Production of Diglyceride from Phosphatidylinositol in Activated Human Platelets 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 glyceride 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- lipid 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- arachidonic acid released frolm PI and phosphatidyl- hibits prostaglandin endoperoxide synthesis, does not choline (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 phosphatidic acid and polyphosphoino- platelets exposed to thrombin, it is concluded that sitides (11) and a decrease in the amount of previously neither phosphatidic acid nor triglyceride 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 calcium 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 inositol phosphate, re(luires Ca+2 and Inhibitors of platelet cyclo-oxygenase, such as ace- shows optimal activity at pH 7. The enzyme does not tylsalicylic acid, have iminimal effect on the release act upon phosphatidylcholine, phosphatidylethanol- reaction induced by thrombin, however, N6-02'-dibu- amine, or phosphatidylserine. 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 glycerol, 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 lipids 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. Cell conicenltrationis salicylic acid (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 Phospholipids 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 Phospholipid 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.