Aging is one of the greatest risk factors for thrombotic diseases such as deep vein thrombosis, myocardial infarction and stroke. Platelets are central to thrombus formation through their activation and aggregation. Prior studies show age-dependent increases in platelet activation, but the molecular mechanisms remain unknown. Though platelets are anucleate they contain functional mitochondria and an active ubiquitin-proteosome system (UPS). The UPS removes old and damaged proteins to maintain cellular homeostasis. Our preliminary data suggests that platelets isolated from healthy aged (>75 years) human subjects show increased UPS activity and decreased expression of the key mitochondrial fusion protein mitofusin 1 (MFN1) compared to platelets isolated from healthy young subjects (18-35 years). Platelets from aged individuals also exhibited increased mitochondrial oxidant production and platelet activation. Based on these observations, we will test the hypothesis that age-dependent changes in the UPS lead to increased mitochondrial dysfunction resulting in mitochondrial reactive oxygen species (mtROS) production to stimulate platelet activation and subsequent thrombosis.
In Aim 1, we will characterize the UPS in isolated platelets from young and aged healthy human subjects. Concomitant MFN1 levels and platelet activation measurements will be made to determine how these systems are altered by the age-related changes in the UPS. To establish a murine model for molecular mechanism, we will perform identical measurements in young (8-10 week) and aged (>1.5 year) wildtype (WT) mice. These studies will be complemented by an in vitro cell culture system of human derived CD34+ progenitor cells which can be differentiated into platelets and used for mechanistic studies that utilize lentivirus to specifically target the UPS components.
In Aim 2, we generated platelet-specific MFN1 knockout (KO) mice to determine whether decreased MFN1 in platelets results in increased mtROS and susceptibility to thrombosis. We will measure changes in platelet morphology, mtROS production, and mitochondrial bioenergetics in young and aged platelet-specific MFN1 KO mice and compare it to young and aged WT mice. Adenovirus will be used to overexpress MFN1 in old WT and platelet-specific MFN1 KO mice to determine if it rescues the aged phenotype. Additionally, young and aged WT and platelet-specific MFN1 (KO) mice will be subject to laser-induced vascular injury to determine if platelet activation and thrombosis susceptibility increases when MFN1 is absent. Lentivirus knockdown and overexpression of MFN1 in our CD34+ cell culture system will complement these mechanistic studies. Finally, Aim 3 will determine whether the clinically used drugs MitoQ (mtROS scavenger) or Bortezomib (proteasome inhibitor) can attenuate age-dependent platelet activation and thrombosis in WT and platelet-specific MFN1 KO murine models. Successful completion of this project will unveil a novel mechanism by which platelet activation and thrombosis occur during healthy aging and new strategies to repurpose clinically available drugs to mitigate age-related thrombosis.
This study will investigate why the incidence of thrombotic diseases, such as myocardial infarction and stroke, increase as people age. It is known that platelets are central to thrombus formation and have been shown to become dysfunctional with age thus causing increased thrombosis; however, the mechanisms driving this age- related process are not understood. This study will identify how platelet mitochondrial function and protein turnover rate change with age, leading to platelet dysfunction, as well as identify novel drug targets and test potential therapeutics targeting these processes, which could prevent age-dependent platelet dysfunction and thrombosis.
