosphatidylethanoiamine, phosphatidic acid, and ceramide. We have not seen appearance of radioactivity in a comparable area with lipids extracted from thrombin-aggregated
Russell and Deykin
68
TABLE IV. Effect of Prostaglandin Inhibitors o n Thrombin Induced Release of Radioactivity From Prelabeled Platelets Medium Platelets
Zone 1
Zone 2
Zone 3
Total
Control
495,889 * (9 8.4
2,689 (0.5)
Thrombin
282,866 (62.9)
15,333 (3.4)
1,108 (0.2) 120,733 (26.8)
4,387 (0.9) 30,846 (6.9)
8,184 (1.6) 166,912 (37.1)
3 10,558 (61.2)
12,262 (2.4)
145,550 (28.7)
38,761 (7.7)
196,573 (38.8)
350,212 (70.8)
8,214 (1.7)
104,73 3 (21.2)
31,431 (6.3)
144,378 (29.2)
300,369 (59.1)
8,683 (1.7)
21,191 (4.2)
178,084 (35.0)
207,958 (40.9)
Sample
Thrombin
+
Aspirin Thrombin -k
Indomethacin Thrombin
+
ETA**
Total
*DPM/109 platelets. Mean of duplicate experiments. Prelabeled platelets were incubated for 5 min with either buffer or inhibitor before the addition of thrombin. Incubations were continued for 15 min and terminated by extraction of platelets and media. ?%of total in each fraction. **ETA = eicosatetraynoic acid.
TABLE V. Effect of Prostaglandin Inhibitors on Distribution of Zone 3 Radioactivity Sample Thrombin Thrombin and Aspirin Thrombin and ETA
Zone 3B
Zone 3A
9,321* 2,906 6,591
11,824 22,868 139,193
WA
1.3 7.9 21.1
*DPM/109 platelets. Zone 3, Table IV samples resolved by System C.
platelets incubated with other fatty acids, suggesting that the appearance of radioactivity in the unknown lipid reflects a process specific for arachidonic acid. The nature of this compound is under study in our laboratory. The data presented in Table I1 indicate that the release of radioactivity from platelets prelabeled with arachidonic acid is dependent on the concentration of thrombin but that a peak effect is reached. The endogenous pools from which arachidonic acid is released and the mechanism by which thrombin activates platelet phospholipases are not yet known. Our data indicate that only a limited fraction of the lipids labeled by arachidonic acid is available to the thrombin activated phospholipases. The data presented in Table I11 indicate that there is extensive transformation of arachdonic acid into more polar derivatives. Arachidonic acid itself is present in zone 3 in System B used to resolve the lipids in the incubation medium. The compounds present
69
Platelet Arachidonic Acid Metabolism
in zones 1 and 2, though derived from arachidonic acid are clearly distinct from it and from the previously known platelet prostaglandins, PGEz and PGF2,, which would have remained at the origin. Therefore, the data presented in Fig. 1 and Tables 1-111 extend our previous observations of the effect of thrombin on the uptake of fatty acids by platelets. Our data indicate that thrombin causes a complex remodeling of platelet phospholipids. Thrombin-mediated alteration in net incorporation of fatty acids into phospholipids does not depend exclusively on either chain length or degree of unsaturation, but rather reflects the combination of several processes which include impaired de novo formation of complex lipids, enhanced acylation of monoacyl receptors, activation of phospholipases, and transformation of liberated fatty acids into derivatives which are released from the platelet. The data presented in Fig. 3 and Tables IV and V indicate that in our experiments thrombin caused the release of at least four distinct compounds from platelets prelabeled with arachidonic acid, one in zone 1, one in zone 2 , and two in zone 3. The probable nature of these compounds has been clarified by recent observations by Hamberg and Samuelsson (4-7). They have proposed that when arachidonic acid is liberated from phospholipids it is oxidized by one of two pathways. The first, catalyzed by platelet lipoxygenase, results in the formation of 12L-hydroxy-5,8, 10, 14 eicosatetraenoic acid, termed HETE. The second is catalyzed by platelet fatty acid cyclooxygenase. The initial step in the pathway is the addition of molecular oxygen to arachidonic acid forming two endoperoxides, prostaglandin Gz ,with a hydroperoxy group at Carbon-15 or prostaglandin Hz , with a hydroxyl group at Carbon-15. These compounds are unstable in aqueous media and rapidly decompose to two stable compounds, 12-hydroxy-5,8, 10 heptadecatrienoic acid (HHT) and 84 1-hydroxy-3-oxopropyl) 9 , 1 2 dihydroxy-5-10 heptadeca dienoic acid (PHD). The formation of HHT is accompanied by the expulsion of the threecarbon compound, malondialdehyde. Through a series of reactions, minor amounts of these products are converted into prostaglandins Ez and F z a . The main, stable end products of arachidonic acid cohversion therefore are HHT and PHD from the cyclooxygenase pathway and HETE from the lipoxygenase pathway. Hamberg and Samuelsson further showed that aspirin and indomethacin inhibited the cyclooxygenase pathway (6) (and thus PHD, HHT, and malondialdehyde production) but did not block the lipoxygenase pathway. In contrast, eicosatetraynoic acid inhibited both pathways (6). In our experiments, aspirin and indomethicin not only did not suppress radioactivity in zone 2 (Table IV), both actually enhanced it (Fig. 3A). Both did block the appearance of one of the two compounds in zone 3 (Fig. 3B). Eicosatetraynoic acid blocked the appearance of radioactivity in zone 2 and in the first compound in zone 3, but markedly potentiated the radioactivity in the second (Table V). Taken together our data suggest that the radioactivity in the peak of zone 2 reflects the lipoxygenase pathway; that in compound A in zone 3 it reflects the cyclooxygenase activity, and is related to HHT; and that in compound B it reflects arachidonic acid itself, which accumulates when the pathways of its further conversion are inhibited. The formal identification of the prostaglandin intermediates by Hamberg and Samuelsson was achieved by gas chromatography and mass spectrum analyses - techniques not yet duplicated in other laboratories. The conditions of our incubations, extractions, and analysis differed from those of Hamberg and Samuelsson. Racemization or further alteration of our compounds may well have occurred. Therefore, we cannot describe the radioactivity in our compounds explicitly to HHT or HETE. Nevertheless, our data add
70
Russell and Deykin
strong support to the existence of two pathways of thrombin stimulated metabolism of arachidonic acid, only one of which is suppressed by aspirin. Neither aspirin, nor eicosatetraynoic acid inhibited the total release of radioactivity into the medium by thrombin (Table IV). These data support the concept that thrombinmediated phospholipase activity is not impeded by either type of prostaglandin inhibitor. Prostaglandins G2 and H2 and some of their derivatives are themselves potent initiators of platelet aggregation. Indeed, Hamberg and Samuelsson have suggested that the defect in certain patients with primary disorders of platelet function reflect diminished cyclo. oxygenase activity (8). In preliminary experiments with three patients with disorders of platelet function we also have observed impaired cyclooxygenase activity but have found altered phospholipase activity as well. I t is clear that the availability of methods for analyzing the pathways of arachidonic acid liberation and metabolism provide new opportunities for understanding platelet function and for classification of hereditary and acquired disorders of primary hemostasis.
REFERENCES 1. Smith, JB, Willis, AL: Aspirin selectively inhibits prostaglandin production in human platelets. Nat New Biol231:235-237, 1971. 2. Kloeze, J: Relationship between chemical structure and platelet aggregation of prostaglandins. Biochim Biophys Acta 187:285-292,1969. 3. Shio, H, Ramwell, PW: Effects of prostaglandin Ez and aspirin on the secondary aggregation of human platelets. Nature 236:45-46, 1972. 4. Hamberg, M, Samuelsson, B: Detection and isolation of an endoperoxide intermediate in prostaglandin biosynthesis. Proc Nat Acad Sci USA 70:899-903, 1973. 5. Hamberg, M, Svensson, J, Wakabayashi, T, Samuelsson, B: Isolation and structure of two prostaglandin endoperoxides that cause platelet aggregation. Proc Nat Acad Sci USA 7 1 : 345-349, 1974. 6 . Hamberg M, Samuelsson, B: Prostaglandin endoperoxides. Novel transformations of arachidonic acid in human platelets. R o c Nat Acad Sci USA 71 :3400-3404, 1974. 7 . Hamberg, M, Svensson, J, Samuelsson, B: Prostaglandin endoperoxides. A new concept concerning the mode of action and release of prostaglandins. Proc Nat Acad Sci USA 71 :3824-3828,1974. 8. Malmsten, C, Hamberg, M, Svensson, J, Samuelsson, B: Physiological role of an endoperoxide in human platelets: Hemostatic defect due to platelet cyclooxygenase deficiency. Proc Nat Acad Sci USA 72:1446-1450,1975. 9. Deykin, D: Altered lipid metabolism in human platelets after primary aggregation. J Clin Invest 52 :483-492, 19 73. 10. Deykin, D, Desser, RK: The incorporation of acetate and palmitate into lipids by human platelets. J Clin Invest 47:1590-1602, 1968. 11. Spector, AA, Hoak, JC, Warner, ED, Fry, GL: Utilization of long-chain free fatty acids by human platelets. J Clin Invest 49:1489-1496, 1970. 12. Hoak, JC, Spector, AA, Fry, GL, Barnes, BC: Localization of free fatty acids taken up by human platelets. Blood 40:16-22, 1972.
THE EFFECT OF THROMBIN ON THE UPTAKE AND TRANSFORMATION OF ARACHIDONIC ACID BY HUMAN PLATELETS Francis A. Russell, M.S. and Daniel Deykin, M.D. Boston Veterans Administration Hospital, and Departments of Medicine, Boston University and Tufts University Schools of Medicine, Boston, Massachusetts
Washed human platelets take up arachidonic acid from plasma and incorporate the fatty acid into the major classes of complex lipids. Thrombin impairs net incorporation. It activates endogenous phospholipases which liberate arachidonic acid from phospholipids. As a consequence of thrombin induced aggregation platelets release arachidonic acid intermediates formed by the action of platelet fatty acid cyclooxygenase and by platelet fatty acid lipoxygenase. Cyclooxygenase, but not lipoxygenase, is inhibited by aspirin and indomethicin. Analysis of the pathways of arachidonic acid metabolism may furnish new insight into platelet function and into disorders of primary hemostasis. Key words: platelets, arachidonic acid, thrombin, phospholipases, cyclooxygenase, prostaglandins
Prostaglandins are formed and released by platelets during aggregation. ( 1 -3). Recent studies by Hamberg and Samuelsson and their associates (4-8) have indicated that certain prostaglandin intermediates are themselves potent initiators of platelet aggregation. In this study we have extended our previous experiments (9) on the effects of thrombin on the incorporation of longchain free fatty acids into platelet lipids to include arachidonic acid, the direct precursor of platelet prostaglandins. Our data indicate that washed human platelets take up arachidonic acid from plasma and incorporate it into the major classes of complex lipids. Thrombin impairs the net incorporation of arachidonic acid into the major lipid classes. It activates phospholipases which release newly incorporated arachidonic acid from phospholipids, and it causes the time-dependent release into plasma of metabolites of arachidonic acid consistant with the newly described lipoxygenase and fatty acid cyclooxygenase pathways of transformation of arachidonic acid.
Address reprint requests to Daniel Deykin, M.D., Boston VA Hospital, 150 S. Huntington Ave., Jamaica Plain, Mass. 021 30.
59
0 1976 Alan R. Liss, Inc., 150 Fifth Avenue, New York, N.Y. 10011
60
Russell and Deykin
METHODS Preparation of Washed Human Platelets
Suspensions of washed human platelets containing 0.6-1.5 X lo9 plateletsfml were prepared from normal subjects as previously described (9). Lipids
(5,6,8,9, 11, 12, 14, 15 [3H]) arachidonic acid (specific activity 72 Ci/mmole) was obtained from New England Nuclear Corporation, Boston, Mass. Prior to use, the arachidonic acid was dissolved in acetone at a final concentration of 100 ,uCi/ml. 5,8,11,14eicosatetraynoic acid was the gift of Dr. W. E. Scott, Hoffman, La Roche, Inc., Nutley, N. J. Prostaglandin standards were the gift of Drs. J. E. Pike, Upjohn Company, Kalamazoo, Mich. and E. W. Salzman, Beth Israel Hospital, Boston, Mass. Thrombin
Human thrombin was obtained from two sources. Commercial human thrombin was purchased from Ortho Diagnostics, Raritan, New Jersey. Highly purified thrombin (specific activity > 1,500 units/mg) was the gift of Dr. Robert Rosenberg, Beth Israel Hospital, Boston, Mass. In preliminary experiments we observed that at final concentrations of thrombin up to 15 U/ml there was no difference in the effects of either preparation on platelet aggregation or on the metabolism of arachidonic acid. At concentrations in excess of 15 U/ml commercial thrombin, but not the purified thrombin, inhibited aggregation. In experiments in w h c h the concentration of thrombin was 5 U/ml we used either preparation. In dose-response experiments we used the highly purified thrombin. Incubation Techniques
Three types of incubations were conducted. In all experiments in which thrombin was added, massive platelet aggregation occurred within 2 min. In experiments in which thrombin was omitted no aggregation occurred. A. Incorporation of fatty acids into platelets from prelabeled plasma. [3 HI arachidonic acid was complexed t o albumin in defibrinogenated dialyzed plasma, pH 6.8, (prepared as previously described [9]) by introducing the acetone solution (2.5pl/ml of plasma) slowly under the surface of the plasma with constant swirling. The acetone was driven off by incubation for 30 min at 37°C under a stream of nitrogen. Reactions were initiated by the addition of 1.5 ml of the platelet suspension (prewarmed to 37°C for 5 min before addition) to siliconized flasks containing 0.4 ml of the labeled plasma t o which 3.4 pmoles of CaC12 had been added, followed by the addition of either 0.1 ml of buffer or 0.1 ml of thrombin (final concentration 5 U/ml). In certain experiments thrombin was added 15 min after the incubations were begun. Under the conditions of these experiments the final amount of added arachidonic acid was approximately 1.5 X lo-' moles/flask. Incubations were terminated at various time intervals by immersing the flasks in an ice bath for 120 sec and then transferring the chilled contents of the flasks to iced centrifuged tubes. The platelets were then separated from the incubation medium by centrifugation at 2,500 X g for 10 min at 4°C. The platelets were then resuspended in a solution of phosphate buffer: dialyzed (unlabeled) plasma, pH 6.5 (1 :1 vol/vol), and were washed twice to remove unreacted and dissociable fatty acids. The platelets were then suspended finally in 2 ml of the washing solution, and the platelets and the incubation media were each transferred separately to extraction flasks
61
Platelet Arachidonic Acid Metabolism
containing 50 ml of chloroform: methanol (2: 1, vol/vol). Lipids were extracted by a modified Folch procedure (9). The lower (chloroform) phase was washed three times with 0.154 M NaC1.
B. Release of radioactive metabolitesfrom prelabeled platelets. [3 HI arachdonic acid was complexed to albumin as described in (A) except that 5 or 10 p1 of the acetone solution were added /ml of defibrinogenated plasma. Equal volumes of platelet suspensions and labeled plasma were incubated for 15 min at 37°C. The platelets were separated from the plasma by centrifugation. They were washed once with phosphate buffer, pH 6.5, and once again with the buffer-plasma solution as in (A) and resuspended in phosphate buffer, pH 6.8. One ml of the suspension was added to siliconized flasks containing 0.9 ml defibrinogenated plasma, pH 6.8, t o which 3.4 ymoles of CaC12 had been added. Incubations were initiated by the addition of 0.1 ml of buffer or thrombin. In one set of flasks platelets were immediately separated from the medium. The platelets were then washed, and the platelets and the medium were extracted separately. A second set of flasks was incubated with buffer for 15 min at 37°C and then processed, and a third set was incubated with thrombin for 1 5 min and then processed. In some experiments, the time of incubation with thrombin or the concentration of thrombin were varied. C. Release of radioactive metabolites from prelabeled platelets in the presence of inhibitors.Preliminary incubations, washing, and resuspension of the platelets were per-
formed as in (B). Prior to the addition of thrombin or buffer indomethacin (final concentration 150 pg/ml), aspirin (150 pg/ml), or eicosatetraynoic acid (final concentration 5 yg/ml were incubated with the platelets at 37°C for 5 min. Thrombin (final concentration 5 U/ml) was then added and the incubations were continued for an additional 15 min. Lipid Analysis
Phospholipids in the platelet samples were resolved by bi-directional thin-layer chromatography. (System A) Thin layer plates were coated (0.5 mm) with a slurry prepared from 50 gm silica gel H, 0.5 gm (N&),S04 and 110 ml of water. A portion of the chloroform extract of the platelets was evaporated at reduced temperature under vacuum to a volume of 100 p1 and was applied under nitrogen to a corner of the plate. Phospholipids were resolved with chloroform: methanol: acetic acid: water (1 50:90:24: 8.5, vol/vol). When the solvent front had reached a point 1 cm from the top of the plate, the plate was removed and dried at room temperature for 60 min. It was then rotated 90” and rechromatographed in a tank newly equilibrated with the same solvent system. Phospholipids were visualized by iodine vapor. With this system sphmgomyelin, lecithin (PC), phosphatidylinositol (PI), phosphatidylserine ( P S ) , and phosphatidylethanolamine (PE) were well resolved. An additional, as yet unidentified lipid (“X”) distinct from lysophosphatidylethanolamine was present between PS and PE. Platelet neutral lipids from a separate portion of the chloroform extract were resolved by one-dimensional chromatography (System B) using plates coated (0.25 mm) with a slurry made from 30 gm silica gel H and 65 ml of water. The solvent system was petroleum ether: diethyl ether: acetic acid (150:50:2 vol/vol). With this system phospholipids remained at the origin, and diglycerides (DG), free fatty acids (FFA), triglycerides (TG), and cholesteryl esters (CE) were well resolved. Medium lipids were separated by two methods. Routinely, the chloroform extract
62
Russell and D e y k i n
was concentrated by evaporation as described above, and the lipids were resolved by System B. The plates were marked into 4 zones: l(r.f., 0.0-0.09); 2(r.f., 0.10-0.31); 3(r.f., 0.32-0.56);4(r.f., 0.57-1 .O). In those experiments in which prostaglandin intermediates and derivatives were to be further examined, medium samples were handled differently. The chloroform extracts were divided into 2 portions, each of which was concentrated to 100 pl, applied to adjacent lanes of thin-layer plates and resolved with System €3 as above. One lane was scraped into zones 1,2,3, and 4 as described above for determination of total radioactivity in each zone. The duplicate adjacent zones were scraped into flasks and the lipids eluted with successive washes of petroleum ether: diethyl ether (1 : 1 vol/vol). The washes from each zone were pooled and concentrated to 100 pl. Methyl esters were formed by the addition of 5.4 X gm of diazomethane in 1.5 ml of dry ethyl ether. The samples were mixed, allowed to stand for 1 hr and reduced to 100 p1 by evaporation as previously described. The methyl esters were separated by System C, a modification of the method of Hamberg and Samuelsson. Thin layer plates were coated (0.5 mm) with a slurry composed of 60 gm silica gel G and 80 ml of water. Lipids were resolved with the upper phase of a mixture of ethyl acetate: 2,2,4-trimethylpentane: HzO (1OO:lOO: 150 vol/vol). The plates were divided into 16 1-cm bands each of w h c h was scraped and counted. In this system methyl arachidonate traveled with the solvent front. Radioactivity
Radioactivity in all samples was determined in a Packard Instruments Tri-Carb model #3385. Absolute activity was determined with an external standard and a computer derived quench calibration curve. RESULTS
Washed human platelets take up labeled arachidonic acid from plasma and incorporate it into all major lipid classes (Table I). Of the total radioactivity, 88%was incorporated into phospholipids and 12% into neutral lipids. Over half of the total radioactivity was present in lecithin. Only 3.8% remained as free fatty acid, and 1.4%was recovered in the area of the unknown lipid. TABLE 1. Platelet Lipid Distribution of Incorporated Arachidonic Acid ~
~~
Lipid class
Radioactivity* (DPM/ 10’ platelets)
PC PI PS PE “X” DG TG FFA
60,447 t 3,033 15,392 t 765 8,650i 359 9,617k 276 1,518i- 561 2,757 t 415 3,345 t 948 4,136 f 1,034
Total
% 54.8 14.0
7.8 8.7 1.4 2.5 3.0 3.8
110,285 i 4,466
*Mean ? SD, 3 experiments. In each experiment points determined in duplicate; 15 min incubations.
63
Platelet Arachidonic Acid Metabolism
The effect of thrombin on the time course of incorporation of arachidonic acid into total platelet lipids and into individual lipid classes is shown in Fig. 1. When thrombin was added at the onset of incubation, incorporation of arachidonic acid into the major lipid classes was reduced at all time points. In contrast thrombin stimulated incorporation of arachidonic acid into the unknown lipid. When thrombin was added after 15 min of incubation, total radioactivity in the platelets declined reaching a minimum withm 3 min. There was concomitant release of radioactivity from all major lipid classes. In contrast there was a sharp, prompt, and sustained stimulation of incorporation of radioactivity into the unknown lipid.
PI
0
PS
10
20
30
0
10
20
30
MINUTES Fig. 1. Effect of thrombin on [ 3 H ] arachidonic acid incorporation into platelet lipids (mean of triplicate experiments, each in duplicate). Abbreviations: PC = lecithin; PI = phosphatidylinositol; PS = phosphatidylserine; PE = phosphatidylethanolamine.
To examine thrombin-induced release of arachidonic acid and its derivatives from washed prelabeled platelets, platelets were first incubated with arachidonic acid, washed, and reincubated in unlabeled plasma in the presence or absence of thrombin. The effect of increasing the concentration of thrombin on release of radioactivity into the medium is shown in Table 11. In the absence of thrombin platelets released 4% of their radioactivity during 15 min. Increasing concentrations of thrombin caused progressively greater release of radioactivity into the medium, reaching a peak at a final concentration of 10 U/ml. The effect of thrombin on the distribution of radioactivity among the major phospholipid classes, the unknown lipid, and in the medium is shown in Table 111. Thrombin (5 Ujml) caused the release of radioactivity from PC, PI, and PS but enhanced the radio-
64
Russell a n d Deykin TABLE 11. Effect of Thrombin Concentration on Release of Radioactivity from Prelabeled Platelets Thrombin (units/ml)
0 0.5 1.o 2 .o 5 .O 10.0 25 .O
Medium radioactivity *
c%)
4.0 5.6 7.5 13.5 21.9 30.0 21.1
1.1 1.3 2.9 f 4.8 t 6.8 t 6.0 k 2.2 f
t t
*Mean f 1 SD of 4 experiments, each performed in duplicate. Initial total radioactivity: 227,550 2 17,443 DPM/109 platelets. % medium radioactivity: (medium radioactivity/platelet radioactivity + medium radioactivity) X 100. Platelets prelabeled with [3 H] a a c h i d n o i c acid, washed, and reincubated with thrombin for 15 min.
activity in the unknown lipid. Thrombin caused a release of 21.5% of the total radioactivity into the medium. When the medium radioactivity was analyzed with solvent System B, radioactivity was recovered in zones 1 , 2 , and 3. In other experiments (not shown) prostaglandins E l , Ez and Fza when added to plasma, extracted, and resolved with solvent System B were recovered exclusively at the origin. The time course of thrombin-induced release of radioactivity into the medium is shown in Fig. 2. The total release was linear for 1 0 min with little net additional release thereafter. The pattern of release in the different zones was not uniform. Radioactivity first appeared in zone 3 , reaching a peak at lOmin,declining thereafter. During the first 5 min, release into zone 2 was less than in zones 1 and 2, but thereafter the rate accelerated so that by 15 min of incubation radioactivity in zone 2 exceeded that in either zone 1 or 3. T o examine further the nature of the derivatives of arachidonic acid released into each zone by thrombin, two types of experiments were performed. In the first, lipids from each zone were extracted, treated with diazomethane, and the methyl esters of the lipids from each zone were separately chromatographed using solvent System C. The radioactivity from zone 1 was recovered near the origin with System C (r.f., 0.06). However, there was marked variability of recovery of methyl esters from this area. Since the radioactivity from this zone rarely exceeded 4% of the total, it was not investigated further. The radioactivity from zone 2 was recovered primarily in a single band; r.f., 0.72 (Fig.3A). The radioactivity from zone 3 was found in two approximately equal peaks, zone 3-A, r.f., 0.41, and zone 3-B,at the solvent front, r.f., 1.0 (Fig. 3B). In the second set of experiments, platelets were incubated with aspirin or indomethacin, both of which have been shown to inhibit platelet cyclooxygenase (5) and with eicosatetraynoic acid, which inhibits both the cyclooxygenase and lipoxygenase pathways of arachidonic acid metabolism. The data shown in Table IV indicate that none of the in-
Platelet Arachidonic Acid Metabolism
65
TABLE 111. The Effect of Thrombin on Distribution of Platelet and Medium Radioactivity 0 min O", no thrombin
15 min 37"C, no thrombin
PC
85,090 +1,631* (57.7)
81,492 t 2,205 (57.1)
56,127 f 6,631 (40.0)
PI
13,712 -f 7,274 (9.3)
16,589 f 2,822 (11.6)
9,715 f 432 (6.9)
PS
11,258 2 4,229 (7.6)
6,383 f 3,561 (4.5)
7,352 f 5,156 (5.2)
PE
17,156 f 1,241 (1 1.6)
18,876 f 2,780 (13.2)
15,348 f 2,140 (10.9)
"X"
1,732 f 923 (1 .2)
1,630 f 561 (1.2)
7,164 f 2,961 (5.1)
Platelet total
144,343 t 27,138 (97.9)
136,986 f 26,256 (95.9)
110,902f 14,134 (78.5)
Zone 1
252 (0.6) 292 f 38 (0.2) 1,549 f 436 (1.1) 327 f 48 (0.2)
1,095 f 75 (0.8) 366 f 86 (0.3) 3,889 f 145 (2.7) 345 t 51 (0.3) 5,689 f 922 (4.1
5,149 f 3,420 (3.6) 13,122 t 4,365 (9.3) 11,731 f 2,500 (8.3) 487 f 145 (0.3) 30,608 f 10,405 (21.5)
142,675 f 27,238 (100)
141,510 ?r 24,539 (100)
Zone 2 Zone 3 Zone 4 Medium total
878
f
3,112 f 647 (2.1)
Total radioactivity 147,455 t 27,785 (100)
15 min 37"C, 5 U/ml thrombin
*Mean f 1 SD 3 experiments; each point in duplicate. DPM/109 platelets. Number in parenthesis: [3 H] -arachidonic acid, washed and reincubated as indicated. %of total radioactivity. Platelets prelabeled with
hibitors prevented the release by thrombin of radioactivity from prelabeled platelets into the medium. Aspirin and indomethicin did not alter the distribution of radioactivity between zones2 and 3. In contrast, eicosatetraynoic acid suppressed the appearance of radioactivity into zone 2 and potentiated release into zone 3. The pattern of radioactivity in zones 2 and 3 was further resolved with System C. None of the inhibitors altered the pattern of radioactivity in zone 2. Whether enhanced by aspirin or suppressed by eicosatetraynoic acid, zone 2 radioactivity remained primarily in a single peak. In contrast, as shown in Table V, aspirin and eicosatetraynoic acid altered the ratio between zone 3A and zone 3B. In the absence of inhibitors, the radioactivity released by thrombin was equally distributed between compounds 3A and 3B. Both aspirin and eicosatetraynoic acid inhibited the appearance of radioactivity in zone 3A and enhanced appearance of radioactivity in zone 3B. The effect of aspirin on the distribution of radioactivity in zones 3, and 3 is shown in Fig. 3.
Russell and Deykin
35 -
Zone 2 Thrombin
-Zone
1
Zone 3 Thrombin 1 Thrombin 0 Control,
Total
I
0
2
4
6
8
10
12
14
16
M/NU TES Fig. 2. Time course of release of radioactivity into the medium lipids analyzed by System B (see Methods). (Mean of duplicate experiments.)
These experiments indicated, therefore, that eicosatetraynoic acid inhibited appearance of radioactivity in zones 2 and 3A, but enhanced appearance of radioactivity in zone 3B. Aspirin impaired appearance of radioactivity in zone 3A but enhanced appearance of radioactivity in zone 2. From these data we have concluded that radioactivity in zone 2 reflects the action of the lipoxygenase pathway of arachidonic acid metabolism, zone 3A reflects the cyclooxygenase pathway, and zone 3B represents methyl arachidonate (previously shown to have an r.f. of 1.O in System C).
DISCUSSION The data presented in Table I indicate that arachidonic acid is taken up by platelets and incorporated into complex lipids. The distribution of labeled arachidonic acid is similar to that which we have previously described for palmitic acid (10). As shown in Fig. 1 , thrombin impairs the net uptake of arachidonic acid, it stimulates the prompt liberation of newly incorporated arachidonic acid from the major phospholipid classes; and it causes the appearance of radioactivity in a lipid we have not previously encountered. Free fatty acids are incorporated into platelet lipids in a series of steps (1 1, 12). They are first taken up by the plasma membrane and then transferred to internal sites within the platelet where they may be either oxidized or incorporated into lipids. We have previously shown (9) that aggregation of platelets by thrombin does not impair the initial binding of free fatty acids by platelets, a rapid, nonenergy dependent process. We found that thrombin impaired the subsequent incorporation of the saturated fatty acids, palmitic
67
Platelet Arachidonic Acid Metabolism
9
9
10
\ $
8
8 6 4
2 0
SOLVENT M/GRAT/ON fcm/ Fig. 3. Effect of aspirin on distribution of lipids in zone 2 and zone 3 radioactivity analyzed by System C (see Methods). (Mean of duplicate determination.)
and stearic, into phospholipids but that it enhanced the uptake of oleic acid, particularly into lecithin. We have postulated that thrombin impairs de novo assembly of platelet complex lipids but that the enhanced incorporation of oleic acid reflects acylation of newly exposed monoacyl receptors. In our present studies we have observed that thrombin causes the net release of arachidonic acid from complex lipids. These findings indicate that for arachidonic acid, in contrast to palmitic, stearic, oleic, and linoleic acids, phospholipase activity plays a major role in determining the net effect of thrombin on fatty acid incorporation into platelet lipids. Thrombin causes the appearance of radioactivity in a lipid we have not previously characterized. The chromatographic migration of the lipid is clearly distinct from lysophosphatidylethanoiamine, phosphatidic acid, and ceramide. We have not seen appearance of radioactivity in a comparable area with lipids extracted from thrombin-aggregated
Russell and Deykin
68
TABLE IV. Effect of Prostaglandin Inhibitors o n Thrombin Induced Release of Radioactivity From Prelabeled Platelets Medium Platelets
Zone 1
Zone 2
Zone 3
Total
Control
495,889 * (9 8.4
2,689 (0.5)
Thrombin
282,866 (62.9)
15,333 (3.4)
1,108 (0.2) 120,733 (26.8)
4,387 (0.9) 30,846 (6.9)
8,184 (1.6) 166,912 (37.1)
3 10,558 (61.2)
12,262 (2.4)
145,550 (28.7)
38,761 (7.7)
196,573 (38.8)
350,212 (70.8)
8,214 (1.7)
104,73 3 (21.2)
31,431 (6.3)
144,378 (29.2)
300,369 (59.1)
8,683 (1.7)
21,191 (4.2)
178,084 (35.0)
207,958 (40.9)
Sample
Thrombin
+
Aspirin Thrombin -k
Indomethacin Thrombin
+
ETA**
Total
*DPM/109 platelets. Mean of duplicate experiments. Prelabeled platelets were incubated for 5 min with either buffer or inhibitor before the addition of thrombin. Incubations were continued for 15 min and terminated by extraction of platelets and media. ?%of total in each fraction. **ETA = eicosatetraynoic acid.
TABLE V. Effect of Prostaglandin Inhibitors on Distribution of Zone 3 Radioactivity Sample Thrombin Thrombin and Aspirin Thrombin and ETA
Zone 3B
Zone 3A
9,321* 2,906 6,591
11,824 22,868 139,193
WA
1.3 7.9 21.1
*DPM/109 platelets. Zone 3, Table IV samples resolved by System C.
platelets incubated with other fatty acids, suggesting that the appearance of radioactivity in the unknown lipid reflects a process specific for arachidonic acid. The nature of this compound is under study in our laboratory. The data presented in Table I1 indicate that the release of radioactivity from platelets prelabeled with arachidonic acid is dependent on the concentration of thrombin but that a peak effect is reached. The endogenous pools from which arachidonic acid is released and the mechanism by which thrombin activates platelet phospholipases are not yet known. Our data indicate that only a limited fraction of the lipids labeled by arachidonic acid is available to the thrombin activated phospholipases. The data presented in Table I11 indicate that there is extensive transformation of arachdonic acid into more polar derivatives. Arachidonic acid itself is present in zone 3 in System B used to resolve the lipids in the incubation medium. The compounds present
69
Platelet Arachidonic Acid Metabolism
in zones 1 and 2, though derived from arachidonic acid are clearly distinct from it and from the previously known platelet prostaglandins, PGEz and PGF2,, which would have remained at the origin. Therefore, the data presented in Fig. 1 and Tables 1-111 extend our previous observations of the effect of thrombin on the uptake of fatty acids by platelets. Our data indicate that thrombin causes a complex remodeling of platelet phospholipids. Thrombin-mediated alteration in net incorporation of fatty acids into phospholipids does not depend exclusively on either chain length or degree of unsaturation, but rather reflects the combination of several processes which include impaired de novo formation of complex lipids, enhanced acylation of monoacyl receptors, activation of phospholipases, and transformation of liberated fatty acids into derivatives which are released from the platelet. The data presented in Fig. 3 and Tables IV and V indicate that in our experiments thrombin caused the release of at least four distinct compounds from platelets prelabeled with arachidonic acid, one in zone 1, one in zone 2 , and two in zone 3. The probable nature of these compounds has been clarified by recent observations by Hamberg and Samuelsson (4-7). They have proposed that when arachidonic acid is liberated from phospholipids it is oxidized by one of two pathways. The first, catalyzed by platelet lipoxygenase, results in the formation of 12L-hydroxy-5,8, 10, 14 eicosatetraenoic acid, termed HETE. The second is catalyzed by platelet fatty acid cyclooxygenase. The initial step in the pathway is the addition of molecular oxygen to arachidonic acid forming two endoperoxides, prostaglandin Gz ,with a hydroperoxy group at Carbon-15 or prostaglandin Hz , with a hydroxyl group at Carbon-15. These compounds are unstable in aqueous media and rapidly decompose to two stable compounds, 12-hydroxy-5,8, 10 heptadecatrienoic acid (HHT) and 84 1-hydroxy-3-oxopropyl) 9 , 1 2 dihydroxy-5-10 heptadeca dienoic acid (PHD). The formation of HHT is accompanied by the expulsion of the threecarbon compound, malondialdehyde. Through a series of reactions, minor amounts of these products are converted into prostaglandins Ez and F z a . The main, stable end products of arachidonic acid cohversion therefore are HHT and PHD from the cyclooxygenase pathway and HETE from the lipoxygenase pathway. Hamberg and Samuelsson further showed that aspirin and indomethacin inhibited the cyclooxygenase pathway (6) (and thus PHD, HHT, and malondialdehyde production) but did not block the lipoxygenase pathway. In contrast, eicosatetraynoic acid inhibited both pathways (6). In our experiments, aspirin and indomethicin not only did not suppress radioactivity in zone 2 (Table IV), both actually enhanced it (Fig. 3A). Both did block the appearance of one of the two compounds in zone 3 (Fig. 3B). Eicosatetraynoic acid blocked the appearance of radioactivity in zone 2 and in the first compound in zone 3, but markedly potentiated the radioactivity in the second (Table V). Taken together our data suggest that the radioactivity in the peak of zone 2 reflects the lipoxygenase pathway; that in compound A in zone 3 it reflects the cyclooxygenase activity, and is related to HHT; and that in compound B it reflects arachidonic acid itself, which accumulates when the pathways of its further conversion are inhibited. The formal identification of the prostaglandin intermediates by Hamberg and Samuelsson was achieved by gas chromatography and mass spectrum analyses - techniques not yet duplicated in other laboratories. The conditions of our incubations, extractions, and analysis differed from those of Hamberg and Samuelsson. Racemization or further alteration of our compounds may well have occurred. Therefore, we cannot describe the radioactivity in our compounds explicitly to HHT or HETE. Nevertheless, our data add
70
Russell and Deykin
strong support to the existence of two pathways of thrombin stimulated metabolism of arachidonic acid, only one of which is suppressed by aspirin. Neither aspirin, nor eicosatetraynoic acid inhibited the total release of radioactivity into the medium by thrombin (Table IV). These data support the concept that thrombinmediated phospholipase activity is not impeded by either type of prostaglandin inhibitor. Prostaglandins G2 and H2 and some of their derivatives are themselves potent initiators of platelet aggregation. Indeed, Hamberg and Samuelsson have suggested that the defect in certain patients with primary disorders of platelet function reflect diminished cyclo. oxygenase activity (8). In preliminary experiments with three patients with disorders of platelet function we also have observed impaired cyclooxygenase activity but have found altered phospholipase activity as well. I t is clear that the availability of methods for analyzing the pathways of arachidonic acid liberation and metabolism provide new opportunities for understanding platelet function and for classification of hereditary and acquired disorders of primary hemostasis.
REFERENCES 1. Smith, JB, Willis, AL: Aspirin selectively inhibits prostaglandin production in human platelets. Nat New Biol231:235-237, 1971. 2. Kloeze, J: Relationship between chemical structure and platelet aggregation of prostaglandins. Biochim Biophys Acta 187:285-292,1969. 3. Shio, H, Ramwell, PW: Effects of prostaglandin Ez and aspirin on the secondary aggregation of human platelets. Nature 236:45-46, 1972. 4. Hamberg, M, Samuelsson, B: Detection and isolation of an endoperoxide intermediate in prostaglandin biosynthesis. Proc Nat Acad Sci USA 70:899-903, 1973. 5. Hamberg, M, Svensson, J, Wakabayashi, T, Samuelsson, B: Isolation and structure of two prostaglandin endoperoxides that cause platelet aggregation. Proc Nat Acad Sci USA 7 1 : 345-349, 1974. 6 . Hamberg M, Samuelsson, B: Prostaglandin endoperoxides. Novel transformations of arachidonic acid in human platelets. R o c Nat Acad Sci USA 71 :3400-3404, 1974. 7 . Hamberg, M, Svensson, J, Samuelsson, B: Prostaglandin endoperoxides. A new concept concerning the mode of action and release of prostaglandins. Proc Nat Acad Sci USA 71 :3824-3828,1974. 8. Malmsten, C, Hamberg, M, Svensson, J, Samuelsson, B: Physiological role of an endoperoxide in human platelets: Hemostatic defect due to platelet cyclooxygenase deficiency. Proc Nat Acad Sci USA 72:1446-1450,1975. 9. Deykin, D: Altered lipid metabolism in human platelets after primary aggregation. J Clin Invest 52 :483-492, 19 73. 10. Deykin, D, Desser, RK: The incorporation of acetate and palmitate into lipids by human platelets. J Clin Invest 47:1590-1602, 1968. 11. Spector, AA, Hoak, JC, Warner, ED, Fry, GL: Utilization of long-chain free fatty acids by human platelets. J Clin Invest 49:1489-1496, 1970. 12. Hoak, JC, Spector, AA, Fry, GL, Barnes, BC: Localization of free fatty acids taken up by human platelets. Blood 40:16-22, 1972.
