Phospholipid Metabolism in Stimulated Human Platelets: CHANGES IN PHOSPHATIDYLINOSITOL, PHOSPHATIDIC ACID, AND LYSOPHOSPHOLIPIDS M. Johan Broekman, … , Jean W. Ward, Aaron J. Marcus J Clin Invest. 1980;66(2):275-283. https://doi.org/10.1172/JCI109854. Endogenous phospholipid metabolism in stimulated human platelets was studied by phosphorus assay of major and minor components following separation by two-dimensional thin-layer chromatography. This procedure obviated the use of radioactive labels. Extensive changes were found in quantities of phosphatidylinositol (PI) and phosphatidic acid (PA) as a consequence of thrombin or collagen stimulation. Thrombin addition was followed by rapid alterations in the amount of endogenous PI and PA. The decrease in PI was not precisely reciprocated by an increase in PA when thrombin was the stimulus. This apparent discrepancy could be explained by removal of a transient intermediate in PI metabolism, such as diglyceride, formed by PI-specific phospholipase C (Rittenhouse-Simmons, S., J. Clin. Invest.63: 580-587, 1979). Diglyceride would be unavailable for PA formation by diglyceride kinase, if hydrolyzed by diglyceride lipase (Bell, R. L., D. A. Kennerly, N. Stanford, and P. W. Majerus. Proc. Natl. Acad. Sci. U. S. A.76: 3238-3241, 1979) to yield arachidonate for prostaglandin endoperoxide formation. Thrombin-treated platelets also accumulated lysophospho-glycerides. Specifically, lysophosphatidyl ethanolamines accumulated within 15s following thrombin addition. Fatty acid and aldehyde analysis indicated phospholipase A2 activity, with an apparent preference for diacyl ethanolamine phosphoglycerides. In the case of collagen, these changes occurred concomitantly with aggregation and consumption of oxygen for prostaglandin endoperoxide formation. These studies of endogenous phospholipid metabolism provide information supporting the existence of […] Find the latest version: https://jci.me/109854/pdf Phospholipid Metabolism in Stimulated Human Platelets CHANGES IN PHOSPHATIDYLINOSITOL, PHOSPHATIDIC ACID, AND LYSOPHOSPHOLIPIDS NI. JOHAN BROEKMAN, JEAN W. WARD, and AARON J. MARCUS, Divisionts of Helmiatology- Otncology, Departments ofMedicine, Netw lYork VeterasisAdnministratiotn Hospital, Newt York, Newv York 10010; Cornell University Medical College, Newc York, Netv York 10021 A B S TR A C T Endogenous phospholipid metabolism These studies of endogenous phospholipid me- in stimulated human platelets was studied by phos- tabolism provide information supporting the existence phorus assay of major and minor components following of two previously postulated pathways for liberation separation by two-dimensional thin-layer chromatog- of arachidoniic acid from platelet phospholipids: (a) raphy. This procedure obviated the use of radio- the coombined action of PI-specific phospholipase C active labels. Extensive changes were found in quanti- plus diglyceride lipase yielding arachidonate derived ties of phosphatidylinositol (PI) and phosphatidic from PI; and (b) a phospholipase A2 acting primarily acid (PA) as a consequence of thrombin or collagen on diacyl ethanolamine phosphoglyceride. stimulation. Thrombin addition was followed by rapid alterations in the amount of endogenous PI and PA. INTRODUCTION The decrease in PI was not precisely reciprocated by an increase in PA when thrombin was the stimulus. Interest in the effects of stimulation upon platelet lipid This apparent discrepancy could be explained by re- metabolism focused initially on inositol-containing moval of a transient intermediate in PI metabolism, phospholipids. As in many secretory tissues (1), platelet such as diglyceride, formed by PI-specific phospho- stimulation increases the turnover of phospha- lipase C (Rittenhouse-Simmons, S., J. Cliii. Invest. tidylinositol (PI)' and related phospholipids, such as 63: 580-587, 1979). Diglyceride would be unavailable phosphatidic acid (PA) and di- and triphosphoinositide for PA formation by diglyceride kinase, if hydrolyzed (2-4). The metabolism of these phospholipids is also by diglyceride lipase (Bell, R. L., D. A. Kennerly, relatively active in unstimulated platelets (5, 6). N. Stanford, and P. W. Majerus. Proc. Natl. Acad. Sci. The turnover of ethanolaminephosphoglycerides (PE), U. S. A. 76: 3238-3241, 1979) to yield arachidonate phosphatidylcholine (PC), and sphingomyelin was for prostaglandin endoperoxide formation. Thrombin- reported to be very low in both stimulated and treated platelets also accumulated lysophospho- unstimulated platelets (5, 6). However, platelet stimu- glycerides. Specifically, lysophosphatidyl ethanola- lation increases de nlovo synthesis of phosphatidyl mines accumutlated within 15 s following thrombin serine (7) and PI (8). addition. Fatty acid and aldehyde analysis indicated Recently the important role of arachidonic acid and phospholipase A2 activity, with an apparent preference its oxygenation products in platelet function has for diacyl ethanolamine phosphoglycerides. In the case initiated new interest in platelet lipid metabolism (for of collagen, these changes occuirred concomitantly with review, 9). In platelets, arachidonic acid is esterified to aggregation and consumption of oxygen for prosta- the 2-position of phospholipids (10-12). Investigations glandin endoperoxide formation. on the mechanism by which arachidonate is made avail- able to cyclooxygenase and lipoxygenase have initially Part of this work was presented at the Annual Meeting of centered on a putative phospholipase A2 (13-17). the American Society for Clinical Investigation, Washington, D. C., May 1979 (Clin. Res. 27: 459A, Abstr.). 1Abbreviations used in this paper: DPG, diphosphatidyl- Dr. Broekman was the recipient of a New York Heart glycerol, cardiolipin; LPC, lysophosphatidylcholine; LPE, Association Fellowship during these studies, and is at present lysophosphatidyl ethanolamine; PA, phosphatidic acid; PC, a Senior Investigator of the New York Heart Association. phosphatidylcholine, lecithin; PE, ethanolamine phospho- Received for publication 15 February 1980 and in revised glycerides; PI, phosphatidylinositol; TLC, thin-layer chro- form 21 April 1980. matography. J. Clin. Invest. © The American Society for Clinical Investigation, Inc. * 0021-9738/80/08/0275/09 $1.00 275 Volume 66 August 1980 275-283 A relationship between two aspects of lipid me- in a flow of nitrogen kept at 55% relative humidity (22). tabolism in stimulated platelets, i.e., PI turnover and Lipid extracts were spotted in this same atmosphere. Between arachidonate liberation, was recently postulated by first (chloroform/methanol/ammonia, 65:35:5.5, vol/vol) and second (chloroform/acetone/methanol/acetic acid/water, Bell et al. (18), and is based upon their demonstration 3:4:1:1:0.5, vol/vol) dimensional developments (12, 22), of a diglyceride lipase in platelets. This enzyme would plates were dried for 1 h in a stream of dry nitrogen. release arachidonate from diglyceride produced by the With this TLC system, separation of major phospholipids, PI-specific phospholipase C identified by Rittenhouse- including PI and phosphatidyl serine, as well as such minor components as PA, diphosphatidylglycerol (DPG), lysophos- Simmons (19). phatidyl ethanolamine (LPE), and lysophosphatidylcholine In the present study we provide evidence for two (LPC), was readily and reproducibly accomplished (Fig. 1) pathways for hydrolysis of the 2-position fatty acid (12, 22). from platelet phospholipids: one involves a phospho- Phosphorus analysis. After two-dimensional TLC, plates lipase A2 of considerable activity, with diacyl ethanola- were air dried and stained in iodine vapor. Areas contain- ing phosphorus (detected in separate experiments with a mine phosphoglycerides as primary substrates; the modified Dittmer-Lester spray reagent [23]) were scraped other involves operation of the aforementioned path- and phosphorus content was determined (24). PI, LPE, PA, way of PI-specific phospholipase C plus diglyceride and DPG were quantitated with high precision, as evidenced lipase. In addition, we have shown that upon collagen by small standard deviations (Table II). Measurements of stimulation PI-PA the phosphorus content of other phospholipids showed larger metabolism, arachidonate oxygena- standard deviations, contributing to the lack of significance tion, and aggregation follow similar time-courses. in changes observed after stimulation (Fig. 2) (Table II). Gas-liquid chromatography. TLC plates with samples for fatty acid and aldehyde analysis were kept in an atmosphere METHODS of dry nitrogen after 2-dimensional TLC until dried. Areas Platelet collection and processing. For each experiment to be analyzed were identified, scraped, methylated (BF3/ 2 U of whole blood were drawn into a plastic pack system methanol), and analyzed as previously described (11, 12, 25). (Fenwal 4R1718, Fenwal Laboratories, Div. Travenol Labora- Results were expressed as weight percent of methylated tories, Inc., Deerfield, Ill.), and washed platelets were pre- components. pared as previously described (20). The blood donors denied Chemicals. Reagents used were obtained as previously having taken medications during the preceding 2 wk. Never- described (20). In addition, albumin was from Sigma Chemical theless, platelet cyclooxygenase activity was checked before Co., St. Louis, Mo. (A6003); silica gel H, type 60 (Merck & platelet processing by stimulating samples of platelet-rich Co., Rahway, N. J.), from Brinkmann Instruments, Inc., plasma with collagen and