PB0427 - The Role of Mitochondrial Calcium Uniporter in Platelet Signaling

Sunday, June 25, 2023

18:30 – 19:30 ET

Room: Exhibition

Presenting Author(s)

Durre Shehwar, MSc MPhil

PhD Student
Department of Biochemistry, Quaid-i-Azam University, Islamabad, Pakistan
Islamabad, Islamabad, Pakistan

Co-Author(s)

Alessandro Aliotta, PhD

Research Associate
Division and Central Laboratory of Hematology, Lausanne University Hospital (CHUV) and University of Lausanne, Lausanne, Switzerland
Lausanne, Vaud, Switzerland
SB

Saima Barki

PhD Student
Department of Biochemistry, Quaid-i-Azam University, Islamabad, Pakistan
Islamabad, Islamabad, Pakistan
Debora Bertaggia Calderara, PhD

Research Associate
Division and Central Laboratory of Hematology, Lausanne University Hospital (CHUV) and University of Lausanne, Lausanne, Switzerland
Lausanne, Vaud, Switzerland
Lucas Veuthey, BSc, MSc (he/him/his)

PhD student
Division and Central Laboratory of Hematology, Lausanne University Hospital (CHUV) and University of Lausanne, Lausanne, Switzerland
Lausanne, Vaud, Switzerland
Cindy Pereira Portela, MD Student

MD student
Division and Central Laboratory of Hematology, Lausanne University Hospital (CHUV) and University of Lausanne, Lausanne, Switzerland
Lausanne, Vaud, Switzerland
MR

Muhammad Rizwan Alam

Assistant Professor
Department of Biochemistry, Quaid-i-Azam University, Islamabad, Pakistan
Islamabad, Islamabad, Pakistan
LA

Lorenzo Alberio, MD (he/him/his)

Chief Physician, Associated Professor
Division and Central Laboratory of Hematology, Lausanne University Hospital (CHUV) and University of Lausanne, Lausanne, Switzerland
Lausanne, Vaud, Switzerland

Background: Platelet signaling triggered by different agonists causes elevation of cytosolic calcium, which contributes to platelet activation. Mitochondria sequester a part of the cytosolic calcium via Mitochondrial Calcium Uniporter (MCU) which has been reported to facilitate the generation of procoagulant platelets upon dual stimulation with thrombin and convulxin. However, its participation in platelet responses upon stimulation with individual agonists remains incompletely understood.

Aims: This study aimed to explore the role of mitochondrial calcium uptake in platelet activation.

Methods: Expression of MCU was confirmed in platelets at mRNA level by using conventional PCR. MCU was blocked with its recently identified inhibitor mitoxantrone (MTX). Lactate dehydrogenase assay, trypan blue dye exclusion assay and annexin-V binding were performed to investigate the cytotoxic effect of MTX. Platelet pre-activation by MTX was assessed by measuring P-selectin and PAC-1 using flow cytometry. Platelet activation induced by single agonists (ADP, convulxin, thrombin) was analyzed by platelet aggregation, surface expression of P-selectin, and binding of PAC-1 and Annexin V.

Results: We demonstrated that MTX did not induce platelet cytotoxicity as indicated by intact plasma membrane and lack of PS externalization; with no impact on platelet pre-activation. Interestingly, our results revealed that MTX significantly decrease ADP-induced platelet aggregation dose-dependently (Fig1A) and this data was further corroborated by reduction in P-selectin expression (Fig1B) and PAC-1 binding (Fig1C). With thrombin or convulxin as single agonists, MTX reduced aggregation, (Fig1D and 1G), P-selectin expression and PAC-1 binding (Fig1E, 1F, 1H, 1I) as well. Remarkably, we also observed an inhibitory impact of MTX on Annexin-V positive platelets upon stimulation with convulxin.

Conclusion(s): Altogether, these data revealed for the first time an activating role of MCU-dependent mitochondrial calcium transport in ADP, thrombin and convulxin stimulated platelet responses.