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Home , Arachidonic acid, Diglyceride

Production of from in Activated Human

S. RITTENHOUSE-SIMMONS, Bostotn Veterans Administration Medical Celnter and the Department of Biochemistry, Boston University School of Medicine, Boston, Massachusetts 02130

A B S T R A C T Human platelets generate diglyceride such as pancreas, cerebral cortex, and parotid gland, within 5 s of exposure to thrombin. Production of di- stimulation by acetylcholinie elicits a breakdown of PI is transient. 15 s after the addition of throin- (3-5). The secretory response of platelets is essenitially bin, the levels of diglyceride have increased up to 30- complete within 30 s (6). Therefore, those chaniges in fold, but decrease thereafter. Prior incubation of plate- metabolism that may participate in promoting se- lets with 2 mM dibutyryl cyclic AMP prevents both the cretioni must be well underway within this period. The generation ofdiglyceride and the secretion of serotonin. rapid generation of cyclic endoperoxides derived from Acetylsalicylic acid (100 ,ug/ml), which completely in- released frolm PI and phosphatidyl- hibits endoperoxide synthesis, does not (PC) is one such change (7-10). Additional block diglyceride production and serotonin secretion short-term effects characterized thus far in platelets induced by thrombin. exposed to agents such as collagen, ADP, and thrombin Based on studies examining the incorporation of have been an increased incorporation of [32P]ortho- [3H]arachidonic acid into diglyceride of prelabeled phosphate into and polyphosphoino- platelets exposed to thrombin, it is concluded that sitides (11) and a decrease in the amount of previously neither phosphatidic acid nor is the source radiolabeled PI (12). of the diglyceride. Phosphatidylinositol appears to be This report describes a rapid, transient generation the most likely source, both because its loss of radio- of diglyceride in human platelets exposed to throinbin. label is sizable and rapid enough to account for the Evidence is presented as well for the existence of a appearance of radiolabel in diglyceride, and because PI-specific phosphodiesterase in platelets which re- a phosphatidylinositol-specific phosphodiesterase, de- (juires ions for full activity and which may be scribed in this report, exists in platelets. The phos- involved in the turnover of PI associated with the se- phatidylinositol-phosphodiesterase, which produces cretory process. diglyceride and phosphate, re(luires Ca+2 and Inhibitors of cyclo-oxygenase, such as ace- shows optimal activity at pH 7. The does not tylsalicylic acid, have iminimal effect on the release act upon , phosphatidylethanol- reaction induced by thrombin, however, N6-02'-dibu- amine, or . tyryl cyclic AMP (dBcAMP) prevents such release. The production ofdiglyceride by platelets was examined in INTRODUCTION thp presence ofacetylsalicylic acid and in the presence of dBcAMP in order to dissociate, if possible, the pro- The human platelet shares with other secretory cells duction of diglyceride from that of cyclic endoperox- a "phosphoinositide effect" (1, 2), broadly defined as an ides. The postthrombin formation of diglyceride ap- alteration in the metabolism of phosphatidylinositol pears to correlate with the occurrence of secretion, (PI)' preceding and paralleling secretion. In tissues rather than with cyclic endoperoxide production. Received for publication 8 September 1978 and in revised form 8 December 1978. METHODS 1 Abbreviations used in this paper: ASA, acetylsalicylic acid; dBcAMP, N6-02'-dibutyryl cyclic AMP; DG, diglyceride; Preparation ofcells and resolution oflipids. Platelet con- DOC, deoxycholate; HHT, 12-L-hydroxy-5,8,10 heptade- centrates were provided by the Red Cross, within 24 h of catrionoic acid; 5-HT, 5-hydroxytryptamine (serotonin); phlebotomy, from normal donors. In some cases, blood was PA, phosphatidic acid; PC, phosphatidylcholine; PE, phos- drawn in the laboratory from normal human volunteers and phatidylethanolamine; PI, phosphatidylinositol; PRP, plate- anticoagulated with acid-citrate-dextrose (13). After a pre- let-rich plasma; PS, phosphatidylserine; TG, triglyceride. liminary centrifugation at 150 g for 1 min (platelet concen-

580 J. Clin. Invest. ©) The American Society forr Clinical Investigation, Inc. 0021-9738/79/04/0580/08 $1.00 Volume 63 April 1979 580-587 trates) or 150 g for 15 min (whole blood), platelet-rich plasmla (101) cells/mil) or erythrocvte-neutrophil suispensions ( 106 (PRP) was mixed wvith EGTA to a final conicenitrationi of 1 mM. cells/ml) from preliminarv cenitrifugation of PRP were iinCu- For studies on the metabolismn of radiolabeled arachi(loniic bated in a fincal voltume of 1.05 ml with and without hu- acid or , PRP was incuibated with [5,6,8,9,11,12,14,15- man thrombini (5-50 U/mI) for periods of up to 120 s. 3H]arachidonic acid (12.5 ,uCi/30 ml PRP, 72 Ci/mmol) which Siliconizedl glassware was used throughout. NaF (10( mNI) had been hound to albuumin as described previously (14), or was included in some mixtures. As a control for cellular vith[(N)-2-3H]glcerol (1 mCi/30 ml PRP, 6.48 Ci/mmol) for damage, lactate dehvdrogenase was assaved as previously 20 min at 37°C. Otherwise, such an incubation was omllitted. described (17, 18). Incubations were terminatedl by the adcli- Platelet suspensions were then washed three times with a tionl of 21 ml ice-coldl CHCI3:MeOH (2:1), and neutral Tris-citrate-bicarbonate lbuffer (15) containing 2 mM1 EGTA were resolved and quantitate(d as described above. at pH 6.5 after centrifugation at 2,500 g for 5 min at 4°C. Care Arachidonic acid oxiclation products in the a(lueous-\leOH wvas takeni to separate platelets from any contaminating eryth- phase vere resolved by high pressure li(quid chromiiatog- rocvtes and(l neuitrophils at the bottom of the pellet by raphv .2 Washed platelets whose lipids containedl [3H]arach- sacrificing part of the platelet pellet. Platelets wvere finally id(onic acid were incubated\with or without dBcAM1P suspended in buffer containiing 1 mrn EGTA, pH 6.8, to a (2 mM.M) for 15 miin at 37°C or with or without acetvl- final concentrationi of 1010 platelets/mIl. conicenltrationis (ASA; 100 1g/ml) for 5 min. Platelets were were determiinied with the aid of a Coulter device (Coulter then incubated as above with 0.17 U thrombin/108 cells. Electronics, Inc., Hialeah, Fla.). Suspensions contained xvere resolved 1y two-dimensioinal silica <0.01% of leukocvtes andl erythrocvtes. For studies examin- paper chromatography (8, 19). Radioactivity contained in ing secretion by platelets, unlabeled platelet suspenisions the lipid spots wvas counted by scintillationi spectrophotom- were then incubated with [14C]5-hydroxytryptamine (creati- etry in H20-AqIuasol (New England Nuclear) (8), and lipid phos- ninle sulfite salt, 1 /ACi/5 ml suispension) for 15 min at 37°C. phorus was assayed after dligestioni of parallel samiiples (19). On the average, 98% of this material was taken up by the Neutral lipids were resolved by thin-laver chromatography platelets in this period, making fuirther washinig unnecessary. in solvent systems I and II. DG spots were charred, Before analysis, platelet lipids were extracted 1y the counted, or digestedl bv KOH in MIeOH as described, and Folch procedlure (16). The lower phase of the Folch extract rechromiiatographed. The altered distril)ution of radlio- was sepatratedl by silica paper chromatography or oni silicic labeled material in 12-visualized spots wvas (letermiinledl. acid (Uni.sil 100-200( mesh, Unisil Corp., Greenivich, Platelets whose lipidls'vere labeled with [3H]glycerol- were Conn.). Neutral lipidls from silicic acid columllns were con- also incubated as described for [3H]arachidonic acid-labeled centrated under vacuum and applied to 0.5i-miml silica G cells. The effect of thrombin on the release of ['4C]5-hvldroxy- plates (A. G. Mierck Darmnstadt, West Germc-any) conitaininlg tryptamine from labeled platelets was also determiinled in 0.4 M (NH4)2SO4. Plates vere placed in equilibrated tanks the presenice and absence of clBcAMP or ASA. Incubations containing dichloroethane:MeOH (196:4 vol/vol), "system were stopped with 2 vol of ice-cold buffer (15), pH 6.5, I," or benzene:(liethyl ether:EtOH:NH3 (100:80:4.0:0.2, conitainiing imipramnine (6.7 ,u\M) anid EGTA (5 mnM). Cells vol/vol), "system II.' Resolved lipidls were madle visible were sedimented at 2,500 g for 5 min at 4°C, and the release with 12 and scraped off the plates for assay or were charred of 14C-labeled material to the medium vwas assessed by onl a heated sand bath. Charred spots were quantitated by scintillationi counting. dlensitometry (Photovolt Corp., New York) in comnparison phosphodiesterase assays. Washed plate- with calibrating amounlts of commercial triglyceride or lets describedl above were suspended to a concentrationi diglyceride derived from human platelets as described of 3 x 109 cells/ml in buffer (15) conitaining 5 mnM EGTA, pH below. 6.5. Platelet suspensions were soniicated in an ice-water bath Diglyceride was generatedl from labelecl platelet phos- wvith a microprobe for periods of 15 s. The total sonication pholipids for use in assessing recovery after the above ex- timne was 2 min. Similarly, suspensions of erythrocytes- traction and chromatographic procedures, and as a standard neutrophils (3 x 105 cells/nil) were sonicated. Sonicates for quantitative determinations on thin-layer plates. MIethanol were spun at 100,000 g for 60 min at 4°C, and the supernatant eluates from silicic acid columniiis (above), containinlg [3H]- solutioni was used in subsequenit incubationis. arachidonic acid or [3H]glecerol-labeled phospholipids, Substrates for assay of phospholipid phosphodiesterase were concentrated and suspenided by sonication in 800 Al consisted of [3H]mnoinositol PI, ['4C]choline PC, phosphati- 50 mni Tris HCI buffer, pH 7.3, containing 0.4% de- dylethanolamnine (PE), or phosphatidylserine (PS). [3H]- lipidated bovine serum albumnin. Sonicate (20 mg) was Mvoinositol PI was prepared according to a method de- mixed with 100 ,ul 50 mM CaCI2 and 25 ,lA phospholi- scril)ed by Paulus and Kennedy (20), with chicken pase C from Bacillus cereuis (50 gg/pul, Sigma Chemical Co., microsome suspensions. [14C]Choline PC, PE, and PS were St. Louis, MIo.) and incubated for 60 min at 37°C. Lipids purchased from. commercial sources. Microsomal suspen- were extracted by the Folch procedure, and the CHCl3 sions were incubated with 1 mM M;InCl2, 10 ,uCi [(N)-2-3H]- phase was resolved into neutral and polar lipidls on silicic myoinositol (12.5 Ci/mmol), and 10 mNI Hepes-NaOH, acid columns. [3H]Diglyceride (DG) in the neutral lipid was pH 7.4, at 37°C for 60 min. Lipids were extracted, identified on silica plates in both solvent systems I and II, washedI of free [3H]myoinositol and resolved on silicic acid in comparisoni with known standards of diolein, monoolein, columns into neutral lipid and labeled phospholipid frac- , and arachidoniic acid. The DG spots of such tions. The phospholipid was chromatographed on silica plates were scraped off and digestecl at 65°C for 2 h in paper (8, 19). The radiolabel and phosphorus contents closedl tubes containing 5% KOH MleOH. After the mnix- of PI were determinevd as were the amounits of other phos- tures were neutralized with HCI, lipids were extracted pholipids present in the eluate (19). The remaining with CHC13 and rechromatographed on plates in solvent radiolabeled eluate was dried under vacuum and suspended system II. The specific activity of the pure 3H-labeled DG with sonicationi in 25 mM Hepes-NaOH-50 mM NaCl, was determined by the use of a Cahn model 4400 electro- balance and scinitillation spectrophotometry (Cahn Instru- ments, Division of Ventron Corp., Cerritos, Calif.). 2 Russell, Francis A., and Daniel Deykin. Manuseript in Metabolic studies with intact cells. Washed platelets preparationi.

Diglyceride Production in Activated Human Platelets 581 pH 7.0, to a final concentration of 0.42 mM [3H]myoinositol PI. Also present in the phosphatide sonicates were 0.4 mM PS, 3.4 mM PC, and 2.1 mM PE. Radiolabeled PC substrate was prepared by mixing un- labeled microsomal lipid extracts with commercial 10 ,uCi [methyl-14C]choline PC, and suspending in buffer as 0 described, yielding a final concentration of 0.42 mM 0 [14C]choline PC. Unlabeled PE and PS were each sonicated to Hepes buffer to a final concentration of 0.42 mM. Assays for phosphatide phosphodiesterase activity con- tained Na deoxycholate (2.1 mg/ml) or Triton X-100 (1 mg/ ml; Rohm and Hass Co., Philadelphia, Pa.); CaCl2 (0-10mM), 50 mM KCI, 21 nmol [3H]myoinositol PI, [14C]choline PC, PE, 0 15 30 45 6071120 or PS, and up to 0.2 mg supematant protein (fresh or heated Time (seconds) at 100°C for 5 min) in a final volume of 200 ,ul. Some mix- tures contained 10 mM NaF. Incubations continued for up FIGURE 1 Generation of DG in response to thrombin. to 60 min at 37°C, and were terminated by the addition of Washed human platelets (10'0/ml) were incubated with 1.0 ml CHCl3:MeOH:HCl (100:100:0.6) and 0.3 ml 1 N thrombin (0.17 U/108 cells) at 37°C. The incubations were HCl/5 mM EGTA. Part of the upper aqueous phase was stopped with 20 vol CHCl3:MeOH (2:1). DG was quantitated assayed for radioactivity by scintillation spectrophotometry by densitometry of charred spots on thin layer plates in in H20-Aquasol (8). Other portions of the upper phase comparison with known amounts of standards. Results are were applied to silica papers and chromatographed in two presented as averages for an experiment performed in directions as before (8). The distribution of radiolabel duplicate. *, +thrombin; 0, -thrombin. among lyso-PI, inositol, and any PI present was deter- mined for incubations that had included [3H]myoinositol PI. Samples of the [3H]myoinositol aqueous phase were platelets (not shown) was unaffected by thrombin. also applied to Whatman no. 1 paper (Whatman, Inc., Clifton, No response was observed for erythrocyte-neutrophil N. J.) and resolved by descending chromatography in EtOH: suspensions similarly incubated. 13.5 M NH3 (3:2) as described by Dawson and Clarke (21), in The time-course for the production of DG was comparison with the migration of inositol phosphate, [3H]- duplicated by the course of labeling of this myoinositol PI, [3H]myoinositol, and glycerol phospho- material inositol. Chromatograms were negatively stained for phos- by [3H]arachidonic acid in platelets which had taken phate with 0.1% ethanolic FeCl3:1% NH4SCN in acetone up the isotope before incubation with thrombin (Fig. (22) and counted by scintillation spectrophotometry. 2A). Thrombin-free controls exhibited no change over The lower CHCl3 phase (for PI, PS, and PE incubations) the period examined. [3H]Arachidonic acid-labeled was concentrated and applied to a 0.5-g column of silicie acid washed with CHCl3. Neutral lipid was resolved on 0.5- triglyceride changed only slightly over this period, mm plates of silica G as described. Lipids were run in sol- with or without thrombin. In the same experiment, vent systems I or II and were made visible by charring. labeled PI decreased, whereas 12-L-hydroxy-5,8,10 DG was quantitated by densitometry in comparison with heptadecatrionoic acid (HHT; an oxidation product of known standards. Other plates containing DG made visible arachidonic acid) and PA increased slightly in 5 s and by I2 were scraped, and the lipid contained digested in KOH and rechromatographed as described. Phospholipid reached a constant level after 30 s (Fig. 2B). Not shown ([3H]PI samples) was resolved by two-dimensional chromatog- is the finding that after 15 s the content of [3H]- raphy. The origin and regions corresponding to known lyso-PI arachidonic acid had not changed significantly in PC, and PI standards were assayed for 3H. Radioisotopes were obtained from New England Nuclear (Boston, Mass.) or Amersham Corp. (Arlington Heights, 7 Ill.). Highly purified human a-thrombin (1 nM = 0.11 .- U/ml) was kindly provided by Dr. J. W. Fenton, II (New 6 York State Department of Health). Lipid standards were supplied by Applied Science Labs (State College, Pa.), Supelco, Inc. (Bellefonte, Pa.), NuCheek Corp. (Elysian, Minn.), and Dr. John Pike of Upjohn Co. (Kalamazoo, Mich.). All solvents employed were spec&rograde or re- 2E ------30- distilled. dBeAMP was purchased from Sigma Chemical Co. 0 20 RESULTS .A B 15 30 45 60 0 15 30 45 60 Metabolic studies on intact cells Time (seconds) Time-dependent changes in human platelet phos- FIGURE 2 Change in content of[3H]arachidonic acid in lipids pholipids induced by thrombin. The production of with time, after exposure of labeled platelets to thrombin. DG by platelets exposed to 0.17 U thrombin/108 plate- Platelets (10'0/ml), preincubated with [3H]arachidonic acid, were incubated with thrombin (0.17 U/108 platelets). lets was rapid. As illustrated in Fig. 1, within 5 s, Thrombin-free control results are not presented, but displayed 69% of the maximum amount of DG produced by the no significant changes. (A) Changes in DG (0) and TG (-) cell had been generated. The triglyceride content of labeling; (B) changes in PI (O), PA(A), and HHT (-) labeling.

582 S. Rittenhouse-Simmons PS, or diacyl-PE. After 30 s of exposure to platelets to 6 thrombin, significant loss of [3H]arachidonic acid _ from PC was detectable. The DG produced in activated platelets was identi- 5 100 fied in comparison with the migration of commercial C-generated standard DG after thin- 0P4 80 H layer chromatography. Triglyceride (TG), 1,2-DG, and I If) arachidonic acid migrated in solvent system I with 4 - 00 60 0 Rfs of 0.86, 0.31, and 0.25, respectively. The Rfs of TG, 1,2-DG, arachidonic acid, and HHT in sol- 2 -A 40Q vent system II were 0.93, 0.71, 0.10, and 0.04. The 0) DG produced by stimulated platelets was labeled either with [3H]glycerol or with [3H]arachidonic acid 20 cr when prelabeled platelets were employed. Such DG tIll I was completely digested by mild akaline treatment, 0 0.1 0.2 0.3 0.4 0.5 as was the DG standard. FFA was produced, con- Thrombin (U/ I08 platelets) taining [3H]arachidonic acid. FIGURE 3 Generation of DG anid release of [14C]5-HT by The changes in specific activity ([3H]carachidonic human platelets in response to thromnbin. In experiments acid) for DG, TG, and PI are correlated in Table I. monitoring DG, washed platelets (10'0/ml) were exposed to Values are calculated for the time of peak generationi thrombin for 15 s at 37°C. Lipids were extracted, resolved, of 15 s. on a and quantitated as described for Fig. 1. Other suspensions DG, If, based weighted average for the of platelets, containing [14C]5-HT vere exposed to thrombin composition of PI in human platelets (23), a and sedimented. Release of [14C]5-HT to the medlinmll was molecular weight of 640 is assigned to the platelet determined. *, DG; 0, percent release [14C]5-HT. DG, a conversion of mass to nanomoles can be made. A molecular weight of 840 is used for platelet TG, based on the fatty acid composition of human platelet A comparison of DG production andl ['4C]5-hydroxv- TG (23). Mass values for PI were arrived at after phos- tryptamine (HT) secretion in human platelets respond- phorus analysis. The calculations on a mole basis ing to various concentrations of thrombin is pre- show that the loss of PI (15 nmol) can easily ac- sented in Fig. 3. Near-maximum responses for 1)oth count for the gain in DG (6.1 nmol) observed. Further, were attained with 0.17 U thrombin/108 platelets. labeled PI could account for the change in specific Contrasting effects of dBcAMP and ASA. Table II activity of DG. The specific activity of TG was low shows the effect of prior incubation of platelet sus- and did not change with time. Specific activity pensions with dBcAMP (2 mM) or ASA (100 ,ug/ml) cvalues for phosphatidic acid (PA) were not de- on serotonin release and DG production as well as on rived as it was not possible to measure PA phos- the generation of [3H]arachidonic acid-labeled prod- phorus on silica papers, although radioactivity was ucts arising ultimately from cyclo-oxygenase activity. conisistently measurable. Such radioactivity migrated It is seen that 1)oth dBcANIP and ASA effectively with known PA standards, and appeared in the nieutral eliminated the formation of such arachidonic acid lipid region wheni the lipid on the spots was iinc'u- products, ordinarily generated in response to thrombin. b1ated un(ler mild alkaline conditions aind rechromiiato- However, only dBcANIP inhibited serotonini secre- graphed. tion and DG production as well. It was also noted

TABLE I TABLE II Specific Activity of [3H]Arachidonic Acid-Labeled Lipid in Inhibitory Effects of dBcAMP and ASA on Changes Intact Platelets Exposed to Thrombin* Induced by Thrornbin*

t=( t = 15s Inhibition

cpttltlninol Inhibitor D(; 5-HT HHT DG 1,157 530 TG 49 51 PI 460 414 dBcAMP 95±2 97±3 96+2 ASA 0+5 0±3 90±3 * Platelets (1010 cells/ml) containing [3H]arachidonic acid- laleled lipids (no free arachidonic acid was present) were * The inhibition by dBcAMP or ASA in comparison with incubated with thrombin (0.17 U/108 platelets) for 15 s. Data controls was derived from the expression 100 - (effect + in- represent the average±1.0% of an experiment performed hibitor/effect - inhibitor)100. Results are the means+SE for in dutplicate. two experiments performed in duplicate.

Diglyceride Production in Activated Humant Platelets 583 401

30 0

0) N 2' 20 E -ou0 II 0

Ia-0 15 30 45 60 Time (minutes) FIGURE 4 of [3H]myoinositol PI as a function of time. [3H]Myoinositol PI was incubated with platelet sonicate supernate (0.5 mg/ml), pH 7.0, in the presence of 5 mM Ca+2 and 2 mg/ml DOC at 37°C. Incubations were concluded Ca"2 (mM) by the addition of CHCl3-MeOH-HCI. The formation of water-soluble [3H]myoinositol was determined after scintilla- FIGURE 5 Hydrolysis of [3H]myoinositol PI as a function of tion counting. Ca+2 concentration. [3H]Myoinositol PI was incubated with platelet sonicate supernate (1 mg/ml), pH 7.0, in the presence of 2 mg/ml DOC and varied amounts of Ca+2. The formation of water-soluble [3H]myoinositol was quantitated after (not shown) that 10 mM NaF, which inhibits phos- incubation for 60 min at 37°C, as described for Fig. 4. Results phatidic acid phosphatase in human platelets and other are the means ±SE of three experiments. cells (24, 25) did not inhibit the formation of DG. Release of lactate dehydrogenase (2.1%) did not differ significantly from control levels. silica paper, <1% of this radioactive material migrated with a lyso-PI standard or with PI. Nearly all of the material was found at the origin. More than 80% Studies on disrupted cells of the radiolabeled material in the aqueous supernate Phospholipid phosphodiesterase activity. Dis- migrated with inositol-l-phosphate after descending rupted cell preparations were used to characterize chromatography, and tested positively for phosphate. the nature of the DG-generating enzyme system in The remainder migrated with free myoinositol. Plate- platelets. Platelet sonicate supernates, incubated with let sonicate supernates incubated with [3H]myoinositol [3H]myoinositol PI, produced a lipid that migrated did not produce labeled inositol-l-phosphate. with Rfs corresponding to those of 1,2-DG in solvent No water-soluble radiolabeled material was pro- systems I and II, and which yielded FFA upon mild duced when ['4C]choline PC was substituted for alkaline hydrolysis. No such material was found in in- [3H]myoinositol PI as a substrate, irrespective of the cubated supernate or in [3H]myoinositol PI solutions pH of the incubation mixture. Further, no DG was incubated in the absence of supernate. The hydrolysis produced when sonicate supernates from platelets of [3H]myoinositol PI by the supernate from platelet were incubated with suspensions of PE or PS. Erythro- sonicates to water-soluble labeled material and DG cyte-neutrophil sonicate preparations did not hydrolyze was dependent upon the presence of both deoxy- [3H]myoinositol PI to water-soluble material. cholate (DOC) and Ca+2. The presence of 10 mM NaF Fig. 6 represents the relationship of the hydrolytic affected neither the production of DG nor that of activity to the pH of the incubation mixture, dis- water-soluble material. In the absence of both DOC played in the presence of 2 mg/ml DOC and 5 mM and Ca+2, no hydrolytic activity was detectable Omis- Ca+2. There was a clear optimum at pH 7 for both the sion of either DOC or Ca+2 from the incubation re- generation of water-soluble radioactivity (represented sulted in little activity. Triton X-100 could not substi- in terms of nanomoles of [3H]myoinositol PI hydro- tute for DOC. Activity was not detectable when lyzed) and the formation of DG. If one takes the molec- supernate that had been heated at 100°C for 5 min was ular weight of chicken liver microsome PI-derived employed. DG to be 590 (liver PI is considerably less rich in The hydrolysis of [3H]myoinositol PI with time is arachidonic acid than is human platelet PI; 26), then shown in Fig. 4. The dependency of the enzymatic it is possible to calculate that the amount of DG pro- activity upon Ca+2 is illustrated in Fig. 5. Simulation duced on a molar basis is comparable both to the by Ca+2 was optimal at 5 mM, whereas 10 mM amount of [3H]myoinositol PI hydrolyzed and to the Ca+2 was inhibitory. The radioactivity detected in the amount of water-soluble [3H]myoinositol-containing aqueous supernate was not removable by lyophiliza- product formed, with a molar ratio of 0.99+0.14 tion. When chromatographed in two directions on (SE): 1.0.

584 S. Rittenhouse-Simmons (20-21), does not inhibit the production of DG. The formation of DG from PC, PS, or PE would require the action of a phosphatide phosphodiesterase acting upon one or all of these substrates. Thus far, Ino such activity has been detectable in humain platelets (28). 10 Further, PS and diacyl-PE are unchanginig with re- E spect to [3H]arachidoniic acid andl [3H]glycerol in the period examined. PC starts to show signiificanit loss 5.0 6.087.08 only after 30 s, and conitiniues to do so well after the 0) time that DG has returnied to normiial levels. Finally, 6O , even in platelets labeled with high specific activity [3H]glycerol, was not found. In con- trast, PI loses mass anid [3H]arachidonic acid with 0~~~~~~~~~~ (D~ ~ ~ p appropriate rapidity. The specific acitivity of PI with respect to [3H]arachidonic acid is in keeping with that calculated for labeled DG. It is also noteworthy that of all the phospholipids of the platelet, PI displays 50 6.0 7.0 8.0 9.0 the highest specific activity when platelets are incu- pH bated with [3H]arachidonic acid (8, 10). The specific FIGURE 6 Hydrolysis of [3H]myoinositol PI and formation of activity of DG is slightly (15%) greater than that of DG as a function ofpH. [3H]Myoinositol PI was incubated with PI, which in turn has a higher specific activity than platelet sonicate supernate (1 mg/mil), 2 mg/ml DOC, and PC, PS, and PE. Finally, the existence of a PI phos- 5 mM Ca+2 for 60 min at 37°C. The pH was varied by the phodliesterase, described here, couldl accounit for the use of 100 mM acetate- and Tris-acetate buffers. The formation of water-soluble [3H]myoinositol was deter- early changes observed. mined as described for Fig. 4. Quantitation of DG in the The data presented in this report constitute direct CHCl3 phase was achieved by densitometry of charred spots evidence for a PI-specific phosphodiesterase in hu- on thin-layer plates in comparison with standards. 0, man platelets. Three sets of findinigs support an argu- DG; *, PI. ment for the specificity of the enzyme for PI. Firstly, the enzyme preparation does not hydrolyze [14C]- DISCUSSION choline PC to water-soluble radiolabeled material. Secondly, [3H]myoinositol PI, in the presence of an The production of DG appears to be one of the earliest eightfold excess of PC, a fivefold excess of PE, and an metabolic events to occur during the activation of equivalent amount of PS, is hydrolyzed to radiolabeled human platelets by thrombin, anteceding the full secre- inositol and DG in equimolar amounts. Were the tory response. The identification of DG as such is sup- enzyme acting comparably upon substrates other than ported by the presence of alkali-hydrolyzable fatty PI, one would expect to observe the production of acid and glycerol, and the observed comigration with significantly greater amounts of DG than radiolabeled known 1,2-DG in two different neutral lipid chroma- inositol. Finally, the enzyme does not hydrolyze pure tography systems. Resting platelets contain very small suspensions of PS or PE to DG. quantities of DG (27). Exposure of platelets to It appears that the enzyme is a , thrombin induces as much as a 30-fold increase in producing myoinositol-l-phosphate and DG. Whether the amount of this material, but the elevation is or not a myoinositol-1:2-cyclic phosphate is generated transitory. Within 2 min, the platelet has essentially in the system, as is the case in certain other cells achieved the low levels of DG that characterize the (21), is unknown because the acidic conditions resting state. employed would cause a hydrolysis of the labile cyclic The source of the DG is most probably PI. Of the compound to the monoester form. other possible candidates for precursor, TG, PA, phos- The existence of platelet PI-phosphodiesterase, and pholipid(s) other than PI, and monoglyceride, none further, its requirement for Ca+2 at physiologic pH, are is a satisfactory alternative. TG levels do not change of interest for the elucidation of a secretory mechanism significantly during the short time interval of rele- in platelets. The platelet contains large stores of vance, nor does the content of [3H]arachidonic acid sequestered Ca+2 which are apparently mobilized in in TG decrease. PA, although difficult to quantitate the cytoplasm during the earliest stages of platelet on a mass basis, does not lose [3H]arachidonic acid activation (29-31). Stimulation by a secretion-inducing with 15 s of exposure to thrombin, but rather, gains agent such as thrombin might promote the hydrolysis this fatty acid. Further, the presence of NaF, which of PI by making Ca+2 available to PI-phosphodiesterase. has been shown to inhibit phosphatidate phosphatase If the activation of PI-phosphodiesterase is an early

Diglyceride Production in Activated Human Platelets 585 event in the sequence leading to secretion by platelets, 4. Lunt, G. G., and M. R. Pickard. 1975. The subcellular as it appears to be, it may provide another control localization of carbamylcholine-stimulated phosphatidyl- inositol turnover in rat cerebral cortex in vivo. J. point for platelet functioning and would thus merit Neurochem. 24: 1203-1208. investigation in human subjects displaying secretory 5. Jones, L. M., and R. H. Michell. 1974. Breakdown of defects. phosphatidyl-inositol provoked by muscarinic cholinergic The elevation of DG and the release of 5-HT from stimulation of rat parotid-gland fragments. Biochem. J. 142: 583-590. storage granules are similarly responsive to increased 6. Charo, I. F., R. D. Feinman, and T. C. Detwiler. 1977. doses ofthrombin. Under conditions in which secretion- Interrelations of platelet aggregation and secretion. inducing arachidonic acid oxidation products are not J. Clin. Invest. 60: 866-873. formed, both the generation of DG and secretion in 7. Hamberg, M., and B. Samuelsson. 1974. Prostaglandin response to are Thus it appears endloperoxides: novel transformations ofarachidonic acid thrombin unimpaired. in human platelets. Proc. Natl. Acad. Sci. U. S. A. 71: that the increase in DG is not dependent upon the 3400-3404. generation of prostaglandin endo-peroxides, which is 8. Rittenhouse-Simmons, S., F. A. Russell, and D. Deykin. blocked by . However, dBcAMP, which is 1977. Mobilization ofarachidonic acid in human platelets: thought to act, in part, to restrict Cal2 mobilization kinetics and Ca+2 dependency. Biochim. Biophys. Acta. 488: 370-380. in platelets (32, 33), effectively inhibits both secretion 9. Russell, F. A., and D. Deykin. 1976. The effect ofthrombin and DG formation. on the uptake and transformation of arachidonic acid The role of DG in the secretory process is as yet by human platelets. Am. J. Hematol. 1: 59-70. unknown. The level of DG has been shown to increase 10. Bills, T. K., J. B. Smith, and M. J. Silver. 1976. in certain other tissues during secretion (34-37), and Metabolism of '4C-arachidonic acid by human platelets. Biochim. Biophys. Acta. 424: 303-314. Allan and Michell have suggested that DG promotes 11. Lloyd, J. V., and J. F. . 1974. Changes in membrane fusibility in such processes (35). Under 32P-content of phosphatidic acid and the phospho- certain conditions, DG facilitates fusion of hen inositides of rabbit platelets during aggregation induced erythrocytes (38). Demel et al. (39) reported that the by collagen or thrombin. Br. J. Haematol. 26: 243-253. 12. Leung, N. L., R. L. Kinlough-Rathbone, and J. F. Mustard. generation of DG in PC monolayers by phospholipase C 1977. Thrombin-induced changes in phosphatidic acid lowered surface pressures, thereby promoting the and phosphoinositides. Thromb. Haemostasis. 38: 7A. breakdown ofthe monolayers. And recently, Chap et al. (Abstr.) (40) observed that the production of diacylglycerols 13. Aster, R. H., and J. H. Jandl. 1964. Platelet sequestration in platelet membranes exposed to Clostridium welchii in man. I. Methods.J. Clin. Invest. 43: 843-856. 14. Rittenhouse-Simmons, E., F. A. Russell, and D. Deykin. phospholipase C was followed by aggregation and cell 1976. Transfer of arachidonic acid to human platelet lysis. plasmalogen in response to thrombin. Biochem. Biophys. It is thus possible that localized DG generated in Res. Commun. 70: 295-301. platelet membranes may assist secretion by promoting 15. Rittenhouse-Simmons, S., and D. Deykin. 1976. Isolation of membranes from normal and thrombin-treated gel- the fusion of membranes. filtered platelets using a lectin marker. Biochim. Biophys. Acta. 426: 588-596. ACKNOWLE ENTS 16. Folch, J., M. Lees, and G. H. S. Stanley. 1957. A simple DGM method for the isolation and purification of total lipids The author thanks Dr. Martin Low for generously providing from tissues. J. Biol. Chem. 226: 497-509. a sample of [3H]myoinositol PI and a method for its prepara- 17. Rittenhouse-Simmons, S., and D. Deykin. 1977. The tion. The author also thanks Dr. George Hauser and Dr. Jacek mobilization of arachidonic acid in platelets exposed to Hawiger for helpful comments regarding this manuscript, and thrombin or ionophore A23187: effects of adenosine Mr. Francis A. Russell for analysis of arachidonic acid triphosphate deprivation.J. Clin. Invest. 60: 495-498. oxidation products. 18. Leathwood, P. D., M. K. Crilford, and D. T. Plummer. This work was supported by grant HL 22502 from the 1972. Enzyme in rat urine: lactate dehydrogenase. National Heart, Lung, and Blood Institute, Department of Enzymologia. 42: 285-301. Health, Education, and Welfare, and by an award from the 19. Wuthier, R. E. 1966. Two-dimensional chromatography Veterans Administration, MRIS 5231822. on silica gel-loaded paper for the microanalysis of polar lipids.J. Lipid. Res. 7: 544-550. 20. Paulus, H., and E. P. Kennedy. 1960. The enzymatic REFERENCES synthesis of inositol monophosphate.J. Biol. Chem. 235: 1303-1311. 1. Hokin, L. E. 1969. Functional activity in glands and 21. Dawson, R. M., and N. Clarke. 1972. D-Myoinositol 1:2 synaptic tissue and the turnover of phosphatidylinositol. cyclic phosphate-2-phosphohydrolase. Biochem. J. 127: Ann. N. Y. Acad. Sci. 165: 696-709. 113-118. 2. Michell, R. H. 1975. Inositol phospholipids and cell 22. Oberleas, D. 1971. The determination of phytate and surface function. Biochim. Biophys. Acta. 415: inositol phosphates. Methods Biochem. Anal. 20: 87- 101. 81-147. 23. Marcus, A. J., L. B. Safier, and H. L. Ullman. 1972. The 3. Hokin-Neaverson, M. R. 1974. Acetylcholine causes a net lipids of human platelets. 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586 S. Rittenhouse-Simmons 24. Call, F. L., and W. J. Williams. 1971. Phosphatidic acid activation by Ca+2 of platelet : effects of metabolism in human platelets. J. Clin. Invest. 50: 17a. dibutyryl cyclic adenosine monophosphate and 8-(N,N- (Abstr.) diethylamino)-octyl-3,4,5-trimethoxy-benzoate. Biochim. 25. Coleman, R., and G. Hubscher. 1962. Metabolism of Biophys. Acta. 543: 409-422. phospholipids. V. Studies of phosphatidic acid phos- 34. Banschbach, M.. W., R. L. Geison, and M. Hokin- phatase. Biochim. Biophys. Acta. 56: 479-490. Neaverson. 1974. Acetylcholine increases the level of 26. White, D. A. 1973. The phospholipid composition of diglyceride in mouse pancreas. Biochem. Biophys. Res. mammalian tissues. In Form and Function of Phospho- Commun. 58: 714-718. lipids. G. B. Ansell, J. N. Hawthorne, and R. M. C. 35. Allan, D., and R. H. Michell. 1977. Ca+2-dependent Dawson, editors. Elsevier Scientific Publishing Co., New di4cylglycerol accumulation in erythrocytes is associated York. 43-65. with microvesiculation but not with efflux ofK+. Biochem. 27. Marcus, A. J., H. L. Ullman, and L. B. Safier. 1969. J. 166: 495-499. Lipid composition of subcellular particles of human 36. Allan, D., and R. H. Michell. 1977. Metabolism of blood platelets. J. Lipid Res. 10: 108-114. phosphatidate at the plasma membrane. Biochem. Soc. 28. Otnaess, A. B., and T. Holm. 1976. The effect of Trans. 5: 55-58. phospholipase C on human blood platelets.J. Clin. Invest. 57: 1419-1425. 37. Allan, D., M. M. Billah, J. B. Finean, and R. H. Michell. 29. Le Breton, G. C., and R. J. Dinerstein. 1977. Effect of 1976. Release of diacylglycerol enriched vesicles from the Ca+2 antagonist TMB-6 on intracellular calcium erythrocytes with increased intracellular- Ca+2 concentra- redistribution associated with platelet shape change. tions. Nature (Lond.). 261: 58-60. Thromb. Res. 10: 521-523. 38. Ahkong, Q. F., D. Fisher, W. Tampion, and J. A. Lucy. 30. Feinman, R. D., and T. C. Detwiler. 1975. Absence of a 1973. Fusion of erythrocytes by fatty acids , retinol, requirement for extracellular calcium for secretion from and a tocopherol. Biochem. J. 136: 147-155. platelets. Thromb. Res. 7: 677-679. 39. Demel, R. A., W. S. M. Geurts Van Kessel, R. F. A. Zwaal, 31. White, J. G., G. H. R. Rao, and J. M. Gerrard. 1974. B. Roelofsen, and L.I.M. Van Deenen. 1975. Relation Effects of the inophore A 23187 on blood platelets. I. between various phospholipase actions on human red cell Influence on aggregation and secretion. Am. J. Pathol. 77: membranes and the interfacial phospholipid pressure in 135- 150. monolayer. Biochim. Biophys. Acta. 406: 97-107. 32. Miller, 0. V., R. A. Johnson, and R. R. Gorman. 1977. 40. Chap, H. J., R. F. A. Zwaal, and L. L. M. Van Deenen. Inhibition of PGE,-stimulated cAMP accumulation in 1977. Action of highly purified on blood human platelets by A2. . platelets: evidence for an asymmetric distribution of 13: 599-607. phospholipids in the surface membrane. Biochim. 33. Rittenhouse-Simmons, S., and D. Deykin. 1978. The Biophys. Acta. 467: 146-164.

Diglyceride Production in Activated Human Platelets 587