Aging-related alterations in mechanistic target of rapamycin signaling promote platelet hyperreactivity and thrombosis

Abstract

Background: Aging is an independent risk factor for the development of cardiovascular, thrombotic, and other chronic diseases. However, mechanisms of platelet hyperactivation in aging remain poorly understood.

Objectives: Here, we examine whether and how aging alters intracellular signaling in platelets to support platelet hyperactivity and thrombosis.

Methods: Quantitative mass spectrometry with tandem mass tag labeling systematically measured protein phosphorylation in platelets from healthy aged (>65 years) and young human (<45 years) subjects. The role of platelet mechanistic target of rapamycin (mTOR) in aging-induced platelet hyperreactivity was assessed using pharmacologic mTOR inhibition and a platelet-specific mTOR-deficient mouse model (mTOR^plt-/-).

Results: Quantitative phosphoproteomics uncovered differential site-specific protein phosphorylation within mTOR, Rho GTPase, and MAPK pathways in platelets from aged donors. Western blot confirmed constitutive activation of the mTOR pathway in platelets from both aged humans and mice, which was associated with increased aggregation compared with that in young controls. Inhibition of mTOR with either Torin 1 in aged humans or genetic deletion in aged mice reversed platelet hyperreactivity. In a collagen-epinephrine pulmonary thrombosis model, aged wild-type (mTOR^plt+/+) mice succumbed significantly faster than young controls, while time to death of aged mTOR^plt-/- mice was similar to that of young mTOR^plt+/+ mice. Mechanistically, we noted increased Rac1 activation and levels of mitochondrial reactive oxygen species in resting platelets from aged mice, as well as increased p38 phosphorylation upstream of thromboxane generation following agonist stimulation.

Conclusion: Aging-related changes in mTOR phosphorylation enhance Rac1 and p38 activation to enhance thromboxane generation, platelet hyperactivity, and thrombosis.

Keywords: aging; phosphoproteome; platelets; tandem mass tag; thrombosis.

Figure 1:. The mTOR pathway is constitutively activated in platelets from aged humans and mice.

(A) Resting, unstimulated platelets isolated from young (Y, <45 years, blue) and old (O, >65 years, red) humans were subjected to western blot analysis for p-Akt, p-p70S6K, and p-4E-BP1. Expression was normalized to β-actin. n=5 per group. **p<0.01. (B) Unstimulated platelets isolated from young (Y, 2–3 month, blue) and old (O, >18 months, red) wild-type mice were subjected to western blot analysis for p-Akt, p-p70S6K, and p-4E-BP1. Expression was normalized to β-actin. n=5 per group. **p<0.01, ***p<0.001 and ****p<0.0001

Figure 2.. Platelets from aged humans and mice are hyperreactive.

(A-C) Representative aggregation traces and maximal aggregation of washed platelets from young (<45 years, blue) and old (>65 years, red) humans stimulated with (A) 2MeSADP, (B) thrombin, or (C) collagen under stirring conditions at 37°C. n=3–5 per group. *p<0.05 and **p<0.01. (D-F) Representative aggregation traces and maximal aggregation of washed platelets from young (2–3 months, blue) and old (>18 months, red) C57BL/6 mice stimulated with (D) 2MeSADP, (E) thrombin, or (F) collagen under stirring conditions at 37°C. n=3–4 per group. *p<0.05 and **p<0.01.

Figure 3:. Inhibition of mTOR suppresses age-dependent platelet hyperreactivity and thrombosis.

(A) Platelets were isolated from young (<45 years, blue) and old (>65 years, red) humans and pre-incubated with vehicle (Veh, solid bars) or Torin 1 (100 nM, clear bars) before inducing aggregation with 2MeSADP. Torin 1 reduced aggregation similar to levels observed with platelets from young humans. n=3 per group. ***p<0.0001. (B) Representative aggregation traces of washed platelets from young (2–3 months, blue) and old (>18 months, red) wild-type (mTOR^plt+/+, solid bars) and platelet-specific mTOR-deleted (mTOR^plt−/−, clear bars) mice stimulated with 2MeSADP. n=4 per group. *p<0.05. (C-E) Platelet JON/A binding was measured in washed platelets by flow cytometry at baseline (BL) and after activation by the indicated platelet agonists 2MeSADP, PAR4-activating peptide (PAR4-AP, AYPGKF) and collagen-related peptide (CRP). *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001. (E) Kaplan-Meier survival curve for the pulmonary thrombosis model induced in young (2–3 months, blue) and old (>18 months, red) wild-type (mTOR^plt+/+, solid lines) and platelet-specific mTOR-deleted (mTOR^plt−/−, dashed lines) mice by intravenous injection of a mixture of collagen (0.4 mg/kg) and epinephrine (30 mg/kg) in 100 μL PBS. n=5–8 per group. Not significantly different between young mTOR^plt+/+ and aged mTOR^plt-/−. p=0.0023 between young mTOR^plt+/+ and mTOR^plt-/−.

Figure 4.. mTOR-dependent Rac1 activity promotes the generation of mitochondrial reactive oxygen species in resting platelets during aging.

Platelets were isolated from young (Y, 2–3 months, blue) and old (O, >18 months, red) wild-type (mTOR^plt+/+, solid bars) and platelet-specific mTOR-deleted (mTOR^plt−/−, clear bars) mice and analyzed for activation of downstream mTOR signaling pathways. (A) Rac1 activation was determined by an active Rac1 pull-down assay and normalized to total Rac1. n=3 per group (B) Washed platelets from young (blue) and old red) wild-type mice were pre-incubated with vehicle (Veh, solid bars) or EHT 1864 (50 μM, clear bars) before inducing aggregation with 2MeSADP. n = 4 per group. (C) Mitochondrial ROS generation in unstimulated platelets was quantified via flow cytometry with MitoSox Red. n=4 per group. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001

Figure 5.. Aging-dependent mTOR activation enhances p38 activation and thromboxane A₂ generation upon low dose agonist stimulation.

Platelets were isolated from young (Y, 2–3 months, blue) and old (O, >18 months, red) wild-type (mTOR^plt+/+, solid bars) and platelet-specific mTOR-deleted (mTOR^plt−/−, clear bars) mice and analyzed for activation of downstream mTOR signaling pathways. (A) Phosphorylation of p38 MAPK and (B) thromboxane (TxB₂) production were evaluated in both unstimulated and 2MeSADP-stimulated platelets. n=3 per group. **p<0.01, ***p<0.001, ****p<0.0001

Figure 6.. Proposed mechanism for mTOR-associated platelet hyperreactivity in aging.

Age-induced activation of the mTOR pathway in resting platelets from older subjects triggers basal activation of Rac1, resulting in the generation of mitochondrial reactive oxygen species (mtROS). Upon low dose agonist stimulation, p38 MAPK phosphorylation is further increased due to elevated basal levels of mtROS. This results in higher levels of thromboxane A₂ (TxA₂) production, amplifying platelet aggregation.