Abstract 119: Aging-related Activation Of Platelet Mtor Promotes Platelet Hyperreactivity And Thrombosis

Background: Aging is an independent risk factor for the development of cardiovascular disease. While aging is associated with increased thrombotic risk, mechanisms of platelet hyperactivation in aging remain undescribed. Aims: We examined the activation of intracellular signaling pathways in platelets from young and old human subjects and mice, where the mammalian target of rapamycin (mTOR) is hypothesized to be a mechanistic regulator of platelet hyperreactivity in aging. Methods: Platelets were isolated from healthy young (<45y) and older (>70y) human subjects. Platelet signaling was examined with quantitative mass spectrometry and Western blot. Platelet function was assessed with aggregometry. Platelet activation was also assessed in young (8-12 weeks) and aged (>18 months) wild-type (WT; mTOR fl/fl ) and platelet-specific mTOR knockout mice (KO; mTOR fl/fl -Pf4-Cre). Results: We observed significant changes in phosphorylation of substrates within and downstream of mTOR/S6K and PI3K/Akt signaling pathways in aged humans compared to young. Western blot analysis confirmed significant (p<0.01) basal activation of the mTOR pathway, including P-AKT, P-p70S6K and P-4EBP1 in both aged humans and mice. Additionally, aged human and murine platelets had significant greater aggregation compared to young controls. Inhibition of mTOR either with torin-1 in aged humans or genetic deletion in aged mice reversed platelet hyperreactivity. In a collagen-epinephrine thrombosis model, aged WT mice succumbed significantly (p<0.001) faster compared to young WT mice, while aged KO mice had similar times to death as young WT mice. Mechanistically, we observed an increase in RAC-GTP (p<0.0001) downstream of mTOR activation in aged WT mice. In addition, we observed increased mitochondrial reactive oxygen species (mtROS) in resting platelets in aged WT mice compared to young WT and young or aged KO mice (p<0.05). Increased ROS led to significantly more P-p38 and thromboxane generation in aged WT mice (p<0.01). P-p38 and thromboxane generation were unchanged in aged and young KO mice. Conclusions: Aging is associated with increased platelet mTOR activation and platelet hyperreactivity mediated through increased activation of p38 and thromboxane generation.