The phospholipase A1 activity of lysophospholipase A-I links platelet activation to LPA production during blood coagulation
2011; Elsevier BV; Volume: 52; Issue: 5 Linguagem: Inglês
10.1194/jlr.m013326
ISSN1539-7262
AutoresAlyssa L. Bolen, Anjaparavanda P. Naren, Sunitha Yarlagadda, Šárka Beranová-Giorgianni, Li Chen, Derek D. Norman, Daniel L. Baker, Meng M. Rowland, Michael D. Best, Takamitsu Sano, Tamotsu Tsukahara, Károly Liliom, Yasuyuki Igarashi, Gábor Tigyi,
Tópico(s)Erythrocyte Function and Pathophysiology
ResumoPlatelet activation initiates an upsurge in polyunsaturated (18:2 and 20:4) lysophosphatidic acid (LPA) production. The biochemical pathway(s) responsible for LPA production during blood clotting are not yet fully understood. Here we describe the purification of a phospholipase A1 (PLA1) from thrombin-activated human platelets using sequential chromatographic steps followed by fluorophosphonate (FP)-biotin affinity labeling and proteomics characterization that identified acyl-protein thioesterase 1 (APT1), also known as lysophospholipase A-I (LYPLA-I; accession code O75608) as a novel PLA1. Addition of this recombinant PLA1 significantly increased the production of sn-2-esterified polyunsaturated LPCs and the corresponding LPAs in plasma. We examined the regioisomeric preference of lysophospholipase D/autotaxin (ATX), which is the subsequent step in LPA production. To prevent acyl migration, ether-linked regioisomers of oleyl-sn-glycero-3-phosphocholine (lyso-PAF) were synthesized. ATX preferred the sn-1 to the sn-2 regioisomer of lyso-PAF. We propose the following LPA production pathway in blood: 1) Activated platelets release PLA1; 2) PLA1 generates a pool of sn-2 lysophospholipids; 3) These newly generated sn-2 lysophospholipids undergo acyl migration to yield sn-1 lysophospholipids, which are the preferred substrates of ATX; and 4) ATX cleaves the sn-1 lysophospholipids to generate sn-1 LPA species containing predominantly 18:2 and 20:4 fatty acids. Platelet activation initiates an upsurge in polyunsaturated (18:2 and 20:4) lysophosphatidic acid (LPA) production. The biochemical pathway(s) responsible for LPA production during blood clotting are not yet fully understood. Here we describe the purification of a phospholipase A1 (PLA1) from thrombin-activated human platelets using sequential chromatographic steps followed by fluorophosphonate (FP)-biotin affinity labeling and proteomics characterization that identified acyl-protein thioesterase 1 (APT1), also known as lysophospholipase A-I (LYPLA-I; accession code O75608) as a novel PLA1. Addition of this recombinant PLA1 significantly increased the production of sn-2-esterified polyunsaturated LPCs and the corresponding LPAs in plasma. We examined the regioisomeric preference of lysophospholipase D/autotaxin (ATX), which is the subsequent step in LPA production. To prevent acyl migration, ether-linked regioisomers of oleyl-sn-glycero-3-phosphocholine (lyso-PAF) were synthesized. ATX preferred the sn-1 to the sn-2 regioisomer of lyso-PAF. We propose the following LPA production pathway in blood: 1) Activated platelets release PLA1; 2) PLA1 generates a pool of sn-2 lysophospholipids; 3) These newly generated sn-2 lysophospholipids undergo acyl migration to yield sn-1 lysophospholipids, which are the preferred substrates of ATX; and 4) ATX cleaves the sn-1 lysophospholipids to generate sn-1 LPA species containing predominantly 18:2 and 20:4 fatty acids. Lysophosphatidic acid (LPA) is a multifunctional phospholipid mediator and second messenger responsible for a wide variety of cellular responses (1Tigyi G. Aiming drug discovery at lysophosphatidic acid targets.Br. J. Pharmacol. 2010; 161: 241-270Crossref PubMed Scopus (137) Google Scholar, 2Mills G.B. Moolenaar W.H. The emerging role of lysophosphatidic acid in cancer.Nat. Rev. Cancer. 2003; 3: 582-591Crossref PubMed Scopus (942) Google Scholar, 3Tigyi G. Parrill A.L. Molecular mechanisms of lysophosphatidic acid action.Prog. 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The rank order of the acyl species of LPA in normal human plasma is 18:2 > 18:1 > 18:0 > 16:0 > 20:4. In contrast, the rank order of the LPA acyl species in serum changes to 20:4 > 18:2 > 16:0 > 18:1 > 18:0 (19Sano T. Baker D. Virag T. Wada A. Yatomi Y. Kobayashi T. Igarashi Y. Tigyi G. Multiple mechanisms linked to platelet activation result in lysophosphatidic acid and sphingosine 1-phosphate generation in blood.J. Biol. Chem. 2002; 277: 21197-21206Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar). The various acyl species of LPA also have differing ligand properties at target LPA GPCR (28Fujiwara Y. Sardar V. Tokumura A. Baker D. Murakami-Murofushi K. Parrill A. Tigyi G. Identification of residues responsible for ligand recognition and regioisomeric selectivity of lysophosphatidic acid receptors expressed in mammalian cells.J. Biol. Chem. 2005; 280: 35038-35050Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). In addition to the differences in carbon chain length and degree of unsaturation, the LPA3 and P2RY5 (LPA6) receptors show a preference for the sn-2 over the sn-1 acyl regioisomer of LPA (29Bandoh K. Aoki J. Taira A. Tsujimoto M. Arai H. Inoue K. Lysophosphatidic acid (LPA) receptors of the EDG family are differentially activated by LPA species. Structure-activity relationship of cloned LPA receptors.FEBS Lett. 2000; 478: 159-165Crossref PubMed Scopus (223) Google Scholar, 30Yanagida K. Masago K. Nakanishi H. Kihara Y. Hamano F. Tajima Y. Taguchi R. Shimizu T. Ishii S. Identification and characterization of a novel lysophosphatidic acid receptor, p2y5/LPA6.J. Biol. Chem. 2009; 284: 17731-17741Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar). However, the sn-2 LPA regioisomer is relatively unstable. At neutral pH, acyl migration begins immediately to yield the more thermodynamically stable sn-1 regioisomer; an equilibrium ratio of 9:1 occurs between the two forms (31Pluckthun A. Dennis E.A. Acyl and phosphoryl migration in lysophospholipids: importance in phospholipid synthesis and phospholipase specificity.Biochemistry. 1982; 21: 1743-1750Crossref PubMed Scopus (220) Google Scholar). The linoleoyl (18:2) and arachidonoyl (20:4) species make up 84% of the LPA found in serum (19Sano T. Baker D. Virag T. Wada A. Yatomi Y. Kobayashi T. Igarashi Y. Tigyi G. Multiple mechanisms linked to platelet activation result in lysophosphatidic acid and sphingosine 1-phosphate generation in blood.J. Biol. Chem. 2002; 277: 21197-21206Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar). The mechanism behind the enrichment and increase in polyunsaturated LPA species upon blood coagulation is still unknown. Because plasma phospholipids containing 18:2 and 20:4 fatty acids are almost exclusively esterified to the sn-2 glycerol carbon, we hypothesized that LPA in serum enriched in these fatty acyl species must be generated by the action of a PLA1 enzyme (19Sano T. Baker D. Virag T. Wada A. Yatomi Y. Kobayashi T. Igarashi Y. Tigyi G. Multiple mechanisms linked to platelet activation result in lysophosphatidic acid and sphingosine 1-phosphate generation in blood.J. Biol. Chem. 2002; 277: 21197-21206Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar). This hypothesis implies that the nascent sn-2 LPL generated by PLA1 will either be rapidly cleaved by ATX and then undergo acyl migration or, alternatively, undergo acyl migration prior to headgroup cleavage. Distinguishing between the latter two possibilities is challenging due to the short half-life of the sn-2 LPLs that precludes the use of classical biochemical separation and analytical techniques. In the present study, we sought to identify the putative PLA1 enzymes released from activated platelets that are responsible for the generation of polyunsaturated LPL. Starting with the supernatants from thrombin-activated human platelets, we isolated lysophospholipase A1/acyl protein thioesterase 1 (LYPLA-I/APT1) using a series of chromatographic steps and fluorophosphonate (FP)-biotin affinity labeling-based proteomics. LYPLA-I/APT1 transcripts were shown to be abundantly expressed in platelets and in megakaryocytes. LYPLA-I/APT1 was found to posses PLA1 activity against plasma phospholipids, did not degrade LPA, and increased LPA production when added to plasma through the production of a pool of LPL, which is further cleaved by ATX. Fluorescently labeled 1-oleoyl-2-{12-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]lauroyl}-sn-glycero-3-phosphoserine (NBD-PS 18:1-12:0), 1-oleoyl-2-[12-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]lauroyl]sn-glycero-3-phosphocholine (NBD-PC 18:1-12:0), dioleoyl phosphatidylserine (PS), linolenoyl phosphatidylcholine (PC), oleoyl LPA, LPC 17:0, and LPA 17:0 were purchased from Avanti Polar Lipids, Inc. (Alabaster, AL). 1-Oleyl-sn-glycero-3-phosphocholine (lyso-PAF 18:1) was purchased from Bachem (Torrance, CA). Fluorophosphonate-rhodamine (FP-RH) and fluorophosphonate-biotin (FP-biotin) were a gift from Dr. Ben Cravatt (Scripps Institute, La Jolla, CA). Freshly expired apheresis platelets were provided by the Regional Medical Center (Memphis, TN), Methodist University Hospital (Memphis, TN), or purchased from Key Biologics, Inc. (Memphis, TN). PGE1 and thrombin were obtained from Sigma-Aldrich (St. Louis, MO). Trizol and Superscript III OneStep RT-PCR were purchased from Invitrogen (Carlsbad, CA). The procedures detailed below were reviewed and approved by the Institutional Review Board of the University of Tennessee Health Science Center. Small-scale batches of platelet-rich plasma (PRP) were prepared by adding 3.6 ml acidic citrate dextrose (ACD, 0.8% citric acid, 2.2% sodium-citrate, 2.45% glucose) to 20 ml of cubital venous blood drawn from a volunteer donor and centrifuged at 180 g for 15min. The top layer of PRP (∼10 ml) was transferred to a new tube. For large-scale purifications, units of freshly expired apheresis platelets in ACD were obtained (approximately 60 ml per unit). Then 10 ml of the PRP from the small-scale purification or 10 ml of the aphersis platelets were diluted with 34 ml buffer A (138 mM NaCl, 3.3 mM NaH2PO4, 2.9 mM KCl, 1 mM MgCl2, 20 mM HEPES, 1 mg/ml glucose, pH 7.5), and 1 µM PGE1 was added. The diluted PRP was centrifuged at 1400 g at room temperature for 15 min. The supernatant was discarded, and the pellet containing the platelets was reconstituted in 2 ml buffer A, and 2 mM Ca2+ was added. The platelets were activated using 1 U thrombin per 1 ml sample at 37°C for 20 min, and the aggregate formed was centrifuged at 9,300 g for 5 min to yield supernatant 1 (Sup1). The Sup1 was then centrifuged again at 100,000 g for 45 min to remove platelet microvesicles. This preparation was designated as supernatant 2 (Sup2). PLA1 activity was measured by determining the amount of lysophosphatidylcholine (LPC) or lysophosphatidylserine (LPS) generated after incubation of sample with phosphatidylcholine (PC) or phosphatidylserine (PS). NBD-PC or NBD-PS (45 ng to 2 µg) was incubated in 10 mM Tris (pH 7.5) or Sup2 (pH 7.5) at 37°C for 1 min to 1 h with or without an equal weight of BSA in water. For quantification, 900 ng, 90 ng, and 9 ng of the fluorescent substrates were incubated without enzyme for construction of a standard curve by plotting fluorescence intensity as a function of substrate mass. Water-saturated butanol (BuOH) was added (30-120 µl) to stop the reaction and extract the lipids. Samples (10 µl) were spotted on Silica Gel 60 TLC plates. The plates were then developed with solvent A consisting of chloroform:methanol:ammonium hydroxide (V/V/V, 6:4:1) for NBD-PS, and solvent B consisting of chloroform:methanol:28% ammonium hydroxide:water (V/V/V/V, 50:40:8:2) for NBD-PC. The products were visualized using a Fotodyne imager (Hartland, WI) and quantified by the TotalLab100 software. The amount of the product generated was determined by interpolation from the standard curve. The Rf values for the various lipids were as follows: NBD-PC, 0.54; NBD-LPC, 0.38; NBD-FA, 0.77; NBD-PS, 0.45; NBD-LPS, 0.33; and NBD-FA, 0.67. For unlabeled substrates, 10 µg PC, PS, or LPA was digested at 37°C for 1 h in the presence of 10 µg BSA. LPC 17:0 and LPA 17:0 (500-2000 ng in DMSO) internal standards were added prior to addition of 60 µl BuOH. The samples were vortexed for 1 min, and the BuOH phase was isolated by centrifugation at 14000 g for 1 min and dried under argon. The lipid extract was reconstituted in 30 µl methanol:acetonitrile:isopropanol:water (V/V/V/V, 1:1:1:1). LPC and LPA concentrations were determined by LC-MS/MS using an Applied Biosystems Sciex ABI 4000 QTRAP tandem mass spectrometer (Foster City, CA) equipped with a Turboionspray™ interface, a Shimadazu LC-10ADvp HLPC pump (Columbia, MD) with a Leap HTS PAL autosampler (Carrboro, NC). Samples (10 µl) were injected onto a Tosoh TSK-ODS-100Z silica column (150 mm × 2 mm; 5 μm particle size) with a solvent consisting of methanol/water (V/V, 95:5) and 5 mM ammonium formate using an isocratic flow rate of 0.22ml/min (32Tokumura A. Carbone L.D. Yoshioka Y. Morishige J. Kikuchi M. Postlethwaite A. Watsky M.A. Elevated serum levels of arachidonoyl-lysophosphatidic acid and sphingosine 1-phosphate in systemic sclerosis.Int. J. Med. Sci. 2009; 6: 168-176Crossref PubMed Scopus (110) Google Scholar). The spectra were processed using Analyst software, version 1.5. The molecular species of LPC and LPA were analyzed by multiple reaction monitoring (MRM) in positive ion mode for LPC and negative ion mode for LPA. The Q3 (product ion) was set at m/z 184.0 for LPC and 153.0 for LPA (glycerophosphate moiety) (32Tokumura A. Carbone L.D. Yoshioka Y. Morishige J. Kikuchi M. Postlethwaite A. Watsky M.A. Elevated serum levels of arachidonoyl-lysophosphatidic acid and sphingosine 1-phosphate in systemic sclerosis.Int. J. Med. Sci. 2009; 6: 168-176Crossref PubMed Scopus (110) Google Scholar). Q1 was set for the neutral molecular ion for all LPLs. Quantification was done by calculating the ratio of peak area to that of the appropriate LPC/LPA 17:0 internal standard and interpolated from the respective standard curve. PLA1 was partially purified using sequential chromatography on an AKTA FPLC system (GE Biosciences, Piscataway, NJ) by loading 330 mg (75 mg/ml) Sup2 to a Q Sepharose Fast Flow ion exchange chromatography column (GE Biosciences, 2 cm × 10 cm). The column was eluted with a NaCl gradient (0-1 M) at a 1 ml/min flow rate over 20 min with buffer C (3.3 mM NaH2PO4, 2.9 mM KCl, 1 mM MgCl26H2O, 20 mM HEPES, pH 7.5). The PLA1 active fractions were combined, and 5 ml (∼30 mg) was loaded onto a Butyl-Sepharose hydrophobic interaction chromatography column (GE Biosciences, 0.7 cm × 2.5 cm, 20 mg/ml medium). The column was eluted using a gradient from 1.7 M to 0 M (NH4)SO4 over 20 min in 0.05 M Na2HPO4 buffer (pH 7.6). The active fractions were combined, and 5 ml (∼2.5 mg) was loaded onto a HiTrap Blue affinity chromatography column (GE Biosciences, 0.7 cm × 2.5 cm). The column was eluted by increasing (NH4)SO4 from 0 M to 1.7 M over 20 min in 0.05 M Na2HPO4 buffer (pH 7.6). An amount of 3 ml fractions were collected at each step. An amount of 100 µl of each fraction was used for PLA1 activity measurement using 1 µg NBD-PS substrate at 37°C for 2 h. An amount of 120 µl water-saturated BuOH was added to stop the reaction and extract the lipids. The samples were vortexed and centrifuged for 1 min at 14,000 g. Then 10 µl of the BuOH phase was spotted to a Silica Gel 60 TLC plate. The plate was developed with solvent A, and the products were visualized. The fractions with PLA1 activity from each chromatographic step were combined, activity was determined as described, and protein concentration was determined by BCA protein assay (Pierce, Rockford, IL) following the manufacturer's protocol. An amount of 50 µg protein of the PLA1 active Butyl-Sepharose pooled fractions (∼100 µl) and 0.9 µg NBD-PS substrate was incubated at 37°C for 3 h with 0.03, 0.1, 0.3, 1, 3, 10, and 30 µM FP-RH. Then 60 µl water-saturated BuOH was used to stop the reaction and extract the lipid products. After mixing, the samples were centrifuged for 1 min at 14,000 g. Then 10 µl of the BuOH phase was spotted to a Silica Gel 60 TLC plate and developed with solvent A. The products were visualized using a Photodyne imager and quantified using standards run alongside the samples on the same TLC plate. The activity-based proteomic probe, FP-biotin, was used to label all serine hydrolases in the PLA1-active HiTrap Blue fractions. An active pool of HiTrap Blue chromatography fractions was concentrated using an Amicon concentrator (Millipore, Billerica, MA) with a 5 kDa molecular weight cutoff. An amount of 1 mg protein (1 mg/ml) was incubated for 2 h with a final concentration of 5 µM FP-biotin. Streptavidin beads (Pierce, Rockford, IL) were prewashed three-times with binding buffer (0.1 M sodium phosphate, 0.15 M NaCl, pH 7.0) followed by centrifugation (3000 g for 1min), and the supernatant was discarded. The protein complex was added to 50 µl resin and incubated with mixing for 1 h at room temperature. Streptavidin-bound FP-biotinylated proteins were washed with binding buffer in the presence of 4 M urea, 0.1% SDS (W/V), and 0.2% TritonX-100 (V/V) (pH 7.2), and then centrifuged for 1 min at 3000 g. The supernatant was removed, and the wash procedure was repeated four times. The beads were reconstituted in 1 ml 50 mM TRIS-HCl (pH 7.2), centrifuged for 4,min at 10,000 g, and the supernatant was decanted. An amount of 200 µl 50 mM TRIS-HCl (pH 7.2) was added to reconstitute the final pellet. The bead-bound FP-biotin-labeled proteins were first reduced with 5 mM dithiothreitol for 30 min at room temperature and alkylated with 10 mM iodoacetamide at room temperature in the dark for 30 min. The sample was centrifuged for 4 min at 10,000 g, and then 100 µl of supernatant was removed. A total of 300 µl 50 mM TRIS (pH 7.2) buffer and 1.5 µl of 0.5µg/µl trypsin (sequencing grade, Sigma-Aldrich, St. Louis, MO) was added and incubated at 37°C for 12 h. The sample was centrifuged for 4 min at 10,000 g, and the supernatant containing the peptide digest was transferred to a new tube; formic acid was added to a final concentration of 5% (V/V). The volume of the peptide sample was reduced to 40 μl in a vacuum centrifuge. The peptides were desalted with a Zip Tip C18 microcolumn (Millipore, Billerica, MA) using the procedure recommended by the manufacturer. Peptides were eluted using 4 μl of 50% acetonitrile/0.1% trifluoroacetic acid (V/V). Four microliters of water/acetic acid (0.5%) were added to the sample. LC-MS/MS experiments were performed on an LTQ linear ion trap mass spectrometer (Thermo Scientific, Waltham, MA) coupled to a nanoflow LC system (Dionex, Sunnyvale, CA). The peptide sample was injected onto a fused-silica capillary column/spray needle (15 cm length, 75 µm ID; New Objective, Woburn, MA) packed in-house with C18 stationary phase (Michrom Bioresources, Auburn, CA). The peptides were separated with a 90 min gradient from 0% to 90% of mobile phase B. The composition of mobile phase B was 90% MeOH/10%water/0.05% HCOOH; the composition of mobile phase A was 2% MeOH/98% water/0.05%HCOOH. Mass spectrometric data acquisition was performed in the data-dependent mode; one cycle encompassed a full-range MS scan followed by seven MS/MS scans on the most abundant ions from the MS scan. The LC-MS/MS data were used to search the SwissProt protein sequence database (subset of human proteins), using the program Bioworks/Sequest (Thermo Scientific). A total of 100 ml of venous blood was drawn into 20 ml ACD, 1 µM PGE1. The blood was centrifuged at 180 g, and the PRP was filtered through a PL6T leukocyte reduction filter (Pall, Inc., Port Washington, NY). Buffer A (68 ml) and 12 ml ACD was added to the sample and centrifuged at 1,400 g. The supernatant was removed, the platelet pellet was resuspended in 1 ml Trizol (Invitrogen), and RNA was extracted following the manufacturer's protocol. Two gene- and species-specific primers for each candidate protein were designed. Each primer was designed to produce a product between 250 and 350 basepairs long and span an intron of more than 1,000
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