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Published on: June 5, 2019
mTOR regulates GPVI-mediated platelet activation
Longsheng Wang1, Gang Liu1,2, Nannan Wu1
1Chronic Disease Research Institute, Department of Nutrition and Food Hygiene, Zhejiang University School of Public Health, 866 Yu-Hang-Tang Road, Hangzhou, 310058, China.
Background:
Due to mTOR (mammalian/mechanistic target of rapamycin) gene-loss mice die during embryonic development, the role of mTOR in platelets has not been evaluated using gene knockout technology.
Methods:
A mouse model with megakaryocyte/platelet-specific deletion of mTOR was established, and be used to evaluate the role of mTOR in platelet activation and thrombus formation.
Results:
mTOR-/- platelets were deficient in thrombus formation when grown on low-concentration collagen-coated surfaces; however, no deficiency in thrombus formation was observed when mTOR-/- platelets were perfused on higher concentration collagen-coated surfaces. In FeCl3-induced mouse mesenteric arteriole thrombosis models, wild-type (WT) and mTOR-/- mice displayed significantly different responses to low-extent injury with respect to the ratio of occluded mice, especially within the first 40 min. Additionally, mTOR-/- platelets displayed reduced aggregation and dense granule secretion (ATP release) in response to low doses of the glycoprotein VI (GPVI) agonist collagen related peptide (CRP) and the protease-activated receptor-4 (PAR4) agonist GYPGKF-NH2; these deficiencies were overcame by stimulation with higher concentration agonists, suggesting dose dependence of the response. At low doses of GPVI or PAR agonist, the activation of αIIbβ3 in mTOR-/- platelets was reduced. Moreover, stimulation of mTOR-/- platelets with low-dose CRP attenuated the phosphorylation of S6K1, S6 and Akt Ser473, and increased the phosphorylation of PKCδ Thr505 and PKCε Ser729. Using isoform-specific inhibitors of PKCs (δ, ɛ, and α/β), we established that PKCδ/ɛ, and especially PKCδ but not PKCα/β or PKCθ, may be involved in low-dose GPVI-mediated/mTOR-dependent signaling.
Conclusion:
These observations indicate that mTOR plays an important role in GPVI-dependent platelet activation and thrombus formation.
Insights
Mammalian target of rapamycin (mTOR) is crucial for platelet activation and thrombus formation, particularly in response to low-dose agonists. Its absence impairs platelet aggregation and dense granule secretion, highlighting mTOR
Area of Science:
- Hematology
- Molecular Biology
- Biochemistry
Background:
- Mammalian target of rapamycin (mTOR) gene-loss in mice causes embryonic lethality, preventing direct study of mTOR's role in platelets via knockout.
- Previous research has not fully elucidated the function of mTOR in platelet activation and thrombus formation due to genetic limitations.
Purpose of the Study:
- To investigate the role of mTOR in platelet activation and thrombus formation using a novel megakaryocyte/platelet-specific mTOR knockout mouse model.
- To determine the impact of mTOR deletion on platelet aggregation, dense granule secretion, and in vivo thrombosis.
Main Methods:
- Generation of a mouse model with megakaryocyte/platelet-specific deletion of mTOR.
- Evaluation of thrombus formation in vitro using low and high concentration collagen-coated surfaces.
- Assessment of FeCl3-induced mesenteric arteriole thrombosis in vivo.
- Analysis of platelet aggregation, dense granule secretion (ATP release), and αIIbβ3 activation in response to various agonists.
- Investigation of signaling pathways, including S6K1, S6, Akt, and PKC phosphorylation, in mTOR-deficient platelets.
Main Results:
- mTOR-deficient platelets showed impaired thrombus formation on low-concentration collagen but not on high-concentration surfaces.
- In vivo thrombosis models revealed significantly different responses in mTOR-deficient mice to low-extent injury.
- Reduced platelet aggregation and ATP release were observed in response to low doses of GPVI and PAR4 agonists, with dose-dependent recovery.
- Low-dose agonist stimulation of mTOR-deficient platelets resulted in decreased αIIbβ3 activation and altered phosphorylation of key signaling molecules (S6K1, S6, Akt, PKCδ, PKCε).
Conclusions:
- mTOR plays a critical role in glycoprotein VI (GPVI)-dependent platelet activation and thrombus formation.
- The findings suggest a dose-dependent involvement of mTOR in platelet function, particularly under conditions of low agonist stimulation.
- PKCδ and PKCε, especially PKCδ, are implicated in low-dose GPVI-mediated and mTOR-dependent platelet signaling.
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