OC 68.5 - Platelet Drp1 phosphorylation is controlled by a dual phosphorylation pathway requiring dense granule release: Implications for evaluating antiplatelet therapy

Tuesday, June 25, 2024

15:45 – 16:00 ICT

Room: 211 A-D

Co-Author(s)

DB

David A. Barrios

Senior Scientist
PlateletDiagnostics, LLC
Watertown, Massachusetts, United States
SK

Secil Koseoglu

Scientist
Division of Hemostasis and Thrombosis, Beth Israel Deaconess Medical Center
Boston, Massachusetts, United States
SG

Shihui Guo

Scientist
Division of Hemostasis and Thrombosis, Beth Israel Deaconess Medical Center
Boston, Massachusetts, United States
SZ

Sabrina Zerbey

Clinical Research Assistant
Division of Hemostasis and Thrombosis, Beth Israel Deaconess Medical Center
Boston, Massachusetts, United States
MP

Matthew Powers

Research Assistant
PlateletDiagnostics
Watertown, Massachusetts, United States
AU

Arielle Urman

Resident
Beth Israel Deaconess Medical Center
Boston, Massachusetts, United States
SK

Somal Khan

Research Assistant
Division of Hemostasis and Thrombosis, Beth Israel Deaconess Medical Center
Boston, Massachusetts, United States
DS

Dominick Svrcek

Research Assistant
Division of Hemostasis and Thrombosis, Beth Israel Deaconess Medical Center
Boston, Massachusetts, United States
HC

Haley Clark

Research Assistant
Division of Hemostasis and Thrombosis, Beth Israel Deaconess Medical Center
Boston, Massachusetts, United States
Rushad Patell, MBBS

Assistant Professor
Beth Israel Deaconess Medical Center
Boston, Massachusetts, United States
Jeffrey I. Zwicker, MD

Chief of Hematology Service
Hematology Service, Department of Medicine, Memorial Sloan Kettering Cancer Center, New York, NY, USA
New York, New York, United States

Presenting Author(s)

Robert Flaumenhaft, MD, PhD

Professor
BIDMC/Harvard Medical School
Boston, Massachusetts, United States

Background: We previously demonstrated a role for the mitochondrial fission protein dynamin-related protein 1 (Drp1) in platelet granule release and thrombus formation. Prior studies in nucleated cells have shown that phosphorylation of Drp1 at S616 promotes mitochondrial fission, while S637 phosphorylation prevents fission. Yet how Drp1 function is controlled in platelets has not previously been evaluated.

Aims: To define mechanisms of platelet Drp1 phosphorylation.

Methods: Platelet function assays with agonist and antagonists, Hermansky-Pudlak syndrome platelets; immunofluorescence microscopy; Western blot analysis, recombinant Drp1; and phospho-Drp1-S616- and phospho-Drp1-S637-specific quantitative ELISAs were used to evaluate platelet Drp1 phosphorylation in vitro and ex vivo from subjects following ingestion of aspirin or clopidogrel.

Results: Drp1 was abundant in platelets (~50K molecules/platelet) and localized to mitochondria, plasma membrane, and cytosol. Platelet Drp1 phosphorylation was sensitive to redox environment (e.g., nitric oxide) and to activation via platelet receptors. Stimulation through protease-activated receptor-1 (PAR1), PAR4, GPVI, P2Y12, and TP receptors resulted in phosphorylation of Drp1 at Ser616. The responsible kinase was p38. Drp1-Ser637 was phosphorylated via protein kinase A (PKA). Inhibition of P2Y12 and use of Hermansky-Pudlak syndrome platelets lacking dense granules showed that these two signaling pathways are connected via negative feedback. PKA-dependent signaling is blocked by release of ADP from platelet dense granules, permitting phosphorylation at Ser616 (Fig. 1A). Forskolin, cilostazol, PGE1, and nitric oxide stimulated Drp1 phosphorylation at S637 (Fig. 1B). Agonist-induced Drp1 phosphorylation in whole blood lysates was inhibited in subjects who ingested aspirin or clopidogrel (Fig. 2). A lateral flow assay to monitor antiplatelet therapy using whole blood was developed using anti-Drp1 antibodies (Fig. 2).

Conclusion(s): Platelet Drp1 phosphorylation is controlled by a dual mechanism. This unusual signaling circuitry renders Drp1 a useful phosphoprotein for monitoring platelet sensitivity to antagonists, leading to the development of the phospho-Drp1 assay as a new method for monitoring antiplatelet therapy.