OC 09.5 - CLEC-2 mediates ferric chloride-induced platelet activation and attenuates ferroptosis in endothelial cells.

Saturday, June 22, 2024

14:00 – 14:15 ICT

Room: 208 A-D

Presenting Author(s)

Nagaharu Tsukiji, PhD

Senior Assistant Professor/Lecturer
Department of Clinical and Laboratory Medicine, Faculty of Medicine, University of Yamanashi
Chuo, Yamanashi, Japan

Co-Author(s)

Ryohei Yokomori

Ph.D student
Department of Clinical and Laboratory Medicine, Faculty of Medicine, University of Yamanashi
Chuo, Yamanashi, Japan
KT

Kazuho Takusagawa

Resident
University of Yamanashi Hospital
Chuo, Yamanashi, Japan
Toshiaki Shirai, PhD

Assisitant Professor
Department of Clinical and Laboratory Medicine, Faculty of Medicine, University of Yamanashi
Chuo, Yamanashi, Japan
SO

Saori Oishi

Assisitant Professor
Department of Clinical and Laboratory Medicine, Faculty of Medicine, University of Yamanashi
Chuo, Yamanashi, Japan
TS

Tomoyuki Sasaki

Associate Professor
Department of Clinical and Laboratory Medicine, Faculty of Medicine, University of Yamanashi
Chuo, Yamanashi, Japan
KT

Katsuhiro Takano

Lecturer
Department of Clinical and Laboratory Medicine, Faculty of Medicine, University of Yamanashi
Chuo, Yamanashi, Japan
Katsue Suzuki-Inoue, MD, PhD

Professor
Department of Clinical and Laboratory Medicine, Faculty of Medicine, University of Yamanashi
Chuo, Yamanashi, Japan

Background: We previously reported hemin (ferric protoporphyrin-IX) as an endogenous ligand for the platelet activation receptors C-type lectin-like receptor-2 (CLEC-2) and glycoprotein VI (GPVI) (Oishi et al. 2021), and protoporphyrin-IX as a competitive inhibitor of CLEC-2 (Tsukiji et al. 2018).　Based on these reports, we hypothesized that ferric ion has an important role in hemin-stimulated platelet activation.

Aims: This study aimed to elucidate platelet activation mechanisms of ferric ion (ferric chloride) including identification of signaling pathways and receptors, and to examine whether platelets regulate ferric chloride/erastin-induced ferroptosis of endothelial cells.

Methods: Platelet aggregometry, platelet activation marker expression, and protein phosphorylation were analyzed in ferric chloride-stimulated human and murine platelets. Specific inhibitors of platelet activation signaling pathways and receptor-deleted murine platelets were used to identify the responsible signaling pathways and receptors. The effect of platelets on ferric chloride/erastin-induced ferroptosis of endothelial cells were investigated in vitro using cell death/viability assays.

Results: Ferric chloride induced platelet aggregation, CD62P/activated integrin αIIbβIII expression and SYK/PLCγ2 phosphorylation in both human and murine platelets. Ferric chloride-induced platelet activation was inhibited by Src family kinase (SFK) inhibitor. In addition, lack of platelet CLEC-2, but not GPVI, almost abolished ferric chloride-induced platelet activation in mice and recombinant CLEC-2 protein inhibited ferric chloride-induced wild type murine platelet aggregation. Furthermore, co-culture of wild type murine platelets, but not CLEC-2-deficient platelets, attenuated ferric chloride/erastin-induced ferroptosis in mouse arterial endothelial cells in vitro.

Conclusion(s): Ferric chloride induced platelet activation through CLEC-2, but not GPVI, and SFK/SYK/PLCγ2 pathway. CLEC-2 in platelets has a protective role in ferroptosis of endothelial cells.