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Sphingosine 1-phosphate (Sph-1-P) has been implicated as an intracellular second messenger in many studies. We investigated the metabolism of Sph-1-P and the mechanism by which Sph-1-P induces activation in enucleated and highly differentiated platelets. Platelets lack Sph-1-P lyase activity, possess persistently active sphingosine (Sph) kinase, and abundantly store Sph-1-P. Although exogenous Sph-1-P activated platelets, intracellular Sph-1-P, formed from exogenously added Sph by cytosolic Sph kinase, failed to do so. To support the notion that exogenous Sph-1-P stimulates platelets from outside, contact of platelet surfaces with immobilized Sph-1-P covalently linked to glass particles resulted in platelet activation. Furthermore, we detected the specific binding sites for radiolabeled Sph-1-P on the platelet surface, suggesting extracellular effects of Sph-1-P on plasma membrane receptors. This specific Sph-1-P binding was inhibited not by other sphingolipids but by lysophosphatidic acid (LPA), and platelet aggregation response to LPA was specifically desensitized by prior addition of Sph-1-P. Finally, internally stored Sph-1-P is released extracellularly upon stimulation, and the release correlated well with protein kinase C activation in intact platelets. These results suggest that Sph-1-P acts not intracellularly but intercellularly, following discharge from activated platelets, and shares a platelet surface receptor with LPA. Sphingosine 1-phosphate (Sph-1-P) has been implicated as an intracellular second messenger in many studies. We investigated the metabolism of Sph-1-P and the mechanism by which Sph-1-P induces activation in enucleated and highly differentiated platelets. Platelets lack Sph-1-P lyase activity, possess persistently active sphingosine (Sph) kinase, and abundantly store Sph-1-P. Although exogenous Sph-1-P activated platelets, intracellular Sph-1-P, formed from exogenously added Sph by cytosolic Sph kinase, failed to do so. To support the notion that exogenous Sph-1-P stimulates platelets from outside, contact of platelet surfaces with immobilized Sph-1-P covalently linked to glass particles resulted in platelet activation. Furthermore, we detected the specific binding sites for radiolabeled Sph-1-P on the platelet surface, suggesting extracellular effects of Sph-1-P on plasma membrane receptors. This specific Sph-1-P binding was inhibited not by other sphingolipids but by lysophosphatidic acid (LPA), and platelet aggregation response to LPA was specifically desensitized by prior addition of Sph-1-P. Finally, internally stored Sph-1-P is released extracellularly upon stimulation, and the release correlated well with protein kinase C activation in intact platelets. These results suggest that Sph-1-P acts not intracellularly but intercellularly, following discharge from activated platelets, and shares a platelet surface receptor with LPA. INTRODUCTIONSphingolipid metabolites have been implicated as modulators of membrane signal transduction systems and shown to be involved in diverse cellular processes (1Spiegel S. Foster D. Kolesnick R. Curr. Opin. Cell Biol. 1996; 8: 159-167Google Scholar, 2Hakomori S. Igarashi Y. J. Biochem. 1995; 118: 1091-1103Google Scholar, 3Hannun Y.A. J. Biol. Chem. 1994; 269: 3125-3128Google Scholar, 4Kolesnick R. Golde D.W. Cell. 1994; 77: 325-328Google Scholar, 5Spiegel S. Milstien S. J. Membr. Biol. 1995; 146: 225-237Google Scholar). The phosphorylated sphingoid base sphingosine 1-phosphate (Sph-1-P) 1The abbreviations used are: Sph-1-Psphingosine-1-phosphateLPAlysophosphatidic acidCerceramideTPA12-O-tetradecanoylphorbol-13-acetateSphsphingosine. is the initial product of catabolism of sphingosine (Sph) by Sph kinase and, generally, is then cleaved by Sph-1-P lyase to ethanolamine phosphate and fatty aldehyde (1Spiegel S. Foster D. Kolesnick R. Curr. Opin. Cell Biol. 1996; 8: 159-167Google Scholar, 5Spiegel S. Milstien S. J. Membr. Biol. 1995; 146: 225-237Google Scholar, 6Van Veldhoven P.P. Mannaerts G.P. J. Biol. Chem. 1991; 266: 12502-12507Google Scholar). Sph-1-P has several important physiologic functions in addition to its role as a metabolite of Sph. Although originally proposed as a mitogenic messenger (7Zhang H. Desai N.N. Olivera A. Seki T. Brooker G. Spiegel S. J. Cell Biol. 1991; 114: 155-167Google Scholar–10Miyake Y. Kozutsumi Y. Nakamura S. Fujita T. Kawasaki T. Biochem. Biophys. Res. Commun. 1995; 211: 396-403Google Scholar), Sph-1-P has been shown to be involved in a variety of cellular functions, including regulation of cell motility (11Sadahira Y. Ruan F. Hakomori S. Igarashi Y. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 9686-9690Google Scholar, 12Bornfeldt K.E. Graves L.M. Raines E.W. Igarashi Y. Wayman G. Yamamura S. Yatomi Y. Sidhu J.S. Krebs E.G. Hakomori S. Ross R. J. Cell Biol. 1995; 130: 193-206Google Scholar), activation of muscarinic K+ current in atrial myocytes (13van Koppen C.J. zu Heringdorf D.M. Laser K.T. Zhang C. Jakobs K.H. Bunemann M. Pott L. J. Biol. Chem. 1996; 271: 2082-2087Google Scholar), mediation of FcϵRI antigen receptor signaling (14Choi O.H. Kim J.-H. Kinet J.-P. Nature. 1996; 380: 634-636Google Scholar), and neurite retraction (15Postma F.R. Jalink K. Hengeveld T. Moolenaar W.H. EMBO J. 1996; 15: 2388-2395Google Scholar). In nonproliferative, terminally differentiated platelets, Sph-1-P induces shape change and aggregation reactions by itself and synergistically elicits aggregation in combination with weak platelet agonists such as epinephrine and ADP (16Yatomi Y. Ruan F. Hakomori S. Igarashi Y. Blood. 1995; 86: 193-202Google Scholar).The Sph-1-P level in cells is generally low because of its degradation by Sph-1-P lyase, and Sph kinase is considered to be the rate-limiting factor in Sph catabolism (7Zhang H. Desai N.N. Olivera A. Seki T. Brooker G. Spiegel S. J. Cell Biol. 1991; 114: 155-167Google Scholar, 8Olivera A. Spiegel S. Nature. 1993; 365: 557-560Google Scholar, 14Choi O.H. Kim J.-H. Kinet J.-P. Nature. 1996; 380: 634-636Google Scholar, 17Stoffel W. Bister K. Hoppe-Seyler's Z. Physiol. Chem. 1973; 354: 169-181Google Scholar). Sph kinase activity is rapidly stimulated, and Sph-1-P level is transiently increased by specific stimuli. Sph-1-P has, therefore, been proposed as an intracellular second messenger (8Olivera A. Spiegel S. Nature. 1993; 365: 557-560Google Scholar, 14Choi O.H. Kim J.-H. Kinet J.-P. Nature. 1996; 380: 634-636Google Scholar), mobilizing Ca2+ from an internal source via an inositol trisphosphate-independent pathway (18Mattie M. Brooker G. Spiegel S. J. Biol. Chem. 1994; 269: 3181-3188Google Scholar). Many studies have supported this notion (an intracellular site of Sph-1-P action). An intracellular Ca2+-permeable channel is reportedly located in the endoplasmic reticulum, which may be gated by Sph-1-P (18Mattie M. Brooker G. Spiegel S. J. Biol. Chem. 1994; 269: 3181-3188Google Scholar, 19Ghosh T.K. Bian J. Gill D.L. Science. 1990; 248: 1653-1656Google Scholar, 20Ghosh T.K. Bian J. Gill D.L. J. Biol. Chem. 1994; 269: 22628-22635Google Scholar, 21Kindman L.A. Kim S. McDonald T.V. Gardner P. J. Biol. Chem. 1994; 269: 13088-13091Google Scholar, 22Mao C. Kim S.H. Almenoff J.S. Rudner X.L. Kearney D.M. Kindman L.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1993-1996Google Scholar). Furthermore, the endoplasmic reticulum contains the kinase that produces Sph-1-P (20Ghosh T.K. Bian J. Gill D.L. J. Biol. Chem. 1994; 269: 22628-22635Google Scholar). However, involvement of pertussis toxin-sensitive GTP-binding proteins in Sph-1-P-induced signaling has been reported recently (9Wu J. Spiegel S. Sturgill T.W. J. Biol. Chem. 1995; 270: 11484-11488Google Scholar, 13van Koppen C.J. zu Heringdorf D.M. Laser K.T. Zhang C. Jakobs K.H. Bunemann M. Pott L. J. Biol. Chem. 1996; 271: 2082-2087Google Scholar, 23Goodemote K.A. Mattie M.E. Berger A. Spiegel S. J. Biol. Chem. 1995; 270: 10272-10277Google Scholar, 24Okajima F. Tomura H. Sho K. Nochi H. Tamoto K. Kondo Y. FEBS Lett. 1996; 379: 260-264Google Scholar). Very recently, it was demonstrated that Sph-1-P only acts from the extracellular surface of the plasma membrane (13van Koppen C.J. zu Heringdorf D.M. Laser K.T. Zhang C. Jakobs K.H. Bunemann M. Pott L. J. Biol. Chem. 1996; 271: 2082-2087Google Scholar, 15Postma F.R. Jalink K. Hengeveld T. Moolenaar W.H. EMBO J. 1996; 15: 2388-2395Google Scholar). These phenomena suggest the existence of a cell surface receptor for Sph-1-P, and that the site of Sph-1-P action is extracellular.We now report here investigation of Sph-1-P biology in platelets, which lack a nucleus and do not proliferate, yet play important roles in physiological or pathophysiological phenomena such as thrombosis, hemostasis, and atherosclerosis. These highly differentiated cells are quite unique in terms of Sph-1-P-related metabolism, and this phospholipid acts not intracellularly but intercellularly. Furthermore, a specific binding site for Sph-1-P exists on the platelet surface, and this is recognized also by lysophosphatidic acid (LPA), a glycerolipid that is similar to Sph-1-P in structure and capable of inducing a multiplicity of biological effects (25Moolenaar W.H. J. Biol. Chem. 1995; 270: 12949-12952Google Scholar, 26Durieux M.E. Lynch K.R. Trends Pharmacol. Sci. 1993; 14: 249-254Google Scholar).DISCUSSIONIn most cells, Sph-1-P is degraded to ethanolamine phosphate and fatty aldehyde by Sph-1-P lyase (1Spiegel S. Foster D. Kolesnick R. Curr. Opin. Cell Biol. 1996; 8: 159-167Google Scholar, 5Spiegel S. Milstien S. J. Membr. Biol. 1995; 146: 225-237Google Scholar, 6Van Veldhoven P.P. Mannaerts G.P. J. Biol. Chem. 1991; 266: 12502-12507Google Scholar). Platelets lack this lyase activity. In contrast, these enucleated cells possess a highly active Sph kinase. Sph kinase is present in platelets as an active enzyme under resting conditions, and its activity is not related to cell activation by physiological agonists. It is not surprising that platelets, which possess high Sph kinase activity and lack Sph-1-P lyase activity, accumulate Sph-1-P abundantly. These findings on Sph-1-P-related metabolism in enucleated platelets contrast with previous findings on nucleated cells (7Zhang H. Desai N.N. Olivera A. Seki T. Brooker G. Spiegel S. J. Cell Biol. 1991; 114: 155-167Google Scholar, 8Olivera A. Spiegel S. Nature. 1993; 365: 557-560Google Scholar, 14Choi O.H. Kim J.-H. Kinet J.-P. Nature. 1996; 380: 634-636Google Scholar, 17Stoffel W. Bister K. Hoppe-Seyler's Z. Physiol. Chem. 1973; 354: 169-181Google Scholar), in which researchers observed fast and extensive metabolism of Sph-1-P with a relative scarcity of cellular Sph-1-P and hypothesized that Sph kinase is the rate-limiting factor in Sph catabolism. The role of Sph-1-P as a mitogenic signaling molecule has been extensively studied in Swiss 3T3 fibroblasts. In these cells, Sph-1-P has been shown to possess properties that qualify it as an intracellular second messenger (7Zhang H. Desai N.N. Olivera A. Seki T. Brooker G. Spiegel S. J. Cell Biol. 1991; 114: 155-167Google Scholar, 8Olivera A. Spiegel S. Nature. 1993; 365: 557-560Google Scholar, 48Olivera A. Zhang H. Carlson R.O. Mattie M.E. Schmidt R.R. Spiegel S. J. Biol. Chem. 1994; 269: 17924-17930Google Scholar). Endogenous Sph-1-P is maintained at a low level, possibly because of degradation by a lyase. Sph kinase is relatively inactive in the resting state (only a small fraction of exogenous Sph is converted to Sph-1-P intracellularly); Sph kinase activity is stimulated and Sph-1-P level is transiently increased by specific growth factors (platelet-derived growth factor and serum). Recently, similar findings were reported for FcϵRI-mediated signal in the rat mast cell line (14Choi O.H. Kim J.-H. Kinet J.-P. Nature. 1996; 380: 634-636Google Scholar). It seems obvious that Sph kinase regulation and hence the functional role of Sph-1-P in nonproliferative, terminally differentiated cells such as platelets differ from those in nucleated cells. The possibility of Sph-1-P playing a pivotal messenger role intracellularly is remote in platelets.Sph-1-P is a platelet activator (16Yatomi Y. Ruan F. Hakomori S. Igarashi Y. Blood. 1995; 86: 193-202Google Scholar). The findings that extracellular but not intracellular Sph-1-P is capable of activating platelets, and that immobilized Sph-1-P mimics free Sph-1-P in terms of activating platelets, indicate that the site of Sph-1-P action resides not inside platelets but on the surface. The more convincing evidence for the site of Sph-1-P action being extracellular is our identification here of specific binding sites for 3HSph-1-P on platelets, the first demonstration of a specific 3HSph-1-P binding site being expressed on the surface of plasma membrane. It has been shown recently that signaling pathways of Sph-1-P are regulated by heterotrimeric GTP-binding proteins (9Wu J. Spiegel S. Sturgill T.W. J. Biol. Chem. 1995; 270: 11484-11488Google Scholar, 13van Koppen C.J. zu Heringdorf D.M. Laser K.T. Zhang C. Jakobs K.H. Bunemann M. Pott L. J. Biol. Chem. 1996; 271: 2082-2087Google Scholar, 23Goodemote K.A. Mattie M.E. Berger A. Spiegel S. J. Biol. Chem. 1995; 270: 10272-10277Google Scholar, 24Okajima F. Tomura H. Sho K. Nochi H. Tamoto K. Kondo Y. FEBS Lett. 1996; 379: 260-264Google Scholar), whose activation is receptor-dependent (49Neer E.J. Cell. 1995; 80: 249-257Google Scholar). Furthermore, during the course of our present study, it was reported that only exogenously (not intracellularly) added Sph-1-P induces biological responses in guinea pig atrial myocytes (13van Koppen C.J. zu Heringdorf D.M. Laser K.T. Zhang C. Jakobs K.H. Bunemann M. Pott L. J. Biol. Chem. 1996; 271: 2082-2087Google Scholar) and N1E-115 neuronal cells (15Postma F.R. Jalink K. Hengeveld T. Moolenaar W.H. EMBO J. 1996; 15: 2388-2395Google Scholar). In addition to intracellular actions after passing the plasma membrane, activation of plasma membrane receptor(s) may be a critical mechanism by which Sph-1-P exerts biological responses in various cells.Another important finding of ours is that the putative Sph-1-P receptor may be shared by LPA, a lysoglycerophospholipid that is similar to Sph-1-P in structure (26Durieux M.E. Lynch K.R. Trends Pharmacol. Sci. 1993; 14: 249-254Google Scholar), capable of inducing platelet aggregation (45Benton A.M. Gerrard J.M. Michiel T. Kindom S.E. Blood. 1982; 60: 642-649Google Scholar), and released from activated platelets (50Eichholtz T. Jalink K. Fahrenfort I. Moolenaar W.H. Biochem. J. 1993; 291: 677-680Google Scholar). In support of this hypothesis, we found that platelet aggregation response to LPA was specifically desensitized by Sph-1-P. These findings are not consistent with several recent studies reporting lack of cross-desensitization between Sph-1-P and LPA in various nucleated cells (13van Koppen C.J. zu Heringdorf D.M. Laser K.T. Zhang C. Jakobs K.H. Bunemann M. Pott L. J. Biol. Chem. 1996; 271: 2082-2087Google Scholar, 15Postma F.R. Jalink K. Hengeveld T. Moolenaar W.H. EMBO J. 1996; 15: 2388-2395Google Scholar, 51Jalink K. Hengeveld T. Mulder S. Postma F.R. Simon M.-F. Chap H. van der Marel G.A. van Boom J.H. van Blitterswijk W.J. Moolenaar W.H. Biochem. J. 1995; 307: 609-616Google Scholar). In this content, it is noted that platelets are shown to possess two different levels of binding sites, a high affinity site (Kd, 110 nM) and a low affinity site (Kd, 2.6 μM), and one can assume that the low affinity site might be functional for platelets, judging from the facts that μM order of Sph-1-P concentrations are needed to induce platelet shape change and aggregation. Although the molecular mechanism of our observation in platelets remains to be solved, one possibility may be that platelets possess a unique receptor for lysophospholipids, including Sph-1-P and LPA, leading to platelet aggregation. It is already established that platelets store a variety of biologically active molecules that are secreted upon stimulation (52Hawiger J. Methods Enzymol. 1989; 169: 191-195Google Scholar). The secreted molecules interact with other platelets, plasma proteins, and the vessel wall. Sph-1-P was released from platelets, as expected given that Sph-1-P activates platelets from outside, but is abundantly stored inside. The Sph-1-P release may be mediated by protein kinase C, which is also highly expressed in platelets (53Kikkawa U. Takai Y. Minakuchi R. Inohara S. Nishizuka Y. J. Biol. Chem. 1982; 257: 13341-13348Google Scholar). We propose that Sph-1-P be added to the list of bioactive molecules stored in platelets and released from them upon stimulation, although, at present, we do not have the direct evidence for platelet activation caused by released Sph-1-P; the fact that not only Sph-1-P but also more potent lipid mediators such as thromboxane A2 are released from activated platelets makes our trial difficult. Previous reports have suggested that sphingolipid metabolites, including Sph-1-P, constitute a new class of intracellular second messengers in cell growth regulation and signal transduction (1Spiegel S. Foster D. Kolesnick R. Curr. Opin. Cell Biol. 1996; 8: 159-167Google Scholar, 3Hannun Y.A. J. Biol. Chem. 1994; 269: 3125-3128Google Scholar, 4Kolesnick R. Golde D.W. Cell. 1994; 77: 325-328Google Scholar, 5Spiegel S. Milstien S. J. Membr. Biol. 1995; 146: 225-237Google Scholar). However, as shown here, a sphingolipid can act intercellularly as a local mediator through its discharge from cells to regulate cellular functions in an autocrine or paracrine fashion. INTRODUCTIONSphingolipid metabolites have been implicated as modulators of membrane signal transduction systems and shown to be involved in diverse cellular processes (1Spiegel S. Foster D. Kolesnick R. Curr. Opin. Cell Biol. 1996; 8: 159-167Google Scholar, 2Hakomori S. Igarashi Y. J. Biochem. 1995; 118: 1091-1103Google Scholar, 3Hannun Y.A. J. Biol. Chem. 1994; 269: 3125-3128Google Scholar, 4Kolesnick R. Golde D.W. Cell. 1994; 77: 325-328Google Scholar, 5Spiegel S. Milstien S. J. Membr. Biol. 1995; 146: 225-237Google Scholar). The phosphorylated sphingoid base sphingosine 1-phosphate (Sph-1-P) 1The abbreviations used are: Sph-1-Psphingosine-1-phosphateLPAlysophosphatidic acidCerceramideTPA12-O-tetradecanoylphorbol-13-acetateSphsphingosine. is the initial product of catabolism of sphingosine (Sph) by Sph kinase and, generally, is then cleaved by Sph-1-P lyase to ethanolamine phosphate and fatty aldehyde (1Spiegel S. Foster D. Kolesnick R. Curr. Opin. Cell Biol. 1996; 8: 159-167Google Scholar, 5Spiegel S. Milstien S. J. Membr. Biol. 1995; 146: 225-237Google Scholar, 6Van Veldhoven P.P. Mannaerts G.P. J. Biol. Chem. 1991; 266: 12502-12507Google Scholar). Sph-1-P has several important physiologic functions in addition to its role as a metabolite of Sph. Although originally proposed as a mitogenic messenger (7Zhang H. Desai N.N. Olivera A. Seki T. Brooker G. Spiegel S. J. Cell Biol. 1991; 114: 155-167Google Scholar–10Miyake Y. Kozutsumi Y. Nakamura S. Fujita T. Kawasaki T. Biochem. Biophys. Res. Commun. 1995; 211: 396-403Google Scholar), Sph-1-P has been shown to be involved in a variety of cellular functions, including regulation of cell motility (11Sadahira Y. Ruan F. Hakomori S. Igarashi Y. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 9686-9690Google Scholar, 12Bornfeldt K.E. Graves L.M. Raines E.W. Igarashi Y. Wayman G. Yamamura S. Yatomi Y. Sidhu J.S. Krebs E.G. Hakomori S. Ross R. J. Cell Biol. 1995; 130: 193-206Google Scholar), activation of muscarinic K+ current in atrial myocytes (13van Koppen C.J. zu Heringdorf D.M. Laser K.T. Zhang C. Jakobs K.H. Bunemann M. Pott L. J. Biol. Chem. 1996; 271: 2082-2087Google Scholar), mediation of FcϵRI antigen receptor signaling (14Choi O.H. Kim J.-H. Kinet J.-P. Nature. 1996; 380: 634-636Google Scholar), and neurite retraction (15Postma F.R. Jalink K. Hengeveld T. Moolenaar W.H. EMBO J. 1996; 15: 2388-2395Google Scholar). In nonproliferative, terminally differentiated platelets, Sph-1-P induces shape change and aggregation reactions by itself and synergistically elicits aggregation in combination with weak platelet agonists such as epinephrine and ADP (16Yatomi Y. Ruan F. Hakomori S. Igarashi Y. Blood. 1995; 86: 193-202Google Scholar).The Sph-1-P level in cells is generally low because of its degradation by Sph-1-P lyase, and Sph kinase is considered to be the rate-limiting factor in Sph catabolism (7Zhang H. Desai N.N. Olivera A. Seki T. Brooker G. Spiegel S. J. Cell Biol. 1991; 114: 155-167Google Scholar, 8Olivera A. Spiegel S. Nature. 1993; 365: 557-560Google Scholar, 14Choi O.H. Kim J.-H. Kinet J.-P. Nature. 1996; 380: 634-636Google Scholar, 17Stoffel W. Bister K. Hoppe-Seyler's Z. Physiol. Chem. 1973; 354: 169-181Google Scholar). Sph kinase activity is rapidly stimulated, and Sph-1-P level is transiently increased by specific stimuli. Sph-1-P has, therefore, been proposed as an intracellular second messenger (8Olivera A. Spiegel S. Nature. 1993; 365: 557-560Google Scholar, 14Choi O.H. Kim J.-H. Kinet J.-P. Nature. 1996; 380: 634-636Google Scholar), mobilizing Ca2+ from an internal source via an inositol trisphosphate-independent pathway (18Mattie M. Brooker G. Spiegel S. J. Biol. Chem. 1994; 269: 3181-3188Google Scholar). Many studies have supported this notion (an intracellular site of Sph-1-P action). An intracellular Ca2+-permeable channel is reportedly located in the endoplasmic reticulum, which may be gated by Sph-1-P (18Mattie M. Brooker G. Spiegel S. J. Biol. Chem. 1994; 269: 3181-3188Google Scholar, 19Ghosh T.K. Bian J. Gill D.L. Science. 1990; 248: 1653-1656Google Scholar, 20Ghosh T.K. Bian J. Gill D.L. J. Biol. Chem. 1994; 269: 22628-22635Google Scholar, 21Kindman L.A. Kim S. McDonald T.V. Gardner P. J. Biol. Chem. 1994; 269: 13088-13091Google Scholar, 22Mao C. Kim S.H. Almenoff J.S. Rudner X.L. Kearney D.M. Kindman L.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1993-1996Google Scholar). Furthermore, the endoplasmic reticulum contains the kinase that produces Sph-1-P (20Ghosh T.K. Bian J. Gill D.L. J. Biol. Chem. 1994; 269: 22628-22635Google Scholar). However, involvement of pertussis toxin-sensitive GTP-binding proteins in Sph-1-P-induced signaling has been reported recently (9Wu J. Spiegel S. Sturgill T.W. J. Biol. Chem. 1995; 270: 11484-11488Google Scholar, 13van Koppen C.J. zu Heringdorf D.M. Laser K.T. Zhang C. Jakobs K.H. Bunemann M. Pott L. J. Biol. Chem. 1996; 271: 2082-2087Google Scholar, 23Goodemote K.A. Mattie M.E. Berger A. Spiegel S. J. Biol. Chem. 1995; 270: 10272-10277Google Scholar, 24Okajima F. Tomura H. Sho K. Nochi H. Tamoto K. Kondo Y. FEBS Lett. 1996; 379: 260-264Google Scholar). Very recently, it was demonstrated that Sph-1-P only acts from the extracellular surface of the plasma membrane (13van Koppen C.J. zu Heringdorf D.M. Laser K.T. Zhang C. Jakobs K.H. Bunemann M. Pott L. J. Biol. Chem. 1996; 271: 2082-2087Google Scholar, 15Postma F.R. Jalink K. Hengeveld T. Moolenaar W.H. EMBO J. 1996; 15: 2388-2395Google Scholar). These phenomena suggest the existence of a cell surface receptor for Sph-1-P, and that the site of Sph-1-P action is extracellular.We now report here investigation of Sph-1-P biology in platelets, which lack a nucleus and do not proliferate, yet play important roles in physiological or pathophysiological phenomena such as thrombosis, hemostasis, and atherosclerosis. These highly differentiated cells are quite unique in terms of Sph-1-P-related metabolism, and this phospholipid acts not intracellularly but intercellularly. Furthermore, a specific binding site for Sph-1-P exists on the platelet surface, and this is recognized also by lysophosphatidic acid (LPA), a glycerolipid that is similar to Sph-1-P in structure and capable of inducing a multiplicity of biological effects (25Moolenaar W.H. J. Biol. Chem. 1995; 270: 12949-12952Google Scholar, 26Durieux M.E. Lynch K.R. Trends Pharmacol. Sci. 1993; 14: 249-254Google Scholar).
Yatomi et al. (Sat,) studied this question.
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