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Arachidonate mobilization in diacyl, alkylacyl and alkenylacyl phospholipids on stimulation of rat platelets by thrombin and the Ca2+ ionophore A23187.

ABSTRACT: Platelet stimulation by thrombin or Ca2+ ionophore induces mobilization of arachidonate from lipid stores. We have previously shown that, in [14C]arachidonic acid-prelabelled resting platelets, [14C]arachidonate was transferred from diacyl-sn-glycerophosphocholine to ethanolamine and choline-containing ether phospholipids. This transfer reached an equilibrium after 5 h incubation [Colard, Breton & Bereziat (1984a) Biochem. J. 222, 657-662]. [14C]Arachidonate-prelabelled platelets having reached this transfer equilibrium were used to study the mobilization of arachidonate in etheracyl and diacyl phospholipids. Upon thrombin stimulation, arachidonate decreased in diacyl-sn-glycero-3-phosphoinositol, in alkylacyl- and diacyl-sn-glycero-3-phosphocholine and increased in alkenylacyl- and diacyl-sn-glycero-3-phosphoethanolamine. Upon challenge with Ca2+ ionophore A23187, arachidonate decreased in diacyl-sn-glycero-3-phosphoethanolamine, in diacyl- and alkylacyl-sn-glycero-3-phosphocholine and increased in alkenylacyl-sn-glycero-3-phosphoethanolamine. We also compared arachidonate mobilization in platelets stimulated immediately after [14C]arachidonic acid chase with platelets stimulated after 5 h reincubation. We observed that the arachidonate newly incorporated into diacyl-sn-glycero-3-phosphocholine and triacylglycerols was rapidly released upon stimulation. This suggests the presence in these two lipids of a rapidly-turning-over arachidonate pool.

SUBMITTER: Colard O

PROVIDER: S-EPMC1153087 | biostudies-other | 1986 Feb

REPOSITORIES: biostudies-other

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Project description:High levels of ether phospholipids were found in rat platelets. Alkylacyl compounds constituted 18 and 29% of glycerophosphocholine (GPC) and glycerophosphoethanolamine (GPE). Alkenylacyl compounds, not detected in GPC, represented 40% of GPE. Arachidonate comprised 60%, 42% and 26% of the acyl residues in the sn-2 position of alkenylacyl-GPE, alkylacyl-GPE and alkylacyl-GPC respectively. Based on all arachidonate being linked to the sn-2 position of the diacyl species, the arachidonate level was 47% in diacyl-GPE and 30% in diacyl-GPC. The incorporation and metabolic fate of arachidonate in various phospholipid classes of resting platelets was examined. Arachidonate was essentially recovered in the diacyl phospholipids and very poorly in alkylacyl- and alkenylacyl-GPE and GPC after 30 min incubation in the presence of [14C]arachidonic acid. Upon reincubation of the platelets after removal of free arachidonate, the radioactivity was gradually lost from diacyl-GPC. Concomitantly, the radioactivities in alkylacyl-GPC, alkylacyl-GPE, alkenylacyl-GPE and to a lower extent in diacyl-GPE were increased. Labelling of glycerophosphoinositol was not changed. This labelling transfer was linear up to 5-6 h, except for alkylacyl-GPC; then labelling remained constant. These data strongly suggest that free arachidonate incorporation through the Lands pathway occurs only for diacyl species and that arachidonate incorporation into the ether phospholipids is achieved by exchange from diacyl-GPC. Based on specific activities related to phosphorus content, the arachidonate incorporation rates into diacyl-GPE and diacyl-GPC were approximately equivalent. The very large differences between specific radioactivities related to arachidonate observed at the starting reincubation time were strongly attenuated when labelling equilibrium was reached. The turnover rate by this exchange pathway was higher in alkylacyl-GPC than in alkyl- and alkenylacyl-GPE. This finding agrees with the selectivity for arachidonate observed in the acylation of PAF-acether in human neutrophils [Chilton, O'Flaherty, Ellis, Swendsen & Wykle (1983) J. Biol. Chem. 258, 7268-7271].

Project description:Phosphoproteomic analysis of platelets activated by pro-thrombotic oxidized phospholipids and thrombin Alejandro Zimman, Bjoern Titz, Evangelia Komisopoulou, Sudipta Biswas, Thomas G. Graeber, and Eugene A. Podrez ABSTRACT Specific oxidized phospholipids (oxPCCD36) promote platelet hyper-reactivity and thrombosis in hyperlipidemia via the scavenger receptor CD36, however the signaling pathway(s) induced in platelets by oxPCCD36 are not well defined. We have now employed mass spectrometry-based tyrosine, serine, and threonine global phosphoproteomics for the unbiased analysis of platelet signaling pathways induced by oxPCCD36 as well as by the strong physiological agonist thrombin. oxPCCD36 and thrombin induced differential phosphorylation of 115 proteins (162 phosphorylation sites) and 181 proteins (334 phosphorylation sites) respectively. Most of the phosphoproteome changes induced by either agonist have never been reported in platelets; thus they provide a new resource to study platelet signaling. Bioinformatic analyses of protein phosphorylation dependent responses were used to categorize preferential motifs for (de)phosphorylation, predict pathways and kinase activity, and construct a phosphoproteome network regulating integrin activation. A putative signaling pathway involving Src-family kinases, SYK, and PLCy2 was identified in platelets activated by oxPCCD36. Subsequent ex vivo studies in human platelets demonstrated that this pathway is downstream of the scavenger receptor CD36 and is critical for platelet activation by oxPCCD36. Our results provide multiple novel insights into the mechanism of platelet activation and specifically in platelet regulation by oxPCCD36. Methods Human platelets isolated by gel filtration (2.7 x 108 / mL) were incubated in Tyrode's buffer with 50 uM KODA-PC or PLPC (as control) for 30 min at 37 degrees C. Platelets were then centrifuged at 3,700 g (10 min, 35 degrees C) and lysed in 8 M urea, 50 mM Tris-HCl (pH 7.4), 1 mM Na3VO4, and 1 mM NaF with sonication. Phosphoproteome changes induced by the agonists were assessed based on trypsin digestion of the protein lysate, phosphopeptide enrichment, mass spectrometry analysis, chromatography alignment, and label-free quantitation as described in the Supplemental Materials and Methods of the publication. The same protocol was applied when studying platelet activation by thrombin, where gel-filtered platelets were incubated in Tyrode's buffer with 0.05 U/mL thrombin or in buffer alone (Resting) for 3 min at 37 degrees C.

Project description:BackgroundIntracytoplasmic sperm injection (ICSI) has tremendous advantages for resolving the problem of male infertility. However, ICSI fertilization can fail in some patients because of various reasons, primarily because of the failure of oocyte activation. Oocytes have been activated using calcium ionophore (A23187) in previous clinical cases of ICSI fertilization failure. However, studies on the efficiency of calcium ionophore (A23187) activation, its effects on the developmental potential of embryos, and its effects on pregnancy outcomes after embryo transfer are relatively limited.MethodsIn this study, we investigated the safety and long-term efficacy of calcium ionophore (A23187) by analyzing its effects on fertilization, embryonic development, aneuploidy, and pregnancy outcomes in patients undergoing preimplantation genetic testing (PGT) cycles.ResultsComparative analyses of the activation followed by PGT (A-PGT) and PGT groups revealed no significant differences between the oocyte cleavage rate and high-quality embryo rate (98.19% vs. 98.63% and 63.13% vs. 68.39%, respectively, p > 0.05). Although the blastocyst formation rate was significantly lower in the A-PGT group than that in the PGT group (52.22% vs. 59.90%, p < 0.05), no significant difference was observed in the blastocyst aneuploidy rates of the two groups (24.49% vs. 24.55%, p > 0.05). Furthermore, no significant differences were observed between the two groups in terms of the live birth rate (43.75% vs. 52.99%), week of delivery, and birth weight of the infants after transfer of euploid blastocysts (p > 0.05). Furthermore, the 2PN rate, oocyte cleavage rate, blastocyst formation rate, and live birth rate were found to be significantly lower in the A-ICSI group than those in the ICSI group (p < 0.01), but there was no significant difference between the two groups in the week of delivery and birth weight of live births (p > 0.05).DiscussionThese results suggest that the use of calcium ionophore (A23187) activation as an option in cases of ICSI fertilization failure does not affect the ploidy of developing blastocysts and has no significant effects on the week of delivery or birth weight after transfer. Thus, we provide a scientific basis for the clinical safety of oocyte activation using calcium ionophore (A23187).

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Arachidonate mobilization in diacyl, alkylacyl and alkenylacyl phospholipids on stimulation of rat platelets by thrombin and the Ca2+ ionophore A23187.

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