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Integrin ?IIb-mediated PI3K/Akt activation in platelets.

ABSTRACT: Integrin ?IIb?3 mediated bidirectional signaling plays a critical role in thrombosis and haemostasis. Signaling mediated by the ?3 subunit has been extensively studied, but ?IIb mediated signaling has not been characterized. Previously, we reported that platelet granule secretion and TxA2 production induced by ?IIb mediated outside-in signaling is negatively regulated by the ?3 cytoplasmic domain residues R(724)KEFAKFEEER(734). In this study, we identified part of the signaling pathway utilized by ?IIb mediated outside-in signaling. Platelets from humans and gene deficient mice, and genetically modified CHO cells as well as a variety of kinase inhibitors were used for this work. We found that aggregation of TxA2 production and granule secretion by ?3?724 human platelets initiated by ?IIb mediated outside-in signaling was inhibited by the Src family kinase inhibitor PP2 and the PI3K inhibitor wortmannin, respectively, but not by the MAPK inhibitor U0126. Also, PP2 and wortmannin, and the palmitoylated ?3 peptide R(724)KEFAKFEEER(734), each inhibited the phosphorylation of Akt residue Ser473 and prevented TxA2 production and storage granule secretion. Similarly, Akt phosphorylation in mouse platelets stimulated by the PAR4 agonist peptide AYPGKF was ?IIb?3-dependent, and blocked by PP2, wortmannin and the palmitoylated peptide p-RKEFAKFEEER. Akt was also phosphorylated in response to mAb D3 plus Fg treatment of CHO cells in suspension expressing ?IIb?3-?724 or ?IIb?3E(724)AERKFERKFE(734), but not in cells expressing wild type ?IIb?3. In summary, SFK(s) and PI3K/Akt signaling is utilized by ?IIb-mediated outside-in signaling to activate platelets even in the absence of all but 8 membrane proximal residues of the ?3 cytoplasmic domain. Our results provide new insight into the signaling pathway used by ?IIb-mediated outside-in signaling in platelets.

SUBMITTER: Niu H

PROVIDER: S-EPMC3474815 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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Integrin αIIb-mediated PI3K/Akt activation in platelets.

Niu Haixia H Chen Xue X Gruppo Ralph A RA Li Ding D Wang Yanhua Y Zhang Lin L Wang Kemin K Chai Weiran W Sun Yueping Y Ding Zhongren Z Gartner T Kent TK Liu Junling J

PloS one 20121017 10

Integrin αIIbβ3 mediated bidirectional signaling plays a critical role in thrombosis and haemostasis. Signaling mediated by the β3 subunit has been extensively studied, but αIIb mediated signaling has not been characterized. Previously, we reported that platelet granule secretion and TxA2 production induced by αIIb mediated outside-in signaling is negatively regulated by the β3 cytoplasmic domain residues R(724)KEFAKFEEER(734). In this study, we identified part of the signaling pathway utilized ...[more]

PMID: 23082158

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Project description:Intestinal epithelial (IEC-6) cells are resistant to apoptosis following the inhibition of ODC (ornithine decarboxylase) and subsequent polyamine depletion. The depletion of polyamines rapidly activates NF-kappaB (nuclear factor kappaB) and STAT3 (signal transducer and activator of transcription 3), which is responsible for the observed decrease in apoptosis. Since both NF-kappaB and STAT3 signalling pathways can be activated by Src kinase, we examined its role in the antiapoptotic response. Inhibition of ODC by DFMO (alpha-difluoromethylornithine) increased the activity of Src and ERK1/2 (extracellular-signal-regulated kinase 1/2) within 30 min, which was prevented by exogenous polyamines added to the DFMO-containing medium. Conversely, epidermal growth factor-mediated Src and ERK1/2 activation was not prevented by the addition of polyamines. Inhibition of Src with PP2 {4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine} and a DN-Src (dominant-negative Src) construct prevented the activation of Akt, JAK (Janus kinase) and STAT3. Spontaneous apoptosis was increased in DN-Src-expressing cells and the protective effect of polyamine depletion was lost. Polyamine depletion by DFMO increased integrin beta3 Tyr785 phosphorylation. Cells plated on fibronectin had significantly higher beta3 phosphorylation and Src activation compared with plastic. Exogenous polyamines added to the fibronectin matrix prevented Src activation. Arg-Gly-Asp-Ser inhibited beta3, Src and Akt phosphorylation and sensitized polyamine-depleted cells to tumour necrosis factor alpha/cycloheximide-mediated apoptosis. Fibronectin activated Src and subsequently protected cells from apoptosis. Together, these results suggest that the inhibition of ODC rapidly removes a small pool of available polyamines triggering the activation of beta3 integrin, which in turn activates Src. The subsequent Akt and JAK activation is accompanied by translocation of NF-kappaB and STAT3 to the nucleus and the synthesis of antiapoptotic proteins.

Dataset Information

Integrin ?IIb-mediated PI3K/Akt activation in platelets.

Publications

Integrin αIIb-mediated PI3K/Akt activation in platelets.

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