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2547 Declining Autophagic Flux Underlies Mitochondrial and Platelet Defects in Aging

Program: Oral and Poster Abstracts
Session: 301. Platelets and Megakaryocytes: Basic and Translational: Poster II
Hematology Disease Topics & Pathways:
Research, Fundamental Science, Translational Research
Sunday, December 8, 2024, 6:00 PM-8:00 PM

Guadalupe Rojas-Sanchez, PharmB, MSc, PhD1, Jorge Calzada-Martinez, MSc2*, Orlando Esparza, MD3*, Giovanny Hernandez, BA3*, Gabriela Dveksler4*, Ken Jones, PhD5*, Lucas Ting6*, Catherine Jankowski7*, Marguerite Kelher8*, Marilyn Manco-Johnson, MD9, Andrew Thorburn, PhD10*, Paola Maycotte, PhD11*, José A López, MD, BS12* and Pavel Davizon-Castillo, MD13

1Bloodworks NW Research Institute, Seattle, WA
2Bloodworks Northwest, Seattle, WA
3Department of Pediatrics Hematology/Oncology and Bone Marrow Transplantation, University of Colorado Anschutz Medical Campus, Aurora, CO
4Department of Pathology, Uniformed Services Univ of the Health Sciences, Bethesda, MD
5Bioinformatics Solutions, Sheridan, WY
6Stasys Medical, Seattle, WA
7University of Colorado College of Nursing, Aurora, CO
8Univ of Colorado Denver/Bonfils Blood Center, Denver, CO
9Hemophilia & Thrombosis Center, Department of Pediatrics, University of Colorado Anschutz Medical Campus, Aurora, CO
10Department of Pharmacology, University of Colorado Anschutz Medical Campus, Aurora, CO
11Centro de Investigación Biomédica de Oriente, OOAD Puebla, IMSS, Puebla, PU, Mexico
12Bloodworks Research Institute, Seattle, WA
13Research Institute, BloodWorks Northwest/University of Washington, Seattle, WA

Background: Aging is a time-dependent, accumulative, and irreversible decline in cellular functions. It is also a significant independent risk factor for thrombosis and, to a lesser extent, bleeding. We have previously demonstrated that in aged mice, a higher platelet mitochondrial mass is associated with hyperreactivity. Furthermore, scRNA-seq of native megakaryocytes from these mice revealed autophagy as one of the top differentially expressed pathways between young and old mice. Autophagy is a crucial catabolic process that removes damaged or long-lived cytoplasmic contents, facilitating cellular homeostasis and providing metabolic substrates essential for sustaining cellular metabolism. Mitophagy, a selective form of autophagy, is critical for eliminating damaged mitochondria and maintaining a pool of healthy mitochondria. Age-related declines in autophagy and mitophagy impair metabolic efficiency and result in the accumulation of damaged molecules and mitochondria, contributing to diseases such as neurodegeneration, metabolic disorders, and cancer. While declining autophagy is a hallmark of aging, its status in human platelets and the impact on their hemostatic functions during this process remain to be established. Given its role in regulating cellular metabolism and mitochondrial mass, we hypothesize that reduced autophagy and mitophagy in aging impair mitochondrial metabolism, thereby altering platelet function and potentially affecting hemostasis.

Methods: We studied platelets from healthy younger (<45 years) and older volunteers (>50 years), as well as from young (<3 months) and old (>18 months) mice. The autophagic and mitophagic fluxes were assessed using chloroquine (CQ) as per the current guidelines for monitoring autophagy. Platelet bioenergetics were analyzed with a Seahorse analyzer (Agilent). Thrombus formation was assessed with a microfluidics assay (T-TAS analyzer). Platelet contractile forces were quantified using the ATLAS system. Clot contraction was evaluated with a thrombin-based assay.

Results: Both autophagy and mitophagy were blocked in platelets from aged individuals, while platelets from younger adults exhibited robust flux (p<0.05, 0.05). Similar results were observed in mouse platelets (p<0.01, 0.05). To determine the metabolic impact of these differences, we evaluated the bioenergetic profiles of younger and older platelets. Compared to those from younger individuals, aged platelets displayed significantly decreased basal, maximal, and ATP-linked respiration (p<0.001, 0.05, 0.05). These metabolic abnormalities were accompanied by lower clot formation under flow conditions (p<0.01), decreased contractile forces (p<0.05), and reduced clot contraction (p<0.01). Pharmacological inhibition of these pathways in platelets from younger individuals with either CQ or EACC recapitulated the metabolic and functional deficits observed in platelets from older adults. These inhibitors significantly decreased mitochondrial respiration (p< 0.01, 0.05), clot formation (p<0.01, p<0.05), and contraction (p<0.05, 0.001).

Conclusion: This research is the first to demonstrate an age-related decline in autophagy and mitophagy in both human and mouse platelets, establishing a link between these deficits and metabolic as well as functional alterations critical for effective clot formation and contraction. Considering the association between impaired clot contraction and thrombotic and hemorrhagic events, these findings suggest that declining platelet autophagy and mitophagy may significantly contribute to the increased incidence of these events with aging. This work not only opens new avenues for understanding the role of platelets in hemostasis and thrombosis in aging but also paves the way for developing targeted interventions, such as modulators of these autophagic pathways, to mitigate these age-related complications.

Disclosures: Ting: Stasys: Current Employment. Manco-Johnson: BioMarin: Honoraria; CSL Behring: Honoraria; Genentech/Roche: Honoraria; NovoNordisk: Honoraria; Spark/Pfizer: Honoraria.

*signifies non-member of ASH