SDF-1α/CXCR4 Signaling in Lipid Rafts Induces Platelet Aggregation via PI3 Kinase-Dependent Akt Phosphorylation

Stromal cell-derived factor-1α (SDF-1α)-induced platelet aggregation is mediated through its G protein-coupled receptor CXCR4 and phosphatidylinositol 3 kinase (PI3K). Here, we demonstrate that SDF-1α induces phosphorylation of Akt at Thr308 and Ser473 in human platelets. SDF-1α-induced platelet aggregation and Akt phosphorylation are inhibited by pretreatment with the CXCR4 antagonist AMD3100 or the PI3K inhibitor LY294002. SDF-1α also induces the phosphorylation of PDK1 at Ser241 (an upstream activator of Akt), GSK3β at Ser9 (a downstream substrate of Akt), and myosin light chain at Ser19 (a downstream element of the Akt signaling pathway). SDF-1α-induced platelet aggregation is inhibited by pretreatment with the Akt inhibitor MK-2206 in a dose-dependent manner. Furthermore, SDF-1α-induced platelet aggregation and Akt phosphorylation are inhibited by pretreatment with the raft-disrupting agent methyl-β-cyclodextrin. Sucrose density gradient analysis shows that 35% of CXCR4, 93% of the heterotrimeric G proteins Gαi-1, 91% of Gαi-2, 50% of Gβ and 4.0% of PI3Kβ, and 4.5% of Akt2 are localized in the detergent-resistant membrane raft fraction. These findings suggest that SDF-1α/CXCR4 signaling in lipid rafts induces platelet aggregation via PI3K-dependent Akt phosphorylation.


Introduction
Lipid rafts are dynamic assemblies of sphingolipids, cholesterol, and proteins that can be stabilized into platforms involved in the regulation of cellular proliferation and differentiation [1]. Lipid rafts have been implicated in signal transduction because various signaling molecules, such as heterotrimeric G proteins, are associated with them [2,3]. A number of studies provide a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 considerable evidence that rafts are integral to the regulation of immune and neuronal signalings. Lipid rafts are also involved in hemostasis and thrombosis. Among blood cells, platelets are critical for maintaining the integrity of the blood coagulation system. Platelet rafts are critical membrane domains in physiological responses such as adhesion and aggregation [4]. The localization of the adhesion receptor GPIb-IX-V complex to membrane rafts is required for platelet adhesion to the vessel wall by binding the von Willebrand factor [5]. Lipid rafts are required for platelet aggregation via the collagen receptor GPVI [6,7], the ADP receptor P2Y12 [8], the Fcγ receptor FcγRIIa [9], and the thromboxane A 2 receptor [10]. Recently, we have reported that lipid rafts are also required for fibrin clot retraction [11]. Fibrin is translocated to lipid rafts of thrombin-stimulated platelets, and lipid rafts act as platforms where extracellular fibrin and intracellular actomyosin efficiently join via integrin αIIbβ3 to promote outside-in signals, leading to clot retraction.
Platelets are a major source of the chemokine stromal cell-derived factor-1α (SDF-1α, also termed CXCL12), which is stored as part of their α-granule secretome. Platelet-derived SDF-1α modulates paracrine mechanisms such as chemotaxis and differentiation [12]. Plateletderived SDF-1αenhances recruitment of hematopoietic progenitor cells to the sites of vascular injury and supports their differentiation to endothelial progenitor cells in vivo to facilitate vascular remodeling and repair. Platelet-derived SDF-1αis also an autocrine activator of platelets operating through its receptor CXCR4 [13][14][15][16]. Pertussis toxin inhibits SDF-1α-induced platelet aggregation, suggesting that its effect is mediated by a pertussis-toxin-sensitive G protein such as Gαi. SDF-1α induces platelet aggregation in a phosphatidylinositol 3 kinase (PI3K)dependent manner [17]. SDF-1αis highly expressed in atherosclerotic plaques [17], suggesting that SDF-1α-induced platelet aggregation contributes to the pathogenesis of atherosclerosis. Surface expression of SDF-1α on platelets is a prospective biomarker in ischemic events [12]. The SDF-1α expression level on platelets is elevated in patients with acute myocardial infarction [18], acute coronary syndrome [19,20], coronary artery disease [21], valvular aortic stenosis [22], and congestive heart failure [23].
Both SDF-1α and its heterotrimeric G protein-coupled receptor CXCR4 are expressed in the developing cerebellum. SDF-1αalso triggers the chemoattraction of cerebellar granule cells [24]. Previously, we reported that SDF-1α induces transmembrane signaling into cerebellar granule cells via lipid rafts [25]. In this study, we demonstrated that lipid rafts are involved in platelet aggregation and Akt phosphorylation via SDF-1α.

Platelet preparation
Blood was collected into test tubes with 3.8% sodium citrate at a ratio of 9:1. The blood was centrifuged (140 x g) to prepare platelet-rich plasma (PRP

Measurement of platelet aggregation
Platelet aggregation was measured as the optical density of PRP during aggregation using a platelet aggregometer (NBS HEMA TRACER 601), with the platelet-poor plasma 100% standard for change in optical density.

Sucrose density gradient analysis
Lipid rafts were obtained as detergent-resistant membranes as previously described [11] with some modifications. Platelets (6 x 10 8 ) were homogenized using a Teflon glass homogenizer in 2 ml of TNE/Triton buffer (0.05% Triton X-100, 25 mM Tris-HCl, pH 7.5, 150 mM NaCl, and 1 mM EGTA). The sucrose content of the homogenate was then adjusted to 40% by adding 80% sucrose, placed at the bottom of an ultracentrifuge tube, and overlaid with 35% and then 5% sucrose in 2 ml of TNE without Triton X-100. The discontinuous gradient was centrifuged for 17 h at 39,000 rpm at 4˚C in a Hitachi RPS40T rotor (Tokyo, Japan). Lipid rafts accumulating at the 5-35% sucrose interface were carefully collected. The detergent-resistant membrane raft fraction and non-raft fraction (40% sucrose fraction) were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting (n = 2). The percentage of the total in the raft fraction was estimated by densitometry using ImageJ.

Statistical analyses
Densitometric data were presented as the mean+/-SD of triplicates and analyzed using oneway analysis of variance (ANOVA) followed by a Tukey-Kramer post hoc test in Supporting Information. P values <.05 were considered statistically significant.

SDF-1α-induced platelet aggregation is mediated by Akt
SDF-1α induced platelet aggregation in a dose-dependent manner (Fig 1A). The SDF-1αinduced platelet aggregation was inhibited by pretreatment with AMD3100, a CXCR4 antagonist ( Fig 1B). Pretreatment with LY294002, a PI3K inhibitor, also inhibited the SDF-1αinduced platelet aggregation (Fig 1C). These observations suggest that SDF-1α-induced platelet aggregation is mediated by the SDF-1α receptor CXCR4 and PI3K, which is consistent with previous findings [17]. Serine/threonine kinase Akt (also termed protein kinase B) is activated by G protein-coupled receptors that induce the production of phosphatidylinositol (3,4,5) trisphosphate (PIP 3 ) by PI3K [26] These lipids serve as plasma membrane docking sites for pleckstrin-homology domain containing proteins, such as Akt or its upstream activator PDK1. Akt is activated by phosphorylation at Thr308 in the kinase domain by PDK1 [27] and by phosphorylation at Ser473 in the carboxy terminal domain. Akt activation in platelets depends on heterotrimeric G protein Gi signaling pathways [28]. Moreover, a recent report shows that there is also a novel PIP 3 -independent and Gq-dependent Akt translocation mechanism in platelets [29]. Treatment of human platelets with SDF-1α induced the phosphorylation of Akt at Thr308 and Ser473 (Fig 2A). The phosphorylation was dose-dependent (Fig 2Ba, 2Bb and 2Bc) and transient (Fig 2Bd, 2Be and 2Bf) on SDF-1α treatment (Table A in S1 File). The SDF-1α-induced Akt phosphorylation at Thr308 (Fig 2Ca and 2Cb) and Ser473 (Fig 2Ca and 2Cc) were inhibited by pretreatment with AMD3100 ( Fig 2C lane 3), or LY294002 (Fig 2C lane 4) (Table B in S1 File). Furthermore, SDF-1α-induced platelet aggregation was inhibited by pretreatment with MK-2206, an Akt inhibitor, in a dose-dependent manner (Fig 2D). These observations suggest that SDF-1 α-induced platelet aggregation is mediated via Akt activation. Consistent with this idea, SDF-1α induced phosphorylation of PDK1 on the activation loop Ser241 and GSK3β at Ser9 (a downstream substrate of Akt [27]) and myosin light chain (MLC) at Ser19 (a downstream element of the PI3K/Akt signaling pathway in platelets [30]) (Fig 3A). The SDF-1α-induced phosphorylation of GSK3β, but not PDK1 and MLC, was inhibited by pretreatment with MK-2206 ( Fig 3B). The MLC phosphorylation might be due to multiple signaling pathways. Lipid rafts are involved in SDF-1α-induced platelet aggregation and Akt phosphorylation Lipid rafts are involved in signal transduction by several G protein-coupled receptors such as the thrombin receptor PAR1, the ADP receptor P2Y12 and the thromboxane A 2 receptor in platelets [31,8,10]. The pretreatment of platelets with the cholesterol-depleting and raft-disrupting agent MβCD [11] inhibited the SDF-1α-induced platelet aggregation ( Fig 4A) and Akt phosphorylation at Thr308 (Fig 4B and 4C), Ser473 (Fig 4B and 4D)(Table C in S1 File). These observations suggest that lipid rafts are mediated in SDF-1α-induced platelet aggregation and Akt activation. To investigate whether SDF-1α-mediated signaling molecules exist in lipid rafts, we isolated the lipid raft fraction by treating platelets with cold Triton X-100 and  2) and SDF-1α-treated platelets (lanes 3,4). The signals for PI3Kβ and Akt2 in the raft fractions were weakly identified. To determine clearly the presence of those proteins in the raft fraction, 10-fold increased amount of the resting and SDF-1α-treated raft fractions were also immunoblotted. The clear bands of PI3Kβ and Akt2, but not Akt1 and vinculin, were detected in the raft fractions of resting (lane 5) and SDF-1α-treated platelets (lane 6). The summary of the data obtained by densitometry: 35% and 13% of CXCR4, 93% and 93% of Gαi-1, 91% and 89% of Gαi-2, 50% and 62% of Gβ, 4.0% and 4.1% of PI3Kβ, 4.5% and 4.1% of Akt2 are localized in the raft fraction of resting and SDF-1α-treated platelets, respectively. Taken together, these results suggest that CXCR4-signaling by SDF-1α on lipid rafts leading to platelet aggregation occurs through PI3K-dependent Akt phosphorylation.

Discussion
In the present study, we demonstrated that the chemokine SDF-1α induces platelet aggregation and Akt phosphorylation at Thr308 and Ser473 through CXCR4 and PI3K. The SDF-1α-  induced platelet aggregation is inhibited by the Akt inhibitor MK-2206, suggesting that SDF-1 α-induced platelet aggregation is mediated by Akt activation. There is substantial evidence suggesting that Akt has an indirect role in regulating platelet aggregation [32]. Our observations suggest that SDF-1α induces Akt activation and phosphorylation of GSK3β, a putative Akt effector. Akt-mediated phosphorylation of GSK3β at Ser9 inhibits it's constitutive kinase activity. Mouse platelets lacking a single allele of GSK3β are hypersensitive to agonist-induced aggregation and secretion, suggesting that GSK3β plays a negative role in regulating platelet activation [33]. Therefore, SDF-1α-induced GSK3β phosphorylation at Ser9 might contribute to platelet aggregation. However, precise mechanism of SDF-1α-induced platelet aggregation via Akt remains to be explored.
Lipid rafts are the major determinant for correct chemokine receptor function [34]. Incorporation of CXCR4 into lipid rafts is responsible for the homing effect in human bone marrow CD34+ cells [35]. SDF-1αinduces CXCR4 association with flotillin-1 in lipid rafts of T cells [36]. Flotillin-1, a raft-associated integral membrane protein, a putative adapter protein recruiting signaling molecules. Down-regulation of flotillin-1 expression using RNAi technology inhibits SDF-1α/CXCR4 signaling and function. The PI3K/Akt pathway is compartmentalized within lipid rafts [37]. Production of PIP 3 in platelet rafts is critical in platelet activation [38]. Lipid rafts play a crucial role in triggering the Akt signaling pathway by facilitating Akt recruitment and activation in the plasma membrane [39]. Lipid raft disruption impairs the Akt signaling pathway in epidermal keratinocytes [40]. Suppressing the formation of raft-associated Akt inhibits SDF-1α-induced invasion of esophageal carcinoma cells [41]. In the present study, we demonstrated that SDF-1α-induced platelet aggregation and Akt phosphorylation are inhibited by the raft disrupting agent MβCD. Sucrose density gradient analysis showed that CXCR4, heterotrimeric G proteins, and part of PI3K, Akt are present in platelet detergent-resistant membrane rafts, suggesting that lipid rafts are involved in Akt-mediated platelet aggregation by SDF-1α.
We have investigated the functions of sphingolipids in raft-mediated signal transduction using platelets and neurons [2,3,11,25,[46][47][48][49][50][51][52]. SDF-1αinduces the activation and translocation of the heterotrimeric G protein Goα to ganglioside GD3-rich rafts, resulting in growth cone collapse of cerebellar granule neurons [25]. The growth cone collapse is prevented by the raft-disrupting agent MβCD. We also demonstrated that sphingomyelin in platelet rafts is involved in outside-in signals, leading to clot retraction [11]. Thrombin induces the translocation of fibrin to sphingomyelin-rich rafts of platelets and enhances outside-in signals, contributing to efficient clot retraction. Furthermore, clot retraction of sphingomyelin-rich raft-depleted platelets from sphingomyelin synthase knockout mice is delayed. The nature of sphingolipids involved in the SDF-1α/CXCR4 pathway in platelets is the subject of future research.
Supporting Information S1 File. Statistical analysis of dose-and time-dependent phosphorylation of Akt in platelets on SDF-1α treatment (Table A). Statistical analysis of effect of CXCR4 antagonist and PI3 kinase inhibitor on SDF-1α-induced Akt phosphorylation (Table B). Statistical analysis of inhibition of SDF-1α-induced Akt phosphorylation by raft disruption with methyl-β-cyclodextrin (Table C)