Abstract
Blood platelets play an essential role in ischemic heart disease and stroke contributing to acute thrombotic events by release of potent inflammatory agents within the vasculature. Lysophosphatidic acid (LPA) is a bioactive lipid mediator produced by platelets and found in the blood and atherosclerotic plaques. LPA receptors on platelets, leukocytes, endothelial cells, and smooth muscle cells regulate growth, differentiation, survival, motility, and contractile activity. Definition of the opposing pathways of synthesis and degradation that control extracellular LPA levels is critical to understanding how LPA bioactivity is regulated. We show that intact platelets and platelet membranes actively dephosphorylate LPA and identify the major enzyme responsible as lipid phosphate phosphatase 1 (LPP1). Localization of LPP1 to the platelet surface is increased by exposure to LPA. A novel receptor-inactive sn-3-substituted difluoromethylenephosphonate analog of phosphatidic acid that is a potent competitive inhibitor of LPP1 activity potentiates platelet aggregation and shape change responses to LPA and amplifies LPA production by agonist-stimulated platelets. Our results identify LPP1 as a pivotal regulator of LPA signaling in the cardiovascular system. These findings are consistent with genetic and cell biological evidence implicating LPPs as negative regulators of lysophospholipid signaling and suggest that the mechanisms involve both attenuation of lysophospholipid actions at cell surface receptors and opposition of lysophospholipid production.
Highlights
Blood platelets play an essential role in ischemic heart disease and stroke contributing to acute thrombotic events by release of potent inflammatory agents within the vasculature
We show that intact platelets and platelet membranes actively dephosphorylate Lysophosphatidic acid (LPA) and identify the major enzyme responsible as lipid phosphate phosphatase 1 (LPP1)
Characterization of lipid phosphate phosphatases (LPPs) Activities in Intact Platelets and Platelet-derived Fractions—When gel-filtered human platelets were incubated with 10 M [32P]oleyl-LPA in a final concentration of 0.1% fatty acid-free BSA, we observed rapid timedependent release of ammonium molybdate-extractable [32P]PO42Ϫ, which occurred without significant uptake of the substrate by the platelets
Summary
We show that intact platelets and platelet membranes actively dephosphorylate LPA and identify the major enzyme responsible as lipid phosphate phosphatase 1 (LPP1). One pathway for LPA synthesis involves the actions of a secretory phospholipase A2 on phosphatidic acid (PA)-enriched membrane microparticles that are released from activated platelets [9]. The major activities responsible are lipid phosphate phosphatases (LPPs), which are integral membrane enzymes with specificity for LPA, S1P, and related lipid monophosphates [15,16,17,18]. Genetic and cell biological evidence identifies roles for LPPs in both intracellular lipid metabolism and as negative regulators of lysophospholipid signaling, the mechanisms involved remain to be firmly established [19, 21,22,23,24,25]. The dual function of LPP1 as a regulator of both LPA production and responsiveness may explain the complex and pleiotropic phenotypes associated with overexpression, misexpression, or down-regulation of this class of enzymes in many systems [19, 21, 25,26,27,28]
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