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

<div><p>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.</p></div

Inhibition of SDF-1α-induced platelet aggregation and Akt phosphorylation by raft disruption with methyl-β-cyclodextrin.

<p>(A) 500 ng/ml SDF-1α-induced platelet aggregation with or without pretreatment with 2% methyl-β-cyclodextrin (MβCD) for 15 min (2 donors, n = 4: a duplicated measurement from 2 donors on the same day). Bar represents 1 min. Western blotting (B) and measurement of Akt phosphorylation at Thr308 (C) and Ser473 (D) in resting platelets (lane 1), 500 ng/ml SDF-1α-treated platelets (lane 2), and 500 ng/ml SDF-1α-treated platelets with pretreatment with 2% MβCD for 15 min (lane 3). Phosphorylation was quantified by densitometry. Data are presented as the mean +/- SD of triplicates. **Statistically significant difference (<i>P</i>< .01).</p

SDF-1α-induced Akt phosphorylation at Thr308 and Ser473 and Akt-dependent platelet aggregation.

<p>(A) Washed human platelets were treated with 500 ng/ml SDF-1α for 10 min at 37^°C. Resting platelets (left lane) and SDF-1α-treated platelets (right lane) were lysed in the Laemmli sample buffer containing phosphatase inhibitors and separated by SDS-PAGE. Phosphorylations of Akt at Thr308 (upper panel), Akt at Ser473 (middle panel), and Akt protein (lower panel) were detected by western blotting using specific Akt antibodies. (B) Dose- and time-dependent phosphorylation of Akt in platelets on SDF-1α treatment. Washed human platelets were treated for 10 min at the indicated concentrations (a-c) or at 500 ng/ml SDF-1α for the indicated times (d-f). Cells were lysed in the Laemmli sample buffer containing phosphatase inhibitors and separated by SDS-PAGE. Phosphorylations of Akt at Thr308 and Ser473 were detected by western blotting (a,d). Phosphorylation of Akt at Thr308 (b,e) and Ser473 (c,f) was quantified by densitometry. Data are presented as the mean +/- SD of triplicates. Statistically significant differences (<i>P</i>< .05 shown by *, <i>P</i>< .01 shown by **). (C) Effect of CXCR4 antagonist and PI3 kinase inhibitor on SDF-1α-induced Akt phosphorylation. Western blotting (a) and measurement of Akt phosphorylation at Thr308 (b) and Ser473 (c) in resting platelets (lane 1), 500 ng/ml SDF-1α-treated platelets (lane 2), and 500 ng/ml SDF-1α-treated platelets with pretreatment with 5 μg/ml AMD3100 (lane 3), or 30 μM LY294002 (lane 4) for 10 min. Data are presented as the mean +/- SD of triplicates. **Statistically significant differences (<i>P</i>< .01). (D) Inhibition of SDF-1α-induced platelet aggregation by Akt inhibitor. SDF-1α(200 ng/ml)-induced platelet aggregation without or with pretreatment with 0.3, 1, or 3 μM MK-2206, an Akt inhibitor, for 10 min (2 donors, n = 3: a single measurement from one donor and a duplicate measurement from another donor on the same day). Bar represents 2 min.</p

SDF-1α-induced phosphorylation of PDK1, GSK3β, and myosin light chain in platelets.

<p>(A) Washed human platelets were treated with 500 ng/ml SDF-1α for 10 min. Resting platelets (left lane) and SDF-1α-treated platelets (right lane) were lysed in the Laemmli sample buffer containing phosphatase inhibitors and separated by SDS-PAGE. Phosphorylations of PDK1 at Ser241 (first panel) and GSK3β at Ser9 (second panel) and myosin light chain (MLC) at Ser19 (third panel) were detected with western blotting. Akt proteins were detected as loading controls (fourth panel). (B) 500 ng/ml SDF-1α-induced phosphorylations of PDK1, GSK3β, and MLC without (left lane) or with pretreatment with 3 μM MK-2206 (right lane).</p

Distribution of SDF-1α-mediated signaling molecules in raft and non-raft fractions.

<p>The raft (lanes 1,3) and non-raft (lanes 2,4) fractions and 10-fold increased amount of raft fractions (lanes 5,6) of resting (lanes 1,2,5) and SDF-1α-treated (lanes 3,4,6) platelets were subjected to SDS-PAGE. CXCR4, Gαi-1, Gαi-2, Gβ, PI3Kβ, Akt1, Akt2, and vinculin were detected by western blotting using specific antibodies. Lyn and flotillin-1 (Flot1) are marker proteins of lipid rafts [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169609#pone.0169609.ref053" target="_blank">53</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169609#pone.0169609.ref031" target="_blank">31</a>].</p