Inhibition Of Platelets MTORC1 Activity Retards The Formation Of Venous Thrombosis In Mice And Its Mechanism

Background:Global population aging is accelerating,which has been predicted to be a great challenge for our society in the twenty-first century.Aging is associated with increased incidence of venous thromboembolism diseases,such as deep vein thrombosis(DVT),resulting in significant morbidity and mortality in the elderly.Platelet hyperactivation is linked to aging-related venous thrombosis.However,the mechanisms through which aging enhances platelet activation and susceptibility to venous thrombosis are poorly understood at present.Methods and Results:1:To investigate the role of platelet mTORC1 in age-related venous thrombosis,we initially examined the relationship between age and platelet and MK mTORC1 activity in mice.Thrombin-induced platelet mTORCl activation P-P70S6K at threonine 389 and PS6 at serine 235/236 in aged(16-month-old)mice was significantly enhanced,as compared with that in young(4-month-old)mice.FACS analysis of bone marrow cells revealed that P-P70S6K and P-S6 were also increased in MKs of aged mice.These findings collectively suggest that mTORC1 activity in MKs and platelets increases with age in mice.2:Next,we examined the effects of rapamycin administration on experimental DVT in young(4-month-old)and aged(16-month old)C57BL/6 mice.We found that aged mice developed significantly larger and heavier IVC thrombi,as compared with young mice.Interestingly,4-month-old mice with 2 months of rapamycin treatment or not developed thrombi of similar lengths and weights.However,in 16-month-old mice,rapamycin protected against the age-dependent effect,as evident from the significantly smaller thrombi in treated animals relative to age-matched vehicles.Our findings suggest that inhibition of mTORC1 by rapamycin reduces the susceptibility of aged mice to experimental thrombosis.3:To identify the specific role(s)of platelet mTORCl activation in VTE,we generated mice with conditional ablation of the mTORC1-specific component,Raptor,in MKs and platelets.KO mice subjected to IVC ligation developed significantly smaller and lighter thrombi,as compared with their WT littermates.These results clearly implying that platelet Raptor deletion provides protection against stasis-induced VTE development in mice.4:To further ascertain the mechanism through which platelet mTORCl promotes venous thrombosis,we examined the effects of rapamycin on MPV.Both MPV and platelet count were markedly increased in 16-month-old mice,compared with 4-month-old mice.After rapamycin treatment of 2 months,MPV of aged,but not young,mice was significantly reduced.These findings indicate that specific inhibition of mTORCl via long-term administration of rapamycin can suppress MPV in mice.We also observed an age-dependent increase in the secretion of PF4 and P-selectin,a?bb3 activation,and platelet aggregation.Rapamycin treatment suppressed PF4 production,a?bb3 activation,and platelet aggregation in aged mice.Collectively,these findings demonstrate that inhibition of mTORC1 prevents age-related elevation of MK size and MPV and platelet activation in mice.5:To investigate the consequences of mTORCl inhibition specifically in MKs and platelets,we compared MKs and platelets formation and activation in Raptor-deletion and WT mice.We found that the average size of bone marrow MKs and MPV were smaller than that of WT mice.In addition,the activation of a?bb3,platelet aggregation,spreading,and secretion of PF4 were reduced in Raptor KO mice.These results suggest that dletion of mTORC1 leads to the generation of small-sized platelets,resulting in defects in production of thrombogenic factors and platelet activation.6:Next,we examined the potential mechanisms leading to aging dependent activation of mTORCl in MKs and platelets.Higher levels of ROS were observed both in platelets and in MKs from 16-month-old mice.H2O2 induced mTORCl activation in platelets from WT mice,but not in Raptor-null platelets,in vitro.To establish the correlations between ROS with mTORCl activation and platelet formation in vivo,aged mice were administered the ROS scavenger NAC for 2 months.Notably,platelet P-S6 levels,MPV,average MK size,PF4 level,a?bb3 activation,and venous thrombus size of 16-month-old mice were significantly reduced after NAC treatment.Based on these findings,we propose that ROS contribute to mTORCl-dependent elevation of MPV and platelet activation during aging.Conclusions:Taken together,our findings collectively demonstrate that ROS production with aging activates mTORC1 to elevate MPV and platelet activation and promote venous thrombosis.Elucidation the role of mTORC1 signaling pathway in venous thrombosis may provide new therapeutic strategies for the treatment of venous thromboembolism and other diseases involving platelet over-activation.