Multiple Mechanisms Linked to Platelet Activation Result in Lysophosphatidic Acid and Sphingosine 1-Phosphate Generation in Blood

Lysophosphatidic acid (LPA) and sphingosine 1-phosphate (Sph1P) production was examined in vitro under conditions that simulated blood clotting. Several approaches were utilized to elucidate the metabolic pathways. 1) Platelet phospholipids were labeled using [32P]orthophosphate, and the production of [32P]Sph1P and LPA was examined. Thrombin stimulation of platelets resulted in rapid secretion of Sph1P stored within the platelet. In contrast, LPA was neither stored within nor secreted from platelets. Nonetheless, extracellular levels of LPA gradually increased following stimulation. 2) Stable-isotope dilution mass spectrometry was used to quantify the molecular species of LPA generated from plateletsin vitro. Only 10% of the LPA generated following thrombin stimulation was associated with platelets, the remaining 90% was contained within the extracellular medium. The acyl composition of LPA produced by platelets differed depending on the presence or absence of plasma in the incubation. 3) The fate of exogenously added fluorescent phospholipid analogs was determined. Incubation of [(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]dodecanoyl-(NBD)-labeled phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine with the supernatant fractions from thrombin-stimulated platelets yielded no LPA production. However, these lipids were converted to the corresponding lysolipids by released PLA1 and PLA2 activities. When incubated with plasma or serum the NBD-labeled lysophospholipids were readily converted to LPA. Inhibitors of lysophospholipase D and the biological activity of LPA were detected in plasma. These results suggest that the bulk of LPA produced through platelet activation results from the sequential cleavage of phospholipids to lysophospholipids by released phospholipases A1 and A2 and then to LPA by plasma lysophospholipase D. Lysophosphatidic acid (LPA) and sphingosine 1-phosphate (Sph1P) production was examined in vitro under conditions that simulated blood clotting. Several approaches were utilized to elucidate the metabolic pathways. 1) Platelet phospholipids were labeled using [32P]orthophosphate, and the production of [32P]Sph1P and LPA was examined. Thrombin stimulation of platelets resulted in rapid secretion of Sph1P stored within the platelet. In contrast, LPA was neither stored within nor secreted from platelets. Nonetheless, extracellular levels of LPA gradually increased following stimulation. 2) Stable-isotope dilution mass spectrometry was used to quantify the molecular species of LPA generated from plateletsin vitro. Only 10% of the LPA generated following thrombin stimulation was associated with platelets, the remaining 90% was contained within the extracellular medium. The acyl composition of LPA produced by platelets differed depending on the presence or absence of plasma in the incubation. 3) The fate of exogenously added fluorescent phospholipid analogs was determined. Incubation of [(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]dodecanoyl-(NBD)-labeled phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine with the supernatant fractions from thrombin-stimulated platelets yielded no LPA production. However, these lipids were converted to the corresponding lysolipids by released PLA1 and PLA2 activities. When incubated with plasma or serum the NBD-labeled lysophospholipids were readily converted to LPA. Inhibitors of lysophospholipase D and the biological activity of LPA were detected in plasma. These results suggest that the bulk of LPA produced through platelet activation results from the sequential cleavage of phospholipids to lysophospholipids by released phospholipases A1 and A2 and then to LPA by plasma lysophospholipase D. lysophosphatidic acid stable isotope-dilution liquid chromatography mass spectrometry sphingosine 1-phosphate lysophospholipase D l-α-lysophosphatidylcholine l-α-lysophosphatidylethanolamine l-α-lysophosphatidylserine bovine serum albumin dioleoyl phosphatidic acid phosphatidylcholine phosphatidylethanolamine phosphatidylserine [(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]dodecanoyl 2DTLC, one- and two-dimensional TLC phospholipase A Lysophosphatidic acid (LPA)1 and sphingosine 1-phosphate (Sph1P) are phospholipid mediators with pleiotropic growth factor properties that elicit their actions via the activation of G protein-coupled receptors encoded by the endothelial differentiation gene family (1Goetzl E.J. Lee H. Tigyi G. Oppenheim J.J. Feldmann M. Cytokine Reference. Academic Press., New York, London2000: 1407-1418Google Scholar, 2Tigyi G. Prostaglandins Lipid Mediators. 2001; 64: 47-62Crossref PubMed Scopus (92) Google Scholar). Several investigators have identified platelets as the source of Sph1P and LPA. However, contradictions exist in the literature concerning the mechanisms by which these mediators are generated. Although some investigators found no Sph1P generation in thrombin-activated platelets (3Eicholtz T. Jalink K. Fahrenfort I. Moolenaar W.H. Biochem. J. 1993; 291: 677-680Crossref PubMed Scopus (577) Google Scholar), others reported as much as 0.5 μm Sph1P in human serum (4Yatomi Y. Igarashi Y. Yang L. Hisano N., Qi, R. Asazuma N. Satoh K. Ozaki Y. Kume S. J Biochem. (Tokyo). 1997; 121: 969-973Crossref PubMed Scopus (412) Google Scholar). Although it is generally agreed that LPA is generated in thrombin-activated platelets (3Eicholtz T. Jalink K. Fahrenfort I. Moolenaar W.H. Biochem. J. 1993; 291: 677-680Crossref PubMed Scopus (577) Google Scholar, 5Lapetina E. Billah M. Cuatrecasas P. J. Biol. Chem. 1981; 256: 11984-11987Abstract Full Text PDF PubMed Google Scholar, 6Gerrard J.M. Robinson P. Biochem. Biophys. Acta. 1989; 1001: 282-285Crossref PubMed Scopus (179) Google Scholar), the rate of production found at 0.02 nmol/min/109 platelet cannot account for the 5–10 μm concentration detected in human serum (7Baker D.L. Desiderio M.D. Miller D.D. Tolley B. Tigyi G. Anal. Biochem. 2001; 292: 287-295Crossref PubMed Scopus (200) Google Scholar). During the first hour of blood clotting the concentration of LPA increases ∼300 nm; however, its production continues and an additional 5 μm is added to serum during the first 24 h, a time course that is hard to reconcile with that of platelet activation and consequently of platelet only origin. Gerrard and Robinson (6Gerrard J.M. Robinson P. Biochem. Biophys. Acta. 1989; 1001: 282-285Crossref PubMed Scopus (179) Google Scholar) quantified the molecular species of acyl-LPA in resting and thrombin-stimulated platelets, with a rank order of 16:0 > 18:0 > 20:4 > 18:1 > 18:2. In contrast, in plasma, the rank order is 18:2 > 18:1 ≥ 18:0 > 16:0 > 20:4, whereas in serum the order is 20:4 > 18:2 > 16:0 ≥ 18:1 > 18:0 (7Baker D.L. Desiderio M.D. Miller D.D. Tolley B. Tigyi G. Anal. Biochem. 2001; 292: 287-295Crossref PubMed Scopus (200) Google Scholar). Hence, the bulk of LPA present in serum is likely generated from a precursor distinct from that of LPA present in plasma. Tokumura et al. (8Tokumura A. Harada K. Fukuzawa K. Tsukatani H. Biochim. Biophys. Acta. 1986; 875: 31-38Crossref PubMed Scopus (154) Google Scholar), described the presence of a lysophospholipase D (LPLD) activity in plasma that was capable of generating LPA with a substrate preference for unsaturated lysophosphatidylcholine (LPC) and required metal ions for its activity (9Tokumura A. Miyake M. Yoshimito O. Shimizu M. Fukuzawa K. Lipids. 1998; 33: 1009-1015Crossref PubMed Scopus (52) Google Scholar). In the present study, we sought to clarify the mechanisms that contribute to the production of Sph1P and LPA in plasma and serum. [32P]Orthophosphate labeling of platelets was utilized to determine the generation, storage, and release of Sph1P and LPA in isolated platelets. Stable-isotope dilution mass spectrometry was used to quantify individual LPA species inside and outside thrombin-stimulated platelets. Finally, the ability to produce LPA from exogenous, fluorescently labeled phospholipids was determined upon incubation with supernatant fractions from stimulated platelets, plasma, and serum. Our results indicate that, in platelets, Sph1P is stored and rapidly released after thrombin stimulation, whereas LPA is generated through two different mechanisms. A minor portion of serum LPA originates within platelets, whereas the majority of LPA is the product of released PLA1 and PLA2 and of plasma lysophospholipase(s) D through sequential cleavage of serum and membrane phospholipids first to lysophospholipids and then to LPA. The following materials were obtained from the indicated suppliers: apyrase, PLA2 from Crotalus adamanteus venom PLD Type VII from Streptomyces species PLA1 type XI from Rhizopus arrhizus, human thrombin, prostaglandin E1, staurosporine, LPA,l-α-lysophosphatidylcholine (LPC),l-α-lysophosphatidylethanolamine (LPE),l-α-lysophosphatidylserine (LPS), bovine serum albumin (BSA, Fraction V, fatty-acid-free), and octyl-glucoside were from Sigma Chemical Co. (St. Louis, MO); d-erythro-Sph1P and dioleoyl phosphatidic acid (PA) were from Matreya, Inc. (Pleasant Gap, PA); 1-oleoyl-2-NBD-sn-glycero-3-phosphate (2-NBD-PA), 1-oleoyl-2-NDB-sn-glycero-3-phosphocholine, 1-oleoyl-2-NBD-sn-glycero-3-phosphoethanolamine, and 1-oleoyl-2-NBD-sn-glycero-3-phosphoserine were obtained from Avanti Polar Lipids, Inc. (Alabaster, AL); Silica Gel 60 high performance TLC and Silica Gel 60 TLC plates were purchased from the Merck Chemical Co. (Darmstadt, Germany); [32P]orthophosphoric acid with a specific activity of 314–337 TBq/mmol was from PerkinElmer Life Sciences (Boston, MA); TLC solvents were all of high performance liquid chromatography grade. Platelets were isolated from cubital venous blood of healthy adult volunteers of both genders as described previously (10Yatomi Y. Ohmori T. Rile G. Kazama F. Okamoto H. Sano T. Satoh K. Kume S. Tigyi G. Igarashi Y. Ozaki Y. Blood. 2000; 96: 3431-3438Crossref PubMed Google Scholar). Washed platelets were suspended in buffer A consisting of 20 mm HEPES (pH 7.4), 138 mm NaCl, 3.3 mm NaH2PO4, 2.9 mm KCl, 1.0 mm MgCl2, 1 mg/ml glucose, and 0.1% (w/v) BSA unless stated otherwise. Platelet numbers were determined using a Coulter counter (Hialeah, FL). Platelets suspended in buffer A without added NaH2PO4 at a cell density of 109/ml were incubated with 2.5 mCi/ml [32P]orthophosphoric acid for 2 h at 37 °C. Labeled platelets were diluted, washed twice in buffer A containing prostaglandin E1 (1 μm) and 3 units/ml apyrase, and finally adjusted to a density of ∼3 × 108/ml. [32P]Orthophosphate-labeled platelets were treated with 1 unit/ml thrombin in 0.5-ml aliquots for various times in the presence or absence of 2 mm Ca2+. The incubation was terminated by 1-min centrifugation at 10,000 rpm in a microcentrifuge to separate a supernatant fraction from the platelet pellet. Lipids from the supernatant and the cell pellet were extracted by either the protocol described by Yatomi et al. (10Yatomi Y. Ohmori T. Rile G. Kazama F. Okamoto H. Sano T. Satoh K. Kume S. Tigyi G. Igarashi Y. Ozaki Y. Blood. 2000; 96: 3431-3438Crossref PubMed Google Scholar) or that of Eicholtzet al. (3Eicholtz T. Jalink K. Fahrenfort I. Moolenaar W.H. Biochem. J. 1993; 291: 677-680Crossref PubMed Scopus (577) Google Scholar). One of the duplicate samples was extracted with a stepwise addition of 3 ml of ice-cold chloroform:methanol (1:2), followed by 2 ml of chloroform, 2 ml of 1 m KCl, and 40 μl of 7 n NH4OH, with vigorous shaking after each step (10Yatomi Y. Ohmori T. Rile G. Kazama F. Okamoto H. Sano T. Satoh K. Kume S. Tigyi G. Igarashi Y. Ozaki Y. Blood. 2000; 96: 3431-3438Crossref PubMed Google Scholar). After centrifugation for 5 min at 3000 ×g, the upper phase was supplemented with 3 ml of chloroform and 160 μl of concentrated HCl, and the phases were separated by centrifugation. The lower phase was transferred to a new tube, and the solvent was evaporated in vacuo. The second of each duplicate sample was extracted by the method of Eicholtz et al. (3Eicholtz T. Jalink K. Fahrenfort I. Moolenaar W.H. Biochem. J. 1993; 291: 677-680Crossref PubMed Scopus (577) Google Scholar) using acidic butanol. The extracted phospholipids were analyzed by either one-dimensional TLC (1DTLC) or 2DTLC, using a variety of solvent mixtures described under “Results.” Autoradiography was performed using X-Omat AR-5 (Kodak, Rochester, NY) or BAS-MS2025 (FUJI Corp.) X-ray films after overnight exposure or by a Model FLA2000 phosphorimaging system (FUJI Corp.) Platelets suspended in buffer A were incubated in the presence or absence of 2 mm CaCl2 and 1 unit/ml thrombin for 1 h at 37 °C. A low speed supernatant (Sup1) was separated from the platelets by centrifugation at 104 ×g for 2 min, and the platelet pellet was designated P1. In several experiments, Sup1, which contains microvesicles shed by activated platelets (11Heijnen H.F.G. Schiel A.E. Fijnheer R. Geuze H.J. Sixma J.J. Blood. 1999; 94: 3791-3799Crossref PubMed Google Scholar), was centrifuged at 105 ×g for 45 min to obtain a particle-free supernatant (Sup2) and the microvesicle pellet (P2). The four fractions served as the source of the enzymes used to examine the metabolism of NBD-labeled phospholipids. Micelles containing 5 nmol ofsn2-NBD-labeled PC, PE, PS, or PA in buffer (500 μm octyl-glucoside, 400 μm NaCl, 67 mm HEPES; pH 7.4) were mixed with 20 μl of supernatant or pellet fraction in a 500-μl final volume for up to 6 h at 37 °C, essentially as described by Ella et al. (12Ella K.M. Meier G.P. Bradshaw C.D. Huffman K.M. Spivey E.C. Meier K.E. Anal. Biochem. 1994; 218: 136-142Crossref PubMed Scopus (29) Google Scholar). The incubation mixture was centrifuged for 5 min at 104 ×g, and lipids were extracted from the supernatant after acidification with 50 μl of 0.2 n HCl, using 275 μl of water-saturated butanol. The butanol phase was obtained after brief centrifugation and evaporated in vacuo before TLC analysis of the metabolic products. One milligram of sn2-NBD-labeled PC, PE, or PS was hydrolyzed with 8000 units of Rhizopus PLA1 in 0.1m borate buffer (pH 6.5) in the presence of 3 mg/ml sodium deoxycholate, 4 mg/ml BSA, and 5 mm CaCl2. The reaction mixture was shaken for 6 h, and lipids were extracted according to the method of Kates (13Kates M. Burdon R.H. van Knippenberg P.H. Techniques in Lipidology. 2nd Ed. Elsevier, Amsterdam, New York, Oxford1986: 404-411Google Scholar). To promote acyl migration, lipids were incubated at pH 8.5 in 0.1 m sodium tetraborate for at least 2 h. The sn2- and sn1-NBD-labeled lysolipids were separated using 1DTLC. Fluorescent spots corresponding to sn1-NBD-lysolipid were scraped, eluted with methanol, dried under nitrogen, and stored at −20 °C. Acyl-chain position was confirmed by resistance to PLA2 cleavage (data not shown). In some experiments, sn1-NBD-labeled LPC, LPE, and LPS were used to monitor their metabolism in the supernatant and pellet fractions as described forsn2-labeled phospholipids above. Cubital venous blood was mixed with a 0.01 volume of heparin solution to a final concentration of 10 IU/ml. Platelet-rich plasma was obtained by centrifugation at 112 × g for 15 min and immediately mixed with 1 mm prostaglandin E1. Platelets were removed by centrifugation at 104 × g for 10 min, and the supernatant was collected. Blood taken without anticoagulant from the same donors was either centrifuged immediately at 10,000 × g for 2 min to yield a supernatant designated as activated plasma or allowed to clot at 37 °C for 1 h. Serum was collected by centrifugation at 1100 ×g for 10 min, followed by recentrifugation at 10,000 ×g for 2 min. Serum and plasma were filtered through a 0.2-μm membrane filter, aliquoted, and stored at −20 °C. For time-course studies, serum or plasma (100 μl) was incubated with 20 nmol of substrate containing a 1:99 ratio of 1-NBD-labeled to unlabeled lysophospholipid. The reaction was terminated by adding a 0.3 volume of 0.1 m citric acid and 5.4 volumes of chloroform:methanol (2:1). After vigorous shaking for 5 min, samples were centrifuged at 1400 × g for 5 min. The upper phase was re-extracted with chloroform:methanol (2:1, v/v), and the lower phases were pooled, dried, and assayed for the generation of NBD-labeled LPA by TLC. For experiments to determine the effects of dilution of plasma, activated plasma, and serum on the conversion ofsn1-NBD-LPC or LPS to LPA, the amount and composition of lipids present in the reaction mixture had to be kept constant while the amount of serum protein was varied. To achieve this in, for example, a 100-μl reaction volume in the case of a 1:1 dilution, lipids were extracted twice from 50 μl of plasma (or serum) and the extract was dried under N2 gas. The extracted lipids were resuspended in 50 μl of phosphate-buffered saline, and 0.2 nmol of sn1-NBD-lysophospholipid was added. For the final reaction mixture, 50 μl plasma or serum was mixed with 50 μl of phosphate-buffered saline buffer containing the lipids extracted from plasma (serum) with the NBD-lysophospholipid tracer and incubated for 3 h at 37 °C, essentially yielding a 1:1 dilution for plasma protein, whereas the total amount of lipid remained equivalent to that present in 100 μl of undiluted plasma. For a 1:2 dilution, the amount of plasma extracted was adjusted to 67 μl, for a 1:4 dilution to 80 μl, etc., whereas the volume of plasma used in the 100-μl reaction mixture was decreased to 33 and 20 μl, respectively. At the end of the incubation, the reaction was terminated by the addition of 35 μl of 0.1 m citrate and 540 μl of chloroform:methanol (2:1, v:v), and lipids were extracted twice. The lipid extract was taken up in 20 μl of chloroform:methanol (2:1, v:v) and applied in toto to a single lane of a TLC plate. The developed TLC plate was scanned using a Model FLA2000 scanner (FUJI Corp.), and the fluorescence intensity at the position of the authentic LPA standard was quantified. The extraction of lipids and the quantification of the molecular species of 16:0, 18:0, 18:1, 18:2, and 20:4 LPA, which constitute over 90% of the total LPA detectable in plasma, were done as previously described (7Baker D.L. Desiderio M.D. Miller D.D. Tolley B. Tigyi G. Anal. Biochem. 2001; 292: 287-295Crossref PubMed Scopus (200) Google Scholar). Purified platelets (1.7–1.9 × 109/ml) were taken up in 500 μl of human plasma or in buffer A containing 1% fatty acid-free BSA. Platelets used for negative control received an additional 5 mm EGTA and 1 μm staurosporine to block activation and spontaneous release. Another set of platelets was stimulated to secrete by using 1 unit/ml thrombin in the presence of 5 mm EGTA. The third set of samples was treated with 1 unit/ml thrombin in the presence of 2 mm CaCl2. The treatments were allowed to proceed for 15 min at 37 °C. To separate platelets from the supernatant, centrifugation at 104 × g was performed for 5 min, and the specimens were snap-frozen in liquid nitrogen and stored at −80 °C until lipid extraction. Electrospray ionization LC-MS was performed on a Bruker Esquire ion trap fitted with an Econosphere 3-μm 50- × 4.6-mm silica column (Alltech Associates, Deerfield, IL). Compounds were eluted using a mobile phase of chloroform:methanol:water:28% ammonium hydroxide (250:100:15:0.3, v/v) at 0.5 ml/min. The source was maintained at 250 °C with a drying gas flow of 10 liters/h; data were collected in the negative ion mode from 100 to 1000 m/z at 13,000 atomic mass units/s. Masses corresponding to the molecular anions [M-H]− for each of the LPA species were plotted versus time, and the peak areas were integrated. The procedures for the collection of and voltage clamp recording from oocytes have been described elsewhere (14Tigyi G.J. Liliom K. Fischer D.J. Guo Z. Laychock S.G. Rubin R.P. Phospholipid Growth Factors: Identification and Mechanism of Action. Lipid Second Messengers. CRC Press, Boca Raton1999: 51-81Google Scholar). The biological fluids collected from three healthy donors were directly diluted 100-fold with Ringer's solution, and peak current amplitudes were measured. Student's t test and repeated analysis of variance were used to test the null hypotheses, and data were considered statistically significantly different atp < 0.05. The first objective of this study was to clarify whether LPA and Sph1P are biosynthetically labeled, i.e. constitutively generated in nonstimulated platelets. For this, we extracted phospholipids from platelets following incubation for 2 h with [32P]orthophosphate. This protocol is essentially the same as that originally used by Eicholtz et al. (3Eicholtz T. Jalink K. Fahrenfort I. Moolenaar W.H. Biochem. J. 1993; 291: 677-680Crossref PubMed Scopus (577) Google Scholar). We compared the acidic butanol extraction protocol used by these authors with that of Yatomi et al. (10Yatomi Y. Ohmori T. Rile G. Kazama F. Okamoto H. Sano T. Satoh K. Kume S. Tigyi G. Igarashi Y. Ozaki Y. Blood. 2000; 96: 3431-3438Crossref PubMed Google Scholar) to determine if a difference between the extraction procedures could have caused the lack of Sph1P detection by Eicholtz et al. The extracts were analyzed by 2DTLC using chloroform:methanol:28% ammonium hydroxide (12:12:1) in the first dimension, taken from the report by Eicholtzet al., and butanol:glacial acetic acid:water (3:1:1) in the second dimension (Fig. 1). The latter solvent has been shown to resolve LPA and Sph1P (10Yatomi Y. Ohmori T. Rile G. Kazama F. Okamoto H. Sano T. Satoh K. Kume S. Tigyi G. Igarashi Y. Ozaki Y. Blood. 2000; 96: 3431-3438Crossref PubMed Google Scholar). At the position of the LPA standard, autoradiographs of lipid extracts derived from supernatants and pellets of nonstimulated platelets prepared by both extraction protocols showed trace amounts of radiolabeled LPA, detectable only by phosphorimaging. In contrast, a clearly labeled spot, comigrating with the Sph1P standard, was detectable in these samples, suggesting that a platelet Sph1P pool is biosynthetically active in nonstimulated platelets and that under these conditions a small fraction of this pool was released into the supernatant in the absence of stimulation with exogenous thrombin. When thrombin was added in the presence of 2 mm extracellular Ca2+, a robust release of Sph1P was detected in the supernatant (Fig.1D). Under these conditions, labeled LPA became detectable in the platelet pellet and supernatant (Fig. 1, C and D). With regard to the production of Sph1P and LPA, identical findings were obtained with both methods of extraction (data not shown). These results confirmed that Sph1P and LPA are both present in the medium of thrombin-stimulated platelets when a physiological concentration of Ca2+ is present. To determine the time course of Sph1P and LPA production from isolated platelets stimulated to secrete and/or to aggregate, we utilized the 1DTLC system of Yatomi et al. (10Yatomi Y. Ohmori T. Rile G. Kazama F. Okamoto H. Sano T. Satoh K. Kume S. Tigyi G. Igarashi Y. Ozaki Y. Blood. 2000; 96: 3431-3438Crossref PubMed Google Scholar) and quantified the radioactivity incorporated into these lipids in the supernatant and platelet pellets (Fig. 2). Measurements of LPA and Sph1P present in the supernatant are a reliable measure of these lipids, because 2DTLC analysis (see Fig. 1, B and D) revealed no other 32P-labeled lipids with similar mobility. However, this 1DTLC system could not be applied to the quantification of LPA and Sph1P present in the platelet pellet, because other unidentified lipids were also detected by 2DTLC with identical mobilities (Fig. 1, A and C). Stimulation with thrombin in the absence of Ca2+ and in the presence of 5 mm EGTA activates secretion from platelets, but not platelet aggregation, and prevents the action of Ca2+-dependent enzymes released into the medium. We performed lipid extractions under these conditions and found a substantial increase in the amount of Sph1P in the supernatant (Fig.2, A and B). In contrast, only a trace of labeled LPA was detectable in the supernatant and the platelet pellets. The findings were again identical for both methods of extraction (Fig. 2, compare A and B). In contrast, when thrombin stimulation was performed in the presence of physiological concentrations of Ca2+, the amount of released Sph1P did not increase substantially over that for platelets stimulated to secrete, whereas LPA became extensively labeled in the platelet pellet and supernatant. Quantitative analysis of the phosphorimaging signals did not detect any increase over unstimulated levels in biosynthetic labeling of LPA in response to a secretory stimulus (Fig.2C). In contrast, thrombin-stimulated secretion increased Sph1P release ∼4-fold (Fig. 2D). When thrombin stimulation was elicited in the presence of Ca2+, a rapid and intense labeling of PA and LPA was detected in the platelets (Fig. 2, A and B). This was accompanied by the release of LPA into the supernatant, where ∼27% of the total label was recovered after a 10-min stimulation. Under these conditions, Sph1P release did not significantly exceed that observed after a secretory stimulus (Fig. 2D). Approximately 25% of the total Sph1P label was recovered from the supernatant. The time course of labeled Sph1P and LPA accumulation in the medium was followed for up to 3 h under conditions that permitted aggregation (Fig. 3). Sph1P reached its maximal release within 15 min and slowly decreased to a steady-state level between 90 and 180 min. LPA production in the supernatant showed a steady increase up to 60 min; thereafter it decreased slightly to reach a steady-state level up to 180 min. Several investigators have shown a rapid increase in the labeling of PA in thrombin-stimulated platelets that reached a maximum at 5 min (15Lloyd J.V. Nishizawa E.E. Hladar J. Mustard J.F. Br. J. Haematol. 1972; 23: 571-585Crossref PubMed Scopus (49) Google Scholar, 16Lloyd J.V. Mustard J.F. Br. J. Haematol. 1974; 26: 243-253Crossref PubMed Scopus (61) Google Scholar, 17Mauco G. Chap H. Simon M.-F. Douste-Blazy L. Biochemie. 1978; 60: 653-661Crossref PubMed Scopus (125) Google Scholar, 18Lapetina E. Cuatrecasas P. Biochim. Biophys. Acta. 1979; 573: 394-402Crossref PubMed Scopus (187) Google Scholar). Lapetina et al.(5Lapetina E. Billah M. Cuatrecasas P. J. Biol. Chem. 1981; 256: 11984-11987Abstract Full Text PDF PubMed Google Scholar, 19Lapetina E. Billah M. Cuatrecasas P. J. Biol. Chem. 1981; 256: 5037-5040Abstract Full Text PDF PubMed Google Scholar, 20Lapetina E. Billah M.M. Cuatrecasas P. Nature. 1981; 292: 367-369Crossref PubMed Scopus (157) Google Scholar) reported that this early rise in PA is followed by a rise in labeled LPA and hence proposed that the labeled PA pool was converted to LPA via Ca2+-activated intracellular PLA2 enzymes. Our results confirm a peak of PA labeling at around 5 min; beyond this point, however, we could not detect a correlation between PA and LPA labeling. Labeled LPA continued to increase in the supernatant, reaching a peak at 60 min, whereas no labeled PA was detectable after 15 min. Biosynthetic labeling of platelet phospholipids yields no quantitative information about the individual molecular species of LPA generated. To circumvent this deficiency we applied SID-LC-MS to determine the concentration of five molecular species of LPA present in platelet pellets and supernatant fractions from nonstimulated and thrombin-stimulated platelets obtained from five healthy donors (Table I). The isolated platelets were divided into two groups, the first of which was incubated in buffer A containing 0.5% BSA to capture released LPA. A second sample set was incubated in human plasma to mimic in vivo conditions. LPA was not found in the supernatant of nonstimulated platelets, whereas its concentration was 190 ± 40 nm in the presence of plasma after 15 min incubation at 37 °C. This latter concentration corresponds to the basal LPA level in plasma (7Baker D.L. Desiderio M.D. Miller D.D. Tolley B. Tigyi G. Anal. Biochem. 2001; 292: 287-295Crossref PubMed Scopus (200) Google Scholar). The corresponding platelet pellets had LPA concentrations of 130 ± 20 and 100 ± 40 nm in the presence of buffer and plasma, respectively. When platelets were stimulated with thrombin in the presence of 5 mm EGTA, no significant change in LPA content was detected in either the platelet pellet or supernatant as compared with nonstimulated controls. This reaffirms our findings obtained from the biosynthetic labeling experiments (Fig. 2). When platelets were stimulated with thrombin in the presence of 2 mmCa2+, there was a substantial increase in LPA concentration in the supernatant. In the presence of buffer A, the combined concentration of the five LPA species increased to 670 ± 140 nm, whereas in the presence of plasma it increased to 560 ± 300 nm. The increase of LPA in the supernatant fraction was accompanied by a slight decrease in LPA concentration in the platelet pellet that amounted to 50 ± 20 nm in buffer A and with no change (30 ± 40 nm) in plasma (Table I). This observation indicates that platelet activation resulted in a marginal change in the labeling of the intracellular pool of LPA. The distribution of acyl species of LPA determined in platelet pellets and supernatants showed striking differences depending on the presence or absence of plasma in the platelet preparation. The rank order of molecular species of LPA detected in the supernatants from platelets stimulated with thrombin and Ca2+ in buffer A was 18:0 ≫ 20:4 > 18:1 > 16:0 ≥ 18:2. In contrast, when platelets were incubated with plasma and stimulated with thrombin and Ca2+, the rank order of the LPA species in the supernatant was 20:4 > 18:2 = 18:0 > 16:0 = 18:1. The acyl species distribution found in the platelet pellet was 18:0 > 20:4 > 16:0 = 18:2 ≥ 18:1 in the absence of plasma, whereas it was 20:4 > 16:0 >