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1201 Mitochondrial Fission Protein Drp1 Regulates Platelet Function

Program: Oral and Poster Abstracts
Session: 301. Vasculature, Endothelium, Thrombosis and Platelets: Basic and Translational: Poster I
Hematology Disease Topics & Pathways:
Research, Fundamental Science
Saturday, December 9, 2023, 5:30 PM-7:30 PM

Shancy Jacob1*, Marina Tistao, PhD1*, Abigail Ajanel Gomez, MS, BS1, Frederik Denorme, PhD, MSc1,2, Yasuhiro Kosaka, PhD1*, Bhanu Kanth Manne, PhD1*, Li Guo, MD, PhD3, Robert A. Campbell, PhD1,4,5, Matthew T. Rondina, MD1,4,6,7,8 and Jesse W Rowley, PhD1,6

1Molecular Medicine Program, University of Utah, Salt Lake City, UT
2Department of Neurology, Division of Vascular Neurology, University of Utah, Salt Lake City, UT
3Bloodworks Research Institute, Hematology Division, University of Washington, Salt Lake City, UT
4Department of Pathology, University of Utah, Salt Lake City, UT
5Division of Hematology and Hematologic Malignancies, Department of Internal Medicine, University of Utah School of Medicine, University of Utah, Salt Lake City, UT
6Division of Respiratory, Critical Care, and Occupational Pulmonary Medicine, Department of Internal Medicine, University of Utah School of Medicine, Salt Lake City, UT
7Molecular Medicine Program, Department of Internal Medicine, and Division of Hematology and Hematologic Malignancies, University of Utah, Salt Lake City, UT
8Department of Internal Medicine and Geriatric Research and Education Clinical Center, George E. Wahlen VAMC, Salt Lake City, UT

Background: Mitochondria in platelets contribute to platelet function and are altered by diseases that involve dysregulated hemostasis or thrombosis. Mitochondrial integrity, governed by its size, number and distribution is modulated by fusion and fission proteins. Pharmacological inhibition of the mitochondrial fission protein Dynamin-related protein 1 (Drp1) has been shown to inhibit granule release in human platelets and impair thrombus formation in a murine model of thrombosis. However, the mitochondrial roles for Drp1 in platelet function are still largely unknown.

Objective: To determine the mitochondrial roles of Drp1 in regulating platelet function.

Methods: PF4-Cre mediated MK/platelet specific Drp1 null mice (Drp1-/-) were used. Platelet morphology in Drp1-/- and wild type (WT) platelets was analyzed by confocal microscopy. Procoagulant platelet formation (PS+ve platelets) after thrombin and the GPVI agonist convulxin (THR+CVX) stimulation was measured using Annexin V staining. Mitochondrial potential (ΔΨm) and mass at baseline and after stimulation was measured using TMRM and mitotracker green. Washed platelet activation in WT and Drp1-/- platelets in response to convulxin was measured by flow cytometry. Platelet cytoplasmic calcium transients were measured using FURA-2AM after stimulation with THR+CVX. Phosphorylation of Drp1 (pDrp1 S616) in washed WT platelets in response to dual agonist THR+CVX was measured by western blotting.

Results: Drp1-/- platelets were bigger in size than WT platelets as measured by microscopy and had increased mitochondrial mass as determined by flow cytometry. Absence of Drp1 reduced activation of the αIIb/β3 fibrinogen binding receptor (P=0.035; n=5-6 per group) and reduced alpha granule release (P=0.025; n=5-6 per group) in response to CVX.Dual agonist stimulation resulted in reduced PS+ve platelets in Drp1-/- compared to WT platelets (P=0.0035; n=7 per group). Since depolarization precedes PS exposure, we measured ΔΨm using TMRM in resting and dual agonist stimulated WT and Drp1-/- platelets. No difference in ΔΨm was observed in resting Drp1-/- vs WT platelets whereas, stimulated Drp1-/- platelets exhibited significantly higher ΔΨm (P=0.038; n=4 per group) than stimulated WT platelets. Interestingly, in response to dual agonists, Drp1-/- platelets also showed a significant increase (P<0.001) in cytoplasmic calcium transients than WT platelets. Western blot analyses of washed WT platelets stimulated with dual agonist showed phosphorylation of Drp1 at S616 (S616), indicating Drp1 activation.

Conclusion: These findings highlight a role for Drp1 in murine platelet mitochondrial function, GPVI activation, and procoagulant platelet formation.

Disclosures: No relevant conflicts of interest to declare.

*signifies non-member of ASH