Sphingosine 1-Phosphate Induces Platelet Activation through an Extracellular Action and Shares a Platelet Surface Receptor with Lysophosphatidic Acid*

(Received for publication, September 5, 1996, and in revised form, December 6, 1996)

Yutaka Yatomi Dagger §, Soichiro Yamamura Dagger par , Fuqiang Ruan and Yasuyuki Igarashi **

From The Biomembrane Institute, Seattle, Washington 98119 and  Department of Pathobiology, University of Washington, Seattle, Washington 98195

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
Acknowledgments
REFERENCES


ABSTRACT

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.


INTRODUCTION

Sphingolipid metabolites have been implicated as modulators of membrane signal transduction systems and shown to be involved in diverse cellular processes (1-5). The phosphorylated sphingoid base sphingosine 1-phosphate (Sph-1-P)1 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 (1, 5, 6). 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 (7-10), Sph-1-P has been shown to be involved in a variety of cellular functions, including regulation of cell motility (11, 12), activation of muscarinic K+ current in atrial myocytes (13), mediation of Fcepsilon RI antigen receptor signaling (14), and neurite retraction (15). 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 (16).

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 (7, 8, 14, 17). 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 (8, 14), mobilizing Ca2+ from an internal source via an inositol trisphosphate-independent pathway (18). 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 (18-22). Furthermore, the endoplasmic reticulum contains the kinase that produces Sph-1-P (20). However, involvement of pertussis toxin-sensitive GTP-binding proteins in Sph-1-P-induced signaling has been reported recently (9, 13, 23, 24). Very recently, it was demonstrated that Sph-1-P only acts from the extracellular surface of the plasma membrane (13, 15). 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 (25, 26).


EXPERIMENTAL PROCEDURES

Materials

Sph-1-P was prepared from sphingosylphosphocholine with bacterial phospholipase D, as described previously (27). C2-ceramide (Cer) (28) and N,N-dimethylsphingosine (29) were synthesized as described previously. [3-3H]Sph-1-P was prepared by ATP-dependent phosphorylation of [3-3H]Sph catalyzed by Sph kinase obtained from Balb/c 3T3 fibroblasts (30).

CMK cells were kindly provided by Dr. T. Sato (Chiba University, Chiba, Japan). K562 cells were from the American Type Culture Collection (Rockville, MD). Bovine and rat livers were obtained from Pelfreez (Rogers, AR).

The following materials were obtained from the indicated suppliers: [3-3H]Sph (22.0 Ci/mmol) and [3H]acetic anhydride (50 mCi/mmol) (DuPont NEN); Sph, Cer (type III), 12-O-tetradecanoylphorbol 13-acetate (TPA), 1-oleoyl-2-acetyl-glycerol, and bovine serum albumin (essentially fatty acid free) (Sigma); thrombin, collagen, and epinephrine (Chrono-Log, Havertown, PA); staurosporine and prostaglandin E1 (Biomol, Plymouth Meeting, PA); and Fura-2/AM (Molecular Probes, Eugene, OR).

Preparation of Platelets

Citrated venous blood was obtained from healthy adult volunteers. Washed platelets were prepared and handled as described previously (16), unless stated otherwise. Bovine serum albumin (fatty acid-free) (1%) was added when indicated. For Sph-1-P lyase activity assay, outdated platelet concentrates obtained from the Oregon Red Cross (Portland, OR) were used.

Sph-1-P Lyase Activity Assay

Sph-1-P lyase activity was measured as described previously (6) except that [3-3H]Sph-1-P instead of [4,5-3H]dihydrosphingosine-1-phosphate was used as a substrate. The reaction products were applied to silica gel 60 HPTLC plates (Merck, Darmstadt, Germany), and the plates were developed in chloroform:methanol:acetic acid (50:50:1). In this polar solvent, all radioactive lyase metabolites (palmitaldehyde, palmitic acid, and palmitol) ran closely together near the front, and the whole region was scraped off and counted by liquid scintillation counting.

Metabolism of [3H]Sph in Platelets

Platelet suspensions (0.5 ml) were incubated with 1 µM [3H]Sph (0.2 µCi). At the indicated time points, the reaction was terminated by the addition of 1.875 ml of ice-cold chloroform:methanol:concentrated HCl (100:200:1), and lipids were extracted from the cell suspensions and analyzed for [3H]Sph metabolism as described previously (16). Portions of lipids obtained from the lower chloroform phase were applied to silica gel 60 HPTLC plates, and the plates were developed in butanol:acetic acid:water (3:1:1), followed by autoradiography. Silica gel areas containing radiolabeled sphingolipids were scraped off and counted by liquid scintillation counting.

Quantitative Measurement of Sph and Sph-1-P

Sph (31) and Sph-1-P (32) were quantitatively measured by N-acylation with [3H]acetic anhydride into [3H]C2-Cer (N-[3H]acetylated Sph) and [3H]C2-Cer-1-P (N-[3H]acetylated Sph-1-P), respectively, as described previously.

Platelet Shape Change

Platelet shape change and aggregation were determined turbidometrically (33), as described (16). Calibration was performed with zero light transmission defined for platelet suspension and 100% transmission for the buffer.

Measurement of Intracellular Ca2+ Concentration

Intracellular Ca2+ concentration was measured using the Ca2+-sensitive fluorophore Fura-2 as described (16). Values of peak increases after addition of an agent were quantified.

Preparation and Application of Sph-1-P or Sph Immobilized on a Solid Support

Sph or Sph-1-P was immobilized on the surface of controlled-pore glass particles with a long chain alkylamine (CPG, Inc., Fairfield, NJ). Sph-1-P or Sph derivative possessing omega -carboxyl group was synthesized and linked to a long chain alkylamine of the glass particles through an amide linkage, as described previously (34). The long chain alkylamine acts as a spacer arm and avoids low steric availability of Sph-1-P or Sph immobilized on glass, enabling them to mimic parent (free) Sph-1-P or Sph. For control glass particles, the amino group was blocked by N-acetylation. Sph-1-P and Sph contents of the conjugates were 22 and 39 µmol/g of dry glass particles, respectively. Extensive washing of the Sph-1-P- and Sph-attached glass particles with chloroform:methanol (1:2) did not result in dissolution of free lipids, which was confirmed by thin layer chromatography. The glass particles were added into platelet suspensions under constant stirring at 1000 rpm, with the Sph-1-P or Sph concentration at 40 µM, followed by scanning electron microscopy (16) and measurement of intracellular Ca2+ concentration.

[3H]Sph-1-P Binding Assays

Binding assays were performed by incubating intact platelets (1 × 108 cells), suspended in phosphate-buffered saline, with 2.1 nM [3H]Sph-1-P. Reactions were initiated by the addition of the ligand and were incubated at 4 °C for 1 h unless stated otherwise. The platelets were then washed with 1 mg/ml of bovine serum albumin, 150 mM NaCl, and 50 mM Tris-HCl, pH 7.5, three times. Radioactivity of the platelets was counted by liquid scintillation counting. Under those conditions, [3H]Sph-1-P added remained intact, which was confirmed by separation with thin layer chromatography.

To examine the effect of a protease on [3H]Sph-1-P binding, platelets were treated with 0.02% of the protease (type XXV) (Sigma) at room temperature for 30 min, and assays were carried out as described above.

Total binding was defined as the amount of radioactivity bound to platelets in the absence of competing ligand. Nonspecific binding was defined as the amount of [3H]Sph-1-P binding that occurred in the presence of 50 µM Sph-1-P. Specific binding was defined as the difference between total and nonspecific binding.

Sph-1-P Release from Platelets Labeled with [3H]Sph

[3H]Sph-labeled platelet suspensions, to which 1% bovine serum albumin had been added, were centrifuged for 15 s at 12,000 × g. Lipids were then extracted from the resultant medium supernatant and cell pellet and analyzed as described above. Albumin was included in the medium to prevent released Sph-1-P, a lipophilic molecule, from being nonspecifically attached to the plasma membrane surface and consequently underestimated. The percentage of Sph-1-P release into medium was calculated as 100 × ([3H]Sph-1-P in medium)/(total [3H]Sph-1-P in medium plus cell pellet).

Protein Kinase C Activation in Intact Platelets

Protein kinase C activation in intact platelets was evaluated by pleckstrin (47-kDa protein) (35) phosphorylation as described (36).


RESULTS

Absence of Sph-1-P Lyase Activity in Platelets

When [3H]Sph was added into platelet homogenates under established assay conditions for Sph-1-P lyase (6), no lyase products (palmitaldehyde, palmitic acid, and palmitol) were formed. Under the same conditions, high lyase activity was detected in bovine (specific activity, 11.2 ± 0.2 pmol/min·mg of protein, mean ± S.D., n = 3) and rat livers, as reported previously (6). Low but significant Sph-1-P lyase activity was detected in CMK and K562 cells (specific activities, 0.25 ± 0.04 and 0.54 ± 0.03 pmol/min·mg of protein, mean ± S.D., n = 3, respectively). CMK is a human megakaryoblastic cell line (37); K562 is a human erythroleukemia cell line capable of megakaryocytic differentiation (38). Our results confirm the absence of Sph-1-P lyase in platelets, as suggested by earlier studies (39, 40). It is likely that blood stem cells lose Sph-1-P lyase activity during late-stage differentiation into platelets.

Independence of Sph Kinase Activity from Cell Activation in Platelets

When 1 µM [3H]Sph was added exogenously to platelet suspensions, the label was efficiently removed from the medium; uptake of [3H]Sph was 96% at 5 min after the label addition. [3H]Sph incorporated into platelets was rapidly converted to [3H]Sph-1-P (Fig. 1A). Conversion was observed as early as at 10 s. Within 5 min, the level of Sph-1-P approached a plateau, with 45% conversion of [3H]Sph to [3H]Sph-1-P. At 3 h (the latest time point observed), 42% of the [3H]Sph originally added remained as [3H]Sph-1-P (Fig. 1A), indicating the stability of Sph-1-P in platelets. Sph at concentrations below 10 µM did not activate platelets at all (Fig. 2B). Sph kinase, which converts [3H]Sph into [3H]Sph-1-P, is therefore considered to be present in platelets as an active enzyme under resting conditions. [3H]Sph was also converted by N-acylation into Cer at later time points (Fig. 1A); 3, 7, and 10% of added [3H]Sph was converted into Cer at 1, 2, and 3 h, respectively. The ratio of [3H]Sph-1-P to [3H]Sph increased rapidly to approximately 5:1 at 20 min and stayed relatively constant at this value until 3 h. This suggests that the cellular content of Sph-1-P is much higher than that of Sph in resting platelets.


Fig. 1. [3H]Sph metabolism in intact platelets. A, rapid conversion of exogenously added [3H]Sph into [3H]Sph-1-P in human platelets. Platelet suspensions were incubated with 1 µM [3H]Sph for various durations, and the metabolic fate of the label was determined. The radioactivity of Sph (open circle ), Sph-1-P (square ), and Cer (triangle ) is expressed as a percentage of the value for [3H]Sph at time 0. B, failure of thrombin to affect the[3H]Sph-1-P formation from [3H]Sph. Platelets were incubated with 0.5 unit/ml of thrombin for 5 min and with [3H]Sph for 15 s (a), 1 min (b), or 10 min (c) before analysis of [3H]Sph metabolism. For comparison, platelets were incubated with 20 µM N,N-dimethylsphingosine for 10 min and [3H]Sph for 5 min (d). [3H]Sph-1-P formed was expressed as a percentage of control (without thrombin or N,N-dimethylsphingosine treatment). Data are the means (bars, S.D.; n = 3).
[View Larger Version of this Image (23K GIF file)]



Fig. 2. Platelet activation by exogenously added Sph-1-P, but not Sph, in spite of extensive conversion of added Sph into Sph-1-P. A, platelet suspensions (0.5 ml) were incubated with 10 µM (5 nmol) Sph for 0 (a), 1 (b), 5 (c), or 30 (d) min. N-Acylation with [3H]acetic anhydride into [3H]C2-Cer (upper, left) and [3H]C2-Cer-1-P (lower, left) was performed for quantification of Sph and Sph-1-P, respectively. Radioactive spots corresponding to [3H]C2-Cer and [3H]C2-Cer-1-P on the thin layer chromatography plates shown (left) were scraped and counted for measurement of Sph (upper, right) and Sph-1-P (lower, right) levels, respectively. Note that over 5 µM Sph-1-P was formed in platelets within 5 min after the addition of 10 µM Sph. B, platelets were challenged with Sph (10 µM) or Sph-1-P (5 or 10 µM), and shape change reaction (left) and intracellular Ca2+ concentration (right) were monitored. Data are means (bars, S.D.; n = 3).
[View Larger Version of this Image (32K GIF file)]


The rapid conversion of [3H]Sph into [3H]Sph-1-P was not affected by the potent platelet activator thrombin (41) (Fig. 1B), which, under similar conditions, strongly induced platelet activation, including intracellular Ca2+ mobilization and protein kinase C activation (data not shown). Other platelet activators (collagen, TPA, and epinephrine) and the platelet activation inhibitor prostaglandin E1 (41) also had no effect on the [3H]Sph conversion (data not shown). N,N-Dimethylsphingosine, which strongly inhibits Sph kinase, suppressed conversion of [3H]Sph to [3H]Sph-1-P (Fig. 1B), as we reported previously (42).

In summary, Sph kinase is highly active, even in resting platelets, and its activity is independent of intracellular events involved in platelet activation.

Determination of Sph and Sph-1-P Levels in Platelets

We determined the mass levels of Sph-1-P and Sph extracted from platelets. The content of Sph-1-P and Sph was calculated as 1.41 ± 0.04 nmol and 374 ± 61 pmol (mean ± S.D., n = 3 and 5), respectively, per 109 platelets. The finding that the cellular content of Sph-1-P is much higher than that of Sph is consistent with the high Sph-1-P:Sph ratio obtained in [3H]Sph labeling studies (Fig. 1A).

We measured the time course of mass changes of Sph-1-P and Sph in platelets incubated with nonradioactive Sph, the substrate of Sph kinase. Exogenous Sph was rapidly converted into Sph-1-P (Fig. 2A), as was also seen in the [3H]Sph labeling studies (Fig. 1A). Consistent with our previous finding (16), exogenous Sph-1-P induced platelet shape change and intracellular Ca2+ mobilization (Fig. 2B). These positive reactions induced by Sph-1-P were not mimicked by Sph (Fig. 2B), although Sph was rapidly converted to Sph-1-P (Figs. 1A and 2A). Sph-1-P (5 µM), but not 10 µM Sph, activated platelets (Fig. 2B), despite the fact that over 5 µM Sph-1-P was rapidly formed in platelets incubated with 10 µM Sph (Fig. 2A) by cytoplasmic Sph kinase (20, 43).

Platelet Activation Induced by Sph-1-P Immobilized on a Solid Support

The finding that only exogenous Sph-1-P activates platelets raises the possibility that Sph-1-P acts on these highly differentiated cells from outside. To test this possibility, we synthesized Sph-1-P immobilized on a solid support; a Sph-1-P derivative possessing an omega -carboxyl group was covalently linked to alkylamine-glass particles through amide linkage. Upon the addition of the immobilized Sph-1-P, platelets underwent shape change and aggregate formation, as determined by scanning electron microscopy (Fig. 3, C and D), and intracellular Ca2+ mobilization (Fig. 4). Sph-bound glass particles (Fig. 3, A and B, and Fig. 4) or control glass particles (data not shown) had no effect on platelets. The fact that immobilized Sph-1-P mimics free Sph-1-P in terms of activating platelets strongly suggests that the site of Sph-1-P action resides not inside platelets but on the surface. This hypothesis would explain why exogenous addition of Sph-1-P but not Sph results in platelet activation in spite of rapid Sph conversion into Sph-1-P (which occurs intracellularly). The hypothesis is consistent with the observation that extracellular, but not intracellular (by microinjection), addition of Sph-1-P elicits intracellular Ca2+ mobilization in Xenopus laevis oocytes (44).


Fig. 3. Platelet activation induced by Sph-1-P immobilized on a solid support. A and B, platelets were treated for 1 min with Sph-bound glass particles and examined by scanning electron microscopy. Platelets neither changed their shape nor formed clusters upon challenge with the Sph-bound glass. Note the presence of dispersed platelets and free glass particles without platelet attachment (arrows). C and D, platelets were treated for 1 min with Sph-1-P-bound glass particles and examined by scanning electron microscopy. Platelets underwent shape change with pseudopod formation and formed aggregates on Sph-1-P-bound particles. Note the scarcity of free platelets. Bars: A, B, and C, 10 µm; D, 1 µm.
[View Larger Version of this Image (85K GIF file)]



Fig. 4. Platelet intracellular Ca2+ mobilization induced by Sph-1-P immobilized on a solid support. Platelets were challenged with Sph- or Sph-1-P-bound glass particles (arrow), and intracellular Ca2+ concentration was monitored. Sph-1-P-bound particles elicited intracellular Ca2+ mobilization (solid line), whereas Sph-bound particles did not (dotted line).
[View Larger Version of this Image (16K GIF file)]


[3H]Sph-1-P Binding Studies

The above finding that Sph-1-P only acts from the extracellular face of platelet surface membranes can be best explained by the hypothesis that exogenous Sph-1-P acts on platelets via interaction with a plasma membrane receptor. Consequently, we performed [3H]Sph-1-P binding studies using intact platelets. Fig. 5A shows the time course of specific binding of [3H]Sph-1-P to platelets. The specific binding was time dependent, reached equilibrium by 1 h, and remained constant for at least 1 h. Saturation binding experiments were performed with [3H]Sph-1-P (Fig. 5B). The specific binding was saturated around 2 nM of the ligand when platelets were incubated at 4 °C for 1 h with or without cold 50 µM Sph-1-P. The data were transformed by Scatchard analysis (Fig. 5C). Platelets were found to possess two binding sites for Sph-1-P; the Kd values were estimated to be 110 nM and 2.6 µM, and the numbers of the binding sites approximately 200/cell and 2400/cell, respectively. We examined the effect of a protease on the specific binding of [3H]Sph-1-P. Treatment of platelets with a protease (type XXV) disrupted the specific binding of [3H]Sph-1-P almost completely (data not shown), indicating that the binding sites are proteins that are located on the surface of platelets. The specific [3H]Sph-1-P binding was not detected on K562 cells, which are capable of megakaryocytic differentiation but do not respond to Sph-1-P in terms of Ca2+ mobilization (data not shown). These results indicate the presence of a cell-surface receptor for Sph-1-P on platelets.


Fig. 5. Specific binding of [3H]Sph-1-P to platelets. A, intact platelets were incubated with [3H]Sph-1-P for various times, and the specific binding to platelets was determined as described under "Experimental Procedures." B, platelets were incubated with various concentrations of [3H]Sph-1-P. C, [3H]Sph-1-P binding to platelets is plotted by the method of Scatchard (54). The data indicate the representative of three separate experiments.
[View Larger Version of this Image (13K GIF file)]


Competition binding experiments were performed using various sphingolipids and LPA (Fig. 6). Sph and C2-Cer were unable to compete for [3H]Sph-1-P binding sites on platelets. Sphingosylphosphocholine only slightly inhibited the binding of [3H]Sph-1-P. In contrast, LPA, which is a glycerophospholipid with a very similar structure to Sph-1-P (26), reduced the binding of [3H]Sph-1-P to platelets as much as unlabeled Sph-1-P did.


Fig. 6. Inhibition of [3H]Sph-1-P binding to platelets by LPA. Platelets were incubated with [3H]Sph-1-P alone or in the presence of various concentrations of unlabeled Sph-1-P (open circle ), LPA (square ), Sph (triangle ), C2-Cer (bullet ), or sphingosylphosphocholine (black-square). Specific [3H]Sph-1-P binding to platelets was expressed as a percentage of control (without unlabeled ligand).
[View Larger Version of this Image (19K GIF file)]


Desensitization of Platelet Aggregation Response to LPA by Sph-1-P

LPA is a well established platelet agonist capable of inducing strong and irreversible aggregation response (45). Sph-1-P is also a platelet-aggregating agent, although its effect is weaker (16). Because the possibility that Sph-1-P and LPA may share a surface receptor on platelets was raised, desensitization studies in platelet aggregation were performed (Fig. 7). LPA (5 µM) induced marked platelet aggregation, which was comparable with that induced by 5 µg/ml of collagen (41). Prior addition of subthreshold concentrations of Sph-1-P inhibited the platelet aggregation response to LPA. In contrast, Sph-1-P did not affect the response to collagen. The specific desensitization of LPA-induced platelet aggregation by Sph-1-P might be related to the fact that Sph-1-P and LPA share a platelet surface receptor, although we cannot completely rule out the desensitization mechanism resulting from the modification of intracellular signaling pathways involved.


Fig. 7. Desensitization of platelet aggregation response to LPA by prior addition of Sph-1-P. Platelets were preincubated with various concentrations of Sph-1-P for 1 min and then challenged with 5 µM LPA (open circle ) or 5 µg/ml collagen (bullet ). Platelet aggregation was analyzed as described under "Experimental Procedures." Under the conditions used, Sph-1-P at concentrations below 20 µM did not induce platelet aggregation.
[View Larger Version of this Image (15K GIF file)]


Sph-1-P Release from Activated Platelets

In view of the fact that Sph-1-P activates platelets from outside but is abundantly stored inside, we examined the possibility that Sph-1-P is released from platelets and acts intercellularly as a local mediator. When platelets were stimulated with 1 µM TPA, which can act as a substitute for diacylglycerol and directly activates protein kinase C (41, 46), 8, 35, 53, 57, and 60% of stored Sph-1-P was released extracellularly 2, 5, 20, 60, and 120 min after challenge, respectively. This protein kinase C activator was also found to release Sph-1-P in a dose-dependent manner (Fig. 8A). The release correlated well with its effect on protein kinase C activation in intact platelets (Fig. 8B). Under the same conditions, thrombin, which produces diacylglycerol and activates protein kinase C as a result of phosphatidylinositol-4,5-bisphosphate hydrolysis (41, 46), and 1-oleoyl-2-acetyl-glycerol, a membrane-permeable diacylglycerol (46), also caused marked Sph-1-P release (Fig. 9). The Sph-1-P release induced by these protein kinase C activators was inhibited by staurosporine (Fig. 9), an inhibitor of protein kinases, including protein kinase C (47). Furthermore, epinephrine, a weak platelet stimulator incapable of activating protein kinase C by itself (41), did not induce Sph-1-P release (data not shown). These findings indicate that Sph-1-P can be released from activated platelets and suggest that protein kinase C activation may be the mechanism involved.


Fig. 8. TPA-induced Sph-1-P release (A) and protein kinase C activation (B) in platelets. A, platelets labeled with [3H]Sph were stimulated with various concentrations of TPA for 5 min, and the percentage of Sph-1-P release into the medium was determined as described under "Experimental Procedures." B, 32Pi-loaded platelets were stimulated with 0 (a), 1 (b), 10 (c), 100 (d), or 1000 (e) nM TPA, and protein kinase C activation in intact platelets was evaluated by phosphorylation of pleckstrin, a protein kinase C substrate in platelets (25). The location of pleckstrin is indicated by an arrow.
[View Larger Version of this Image (39K GIF file)]



Fig. 9. Thrombin-, TPA-, or 1-oleoyl-2-acetyl-glycerol-induced Sph-1-P release from platelets and its inhibition by staurosporine. The percentage of Sph-1-P release into the medium was measured in platelets treated with 0.5 unit/ml of thrombin, 1 µM TPA, or 25 µg/ml of 1-oleoyl-2-acetyl-glycerol for 5 min in the absence (-) or presence (+) of 1 µM staurosporine. Data are the means (bars, S.D.) of three determinations.
[View Larger Version of this Image (15K GIF file)]



DISCUSSION

In most cells, Sph-1-P is degraded to ethanolamine phosphate and fatty aldehyde by Sph-1-P lyase (1, 5, 6). 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 (7, 8, 14, 17), 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 (7, 8, 48). 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 Fcepsilon RI-mediated signal in the rat mast cell line (14). 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 (16). 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 [3H]Sph-1-P on platelets, the first demonstration of a specific [3H]Sph-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 (9, 13, 23, 24), whose activation is receptor-dependent (49). 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 (13) and N1E-115 neuronal cells (15). 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 (26), capable of inducing platelet aggregation (45), and released from activated platelets (50). 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 (13, 15, 51). 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 (52). 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 (53). 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 (1, 3-5). 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.


FOOTNOTES

*   This study was supported by funds from The Biomembrane Institute and in part under a research contract with Otsuka Pharmaceutical Co. and Seikagaku Corp. The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Dagger    These two authors contributed equally to the study.
§   Present address: Dept. of Laboratory Medicine, Yamanashi Medical University, Yamanashi, Japan.
par    Present address: Pacific Northwest Research Foundation, Division of Biomembrane Research, Seattle, WA 98122.
**   To whom correspondence should be addressed: Fred Hutchinson Cancer Research Center, 1124 Columbia, M621, Seattle, WA 98104. Tel.: 206-667-2844; Fax: 206-667-6519. E-mail: yigarash{at}fhcrc.org.
1    The abbreviations used are: Sph-1-P, sphingosine-1-phosphate; LPA, lysophosphatidic acid; Cer, ceramide; TPA, 12-O-tetradecanoylphorbol-13-acetate; Sph, sphingosine.

Acknowledgments

We thank Prof. S. Hakomori of University of Washington for his encouragement throughout this study. We also thank L. E. Caldwell (Fred Hutchinson Cancer Research Center) for scanning electron microscopy and Drs. S. Anderson and E. A. Sweeney for scientific editing of the manuscript.


REFERENCES

  1. Spiegel, S., Foster, D., and Kolesnick, R. (1996) Curr. Opin. Cell Biol. 8, 159-167 [CrossRef][Medline] [Order article via Infotrieve]
  2. Hakomori, S., and Igarashi, Y. (1995) J. Biochem. 118, 1091-1103 [Abstract]
  3. Hannun, Y. A. (1994) J. Biol. Chem. 269, 3125-3128 [Free Full Text]
  4. Kolesnick, R., and Golde, D. W. (1994) Cell 77, 325-328 [Medline] [Order article via Infotrieve]
  5. Spiegel, S., and Milstien, S. (1995) J. Membr. Biol. 146, 225-237 [Medline] [Order article via Infotrieve]
  6. Van Veldhoven, P. P., and Mannaerts, G. P. (1991) J. Biol. Chem. 266, 12502-12507 [Abstract/Free Full Text]
  7. Zhang, H., Desai, N. N., Olivera, A., Seki, T., Brooker, G., and Spiegel, S. (1991) J. Cell Biol. 114, 155-167 [Abstract]
  8. Olivera, A., and Spiegel, S. (1993) Nature 365, 557-560 [CrossRef][Medline] [Order article via Infotrieve]
  9. Wu, J., Spiegel, S., and Sturgill, T. W. (1995) J. Biol. Chem. 270, 11484-11488 [Abstract/Free Full Text]
  10. Miyake, Y., Kozutsumi, Y., Nakamura, S., Fujita, T., and Kawasaki, T. (1995) Biochem. Biophys. Res. Commun. 211, 396-403 [CrossRef][Medline] [Order article via Infotrieve]
  11. Sadahira, Y., Ruan, F., Hakomori, S., and Igarashi, Y. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 9686-9690 [Abstract]
  12. Bornfeldt, K. E., Graves, L. M., Raines, E. W., Igarashi, Y., Wayman, G., Yamamura, S., Yatomi, Y., Sidhu, J. S., Krebs, E. G., Hakomori, S., and Ross, R. (1995) J. Cell Biol. 130, 193-206 [Abstract]
  13. van Koppen, C. J., zu Heringdorf, D. M., Laser, K. T., Zhang, C., Jakobs, K. H., Bunemann, M., and Pott, L. (1996) J. Biol. Chem. 271, 2082-2087 [Abstract/Free Full Text]
  14. Choi, O. H., Kim, J.-H., and Kinet, J.-P. (1996) Nature 380, 634-636 [CrossRef][Medline] [Order article via Infotrieve]
  15. Postma, F. R., Jalink, K., Hengeveld, T., and Moolenaar, W. H. (1996) EMBO J. 15, 2388-2395 [Abstract]
  16. Yatomi, Y., Ruan, F., Hakomori, S., and Igarashi, Y. (1995) Blood 86, 193-202 [Abstract/Free Full Text]
  17. Stoffel, W., and Bister, K. (1973) Hoppe-Seyler's Z. Physiol. Chem. 354, 169-181 [Medline] [Order article via Infotrieve]
  18. Mattie, M., Brooker, G., and Spiegel, S. (1994) J. Biol. Chem. 269, 3181-3188 [Abstract/Free Full Text]
  19. Ghosh, T. K., Bian, J., and Gill, D. L. (1990) Science 248, 1653-1656 [Medline] [Order article via Infotrieve]
  20. Ghosh, T. K., Bian, J., and Gill, D. L. (1994) J. Biol. Chem. 269, 22628-22635 [Abstract/Free Full Text]
  21. Kindman, L. A., Kim, S., McDonald, T. V., and Gardner, P. (1994) J. Biol. Chem. 269, 13088-13091 [Abstract/Free Full Text]
  22. Mao, C., Kim, S. H., Almenoff, J. S., Rudner, X. L., Kearney, D. M., and Kindman, L. A. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 1993-1996 [Abstract/Free Full Text]
  23. Goodemote, K. A., Mattie, M. E., Berger, A., and Spiegel, S. (1995) J. Biol. Chem. 270, 10272-10277 [Abstract/Free Full Text]
  24. Okajima, F., Tomura, H., Sho, K., Nochi, H., Tamoto, K., and Kondo, Y. (1996) FEBS Lett. 379, 260-264 [CrossRef][Medline] [Order article via Infotrieve]
  25. Moolenaar, W. H. (1995) J. Biol. Chem. 270, 12949-12952 [Free Full Text]
  26. Durieux, M. E., and Lynch, K. R. (1993) Trends Pharmacol. Sci. 14, 249-254 [CrossRef][Medline] [Order article via Infotrieve]
  27. Van Veldhoven, P. P., Foglesong, R. J., and Bell, R. M. (1989) J. Lipid Res. 30, 611-616 [Abstract]
  28. Vunnam, R. R., and Radin, N. S. (1979) Biochim. Biophys. Acta 573, 73-82 [Medline] [Order article via Infotrieve]
  29. Igarashi, Y., Hakomori, S., Toyokuni, T., Dean, B., Fujita, S., Sugimoto, M., Ogawa, T., El-Ghendy, K., and Racker, E. (1989) Biochemistry 28, 6796-6800 [Medline] [Order article via Infotrieve]
  30. Mazurek, N., Megidish, T., Hakomori, S., and Igarashi, Y. (1994) Biochem. Biophys. Res. Commun. 198, 1-9 [CrossRef][Medline] [Order article via Infotrieve]
  31. Ohta, H., Ruan, F., Hakomori, S., and Igarashi, Y. (1994) Anal. Biochem. 222, 489-494 [CrossRef][Medline] [Order article via Infotrieve]
  32. Yatomi, Y., Ruan, F., Ohta, H., Welch, R. J., Hakomori, S., and Igarashi, Y. (1995) Anal. Biochem. 230, 315-320 [CrossRef][Medline] [Order article via Infotrieve]
  33. Zucker, M. B. (1989) Methods Enzymol. 169, 117-133 [Medline] [Order article via Infotrieve]
  34. Ruan, F., Yamamura, S., Hakomori, S., and Igarashi, Y. (1995) Tetrahedron Lett. 36, 6615-6618 [CrossRef]
  35. Tyers, M., Rachubinski, R. A., Stewart, M. I., Varrichio, A. M., Shorr, R. G. L., Haslam, R. J., and Harley, C. B. (1988) Nature 333, 470-473 [CrossRef][Medline] [Order article via Infotrieve]
  36. Yatomi, Y., Hazeki, O., Kume, S., and Ui, M. (1992) Biochem. J. 285, 745-751 [Medline] [Order article via Infotrieve]
  37. Sato, T., Fuse, A., Eguchi, M., Hayashi, Y., Ryo, R., Adachi, M., Kishimoto, Y., Teramura, M., Mizoguchi, H., Shima, Y., Komori, I., Sunami, S., Okimoto, Y., and Nakajima, H. (1989) Br. J. Haematol. 72, 184-190 [Medline] [Order article via Infotrieve]
  38. Cheng, T., Wang, Y., and Dai, W. (1994) J. Biol. Chem. 269, 30848-30853 [Abstract/Free Full Text]
  39. Stoffel, W., Assmann, G., and Binczek, E. (1970) Hoppe-Seyler's Z. Physiol. Chem. 351, 635-642 [Medline] [Order article via Infotrieve]
  40. Stoffel, W., Heimann, G., and Hellenbroich, B. (1973) Hoppe-Seyler's Z. Physiol. Chem. 354, 562-566 [Medline] [Order article via Infotrieve]
  41. Siess, W. (1989) Physiol. Rev. 69, 58-178 [Free Full Text]
  42. Yatomi, Y., Ruan, F., Megidish, T., Toyokuni, T., Hakomori, S., and Igarashi, Y. (1996) Biochemistry 35, 626-633 [CrossRef][Medline] [Order article via Infotrieve]
  43. Stoffel, W., Hellenbroich, B., and Heimann, G. (1973) Hoppe-Seyler's Z. Physiol. Chem. 354, 1311-1316 [Medline] [Order article via Infotrieve]
  44. Durieux, M. E., Carlisle, S. J., Salafranca, M. N., and Lynch, K. R. (1993) Am. J. Physiol. 264, C1360-C1364 [Abstract/Free Full Text]
  45. Benton, A. M., Gerrard, J. M., Michiel, T., and Kindom, S. E. (1982) Blood 60, 642-649 [Medline] [Order article via Infotrieve]
  46. Nishizuka, Y. (1984) Nature 308, 693-698 [Medline] [Order article via Infotrieve]
  47. Tamaoki, T. (1991) Methods Enzymol. 201, 340-347 [Medline] [Order article via Infotrieve]
  48. Olivera, A., Zhang, H., Carlson, R. O., Mattie, M. E., Schmidt, R. R., and Spiegel, S. (1994) J. Biol. Chem. 269, 17924-17930 [Abstract/Free Full Text]
  49. Neer, E. J. (1995) Cell 80, 249-257 [Medline] [Order article via Infotrieve]
  50. Eichholtz, T., Jalink, K., Fahrenfort, I., and Moolenaar, W. H. (1993) Biochem. J. 291, 677-680 [Medline] [Order article via Infotrieve]
  51. Jalink, 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., and Moolenaar, W. H. (1995) Biochem. J. 307, 609-616 [Medline] [Order article via Infotrieve]
  52. Hawiger, J. (1989) Methods Enzymol. 169, 191-195 [Medline] [Order article via Infotrieve]
  53. Kikkawa, U., Takai, Y., Minakuchi, R., Inohara, S., and Nishizuka, Y. (1982) J. Biol. Chem. 257, 13341-13348 [Free Full Text]
  54. Scatchard, G. (1949) Ann. NY Acad. Sci. 51, 660-672

©1997 by The American Society for Biochemistry and Molecular Biology, Inc.