PB0793 - Continuous flow cytometry analysis of calcium signaling and mitochondrial function in human platelets

Monday, June 24, 2024

13:45 – 14:45 ICT

Room: Exhibition Hall

Presenting Author(s)

AM

Artem A. Mishukov

Junior researcher
Center for Theoretical Problems of Physico-Chemical Pharmacology RAS
Moscow, Moskva, Russia

Co-Author(s)

AT

Anastasia Tarakanova

PhD Candidate
Center for Theoretical Problems of Physico-Chemical Pharmacology RAS, Moscow, Russia
Moscow, Moskva, Russia
AO

Anna Ogneva

Hematologist
Dmitry Rogachev National Research Center of Pediatric Hematology, Oncology, and Immunology, Moscow, Russia
Moscow, Moskva, Russia
Anna Boldova, PhD

PhD student
Center for Theoretical Problems of Physicochemical Pharmacology
Moscow, Moskva, Russia
MP

Mikhail Panteleev

Director
Center for Theoretical Problems of Physico-Сhemical Pharmacology
Moscow, Moskva, Russia
AS

Anna Shcherbina

Deputy Director of the Institute of Hematology, Immunology and Cell Technologies
Dmitry Rogachev National Medical Research Centеr of Pediatric Hematology, Oncology and Immunology
Moscow, Moskva, Russia
AS

Anastasia N. Sveshnikova

Head of Laboratory
Dmitry Rogachev National Medical Research Center Of Pediatric Hematology, Oncology and Immunology, Moscow, Russia
Moscow, Moskva, Russia

Background: Calcium signaling is central to platelet hemostatic function’s regulation. Platelet mitochondria also play essential roles in platelet activation via ATP supply, ROS generation and regulation of platelet cell death. However, there is no reliable method for observation of mitochondria activity in platelets.

Aims: Simultaneous evaluation of calcium signaling and mitochondrial activity in platelets.

Methods: Blood collection via venipuncture into blood collection tubes, supplemented with hirudin (525 ATU/mL). Continuous flow cytometry of platelets, loaded with Fura Red-AM and DiOC3(6) was performed using FACS Canto II device, platelet activation in response to ADP or SFLLRN was observed. 13 healthy donors and 7 patients with Wiskott-Aldrich syndrome (WAS) were included into the study.

Results: Activation of platelets from healthy donors with conventional activators induces mitochondrial hyperpolarization, determined by an elevation of DiOC3(6) fluorescence (H, Figure 1). These parameter strongly correlates (𝜌 = 0.85) with cytoplasmic calcium mobilization (ΔR), so the slope of an approximated line (H = A*ΔR) could be used as a parameter, characterizing mitochondrial function during activation of platelet. Also, relative mitochondrial membrane potential (Δψ) in resting platelets could be recalculated from H before and after CCCP treatment. Platelets from patients with WAS did not show significantly lower A than healthy donors (Figure 2A), however, they demonstrated a significant reduction in Δψ in resting platelets (2.43 ± 0.41 r.u. vs 1.61 ± 0.39 r.u.) (Figure 2B).

Conclusion(s): Platelet mitochondria activity could be observed by means of continues flow cytometry. The level of mitochondrial hyperpolarization strongly correlates with cytosolic calcium mobilization both in healthy donors and patients with WAS. Mitochondria in WAS platelets have decreased Δψ in resting platelets. The study was supported by Russian Science Foundation grant 23-74-00057.