title: Multiple mechanisms linked to platelet activation result in lysophosphatidic acid and sphingosine 1-phosphate generation in blood.
abstract: 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 platelets in 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.
authors.affiliation: Department of Biomembrane and Biofunctional Chemistry, Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo 06-0812, Japan.
authors.forename: Takamitsu
authors.initials: T
authors.lastname: Sano
authors.forename: Daniel
authors.initials: D
authors.lastname: Baker
authors.forename: Tamas
authors.initials: T
authors.lastname: Virag
authors.forename: Atsushi
authors.initials: A
authors.lastname: Wada
authors.forename: Yutaka
authors.initials: Y
authors.lastname: Yatomi
authors.forename: Tetsuyuki
authors.initials: T
authors.lastname: Kobayashi
authors.forename: Yasuyuki
authors.initials: Y
authors.lastname: Igarashi
authors.forename: Gabor
authors.initials: G
authors.lastname: Tigyi
mesh_terms: D000818:Animals; D001681:Biological Assay; D001777:Blood Coagulation; D001792:Blood Platelets / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D002713:Chlorine / Q000378:metabolism; D002853:Chromatography, Liquid; D002855:Chromatography, Thin Layer; D006801:Humans; D050356:Lipid Metabolism; D008055:Lipids / Q000737:chemistry; D008070:Lipopolysaccharides / Q000378:metabolism; D008246:Lysophospholipids / Q000097:blood / Q000378:metabolism*; D013058:Mass Spectrometry; D008954:Models, Biological; D009865:Oocytes / Q000378:metabolism; D010741:Phospholipases A / Q000378:metabolism; D054466:Phospholipases A1; D054467:Phospholipases A2; D010727:Phosphoric Diester Hydrolases / Q000097:blood; D015539:Platelet Activation; D013110:Sphingosine / Q000031:analogs & derivatives* / Q000378:metabolism*; D013917:Thrombin / Q000378:metabolism; D013997:Time Factors; D014981:Xenopus; D000099189:Lysophospholipase D
chemical_list: D002118:Calcium; D002713:Chlorine; D008055:Lipids; D008070:Lipopolysaccharides; D008246:Lysophospholipids; D010741:Phospholipases A; D054466:Phospholipases A1; D054467:Phospholipases A2; D010727:Phosphoric Diester Hydrolases; D013110:Sphingosine; D013917:Thrombin; D000099189:Lysophospholipase D; C060506:sphingosine 1-phosphate