The Contribution of Mitochondrial Calcium Homeostasis in Agonist-Induced Platelet Responses

Abstract

Platelet agonists initiate a signaling cascade that leads to platelet activation, essential for thrombosis and hemostasis. Agonist-induced Ca2+ mobilization from intracellular stores contributes to platelet shape change, thromboxane A2 generation, platelet aggregation, granule secretion, and αIIbβ3 integrin activation. Besides its role in platelet function, cytosolic Ca2+ is also taken up by mitochondria. The influx of mitochondrial Ca2+ is strongly regulated by MCU whereas the efflux of matrix Ca2+ is carried out by NCLX. The role of cytosolic Ca2+ in platelet function is a well-known phenomenon. However, the contribution of mitochondrial Ca2+ transport in agonist-induced platelet responses is poorly explored. In this study we aimed to investigate the role of mitochondrial Ca2+ in agonist-evoked platelet activation and aggregation. Presence of MCU and NCLX was confirmed by mRNA expression showing relatively lower level of NCLX in platelets. MCU and NCLX was blocked by their pharmacological inhibitors MTX and CGP respectively. Cell death assays including Annexin V assay, LDH release and trypan blue exclusion assay were performed to exclude the cytotoxic effect and preactivation of platelets in the presence of MTX and CGP. Platelet aggregation was measured by timelapsed spectrophotometric assay and aggregometer. Platelet activation markers including PAC-1 binding and p-selectin expression and procoagulant platelets using Annexin V were measured by employing flow cytometry. Interestingly, our data revealed a promising inhibitory effect of MTX showing a remarkable decrease on ADP-induced platelet aggregation. Moreover, it significantly reduced platelet activation endpoints measured by p-selectin and PAC-1. On the contrary, we observed a striking result by inhibition of NCLX with CGP, which markedly enhanced ADP-mediated platelet aggregation and activation. Furthermore, in line with ADP-induced platelet response MTX exhibited a remarkable decrease on THR/CVX-stimulated platelet aggregation and activation. On the contrary, THR/CVX-induced platelet activation remained unaltered in the presence of CGP. In addition to this, MTX exhibited a significant effect on THR/CVX-induced procoagulant platelets. The effect of MTX on procoagulant platelets was further validated by Ru265 that is recently developed and more specific inhibitor of MCU. Altogether, the present study revealed a significant involvement of mitochondrial calcium fluxes in agonist-induced platelet responses. Keywords: Mitochondrial Ca2+, MCU, NCLX, procoagulant platelets, mitoxantrone, CGP37157.