Inhibition of mitochondrial calcium transporters alters adp-induced platelet responses

Abstract

Introduction: ADP-stimulated elevation of cytosolic Ca²⁺ is an important effector mechanism for platelet activation. The rapidly elevating cytosolic Ca²⁺ is also transported to mitochondrial matrix via Mitochondrial Ca²⁺ Uniporter (MCU) and extruded via Na⁺/Ca²⁺/Li⁺ Exchanger (NCLX). However, the exact contribution of MCU and NCLX in ADP-mediated platelet responses remains incompletely understood.

Methods and results: The present study aimed to elucidate the role of mitochondrial Ca²⁺ transport in ADP-stimulated platelet responses by inhibition of MCU and NCLX with mitoxantrone (MTX) and CGP37157 (CGP), respectively. As these inhibitory strategies are reported to cause distinct effects on matrix Ca²⁺ concentration, we hypothesized to observe opposite impact of MTX and CGP on ADP-induced platelet responses. Platelet aggregation profiling was performed by microplate-based spectrophotometery while p-selectin externalization and integrin αIIbβ3 activation were analyzed by fluorescent immunolabeling using flow cytometery. Our results confirmed the expression of both MCU and NCLX mRNAs with relatively low abundance of NCLX in human platelets. In line with our hypothesis, MTX caused a dose-dependent inhibition of ADP-induced platelet aggregation without displaying any cytotoxicity. Likewise, ADP-induced p-selectin externalization and integrin αIIbβ3 activation was also significantly attenuated in MTX-treated platelets. Concordantly, inhibition of NCLX with CGP yielded an accelerated ADP-stimulated platelet aggregation which was associated with an elevation of p-selectin surface expression and αIIbβ3 activation.

Conclusion: Together, these findings uncover a vital and hitherto poorly characterized role of mitochondrial Ca²⁺ transporters in ADP-induced platelet activation.

Keywords: CGP37157; Mitochondrial Calcium Uniporter; Mitoxantrone; Platelet Aggregation; Sodium/Calcium/Lithium Exchanger.